Omega-3多不饱和脂肪酸对人肝细胞和巨噬细胞抗炎作用的实验研究
|
摘要
研究背景及目的
危重新生儿、早产儿、先天性消化道畸形、严重消化道疾病以及消化道手术的患儿不能通过胃肠道获得足够营养时,需要用静脉营养液由静脉途径提供各种营养素,称为静脉营养,又名完全胃肠外营养(total parenteral nutrition,TPN)。静脉营养,已被儿科界尤其是新生儿界普遍公认为是一种相对安全、有效的治疗措施,能及时为患儿提供必要的能量、营养,维持患儿的生命,为进一步治疗赢得时间。但是,随着TPN在儿科领域临床应用范围的扩大,其并发症也日见增多,尤其是长期应用的患儿更容易发生。早在1971年,Peden V. H就报道了胃肠外营养相关肝损害(parental nutrition associated liver disease,PNALD),并指出PNALD是TPN常见的严重并发症,甚至可威胁患儿生命；并且随着TPN应用时间的延长,其发生率也相应增高。尤其对于那些生理机能尚未发育成熟的新生儿和婴幼儿,在长期接受TPN治疗时,更容易发生PNALD。1998年,Kelly的研究报道显示,长期接受TPN治疗的患儿中,大约有40%-60%的患儿会发展成不同程度的肝功能紊乱。同年,Sondheimer等做了一项临床研究,在42名接受六周短期TPN治疗的患儿中,约有13%的患儿发展成为肝功能衰竭。PNALD在成人和婴幼儿中的病理基础是不同的,成人以肝脂肪变性为主,而婴幼儿则以胆汁淤积最为常见。进展型PNALD可以导致肝脏损害,肝衰竭,甚至多脏器功能衰竭,一旦发生PNALD,不仅需要中断TPN的治疗,还会对原发病产生不良影响,严重者甚至危及生命。因此,PNALD的预防与治疗显得尤为重要。
目前,PNALD发病的确切机制尚不清楚,但TPN相关胆汁淤积(TPN-associated cholcstasis, PNAC)是其主要的病理基础。PNAC的发生普遍认为是多因素共同作用的结果,诸如早产儿肝脏功能不成熟、早期禁食、胃肠外营养液中营养成分失衡以及某些成分具有肝毒性、胎龄以及早期感染(如败血症、导管相关性感染、坏死性小肠结肠炎)及肠道手术等基础疾病。第一、PNAC与早产儿及新生儿尚未发育成熟的胆汁排泄系统和肝脏功能密切相关,首先,发育不成熟的胆管系统使得肝脏的摄取率降低,胆汁酸再循环障碍；其次,由于早产儿的肝脏转运和胆酸代谢功能均不完善,肝脏摄取、合成胆盐的能力降低导致胆红素的肝肠循环障碍；再次,由于胆汁在肠道内停留时间延长,在肠道细菌作用下的石胆酸形成(毒性胆盐)增多,并重吸收到肝脏,对肝细胞产生毒性作用。第二,接受TPN治疗的患儿由于不能耐受经口喂养,或喂养不足,喂养延迟,缺乏有效的胃肠道刺激,使一些胃肠激素,如胃动素、胃泌素、促胰液素、胰高血糖素、舒血管肠肽等减少,胆囊收缩力下降,导致胆汁淤积。有研究表明,早期肠内喂养的平均摄入量与胆汁淤积的发生率呈负相关。第三,不恰当的TPN及营养成分失衡,首先,例如：高热卡,静脉营养液的热卡过高使肝脏内水分、糖原和脂肪沉积增加,从而引起肝细胞肿胀,胆管堵塞,最终导致胆汁淤积。有研究显示,随着摄入热卡的降低,PNAC的发生率明显下降；其次,必需氨基酸的缺乏,输入氨基酸的量和成分亦与PNAC的发生有关。TPN溶液的肝毒性可能与酪氨酸、半胱氨酸、牛磺酸等必需氨基酸缺乏有关。牛磺酸对早产儿是一种重要的氨基酸,它与多种肝酶的活性有关,可促进胆汁流动和防止石胆酸的毒性,缺乏牛磺酸可引起胆汁淤积。第四,感染,接受静脉营养的患儿发生感染后随之出现了胆汁淤积,原因可能为细菌等病原微生物感染后导致肝酶异常,胆汁分泌减少,致病微生物产生的内毒素抑制了肝细胞膜Na+-K+-ATP酶的活性,肝胆管的转运系统受到破坏、功能失调,从而抑制了肝细胞对胆汁酸的摄取及转运。内毒素的刺激,使肝脏发生了一系列炎症级联反应,首先,肝脏Kupffer细胞产生肝毒性的细胞因子TNF-α、IL-1和IL-6；其次,肠道也作出了相应反应,即正向调节细胞因子TNF-α、IL-1和IL-6的产生,加重了肝脏的损伤。小肠蠕动的减慢和肠内细菌过度滋生,内毒素产生增加,而内毒素可以直接损害肝细胞,减少胆汁的合成及分泌,加重胆汁淤积的程度。反之,胆汁淤积导致胃肠道感染,严重感染促进胃肠道脓毒症的发生,随之而来的是全身系统性炎症反应,产生了大量细胞因子,直接抑制了肝细胞转运蛋白和酶的活性,进而又促进肝脏的纤维化,加重了原有的胆汁淤积,这样就形成了感染和胆汁淤积之间的恶性循环,进一步导致严重的PNALD。
近年来,研究最多的是静脉营养乳化脂类在PNALD的发生、发展过程中所起的重要作用。临床上应用的脂肪乳剂主要成分为脂肪酸和甘油三酯,其中的脂肪酸有多种,根据脂肪酸分子结构中碳链的长度可分为短链脂肪酸(2C-4C)、中链脂肪酸(6C-12C)和长链脂肪酸(14C-24C)。由于人体不能合成亚油酸、亚麻酸和二十碳四烯酸,这三种脂肪酸必须从外界摄取,因此称之为必需脂肪酸,其它脂肪酸称为非必需脂肪酸；脂肪酸碳链中的碳原子间有的完全以共价键结合,称为饱和脂肪酸,有的则含有不饱和双键,称为不饱和脂肪酸,含有一个不饱和双键的称为单不饱和脂肪酸,含有多个不饱和双键的称为多不饱和脂肪酸(polyunsaturated fatty acids,PUFAs)。从碳链的甲基端开始,如第一个不饱和双键位于第三个碳原子上,则称为omega-3多不饱和脂肪酸,同样还有omega-6、omega-7和omega-9多不饱和脂肪酸。长链多不饱和脂肪酸中的omega-3PUFAs和omega-6PUFAs代谢后可产生花生四烯酸类物质,但omega-3PUFAs和omega-6PUFAs的代谢产物有所不同,前者代谢生成的产物为三烯酸环氧化物和五烯酸脂氧化物,后者代谢生成的是二烯酸环氧化物和四烯酸脂氧化物。Omega-6PUFAs的代谢产物具有强烈的促进炎症反应的作用,包括收缩血管和平滑肌,提高毛细血管通透性,促进血小板聚集,白细胞趋化作用和免疫抑制；而omega-3PUFAs代谢产物的化学结构虽然与omega-6PUFAs代谢产物类似,具有相似的生理功能,但其生理作用仅为omega-6PUFAs的1/100左右,为减轻omega-6PUFAs代谢产物对机体免疫功能和炎症反应的不利影响,可以在omega-6PUFAs当中添加适量的omega-3PUFAs,可以降低毛细血管通透性,减轻水肿,抑制过度的炎症反应,减轻或避免脂肪乳剂导致的免疫抑制,改善免疫功能。二十碳五烯酸(Eicosapentaenoic acid,EPA)是一种omega-3多不饱和脂肪酸,是十八碳亚麻酸的分解产物,最初从鱼油和母乳中发现,它存在于细胞膜磷脂脂肪酸结构中。有研究表明,EPA可以改变细胞磷脂膜的成分,影响细胞膜流动性和膜上相关信号分子、酶及受体的功能,从而改变信号转导过程,来调节炎症反应。Omega-3多不饱和脂肪酸的抗炎作用还表现在它能够减少前列腺素(prostaglandins)的合成,进而降低炎性因子的释放,最终实现减少炎症反应,减轻肝脏受损的程度。虽然,临床相关研究已经证实,在静脉营养液中添加适量omega-3多不饱和脂肪酸,对长期接受TPN治疗的患儿有明显的保护作用,但对于omega-3多不饱和脂肪酸确切的作用机制还不清楚。
机体促炎和抗炎平衡的变化,反映了PNALD的状态及进展情况。在PNALD的发病过程中,促炎反应主要是通过巨噬细胞和肝脏中的Kupffer细胞介导的,它们是通过释放炎性因子来实现的,最具有代表性的是肿瘤坏死因子-α (TNF-α)和白介素-6(IL-6)。血浆TNF-α和IL-6的水平与肝脏受损的严重程度正相关。如果损伤持续存在,就会产生大量的TNF-α和IL-6,炎症的持续存在最终会导致肝硬化和瘢痕组织的形成；另一方面,抗炎因子白介素-10(IL-10),在调节过度炎症反应方面有重要的作用,有助于降低机体炎症反应的程度。
我们假设omega-3多不饱和脂肪酸可以作用于相关细胞,从而影响细胞促炎和抗炎因子的产生。本研究通过培养正常人外周血巨噬细胞和正常人肝细胞,观察在模拟外源性(LPS介导)和内源性(PGE2介导)炎症模型中,促炎因子TNF-3和IL-6分泌水平的改变,同时应用omega-3和omega-6多不饱和脂肪酸进行干预,证实omega-3多不饱和脂肪酸在抗炎反应中的重要作用。在omega-3多不饱和脂肪酸干预的巨噬细胞和LPS诱导的肝细胞联合培养的实验中,观察到促炎因子TNF-α、IL-6分泌水平的改变,从而进一步证实了omega-3多不饱和脂肪酸不仅对巨噬细胞和肝细胞有直接的抗炎作用,而且还可以通过激活的巨噬细胞对肝细胞发挥间接的保护作用。
研究方法
1、细胞的分离与培养
(1)人外周血单核细胞(Peripheral blood mononuclear cells)的分离与培养
健康人新鲜全血(购自香港红十字会输血处)通过Ficoll Paque梯度分离法中分离出单核细胞。简单来说,将ACK buffer and Ficoll加入新鲜全血中,室温下静置15分钟,用PBS缓冲液稀释后,2000rpm离心25分钟。细胞分层,将中间层细胞(主要是淋巴细胞、单核细胞和血小板)转移到新的离心管中,室温1200rpm离心7分钟,小心去掉上清液。为了除去血小板,用PBS缓冲液混悬沉淀,800rpm离心7分钟,弃掉上清并重复此过程两次。然后,加入35ml RPMI1640培养液充分混匀,1200rpm离心7分钟。去上清,用50ml巨噬细胞SFM培养液充分混匀后,加入6-well细胞培养板(2mL/well),放置于培养箱中(37℃,5%CO2)7天后,单核细胞演变成巨噬细胞,备用。
(2)正常人肝细胞系(THLE-3)的准备
正常人肝细胞系(THLE-3),购自American Type Culture Collection (ATCC,USA)培养在预先用纤维蛋白处理过的培养板中。按要求在专用的BEGM培养液中加入牛胶原蛋白-1(0.03mg/ml),牛血清白蛋白(0.01mg/ml),培养在细胞培养箱中(37℃,5%CO2),每2-3天换一次培养液,备用。
2、具体实验方案
(1) LPS、PGE2诱导的人巨噬细胞和肝细胞炎性因子TNF-α、IL-6的表达。
在人巨噬细胞和肝细胞的培养液中分别加入LPS(0.1μg/ml)、PGE2(0.1μg/ml),继续孵育8h、16h、24h、32h分别收集各时间点的上清液。用酶联免疫吸附法(ELISA)方法,精确检测TNF-α和IL-6的含量。
(2)预先用EPA、AA及EPA+AA干预巨噬细胞和肝细胞,再给予LPS、PGE2刺激细胞,观察TNF-α和IL-6的表达。
在巨噬细胞和肝细胞的培养液中加入100u M EPA、100μMAA、100μM EPA+AA的混合物(比例为1：1)培养24h后,用PBS洗两次,再加入LPS(0.1μ g/ml)、PGE2(0.1μg/ml)模拟体内外的炎症刺激,孵育24h后,收集上清液。用ELISA方法,精确检测TNF-α和IL-6含量。
(3)同时给予巨噬细胞和肝细胞刺激和保护,观察TNF-α和IL-6表达的变化。
在巨噬细胞和肝细胞的培养液中同时加入诱导炎症发生LPS(0.1μ g/ml)、PGE2(0.1μ g/ml)以及干预试剂100μM EPA、100μMAA、100μM AA EPA+AA (比例为1：1),共同培养24h后,收集上清液。用ELISA方法,精确检测TNF-α和IL-6的含量。
(4)先用LPS、PGE2诱导巨噬细胞和肝细胞,再给予EPA、AA及EPA+AA进行干预,观察TNF-α和IL-6的表达。
在巨噬细胞和肝细胞的培养液中分别加入LPS(0.1μ g/ml)、PGE2(0.1μ g/ml)培养24h后,用PBS洗两次,再向培养液中分别加入100μM EPA、100μM AA、100μM EPA+AA(比例为1：1)孵育24h后,收集上清液。用ELISA方法,精确检测TNF-α和IL-6的含量。
(5)用LPS诱导巨噬细胞,再给予EPA干预后,观察TNF-α、IL-6和IL-10在不同时间点的表达。
在巨噬细胞的培养液中加入LPS(0.1μ g/ml),继续培养24h后,用PBS洗两次,再向培养液中加入100μM EPA,继续孵育8h、16h、24h、32h后,在各个时间点分别收集上清液。用ELISA方法,精确检测TNF-α、IL-6和IL-10含量,观察其变化。
(6)联合培养LPS诱导后经EPA干预的巨噬细胞和LPS诱导的肝细胞,观察促炎因子TNF-α和IL-6表达的变化。
首先,在巨噬细胞的培养液中加入LPS(0.1μg/ml)培养24h后,用PBS洗两次,再加入100μM EPA;其次,在肝细胞的培养液中加入LPS(0.1μg/ml)培养24h；然后,将以上处理过的两种细胞联合培养24h后,收集上清液。用ELISA方法,精确检测TNF-α和IL-6的含量,观察其变化。
3、统计学分析
所有统计资料均采用x±SD描述,统计分析应用方差分析(ANOVA)和Student's t-test检验。P<0.05(*)认为差异有统计学意义。
研究结果
1、LPS对巨噬细胞的刺激比PGE2的刺激更敏感。在0.1μg/ml LPS和0.1μg/ml PGE2的刺激下,巨噬细胞均产生大量的TNF-α和IL-6,甚至比未经处理的巨噬细胞分泌的TNF-α、IL-6高出10倍以上,且在24h时,达到峰值,以后随着时间的延长,变化趋于平坦。相同的刺激作用于肝细胞,也可以观察到类似的结果,但其反应程度要远远低于巨噬细胞。用LPS和PGE2诱导细胞,模拟体内体外的炎性刺激,成功的诱导出细胞的炎症反应,但不同的细胞对不同的炎症刺激的反应程度是不同的。我们观察到,在24h时,反应最为显著。因此,在接下来的实验中,我们以24h作为研究的时间点。
2、在致炎因素刺激之前,对巨噬细胞和肝细胞均分别给予100μM EPA、100μMAA、100μM EPA+AA(比例为1：1)三种方式干预24h,再用0.1μg/ml LPS和0.1μg/ml PGE2刺激24h,检测TNF-α, IL-6的水平。发现相对于未干预组,EPA干预组TNF-α, IL-6的表达水平明显下降(p<0.05)。AA组和EPA+AA的混合物组也观察到了TNF-α、IL-6不同程度的下降,但其下降幅度远不如单独EPA干预组(p<0.05)。
3、当同时给予巨噬细胞和肝细胞刺激因素0.1μ/ml LPS、0.1μg/ml PGE2以及三种不同的干预方式进行保护时,我们可以清楚的观察到,虽然在各个干预组中TNF-α和IL-6的表达都有所下降,但只有EPA干预组变化最为显著。比较两种不同时间的干预(先给予干预还是同时给予刺激和干预),可以得出：先给予干预的效果要好于后者。
4、先给予致炎因素刺激24h后,再分别给予三种不同的干预方式进行处理。我们惊喜的发现,先刺激后干预的处理方式,可以最大程度的降低炎性因子的释放。另外,在三种不同的干预方式中,EPA干预组的效果最好,与其他两种干预方式相比有显著的统计学意义(p<0.01)。
5、既然,EPA能有效地抑制促炎因子的产生；接下来使我们好奇的是,它是否能改变抗炎因子IL-10的生成呢?在这里,进一步实验证实了,用LPS刺激24h后的巨噬细胞,给予100μM EPA进行干预,在不同的时间点8h、16h、24h、32h测出IL-10的分泌水平有着显著的提高,并且在24h时,其值达到了顶点(280.32±11.86pg/m1)。更为有趣的是,在这个时间点上,促炎因子TNF-α和IL-6则恰好降到了最低水平。
6、前面的实验数据提示我们,EPA可以显著降低被LPS刺激后的巨噬细胞分泌细胞因子TNF-α和IL-6的水平,同时提高了IL-10的分泌水平。因此我们会提出疑问：这种作用是否可以间接的通过巨噬细胞作用于肝细胞呢?于是,我们联合培养被LPS刺激后,又用EPA处理过的巨噬细胞和用LPS刺激的肝细胞。结果显示：与未处理组相比,促炎因子TNF-α和IL-6分别降低了85.2%和75.9%。
研究结论
1、用LPS和PGE2模拟体外体内炎症,成功的刺激了正常人巨噬细胞和肝细胞分泌出大量的促炎因子TNF-α和IL-6,但不同的细胞对不同的刺激因素反应程度是不同的。
2、EPA、AA以及EPA+AA(比例为1：1)的混合物都有抑制细胞炎症反应的作用,但其中以单独用EPA的效果最好。
3、不同的干预时机,其治疗效果是不同的。从实验数据中我们可以看到：在先于,同时和后于刺激因素给予三种不同的干预方式中,先刺激后干预的效果最为显著。
4、用EPA处理过的细胞,不仅促炎因子TNF-α和IL-6的水平显著降低了,同时,抗炎因子IL-10的水平也显著提高了。有趣的是,这两种作用均在24h时,达到了峰值。
5、通过联合培养EPA处理过且被LPS刺激的巨噬细胞和LPS诱导的肝细胞,检测其促炎因子TNF-α和IL-6水平的变化,我们得出：EPA不仅对巨噬细胞和肝细胞有直接的抗炎作用,而且,可以通过活化的巨噬细胞对肝细胞发挥间接的抗炎作用。
创新及意义
1、本研究首次成功培养原代人类外周血单核细胞(演变为巨噬细胞)来比较研究omega-3多不饱和脂肪酸在不同治疗阶段的作用。此外,我们模拟活体内肝脏的真实情况：聚集了大量的肝细胞和Kupffer细胞来设计实验,因此,实验结果更加有说服力。
2、本研究阐述了omega-3多不饱和脂肪酸(EPA)在100u M的浓度下,有效地抑制了被LPS和PGE2刺激的巨噬细胞和肝细胞促炎因子的产生,并且其作用要明显好于omega-6多不饱和脂肪酸(AA)及它们的混合物。这些发现帮助我们回答在临床TPN营养液的配制是单独添加omega-3多不饱和脂肪酸,还是以混合物的方式添加的问题。
3、本研究首次通过联合培养omega-3多不饱和脂肪酸干预的巨噬细胞和LPS刺激的肝细胞,证明EPA不仅可以直接作用于这些细胞,发挥抗炎作用,还可以通过激活的巨噬细胞发挥对肝细胞间接的保护作用。EPA通过两种途径抑制炎症的作用可以很好的解释omega-3多不饱和脂肪酸临床上应用于长期接受TPN患儿,可以有效地预防和缓解PNALD.这为omega-3多不饱和脂肪酸在接受TPN患儿的临床应用方面提供了强有力的理论依据。
Backgroud and objectives
Total parenteral nutrition (TPN) is essential for survival of critical premature,congenital digestive tract,serious diseases of tract and pediatric patients with digestive tract operation, and it has been generally acknowledged as an effective and relatively safe method for supplying energy and nutrients to pediatric patients. TPN is one of the major advances of neonatal medicine and can be used successfully for prolonged periods in infants who cannot be fed enterally. Parenteral nutrition minimizes the harmful impact of multiple metabolic complications and helps to maintain an optimal nutrition level. However, long-term application of TPN can cause parental nutrition associated liver disease (PNALD), which was first described by Peden et al in1971. As the duration of total parenteral nutrition increases, so does the incidence of liver complications associated with parenteral nutrition. This incidence of PNALD is higher in neonates and infants, owing to physiological immaturity. In1998, Sondheimer reported that approximately40%-60%of children on long-term TPN will develop hepatic dysfunction. Also, in the same year, Sondheimer et al. performed a study of42infants on total parenteral nutrition and in the short time of six weeks, about13%of the patients progress to liver failure. The manifestation of liver complications differs in adults and in infants. In infants, intrahepatic cholestasis is the most common liver abnormality that manifests when the therapy is total parenteral nutrition. The development of parenteral nutrition-associated liver disease (PNALD) predisposes patients to an increased incidence of sepsis, higher mortality rates, and the potential to develop irreversible liver injury, and even to endanger the life, so the prevention and treatment of PNALD is critical.
Thus far, the etiology of TPN-induced liver disease remains unknown, but TPN-associated cholcstasis (PNAC) is its main pathological basis, PNAC is thought to be the result under the action of various factors, such as liver immature of premature infants, early fasting, imbalance of nutrition composition of the parenteral nutrient solution, some components in lipid emulsion have hepatotoxicity, fetal age and early infection (such as sepsis, catheter-related infection, necrotizing enterocolitis), intestinal surgery and so on. PNAC is related to the immature bile excretion system of premature infants and newborn, the immature bile excretion system lead to the decrease of the uptake rate of liver and the disorder of bile acids recycling. The capacity of uptake and synthesis bile salts of liver is decreased and the disorder of enterohepatic circulation bilirubin because of the imperfect of the function of hepatobiliary transportation and metabolic disorder in premature infants.The decrease of the capacity of uptake and synthesis bile salts of liver and the disorder of bile acids recycling, the time of bile stay in the intestinal is prolonged, result in lithocholic acid formation increased under the effect of intestinal bacteria, and reabsorbed to liver, so that this produced toxicity to hepatocyte. Secondly, children patients receving TPN who can't be tolerated feeding by oral, or feeding deficiency, feeding delay, lack of the effective gastrointestinal stimulation, which cause a decrease in some gastrointestinal hormones, such as motilin, gastrin, secretin, glucagon, porcine Vasoactive Intestinal Peptide (VIP), and then, the contractility of gall bladder is decreased which lead to PNAC. There was a research shows that the PNAC incidence decreased more significantly with caloric intake decreased. Deficiency in essential amino acids, the amount and components of amino acid which are related to the occurrence of PNAC. Hepatotoxicity of nutrient admixture may be related to the deficiency in essential amino acids such as tyrosine, cysteine, taurine and so on. Taurine is an important amino acid for premature infants, which is related to many liver enzyme activities and promote bile flow. The deficiency of taurine will cause PNAC. Premature infants receiving TPN who have been infected, subsequently, PNAC occurred. After infected by bacteria or other pathogens, the liver enzyme became abnormal, and the bile secretion reduced. Lipopolysaccharide produced by pathogens inhibits Na+-K+-ATPase activity in liver plasma membrane. Intrahepatic bile duct transport system were damaged, so to inhibit the uptake and transport of bile acids. In liver biopsy, we can find the existence of the bile stasis, portal inflammation and ductular proliferation. PNALD also involves the downregulation of genes coding for transporters responsible for the bile acid-dependent system in endotoxin-mediated models, inducing cholestasis. Independent of how cholestasis is initiated, a cascade of events in the liver will occur in response to endotoxins. First, Kupffer cells generate hepatoxic cytokines, TNF-a, IL-1and IL-6. The intestines will upregulate the production of the same cytokines but in response to the parenteral nutrition. Fibrosis then occurs. It is caused by the overproduction of collagen by the Kupffer cells of the liver because of the stagnant bile acids caused by endotoxins slowing down the bile acid flow. This reduction in enterohepatic circulation of bile salts may further contribute to the liver injury as intestinal stasis and bacterial overgrowth of the small intestine promotes intraluminal bile salt deconjugation and the increase in the production of lithocholic acid, which impairs bile flow and produces cholestasis. This cholestasis promotes gastrointestinal sepsis, follow systemic inflammatory response includes the production of cytokines that directly inhibit the hepatocellular ion transporters essential to bile generation and also initiate a fibrogenic response by hepatic sinusoidal cells, sepsis aggravate original cholestasis,which creates a vicious cycle of severe cholestasis and sepsis. Furtherly, serious PNALD occurred.
Recently, some study shows that PNALD is related to the lipid material in nutrient admixture. In clinic, parenteral lipid emulsion are used including fatty acid and triglyceride, triglycerides were classified into short-chain fatty acids (2C-4C), medium-chain fatty acids (6C-12C) and long-chain fatty acids (14C-24C). We couldn't synthesize linoleic acid, linolenic acid and arachidonic acid which called necessary fatty acids. Other fatty acids called nonessential fatty acids. Fatty acids were classified into saturated fatty acid and unsaturated fatty acid. Multiple unsaturated double bond content is called polyunsaturated fatty acids (PUFAs). Long-chain PUFAs included omega-3PUFAs, omega-6PUFAs、omega-7PUFAs and omega-9PUFAs on the basis of the position of unsaturated double bond. The metabolic product of omega-3PUFAs and omega-6PUFAs are eicosanoids, but their products are different, the former are mainly triene-acid epoxide and pentaene-acid epoxide, the later are mainly diene-acid epoxide and arachidonic-acid lipid oxide. Omega-6PUFA is a pro-inflammatory mediator, which has potent biological effects upon the immune system, may cause constriction or dilation in vascular smooth muscle cells, they may cause aggregation of platelets, and they have leukocyte chemotaxis. Although, the metabolites of omega-3PUFAs are similar to the metabolites of omega-6PUFAs on the structure, and their physiological function are similar, the physiological function of omega-3PUFAs was one hundredth of that of omega-6PUFAs. We can add appropriate content omega-3PUFAs so that to reduce adverse effect of omega-6PUFAs on the systemic inflammatory response. Eicosapentaenoic acid (EPA) is an omega-3fatty acid, which is a breakdown product of a-linolenic acid, found in fish oils and breast milk. It suppresses the production of arachidonic acid-derived eicosanoids and is also a substrate for the synthesis of an alternative family of eicosanoids, which have many anti-inflammatory effects. Furthermore, a study showed that EPA can replace arachidonic acid (AA) on membrane phospholipid and compete cyclooxygenase and lipoxidase to relieve inflammation. EPA exists in cell membrane phospholipids and it affectes the membrane fluidity and the function of related signal molecules, receptor, enzyme on the membrane to change signal transduction. Furthermore, omega-3fatty acids inhibit the secretion of por-inflammatory factors, regulating the expression of adhesion molecule by affecting the expression of enzymes and cytokines. The protective benefits of omega-3fatty acids may be related to its ability to decrease the production of prostaglandins and subsequently, the release of other inflammatory cytokines. These reductions will inevitably lead to the decrease in the magnitude of inflammation and the severity of insult to the liver. Indeed, there have been a few case series published recently suggesting the advantages of omega-3fatty acids in the formulation of parenteral nutrition in the rescue of babies with PNALD. Despite all these significant and encouraging clinical findings, the exact mechanism of action of omega-3fatty acids in preventing PNALD is still not clear and has not been fully investigated.
As the change from pro-inflammatory to anti-inflammatory state has implications for the status and progression of PNALD in response to the initial cholestatic and steatotic insult, it is likely that both pro-inflammatory and anti-inflammatory cytokines could play a role. The pro-inflammatory state is mediated by macrophages and Kupffer cells in liver through the release of cytokines such as tumor necrosis factor-a (TNF-a) and interleukin-6(IL-6), and the plasma levels of TNF-a and IL-6correlate positively with the degree of underlying liver damage. On the other hand, interleukin-10(IL-10), an anti-inflammatory cytokine, has pleiotropic effects in regulating exaggerated immune response and the eventual termination of inflammation.
We therefore hypothesize that omega-3fatty acid (EPA) could exert its action on cells which produce these pro-inflammatory and anti-inflammatory cytokines. In this study, we observed TNF-a and IL-6secretion pattern in human marophages and Kupffer cells in different inflammatory models, in the same time, treating with omega-3fatty acid and omega-6fatty acid in the expriment, which demonstrated omega-3fatty acid play a role in inflammation. In addition, the treatment effect of omega-3fatty acid on the level of TNF-a and IL-6in a inflammatory model of co-culture human marophages and Kupffer cells was investigated. We try to elucidate that the anti-inflammatory effects of omega-3fatty acids on human marophages and Kupffer cells in order to demonstrate the protective action of the liver against hepatic steatosis and damage.
Methods
1. Cell sepration and culture
(1)Peripheral blood mononuclear cells (PBMC) sepration and culture
Peripheral blood mononuclear cells were obtained from donated blood (Hong Kong Red Cross Blood Transfusion Service) by Ficoll Paque gradient method. Briefly, ACK buffer and Ficoll were added to blood and left in room temperature for15minutes. The samples were then diluted with phosphate buffered solution (PBS) and centrifuged at2000rpm for25minutes.Cells at the interphase (lymphocytes, monocytes, and thrombocytes) were transferred to a new conical tube filled with PBS and centrifuged at1200rpm for7minutes at room temperature. The supernatant was carefully removed completely.For removal of platelets,the cell pellet was re-suspended in PBS and centrifuged at800rpm for7minutes.The supernatant was removed and repeated twice.35ml of RPMI1640medium was added and mixed, then centrifuged at1200rpm for7minutes.The pellet was re-suspended in50ml macrophage SFM medium, supplemented with L-glutamine2ml of the solution was added to6-well plates and cultured at37℃and5%CO2. After7days of incubation, mononuclear cells would change into macrophages,which could be used in further experiments.
(2)Preparation of Liver THLE-3Cells
The human liver cell line THLE-3, was purchased from the American Type Culture Collection (ATCC, USA). The cells were maintained in precoated flasks with a mixture of fibronectin (0.01mg/ml), bovine collagen type1(0.03mg/ml), and bovine serum albumin (0.01mg/ml) dissolved in BEGM medium and incubated at37℃and5%CO2.Medium was changed every2to3days.
2. Experimental Design
Macrophages and THLE-3cells were used when80%confluent.1×106of macrophages or THLE-3was seeded in each well of a6-well plate which could be used in further experiments.
(1) LPS-induced and PGE2-induced IL-6and TNF-a expression in macrophages and THLE-3cell line
Macrophages and THLE-3cells were stimulated with LPS (0.lug/ml) and PGE2(0.1μg/ml),respectively, and continue to incubate for8h,16h,24h,32h. Then the supernatant was collected at different time points for the measurement of TNF-a and IL-6concentrations using enzyme-linked immunosorbent assay (ELISA).
(2) LPS-induced and PGE2-induced IL-6and TNF-a expression in pretreated macrophages and THLE-3cells with EPA, AA and EPA+AA
100μM EPA,100μM AA or100μM EPA+100μM AA (ratio,1:1) was added to the medium of macrophages and THLE-3cells, and were incubated for24hours. Then, the cells were washed twice with PBS. All groups were then stimulated with LPS (0.1μg/ml) or PGE2(0.1μg/ml) to induce an in-vitro inflammatory condition respectively.After24h,the supernatants of each group were collected and for the measurement of TNF-a and IL-6concentrations using enzyme-linked immunosorbent assay (ELISA).
(3) Co-incubation of EPA, AA or EPA+AA with LPS or PGE2in macrophages and THLE-3cells
Simultaneously, macrophages and THLE-3cells were treated with100μM EPA,100μM AA or100μM EPA+100μM AA (ratio,1:1) and LPS (0.1μg/ml) or PGE2(0.1μg/ml) for24h. Then, the supernatants of each group were collected and for the measurement of TNF-a and IL-6concentrations using enzyme-linked immunosorbent assay (ELISA).
(4) Post-incubation with EPA, AA or EPA+AA,24h after stimulation with LPS or PGE2in macrophages and THLE-3cells
Macrophages and THLE-3cells were stimulated with LPS (0.1μg/ml) or PGE2(0.1μg/ml) for24h, then, the cells were washed twice with PBS. All groups were then treated with100μM EPA,100μM AA or100μM EPA+100μM AA (ratio,1:1), respectively. After24h, the supernatants of each group was collected and for the measurement of TNF-a and IL-6concentrations using ELISA.
(5) IL-10expression in EPA-treated macrophages with pre-stimulated with LPS
Macrophages were stimulated with LPS (0.1μg/ml) for24h, then, the cells were washed twice with PBS.100μM EPA was added into each well, and continue to incubate for8h,16h,24h,32h. Then the supernatant was collected at different time points for the measurement of IL-10concentrations using ELISA.
(6) Co-culture of EPA-treated macrophages with pre-stimulated THLE-3
Firstly, macrophages were stimulated with LPS (0.1ug/ml) for24h, after washing twice with PBS,100μM EPA was added into the medium; and then, THLE-3cells were stimulated with LPS (0.1μg/ml) for24h; finally, we co-cultured EPA-treated, LPS pre-stimulated macrophages and LPS pre-stimulated THLE-3for24h.The supernatant was collected for the measurement of TNF-α and IL-6concentrations using ELISA.
3. Statistics
All values are measured as means±SD in experiments. ANOVA and the Student's t-test were used for statistical analysis. Differences were considered significant at P<0.05(*) or P<0.01(**).
Results
1. The reaction was more sensitive under LPS stimulation than under PGE2stimulation in macrophages. In the presence of0.1μg/ml LPS and0.1μg/ml PGE2stimulation, there was an increase in TNF-α and IL-6production in macrophages when compared to the medium alone group, even to nearly ten fold above the baseline, peaking at24h time point. The same stimulation acting on THLE-3cells, a liver cell line. Here, although the variation trend of IL-6and TNF-α production were similar to those seen in macrophages, the overall response of THLE-3was much lower. We used LPS and PGE2to stimulate human peripheral blood mononuclear cells and human liver cell line (THLE-3) to induce an in-vitro inflammatory condition successfully. The level of response of different cell lines was different to different stimulation.We observed the reaction was most strongest at24-h time point, so in the following experiments, we made24-h time point as the investigative time point.
2. Before stimulation, macrophages and THLE-3cells were pre-treated with 100μM EPA,100μM AA,100μM EPA+AA (ratio,1:1) for24h, then were stimulated with0.1μg/ml LPS or0.1μg/ml PGE2for24h, we obersved the level of TNF-a and IL-6decreased significantly in EPA treatment group(p<0.05)compared with control. In AA treatment group and EPA+AA (ratio,1:1) treatment group, we found there was varying decline of the concentration of TNF-a and IL-6.However, it's descensive extent was lower than EPA treatment group(p<0.05).
3. When macrophages and THLE-3cells were given stimulating factor LPS or PGE2and five different treatment measures simultaneously. We observed that the level of TNF-a and IL-6was decreased in all groups, however, EPA treatment group was most significant.Compare the two different time point (pre-incubation or co-incubation), it was obvious that pre-incubation was more better than co-incubation.
4. After stimulated with LPS or PGE2for24h, then macrophages and THLE-3cells were treated with100μM EPA,100μM AA,100μM EPA+AA (ratio,1:1) for24h. We were surprised to find that there was a most significant decline of TNF-a and IL-6in post-incubation groups. In addition, EPA treatment group was the best than the other four treatment measures (p<0.01).
5. Since EPA could effectively suppress the production of pro-inflammatory cytokines, we next asked if it could also alter the production of an anti-inflammatory cytokine, IL-10. Here, a time chase experiment was carried out after EPA had been added to LPS-stimulated macrophages(8h,16h,24h,32h). IL-10secretion was found to be significantly increased after treatment with EPA. At24-h time point, its level reached the peak at280.32±11.86pg/ml. Interestingly, at the same time point, the levels of IL-6(110.72±12.23pg/ml) and TNF-a (170.75±15.76pg/ml) were at the lowest correspondingly.
6. The above data showed that EPA could significantly decrease the production of IL-6and TNF-a when macrophages were stimulated by LPS,with a corresponding increase in IL-10secretion. So we next asked whether this effect could be mediated indirectly through macrophages on liver cells.We co-cultured EPA-treated, LPS pre-stimulated macrophages with LPS pre-stimulated THLE-3cells for24h.Results showed that when compared with untreated group, the production of IL-6and TNF-a decreased by75.9%and by85.2%in the EPA-treated group.
Conclusions
1. We used LPS and PGE2for stimulation to mimic an in-vitro inflammatory condition in liver. Macrophages and THLE-3cells were stimulated to secrete massive pro-inflammatory cytokines TNF-a and IL-6. From the experiment data, we can draw a conclusion that the level of response of different cell lines to different stimulation was various.
2. EPA, AA and the mixture of EPA and AA (ratio,1:1) could control inflammation of macrophages and THLE-3cells stimulated by LPS and PGE2. But, the group of EPA was added alone was best among all groups.
3. Our data indicate that different treatment time had different therapentic efficacy. Among three treatment time, the effect was found to be most dramatic in the post-incubation group.
4. EPA could effectively suppress the production of pro-inflammatory cytokines, and could significantly increase anti-inflammatory cytokine IL-10.In addition, at24-h time point, the two effectes were most effective.
5. We co-cultured EPA-treated,LPS pre-stimulated macrophages and LPS pre-stimulated THLE-3,and detected the level of TNF-a and IL-6. We concluded that EPA not only had anti-inflammatory effect on macrophages and hepatocytes directly, but could indirectly reduce inflammations in hepatocytes through activated macrophages.
Innovation and significance
1. The study firstly used stimulated-human peripheral blood mononuclear cells to make a comparative study on the anti-inflammatory effect of omega-3fatty acids in different treatment time successfully. In addition, in the experiment we imitate real circumstances in liver, a mass of Kupffer cells and liver cells gathered in liver. So, the experiment results have relatively strong theory persuasiveness.
2. The study descrided that EPA (omega-3fatty acids) at100μM concentrations effectively reduced LPS-induced and PGE2-induced TNF-a and IL-6expression in macrophages and THLE-3cells.In addition,AA (omega-6fatty acids) and the mixture of EPA and AA did not seem to have the same effect. These findings may help explain the clinical benefits of EPA in pediatric patients receiving long term TPN.
3. In this study we firstly co-cultured EPA-treated,LPS pre-stimulated macrophages and LPS pre-stimulated THLE-3to carry out experiment. We demonstrated that EPA not only had anti-inflammatory effect on macrophages and hepatocytes directly, but could indirectly reduce inflammations in hepatocytes through activated macrophages.The benefits of omega-3fatty acids in TPN preserves immune function and suppress the inflammatory response.In the future, we hope that omega-3fatty acids will become a standard for both the rescue as well as prevention of PNALD.
危重新生儿、早产儿、先天性消化道畸形、严重消化道疾病以及消化道手术的患儿不能通过胃肠道获得足够营养时,需要用静脉营养液由静脉途径提供各种营养素,称为静脉营养,又名完全胃肠外营养(total parenteral nutrition,TPN)。静脉营养,已被儿科界尤其是新生儿界普遍公认为是一种相对安全、有效的治疗措施,能及时为患儿提供必要的能量、营养,维持患儿的生命,为进一步治疗赢得时间。但是,随着TPN在儿科领域临床应用范围的扩大,其并发症也日见增多,尤其是长期应用的患儿更容易发生。早在1971年,Peden V. H就报道了胃肠外营养相关肝损害(parental nutrition associated liver disease,PNALD),并指出PNALD是TPN常见的严重并发症,甚至可威胁患儿生命；并且随着TPN应用时间的延长,其发生率也相应增高。尤其对于那些生理机能尚未发育成熟的新生儿和婴幼儿,在长期接受TPN治疗时,更容易发生PNALD。1998年,Kelly的研究报道显示,长期接受TPN治疗的患儿中,大约有40%-60%的患儿会发展成不同程度的肝功能紊乱。同年,Sondheimer等做了一项临床研究,在42名接受六周短期TPN治疗的患儿中,约有13%的患儿发展成为肝功能衰竭。PNALD在成人和婴幼儿中的病理基础是不同的,成人以肝脂肪变性为主,而婴幼儿则以胆汁淤积最为常见。进展型PNALD可以导致肝脏损害,肝衰竭,甚至多脏器功能衰竭,一旦发生PNALD,不仅需要中断TPN的治疗,还会对原发病产生不良影响,严重者甚至危及生命。因此,PNALD的预防与治疗显得尤为重要。
目前,PNALD发病的确切机制尚不清楚,但TPN相关胆汁淤积(TPN-associated cholcstasis, PNAC)是其主要的病理基础。PNAC的发生普遍认为是多因素共同作用的结果,诸如早产儿肝脏功能不成熟、早期禁食、胃肠外营养液中营养成分失衡以及某些成分具有肝毒性、胎龄以及早期感染(如败血症、导管相关性感染、坏死性小肠结肠炎)及肠道手术等基础疾病。第一、PNAC与早产儿及新生儿尚未发育成熟的胆汁排泄系统和肝脏功能密切相关,首先,发育不成熟的胆管系统使得肝脏的摄取率降低,胆汁酸再循环障碍；其次,由于早产儿的肝脏转运和胆酸代谢功能均不完善,肝脏摄取、合成胆盐的能力降低导致胆红素的肝肠循环障碍；再次,由于胆汁在肠道内停留时间延长,在肠道细菌作用下的石胆酸形成(毒性胆盐)增多,并重吸收到肝脏,对肝细胞产生毒性作用。第二,接受TPN治疗的患儿由于不能耐受经口喂养,或喂养不足,喂养延迟,缺乏有效的胃肠道刺激,使一些胃肠激素,如胃动素、胃泌素、促胰液素、胰高血糖素、舒血管肠肽等减少,胆囊收缩力下降,导致胆汁淤积。有研究表明,早期肠内喂养的平均摄入量与胆汁淤积的发生率呈负相关。第三,不恰当的TPN及营养成分失衡,首先,例如：高热卡,静脉营养液的热卡过高使肝脏内水分、糖原和脂肪沉积增加,从而引起肝细胞肿胀,胆管堵塞,最终导致胆汁淤积。有研究显示,随着摄入热卡的降低,PNAC的发生率明显下降；其次,必需氨基酸的缺乏,输入氨基酸的量和成分亦与PNAC的发生有关。TPN溶液的肝毒性可能与酪氨酸、半胱氨酸、牛磺酸等必需氨基酸缺乏有关。牛磺酸对早产儿是一种重要的氨基酸,它与多种肝酶的活性有关,可促进胆汁流动和防止石胆酸的毒性,缺乏牛磺酸可引起胆汁淤积。第四,感染,接受静脉营养的患儿发生感染后随之出现了胆汁淤积,原因可能为细菌等病原微生物感染后导致肝酶异常,胆汁分泌减少,致病微生物产生的内毒素抑制了肝细胞膜Na+-K+-ATP酶的活性,肝胆管的转运系统受到破坏、功能失调,从而抑制了肝细胞对胆汁酸的摄取及转运。内毒素的刺激,使肝脏发生了一系列炎症级联反应,首先,肝脏Kupffer细胞产生肝毒性的细胞因子TNF-α、IL-1和IL-6；其次,肠道也作出了相应反应,即正向调节细胞因子TNF-α、IL-1和IL-6的产生,加重了肝脏的损伤。小肠蠕动的减慢和肠内细菌过度滋生,内毒素产生增加,而内毒素可以直接损害肝细胞,减少胆汁的合成及分泌,加重胆汁淤积的程度。反之,胆汁淤积导致胃肠道感染,严重感染促进胃肠道脓毒症的发生,随之而来的是全身系统性炎症反应,产生了大量细胞因子,直接抑制了肝细胞转运蛋白和酶的活性,进而又促进肝脏的纤维化,加重了原有的胆汁淤积,这样就形成了感染和胆汁淤积之间的恶性循环,进一步导致严重的PNALD。
近年来,研究最多的是静脉营养乳化脂类在PNALD的发生、发展过程中所起的重要作用。临床上应用的脂肪乳剂主要成分为脂肪酸和甘油三酯,其中的脂肪酸有多种,根据脂肪酸分子结构中碳链的长度可分为短链脂肪酸(2C-4C)、中链脂肪酸(6C-12C)和长链脂肪酸(14C-24C)。由于人体不能合成亚油酸、亚麻酸和二十碳四烯酸,这三种脂肪酸必须从外界摄取,因此称之为必需脂肪酸,其它脂肪酸称为非必需脂肪酸；脂肪酸碳链中的碳原子间有的完全以共价键结合,称为饱和脂肪酸,有的则含有不饱和双键,称为不饱和脂肪酸,含有一个不饱和双键的称为单不饱和脂肪酸,含有多个不饱和双键的称为多不饱和脂肪酸(polyunsaturated fatty acids,PUFAs)。从碳链的甲基端开始,如第一个不饱和双键位于第三个碳原子上,则称为omega-3多不饱和脂肪酸,同样还有omega-6、omega-7和omega-9多不饱和脂肪酸。长链多不饱和脂肪酸中的omega-3PUFAs和omega-6PUFAs代谢后可产生花生四烯酸类物质,但omega-3PUFAs和omega-6PUFAs的代谢产物有所不同,前者代谢生成的产物为三烯酸环氧化物和五烯酸脂氧化物,后者代谢生成的是二烯酸环氧化物和四烯酸脂氧化物。Omega-6PUFAs的代谢产物具有强烈的促进炎症反应的作用,包括收缩血管和平滑肌,提高毛细血管通透性,促进血小板聚集,白细胞趋化作用和免疫抑制；而omega-3PUFAs代谢产物的化学结构虽然与omega-6PUFAs代谢产物类似,具有相似的生理功能,但其生理作用仅为omega-6PUFAs的1/100左右,为减轻omega-6PUFAs代谢产物对机体免疫功能和炎症反应的不利影响,可以在omega-6PUFAs当中添加适量的omega-3PUFAs,可以降低毛细血管通透性,减轻水肿,抑制过度的炎症反应,减轻或避免脂肪乳剂导致的免疫抑制,改善免疫功能。二十碳五烯酸(Eicosapentaenoic acid,EPA)是一种omega-3多不饱和脂肪酸,是十八碳亚麻酸的分解产物,最初从鱼油和母乳中发现,它存在于细胞膜磷脂脂肪酸结构中。有研究表明,EPA可以改变细胞磷脂膜的成分,影响细胞膜流动性和膜上相关信号分子、酶及受体的功能,从而改变信号转导过程,来调节炎症反应。Omega-3多不饱和脂肪酸的抗炎作用还表现在它能够减少前列腺素(prostaglandins)的合成,进而降低炎性因子的释放,最终实现减少炎症反应,减轻肝脏受损的程度。虽然,临床相关研究已经证实,在静脉营养液中添加适量omega-3多不饱和脂肪酸,对长期接受TPN治疗的患儿有明显的保护作用,但对于omega-3多不饱和脂肪酸确切的作用机制还不清楚。
机体促炎和抗炎平衡的变化,反映了PNALD的状态及进展情况。在PNALD的发病过程中,促炎反应主要是通过巨噬细胞和肝脏中的Kupffer细胞介导的,它们是通过释放炎性因子来实现的,最具有代表性的是肿瘤坏死因子-α (TNF-α)和白介素-6(IL-6)。血浆TNF-α和IL-6的水平与肝脏受损的严重程度正相关。如果损伤持续存在,就会产生大量的TNF-α和IL-6,炎症的持续存在最终会导致肝硬化和瘢痕组织的形成；另一方面,抗炎因子白介素-10(IL-10),在调节过度炎症反应方面有重要的作用,有助于降低机体炎症反应的程度。
我们假设omega-3多不饱和脂肪酸可以作用于相关细胞,从而影响细胞促炎和抗炎因子的产生。本研究通过培养正常人外周血巨噬细胞和正常人肝细胞,观察在模拟外源性(LPS介导)和内源性(PGE2介导)炎症模型中,促炎因子TNF-3和IL-6分泌水平的改变,同时应用omega-3和omega-6多不饱和脂肪酸进行干预,证实omega-3多不饱和脂肪酸在抗炎反应中的重要作用。在omega-3多不饱和脂肪酸干预的巨噬细胞和LPS诱导的肝细胞联合培养的实验中,观察到促炎因子TNF-α、IL-6分泌水平的改变,从而进一步证实了omega-3多不饱和脂肪酸不仅对巨噬细胞和肝细胞有直接的抗炎作用,而且还可以通过激活的巨噬细胞对肝细胞发挥间接的保护作用。
研究方法
1、细胞的分离与培养
(1)人外周血单核细胞(Peripheral blood mononuclear cells)的分离与培养
健康人新鲜全血(购自香港红十字会输血处)通过Ficoll Paque梯度分离法中分离出单核细胞。简单来说,将ACK buffer and Ficoll加入新鲜全血中,室温下静置15分钟,用PBS缓冲液稀释后,2000rpm离心25分钟。细胞分层,将中间层细胞(主要是淋巴细胞、单核细胞和血小板)转移到新的离心管中,室温1200rpm离心7分钟,小心去掉上清液。为了除去血小板,用PBS缓冲液混悬沉淀,800rpm离心7分钟,弃掉上清并重复此过程两次。然后,加入35ml RPMI1640培养液充分混匀,1200rpm离心7分钟。去上清,用50ml巨噬细胞SFM培养液充分混匀后,加入6-well细胞培养板(2mL/well),放置于培养箱中(37℃,5%CO2)7天后,单核细胞演变成巨噬细胞,备用。
(2)正常人肝细胞系(THLE-3)的准备
正常人肝细胞系(THLE-3),购自American Type Culture Collection (ATCC,USA)培养在预先用纤维蛋白处理过的培养板中。按要求在专用的BEGM培养液中加入牛胶原蛋白-1(0.03mg/ml),牛血清白蛋白(0.01mg/ml),培养在细胞培养箱中(37℃,5%CO2),每2-3天换一次培养液,备用。
2、具体实验方案
(1) LPS、PGE2诱导的人巨噬细胞和肝细胞炎性因子TNF-α、IL-6的表达。
在人巨噬细胞和肝细胞的培养液中分别加入LPS(0.1μg/ml)、PGE2(0.1μg/ml),继续孵育8h、16h、24h、32h分别收集各时间点的上清液。用酶联免疫吸附法(ELISA)方法,精确检测TNF-α和IL-6的含量。
(2)预先用EPA、AA及EPA+AA干预巨噬细胞和肝细胞,再给予LPS、PGE2刺激细胞,观察TNF-α和IL-6的表达。
在巨噬细胞和肝细胞的培养液中加入100u M EPA、100μMAA、100μM EPA+AA的混合物(比例为1：1)培养24h后,用PBS洗两次,再加入LPS(0.1μ g/ml)、PGE2(0.1μg/ml)模拟体内外的炎症刺激,孵育24h后,收集上清液。用ELISA方法,精确检测TNF-α和IL-6含量。
(3)同时给予巨噬细胞和肝细胞刺激和保护,观察TNF-α和IL-6表达的变化。
在巨噬细胞和肝细胞的培养液中同时加入诱导炎症发生LPS(0.1μ g/ml)、PGE2(0.1μ g/ml)以及干预试剂100μM EPA、100μMAA、100μM AA EPA+AA (比例为1：1),共同培养24h后,收集上清液。用ELISA方法,精确检测TNF-α和IL-6的含量。
(4)先用LPS、PGE2诱导巨噬细胞和肝细胞,再给予EPA、AA及EPA+AA进行干预,观察TNF-α和IL-6的表达。
在巨噬细胞和肝细胞的培养液中分别加入LPS(0.1μ g/ml)、PGE2(0.1μ g/ml)培养24h后,用PBS洗两次,再向培养液中分别加入100μM EPA、100μM AA、100μM EPA+AA(比例为1：1)孵育24h后,收集上清液。用ELISA方法,精确检测TNF-α和IL-6的含量。
(5)用LPS诱导巨噬细胞,再给予EPA干预后,观察TNF-α、IL-6和IL-10在不同时间点的表达。
在巨噬细胞的培养液中加入LPS(0.1μ g/ml),继续培养24h后,用PBS洗两次,再向培养液中加入100μM EPA,继续孵育8h、16h、24h、32h后,在各个时间点分别收集上清液。用ELISA方法,精确检测TNF-α、IL-6和IL-10含量,观察其变化。
(6)联合培养LPS诱导后经EPA干预的巨噬细胞和LPS诱导的肝细胞,观察促炎因子TNF-α和IL-6表达的变化。
首先,在巨噬细胞的培养液中加入LPS(0.1μg/ml)培养24h后,用PBS洗两次,再加入100μM EPA;其次,在肝细胞的培养液中加入LPS(0.1μg/ml)培养24h；然后,将以上处理过的两种细胞联合培养24h后,收集上清液。用ELISA方法,精确检测TNF-α和IL-6的含量,观察其变化。
3、统计学分析
所有统计资料均采用x±SD描述,统计分析应用方差分析(ANOVA)和Student's t-test检验。P<0.05(*)认为差异有统计学意义。
研究结果
1、LPS对巨噬细胞的刺激比PGE2的刺激更敏感。在0.1μg/ml LPS和0.1μg/ml PGE2的刺激下,巨噬细胞均产生大量的TNF-α和IL-6,甚至比未经处理的巨噬细胞分泌的TNF-α、IL-6高出10倍以上,且在24h时,达到峰值,以后随着时间的延长,变化趋于平坦。相同的刺激作用于肝细胞,也可以观察到类似的结果,但其反应程度要远远低于巨噬细胞。用LPS和PGE2诱导细胞,模拟体内体外的炎性刺激,成功的诱导出细胞的炎症反应,但不同的细胞对不同的炎症刺激的反应程度是不同的。我们观察到,在24h时,反应最为显著。因此,在接下来的实验中,我们以24h作为研究的时间点。
2、在致炎因素刺激之前,对巨噬细胞和肝细胞均分别给予100μM EPA、100μMAA、100μM EPA+AA(比例为1：1)三种方式干预24h,再用0.1μg/ml LPS和0.1μg/ml PGE2刺激24h,检测TNF-α, IL-6的水平。发现相对于未干预组,EPA干预组TNF-α, IL-6的表达水平明显下降(p<0.05)。AA组和EPA+AA的混合物组也观察到了TNF-α、IL-6不同程度的下降,但其下降幅度远不如单独EPA干预组(p<0.05)。
3、当同时给予巨噬细胞和肝细胞刺激因素0.1μ/ml LPS、0.1μg/ml PGE2以及三种不同的干预方式进行保护时,我们可以清楚的观察到,虽然在各个干预组中TNF-α和IL-6的表达都有所下降,但只有EPA干预组变化最为显著。比较两种不同时间的干预(先给予干预还是同时给予刺激和干预),可以得出：先给予干预的效果要好于后者。
4、先给予致炎因素刺激24h后,再分别给予三种不同的干预方式进行处理。我们惊喜的发现,先刺激后干预的处理方式,可以最大程度的降低炎性因子的释放。另外,在三种不同的干预方式中,EPA干预组的效果最好,与其他两种干预方式相比有显著的统计学意义(p<0.01)。
5、既然,EPA能有效地抑制促炎因子的产生；接下来使我们好奇的是,它是否能改变抗炎因子IL-10的生成呢?在这里,进一步实验证实了,用LPS刺激24h后的巨噬细胞,给予100μM EPA进行干预,在不同的时间点8h、16h、24h、32h测出IL-10的分泌水平有着显著的提高,并且在24h时,其值达到了顶点(280.32±11.86pg/m1)。更为有趣的是,在这个时间点上,促炎因子TNF-α和IL-6则恰好降到了最低水平。
6、前面的实验数据提示我们,EPA可以显著降低被LPS刺激后的巨噬细胞分泌细胞因子TNF-α和IL-6的水平,同时提高了IL-10的分泌水平。因此我们会提出疑问：这种作用是否可以间接的通过巨噬细胞作用于肝细胞呢?于是,我们联合培养被LPS刺激后,又用EPA处理过的巨噬细胞和用LPS刺激的肝细胞。结果显示：与未处理组相比,促炎因子TNF-α和IL-6分别降低了85.2%和75.9%。
研究结论
1、用LPS和PGE2模拟体外体内炎症,成功的刺激了正常人巨噬细胞和肝细胞分泌出大量的促炎因子TNF-α和IL-6,但不同的细胞对不同的刺激因素反应程度是不同的。
2、EPA、AA以及EPA+AA(比例为1：1)的混合物都有抑制细胞炎症反应的作用,但其中以单独用EPA的效果最好。
3、不同的干预时机,其治疗效果是不同的。从实验数据中我们可以看到：在先于,同时和后于刺激因素给予三种不同的干预方式中,先刺激后干预的效果最为显著。
4、用EPA处理过的细胞,不仅促炎因子TNF-α和IL-6的水平显著降低了,同时,抗炎因子IL-10的水平也显著提高了。有趣的是,这两种作用均在24h时,达到了峰值。
5、通过联合培养EPA处理过且被LPS刺激的巨噬细胞和LPS诱导的肝细胞,检测其促炎因子TNF-α和IL-6水平的变化,我们得出：EPA不仅对巨噬细胞和肝细胞有直接的抗炎作用,而且,可以通过活化的巨噬细胞对肝细胞发挥间接的抗炎作用。
创新及意义
1、本研究首次成功培养原代人类外周血单核细胞(演变为巨噬细胞)来比较研究omega-3多不饱和脂肪酸在不同治疗阶段的作用。此外,我们模拟活体内肝脏的真实情况：聚集了大量的肝细胞和Kupffer细胞来设计实验,因此,实验结果更加有说服力。
2、本研究阐述了omega-3多不饱和脂肪酸(EPA)在100u M的浓度下,有效地抑制了被LPS和PGE2刺激的巨噬细胞和肝细胞促炎因子的产生,并且其作用要明显好于omega-6多不饱和脂肪酸(AA)及它们的混合物。这些发现帮助我们回答在临床TPN营养液的配制是单独添加omega-3多不饱和脂肪酸,还是以混合物的方式添加的问题。
3、本研究首次通过联合培养omega-3多不饱和脂肪酸干预的巨噬细胞和LPS刺激的肝细胞,证明EPA不仅可以直接作用于这些细胞,发挥抗炎作用,还可以通过激活的巨噬细胞发挥对肝细胞间接的保护作用。EPA通过两种途径抑制炎症的作用可以很好的解释omega-3多不饱和脂肪酸临床上应用于长期接受TPN患儿,可以有效地预防和缓解PNALD.这为omega-3多不饱和脂肪酸在接受TPN患儿的临床应用方面提供了强有力的理论依据。
Backgroud and objectives
Total parenteral nutrition (TPN) is essential for survival of critical premature,congenital digestive tract,serious diseases of tract and pediatric patients with digestive tract operation, and it has been generally acknowledged as an effective and relatively safe method for supplying energy and nutrients to pediatric patients. TPN is one of the major advances of neonatal medicine and can be used successfully for prolonged periods in infants who cannot be fed enterally. Parenteral nutrition minimizes the harmful impact of multiple metabolic complications and helps to maintain an optimal nutrition level. However, long-term application of TPN can cause parental nutrition associated liver disease (PNALD), which was first described by Peden et al in1971. As the duration of total parenteral nutrition increases, so does the incidence of liver complications associated with parenteral nutrition. This incidence of PNALD is higher in neonates and infants, owing to physiological immaturity. In1998, Sondheimer reported that approximately40%-60%of children on long-term TPN will develop hepatic dysfunction. Also, in the same year, Sondheimer et al. performed a study of42infants on total parenteral nutrition and in the short time of six weeks, about13%of the patients progress to liver failure. The manifestation of liver complications differs in adults and in infants. In infants, intrahepatic cholestasis is the most common liver abnormality that manifests when the therapy is total parenteral nutrition. The development of parenteral nutrition-associated liver disease (PNALD) predisposes patients to an increased incidence of sepsis, higher mortality rates, and the potential to develop irreversible liver injury, and even to endanger the life, so the prevention and treatment of PNALD is critical.
Thus far, the etiology of TPN-induced liver disease remains unknown, but TPN-associated cholcstasis (PNAC) is its main pathological basis, PNAC is thought to be the result under the action of various factors, such as liver immature of premature infants, early fasting, imbalance of nutrition composition of the parenteral nutrient solution, some components in lipid emulsion have hepatotoxicity, fetal age and early infection (such as sepsis, catheter-related infection, necrotizing enterocolitis), intestinal surgery and so on. PNAC is related to the immature bile excretion system of premature infants and newborn, the immature bile excretion system lead to the decrease of the uptake rate of liver and the disorder of bile acids recycling. The capacity of uptake and synthesis bile salts of liver is decreased and the disorder of enterohepatic circulation bilirubin because of the imperfect of the function of hepatobiliary transportation and metabolic disorder in premature infants.The decrease of the capacity of uptake and synthesis bile salts of liver and the disorder of bile acids recycling, the time of bile stay in the intestinal is prolonged, result in lithocholic acid formation increased under the effect of intestinal bacteria, and reabsorbed to liver, so that this produced toxicity to hepatocyte. Secondly, children patients receving TPN who can't be tolerated feeding by oral, or feeding deficiency, feeding delay, lack of the effective gastrointestinal stimulation, which cause a decrease in some gastrointestinal hormones, such as motilin, gastrin, secretin, glucagon, porcine Vasoactive Intestinal Peptide (VIP), and then, the contractility of gall bladder is decreased which lead to PNAC. There was a research shows that the PNAC incidence decreased more significantly with caloric intake decreased. Deficiency in essential amino acids, the amount and components of amino acid which are related to the occurrence of PNAC. Hepatotoxicity of nutrient admixture may be related to the deficiency in essential amino acids such as tyrosine, cysteine, taurine and so on. Taurine is an important amino acid for premature infants, which is related to many liver enzyme activities and promote bile flow. The deficiency of taurine will cause PNAC. Premature infants receiving TPN who have been infected, subsequently, PNAC occurred. After infected by bacteria or other pathogens, the liver enzyme became abnormal, and the bile secretion reduced. Lipopolysaccharide produced by pathogens inhibits Na+-K+-ATPase activity in liver plasma membrane. Intrahepatic bile duct transport system were damaged, so to inhibit the uptake and transport of bile acids. In liver biopsy, we can find the existence of the bile stasis, portal inflammation and ductular proliferation. PNALD also involves the downregulation of genes coding for transporters responsible for the bile acid-dependent system in endotoxin-mediated models, inducing cholestasis. Independent of how cholestasis is initiated, a cascade of events in the liver will occur in response to endotoxins. First, Kupffer cells generate hepatoxic cytokines, TNF-a, IL-1and IL-6. The intestines will upregulate the production of the same cytokines but in response to the parenteral nutrition. Fibrosis then occurs. It is caused by the overproduction of collagen by the Kupffer cells of the liver because of the stagnant bile acids caused by endotoxins slowing down the bile acid flow. This reduction in enterohepatic circulation of bile salts may further contribute to the liver injury as intestinal stasis and bacterial overgrowth of the small intestine promotes intraluminal bile salt deconjugation and the increase in the production of lithocholic acid, which impairs bile flow and produces cholestasis. This cholestasis promotes gastrointestinal sepsis, follow systemic inflammatory response includes the production of cytokines that directly inhibit the hepatocellular ion transporters essential to bile generation and also initiate a fibrogenic response by hepatic sinusoidal cells, sepsis aggravate original cholestasis,which creates a vicious cycle of severe cholestasis and sepsis. Furtherly, serious PNALD occurred.
Recently, some study shows that PNALD is related to the lipid material in nutrient admixture. In clinic, parenteral lipid emulsion are used including fatty acid and triglyceride, triglycerides were classified into short-chain fatty acids (2C-4C), medium-chain fatty acids (6C-12C) and long-chain fatty acids (14C-24C). We couldn't synthesize linoleic acid, linolenic acid and arachidonic acid which called necessary fatty acids. Other fatty acids called nonessential fatty acids. Fatty acids were classified into saturated fatty acid and unsaturated fatty acid. Multiple unsaturated double bond content is called polyunsaturated fatty acids (PUFAs). Long-chain PUFAs included omega-3PUFAs, omega-6PUFAs、omega-7PUFAs and omega-9PUFAs on the basis of the position of unsaturated double bond. The metabolic product of omega-3PUFAs and omega-6PUFAs are eicosanoids, but their products are different, the former are mainly triene-acid epoxide and pentaene-acid epoxide, the later are mainly diene-acid epoxide and arachidonic-acid lipid oxide. Omega-6PUFA is a pro-inflammatory mediator, which has potent biological effects upon the immune system, may cause constriction or dilation in vascular smooth muscle cells, they may cause aggregation of platelets, and they have leukocyte chemotaxis. Although, the metabolites of omega-3PUFAs are similar to the metabolites of omega-6PUFAs on the structure, and their physiological function are similar, the physiological function of omega-3PUFAs was one hundredth of that of omega-6PUFAs. We can add appropriate content omega-3PUFAs so that to reduce adverse effect of omega-6PUFAs on the systemic inflammatory response. Eicosapentaenoic acid (EPA) is an omega-3fatty acid, which is a breakdown product of a-linolenic acid, found in fish oils and breast milk. It suppresses the production of arachidonic acid-derived eicosanoids and is also a substrate for the synthesis of an alternative family of eicosanoids, which have many anti-inflammatory effects. Furthermore, a study showed that EPA can replace arachidonic acid (AA) on membrane phospholipid and compete cyclooxygenase and lipoxidase to relieve inflammation. EPA exists in cell membrane phospholipids and it affectes the membrane fluidity and the function of related signal molecules, receptor, enzyme on the membrane to change signal transduction. Furthermore, omega-3fatty acids inhibit the secretion of por-inflammatory factors, regulating the expression of adhesion molecule by affecting the expression of enzymes and cytokines. The protective benefits of omega-3fatty acids may be related to its ability to decrease the production of prostaglandins and subsequently, the release of other inflammatory cytokines. These reductions will inevitably lead to the decrease in the magnitude of inflammation and the severity of insult to the liver. Indeed, there have been a few case series published recently suggesting the advantages of omega-3fatty acids in the formulation of parenteral nutrition in the rescue of babies with PNALD. Despite all these significant and encouraging clinical findings, the exact mechanism of action of omega-3fatty acids in preventing PNALD is still not clear and has not been fully investigated.
As the change from pro-inflammatory to anti-inflammatory state has implications for the status and progression of PNALD in response to the initial cholestatic and steatotic insult, it is likely that both pro-inflammatory and anti-inflammatory cytokines could play a role. The pro-inflammatory state is mediated by macrophages and Kupffer cells in liver through the release of cytokines such as tumor necrosis factor-a (TNF-a) and interleukin-6(IL-6), and the plasma levels of TNF-a and IL-6correlate positively with the degree of underlying liver damage. On the other hand, interleukin-10(IL-10), an anti-inflammatory cytokine, has pleiotropic effects in regulating exaggerated immune response and the eventual termination of inflammation.
We therefore hypothesize that omega-3fatty acid (EPA) could exert its action on cells which produce these pro-inflammatory and anti-inflammatory cytokines. In this study, we observed TNF-a and IL-6secretion pattern in human marophages and Kupffer cells in different inflammatory models, in the same time, treating with omega-3fatty acid and omega-6fatty acid in the expriment, which demonstrated omega-3fatty acid play a role in inflammation. In addition, the treatment effect of omega-3fatty acid on the level of TNF-a and IL-6in a inflammatory model of co-culture human marophages and Kupffer cells was investigated. We try to elucidate that the anti-inflammatory effects of omega-3fatty acids on human marophages and Kupffer cells in order to demonstrate the protective action of the liver against hepatic steatosis and damage.
Methods
1. Cell sepration and culture
(1)Peripheral blood mononuclear cells (PBMC) sepration and culture
Peripheral blood mononuclear cells were obtained from donated blood (Hong Kong Red Cross Blood Transfusion Service) by Ficoll Paque gradient method. Briefly, ACK buffer and Ficoll were added to blood and left in room temperature for15minutes. The samples were then diluted with phosphate buffered solution (PBS) and centrifuged at2000rpm for25minutes.Cells at the interphase (lymphocytes, monocytes, and thrombocytes) were transferred to a new conical tube filled with PBS and centrifuged at1200rpm for7minutes at room temperature. The supernatant was carefully removed completely.For removal of platelets,the cell pellet was re-suspended in PBS and centrifuged at800rpm for7minutes.The supernatant was removed and repeated twice.35ml of RPMI1640medium was added and mixed, then centrifuged at1200rpm for7minutes.The pellet was re-suspended in50ml macrophage SFM medium, supplemented with L-glutamine2ml of the solution was added to6-well plates and cultured at37℃and5%CO2. After7days of incubation, mononuclear cells would change into macrophages,which could be used in further experiments.
(2)Preparation of Liver THLE-3Cells
The human liver cell line THLE-3, was purchased from the American Type Culture Collection (ATCC, USA). The cells were maintained in precoated flasks with a mixture of fibronectin (0.01mg/ml), bovine collagen type1(0.03mg/ml), and bovine serum albumin (0.01mg/ml) dissolved in BEGM medium and incubated at37℃and5%CO2.Medium was changed every2to3days.
2. Experimental Design
Macrophages and THLE-3cells were used when80%confluent.1×106of macrophages or THLE-3was seeded in each well of a6-well plate which could be used in further experiments.
(1) LPS-induced and PGE2-induced IL-6and TNF-a expression in macrophages and THLE-3cell line
Macrophages and THLE-3cells were stimulated with LPS (0.lug/ml) and PGE2(0.1μg/ml),respectively, and continue to incubate for8h,16h,24h,32h. Then the supernatant was collected at different time points for the measurement of TNF-a and IL-6concentrations using enzyme-linked immunosorbent assay (ELISA).
(2) LPS-induced and PGE2-induced IL-6and TNF-a expression in pretreated macrophages and THLE-3cells with EPA, AA and EPA+AA
100μM EPA,100μM AA or100μM EPA+100μM AA (ratio,1:1) was added to the medium of macrophages and THLE-3cells, and were incubated for24hours. Then, the cells were washed twice with PBS. All groups were then stimulated with LPS (0.1μg/ml) or PGE2(0.1μg/ml) to induce an in-vitro inflammatory condition respectively.After24h,the supernatants of each group were collected and for the measurement of TNF-a and IL-6concentrations using enzyme-linked immunosorbent assay (ELISA).
(3) Co-incubation of EPA, AA or EPA+AA with LPS or PGE2in macrophages and THLE-3cells
Simultaneously, macrophages and THLE-3cells were treated with100μM EPA,100μM AA or100μM EPA+100μM AA (ratio,1:1) and LPS (0.1μg/ml) or PGE2(0.1μg/ml) for24h. Then, the supernatants of each group were collected and for the measurement of TNF-a and IL-6concentrations using enzyme-linked immunosorbent assay (ELISA).
(4) Post-incubation with EPA, AA or EPA+AA,24h after stimulation with LPS or PGE2in macrophages and THLE-3cells
Macrophages and THLE-3cells were stimulated with LPS (0.1μg/ml) or PGE2(0.1μg/ml) for24h, then, the cells were washed twice with PBS. All groups were then treated with100μM EPA,100μM AA or100μM EPA+100μM AA (ratio,1:1), respectively. After24h, the supernatants of each group was collected and for the measurement of TNF-a and IL-6concentrations using ELISA.
(5) IL-10expression in EPA-treated macrophages with pre-stimulated with LPS
Macrophages were stimulated with LPS (0.1μg/ml) for24h, then, the cells were washed twice with PBS.100μM EPA was added into each well, and continue to incubate for8h,16h,24h,32h. Then the supernatant was collected at different time points for the measurement of IL-10concentrations using ELISA.
(6) Co-culture of EPA-treated macrophages with pre-stimulated THLE-3
Firstly, macrophages were stimulated with LPS (0.1ug/ml) for24h, after washing twice with PBS,100μM EPA was added into the medium; and then, THLE-3cells were stimulated with LPS (0.1μg/ml) for24h; finally, we co-cultured EPA-treated, LPS pre-stimulated macrophages and LPS pre-stimulated THLE-3for24h.The supernatant was collected for the measurement of TNF-α and IL-6concentrations using ELISA.
3. Statistics
All values are measured as means±SD in experiments. ANOVA and the Student's t-test were used for statistical analysis. Differences were considered significant at P<0.05(*) or P<0.01(**).
Results
1. The reaction was more sensitive under LPS stimulation than under PGE2stimulation in macrophages. In the presence of0.1μg/ml LPS and0.1μg/ml PGE2stimulation, there was an increase in TNF-α and IL-6production in macrophages when compared to the medium alone group, even to nearly ten fold above the baseline, peaking at24h time point. The same stimulation acting on THLE-3cells, a liver cell line. Here, although the variation trend of IL-6and TNF-α production were similar to those seen in macrophages, the overall response of THLE-3was much lower. We used LPS and PGE2to stimulate human peripheral blood mononuclear cells and human liver cell line (THLE-3) to induce an in-vitro inflammatory condition successfully. The level of response of different cell lines was different to different stimulation.We observed the reaction was most strongest at24-h time point, so in the following experiments, we made24-h time point as the investigative time point.
2. Before stimulation, macrophages and THLE-3cells were pre-treated with 100μM EPA,100μM AA,100μM EPA+AA (ratio,1:1) for24h, then were stimulated with0.1μg/ml LPS or0.1μg/ml PGE2for24h, we obersved the level of TNF-a and IL-6decreased significantly in EPA treatment group(p<0.05)compared with control. In AA treatment group and EPA+AA (ratio,1:1) treatment group, we found there was varying decline of the concentration of TNF-a and IL-6.However, it's descensive extent was lower than EPA treatment group(p<0.05).
3. When macrophages and THLE-3cells were given stimulating factor LPS or PGE2and five different treatment measures simultaneously. We observed that the level of TNF-a and IL-6was decreased in all groups, however, EPA treatment group was most significant.Compare the two different time point (pre-incubation or co-incubation), it was obvious that pre-incubation was more better than co-incubation.
4. After stimulated with LPS or PGE2for24h, then macrophages and THLE-3cells were treated with100μM EPA,100μM AA,100μM EPA+AA (ratio,1:1) for24h. We were surprised to find that there was a most significant decline of TNF-a and IL-6in post-incubation groups. In addition, EPA treatment group was the best than the other four treatment measures (p<0.01).
5. Since EPA could effectively suppress the production of pro-inflammatory cytokines, we next asked if it could also alter the production of an anti-inflammatory cytokine, IL-10. Here, a time chase experiment was carried out after EPA had been added to LPS-stimulated macrophages(8h,16h,24h,32h). IL-10secretion was found to be significantly increased after treatment with EPA. At24-h time point, its level reached the peak at280.32±11.86pg/ml. Interestingly, at the same time point, the levels of IL-6(110.72±12.23pg/ml) and TNF-a (170.75±15.76pg/ml) were at the lowest correspondingly.
6. The above data showed that EPA could significantly decrease the production of IL-6and TNF-a when macrophages were stimulated by LPS,with a corresponding increase in IL-10secretion. So we next asked whether this effect could be mediated indirectly through macrophages on liver cells.We co-cultured EPA-treated, LPS pre-stimulated macrophages with LPS pre-stimulated THLE-3cells for24h.Results showed that when compared with untreated group, the production of IL-6and TNF-a decreased by75.9%and by85.2%in the EPA-treated group.
Conclusions
1. We used LPS and PGE2for stimulation to mimic an in-vitro inflammatory condition in liver. Macrophages and THLE-3cells were stimulated to secrete massive pro-inflammatory cytokines TNF-a and IL-6. From the experiment data, we can draw a conclusion that the level of response of different cell lines to different stimulation was various.
2. EPA, AA and the mixture of EPA and AA (ratio,1:1) could control inflammation of macrophages and THLE-3cells stimulated by LPS and PGE2. But, the group of EPA was added alone was best among all groups.
3. Our data indicate that different treatment time had different therapentic efficacy. Among three treatment time, the effect was found to be most dramatic in the post-incubation group.
4. EPA could effectively suppress the production of pro-inflammatory cytokines, and could significantly increase anti-inflammatory cytokine IL-10.In addition, at24-h time point, the two effectes were most effective.
5. We co-cultured EPA-treated,LPS pre-stimulated macrophages and LPS pre-stimulated THLE-3,and detected the level of TNF-a and IL-6. We concluded that EPA not only had anti-inflammatory effect on macrophages and hepatocytes directly, but could indirectly reduce inflammations in hepatocytes through activated macrophages.
Innovation and significance
1. The study firstly used stimulated-human peripheral blood mononuclear cells to make a comparative study on the anti-inflammatory effect of omega-3fatty acids in different treatment time successfully. In addition, in the experiment we imitate real circumstances in liver, a mass of Kupffer cells and liver cells gathered in liver. So, the experiment results have relatively strong theory persuasiveness.
2. The study descrided that EPA (omega-3fatty acids) at100μM concentrations effectively reduced LPS-induced and PGE2-induced TNF-a and IL-6expression in macrophages and THLE-3cells.In addition,AA (omega-6fatty acids) and the mixture of EPA and AA did not seem to have the same effect. These findings may help explain the clinical benefits of EPA in pediatric patients receiving long term TPN.
3. In this study we firstly co-cultured EPA-treated,LPS pre-stimulated macrophages and LPS pre-stimulated THLE-3to carry out experiment. We demonstrated that EPA not only had anti-inflammatory effect on macrophages and hepatocytes directly, but could indirectly reduce inflammations in hepatocytes through activated macrophages.The benefits of omega-3fatty acids in TPN preserves immune function and suppress the inflammatory response.In the future, we hope that omega-3fatty acids will become a standard for both the rescue as well as prevention of PNALD.
引文
[1]Axelrod D, Kazmerski K, Iyer K. Pediatric enteral nutrition. J Parenter Enteral Nutr,2006, (30):S21-26.
[2]Cheung HM, Lam HS, Tam YH,et al.Rescue treatment of infants with intestinal failure and parenteral nutrition-associated cholestasis (PNAC) using a parenteral fish-oil-based lipid. Clin Nutr,2009,28(2):209-212.
[3]Le HD, de Meijer VE, Robinson EM,et al. Parenteral fish-oil-based lipid emulsion improves fatty acid profiles and lipids in parenteral nutrition-dependent children. Am J Clin Nutr,2011,94(3):749-758.
[4]Nehra D, Fallon EM, Puder M. The prevention and treatment of intestinal failure-associated liver disease in neonates and children. Surg Clin North Am,2011, 91(3):543-563.
[5]Andorsky DJ, Lund DP, Lillehei CW, et al. Nutritional and other postoperative management of neonates with short bowel syndrome correlates with clinical outcomes. J Pediatr,2001,139(1):27-33.
[6]Kaufman SS, Atkinson JB, Bianchi A, et al. Indications for pediatric intestinal transplantation:a position paper of the American Society of Transplantation. Pediatr Transplant,2001,5(2):80-87.
[7]Robinson DT, Ehrenkranz RA. Parenteral nutrition-associated cholestasis in small for gestational age infants.J Pediatr,2008,152(1):59-62.
[8]Venigalla S,Gourley GR.Neonatal cholestasis.Semin Perinatol,2004, 28(5):348-355.
[9]Najati N, Gharebaghi MM, Mortazavi F. Underlying etiologies of prolonged icterus in neonates. Pak J Biol Sci,2010,13(14):711-714.
[10]Emerick KM, Whitington PF. Molecular basis of neonatal cholestasis. Pediatr Clin North Am,2002,49(1):221-235.
[11]Kubota A, Yonekura T, Hoki M, et al. Total parenteral nutrition-associated intrahepatic cholestasis in infants:25 years'experience.J Pediatr Surg,2000, 35(7):1049-1051.
[12]Lourenco R, Camilo ME. Taurine:a conditionally essential amino acid in humans? An overview in health and disease. Nutr Hosp,2002,17(6):262-270.
[13]Kosterg A, Karpen S J. The role of inflammation in cholestasis:clinical and basic aspects. Semin Liver Disease,2010,30(2):186-194.
[14]Ghaemi S, Fesharaki RJ, Kelishadi R. Late onset jaundice and urinary tract infection in neonates. Indian J Pediatr,2007,74(2):139-141.
[15]Kelly DA. Liver complications of pediatric parenteral nutrition-epidemiology. Nutrition,1998,4(1):153-157.
[16]Alwayn IP, Gura K, Nose V, et al. Omega-3 fatty acid supplementation prevents hepatic steatosis in a murine model of nonalcoholic fatty liver disease. Pediatr Res, 2005,57(3):445-452.
[17]Gura KM, Lee S, Valim C,et al.Safety and efficacy of a fish-oil-based fat emulsion in the treatment of parenteral nutrition-associated liver disease. Pediatrics, 2008,121(3):e678-686.
[18]Puder M, Valim C, Meisel JA, et al. Parenteral fish oil improves outcomes in patients with parenteral nutrition-associated liver injury.Ann Surg,2009, 250(3):395-402.
[19]Mata Lopez P, Ortega RM. Omega-3 fatty acids in the prevention and control of cardiovascular disease. Eur J Clin Nutr,2003,57(l):S22-25.
[20]Leaf A, Kang JX, Xiao YF et al.Clinical prevention of sudden cardiac death by n-3 polyunsaturated fatty acids and mechanism of prevention of arrhythmias by n-3 fish oils. Circulation,2003,107 (21):2646-2652.
[21]Su KP, Huang SY, Chiu CC, et al. Omega-3 fatty acids in major depressive disorder. A preliminary double-blind,placebo-controlled trial. Eur Neuropsychopharmacol,2003,13 (4):267-271.
[22]Freeman MP. Omega-3 fatty acids in psychiatry:a review. Ann Clin Psychiatry, 2000,12 (3):159-165.
[23]Arvindakshan M, Ghate M, Ranjekar PK, et al. Supplementation with a combination of omega-3 fatty acids and antioxidants improves (vitamins E and C) the outcome of schizophrenia. Schizophr Res,2003,62 (3):195-204.
[24]Simopoulos AP. Omega-3 fatty acids in inflammation and autoimmune diseases. J Am Coll Nutr,2002,21 (6):495-505.
[25]Calder PC. Polyunsaturated fatty acids and inflammatory processes:New twists in an old tale. Biochimie,2009,91(6):791-795.
[26]Simopoulos AP. Human requirement for N-3 polyunsaturated fatty acids.Poult Sci,2000,79(7):961-970.
[27]Christensen JH. n-3 fatty acids and the risk of sudden cardiac death. Emphasis on heart rate variability. Dan Med Bull,2003,50(4):347-367.
[28]Reiner E, Tedeschi-Reiner E, Stajminger G. The role of omega-3 fatty acids from fish in prevention of cardiovascular diseases. Lijec Vjesn,2007,129(10-11):350-355.
[29]Jung UJ, Torrejon C, Tighe AP, et al. n-3 Fatty acids and cardiovascular disease: mechanisms underlying beneficial effects. Am J Clin Nutr,2008,87(6):2003S-2009S.
[30]沈杨,施小燕,吴爱.ω-3多不饱和脂肪酸对急性肺损伤患者血浆TNF-α、IL-6、I L-10的影响,海峡药学,2011,23(6)：247-250.
[31]Engler MM, Engler MB. Omega-3 fatty acids:role in cardiovascular health and disease. J Cardiovasc Nurs,2006,21(1):17-24.
[32]扶志敏,王正.鱼油脂肪乳剂对内毒素致兔急性肺损伤保护作用机制的研究.中国急救医学,2009,29(8)：715-719.
[33]胡翊群,丁磊,夏文权,等.白介素-6和白介素-8对单核细胞组织因子表达的影响.中国免疫学杂志,2000,16(9)：506-507.
[34]Bhatia M, He M, Zhang H, et al. Sepsis as a model of SIRS. Front Biosci,2009, l(14):4703-4711.
[35]Holub M, Cheng CW, et al. Mott S Neutrophils sequestered in the liver suppress the proinflammatory response of Kupffer cells to systemic bacterial infection. J Immunol,2009,183(5):3309-3316.
[36]Gregory SH, Cousens LP, van Rooijen N, et al. Complementary adhesion molecules promote neutrophil-Kupffer cell interaction and the elimination of bacteria taken up by the liver. J Immunol,2002,168(1):308-315.
[37]谢钢,李运景.全胃肠外营养长期应用引起的肝损害.药物不良反应杂志,2007,9(5)：335-338
[38]李丹,任亚娜,范华骅.巨噬细胞的分类及其调节性功能的差异.生命科学,2011,23(3)：149-254.
[39]Sabater J, Masclans JR, Sacanell J,et al. Effects of an omega-3 fatty acid-enriched lipid emulsion on eicosanoid synthesis in acute respiratory distress syndrome (ARDS):A prospective, randomized, double-blind, parallel group study. Nutr Metab (Lond),2011,8(1):22.
[40]Hughes DA, Pinder AC, Piper Z, et al. Fish oil supplementation inhibits the expression of major histocompatibility complex class Ⅱ molecules and adhesion molecules on human monocytes. Am J Clin Nutr,1996,63 (2):267-272.
[41]Kew S, Banerjee T, Minihane AM, et al. Lack of effect of foods enriched with plant-or marine-derived n-3 fatty acids on human immune function. Am J Clin Nutr, 2003,77(5):1287-1295.
[42]Miles EA, Wallace FA, Calder PC. Dietary fish oil reduces intercellular adhesion molecule 1 and scavenger receptor expression on murine macrophages. Atherosclerosis,2000,152 (1):43-50.
[43]Farkas S, Bolder U, Schlittenbauer T, et al. Conditioning of liver grafts with prostaglandins improves bile acid transport. Transplant Proc,2005,37(1):435-438.
[44]Babcock TA, Espat NJ, Helton WS, et al. NF-kappa B inhibition by omega -3 fatty acids modulates LPS-stimulated macrophage TNF-alpha transcription. Am J Physiol Lung Cell Mol Physiol,2003,284 (1):L84-89.
[45]Lo CJ, Chiu KC, Fu M, et al.Fish oil decreases macrophage tumor necrosis factor gene transcription by altering the NF kappa B activity. J Surg Res,1999,82 (2):216-221.
[46]Boutros C, Somasundar P, Razzak A, et al. Omega-3 fatty acids:investigations from cytokine regulation to pancreatic cancer gene suppression. Arch Surg,2010, 145(6):515-520.
[47]Ruan H, Pownall HJ, Lodish HF.Troglitazone antagonizes tumor necrosis factor-alpha-induced reprogramming of adipocyte gene expression by inhibiting the transcriptional regulatory functions of NF-kappaB. J Biol Chem,2003,278 (30):28181-28192
[48]Wall R, Ross RP, Fitzgerald GF, et al. Fatty acids from fish:the anti-inflammatory potential of long-chain omega-3 fatty acids. Nutr Rev,2010, 68(5):280-289.
[49]Rosso GL. Dietary n-6 and n-3 polyunsaturated fatty acids:from biochemistry to clinical implications in cardiovascular prevention.Biochem Pharmacol,2009, 77(6):937-946.
[1]Alwayn IP, Gura K, Nose V, et al. Omega-3 fatty acid supplementation prevents hepatic steatosis in a murine model of nonalcoholic fatty liver disease. Pediatr Res, 2005,57 (3):445-452.
[2]Calder PC, Grimble RF.Polyunsaturated fatty acids, inflammation and immunity. Eur J Clin Nutr,2002,56(3):S14-19.
[3]Grimm H, Mayer K, Mayser P, et al. Regulatory potential of n-3 fatty acids in immunological and inflammatory processes. Br J Nutr,2002, (87) 1:S59-67.
[4]Tsou SS, Chiu WC, Yeh CL, et al. Effects of omega-3 fatty acids on inflammatory mediators and splenocyte cytokine mRNA expressions in rats with polymicrobial sepsis. Nutrition,2008,24 (5):484-491.
[5]Diamond IR, Sterescu A, Pencharz PB, et al. Changing the paradigm:omegaven for the treatment of liver failure in pediatric short bowel syndrome. J Pediatr Gastroenterol Nutr,2009,48(2):209-215.
[6]Ekema G, Falchetti D, Boroni G, et al.Reversal of severe parenteral nutrition-associated liver disease in an infant with short bowel syndrome using parenteral fish oil (Omega-3 fatty acids). J Pediatr Surg,2008,43(6):1191-1195.
[7]Gura KM, Duggan CP, Collier SB, et al. Reversal of parenteral nutrition-associated liver disease in two infants with short bowel syndrome using parenteral fish oil:implications for future management. Pediatrics,2006, 118(1):e197-201.
[8]Chung PHY, Wong KKY, Wong RMS, et al. Clinical experience in managing pediatric patients with ultra short bowel syndrome using omega-3-fatty acid. Eur J Pediatr Surg,2010,20(2):139-142.
[9]Lang R, Patel D, Morris JJ,et al. Shaping gene expression in activated and resting primarymacrophages by IL-10. J Immunol,2002,169(5):2253-2263.
[10]Couper KN, Blount DG, Riley EM. IL-10:the master regulator of immunity to infection. J Immunol,2008,180 (9):5771-5777.
[11]Georgia Perona-Wright, Katja Mohrs,Frank M, et al. Systemic but not local infections elicit immunosuppressive IL-10production by Natural Killer cells.Cell Host Microbe,2009,6(6):503-512.
[12]Anker SD, von Haehling S. Inflammatory mediators in chronic heart failure:an overview. Heart,2004,90 (4):464-470.
[13]Moore K, Malefyt R, Coffman R, et al. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol,2001,19 (1):683-765.
[14]Bolger AP, Sharma R, von Haehling S, et al. Effect of interleukin-10 on the production of tumor necrosis factor-alpha by peripheral blood mononuclear cells from patients with chronic heart failure. Am J Cardiol,2002,90 (4):384-389.
[15]Denys A, Udalova IA, Smith C, et al. Evidence for a dual mechanism for IL-10 suppression of TNF-{alpha} production that does not involve inhibition of p38 mitogen-activated protein kinase or NF-{kappa}B in primary human macrophages. J Immunol,2002,168 (10):4837-4845.
[16]Kaur K, Sharma AK, Dhingra S, et al.Interplay of TNF-alpha and IL-10 in regulating oxidative stress in isolated adult cardiac myocytes. J Mol Cell Cardiol, 2006,41 (6):1023-1030.
[17]Nozaki N, Yamaguchi S, Yamaoka M, et al. Enhanced expression and shedding of tumor necrosis factor (TNF) receptors from mononuclear leukocytes in human heart failure. J Moll Cell Cardiol,1998, (30):2003-2012.
[18]Stumpf C, Lehner C, Yilmaz A,et al. Decrease of serum levels of the anti-inflammatory cytokine interleukin-10 in patients with advanced chronic heart failure. Clin Sci (Lond),2003,105(1):45-50.
[19]De Maria A, Fogli M, Mazza S,et al. Increased natural cytotoxicity receptor expression and relevant IL-10 production in NK cells fromchronically infected viremic HCV patients. Eur J Immunol,2007,37 (2):445-455.
[20]Maroof A, Beattie L, Zubairi S, et al.Posttranscriptional regulation of il-10gene expression allows natural killer cells to express immunoregulatory function. Immunity,2008,29 (2):295-305.
[21]Kelly DA. Liver complications of pediatric parenteral nutrition-epidemiology. Nutrition,1998,14(1):153-157.
[22]Katakura T, Miyazaki M, Kobayashi M, et al. CCL17 and IL-10 as effectors that enable alternatively activated macrophages to inhibit the generation of classically activated macrophages. J Immunol,2004,172(3):1407-1413.
[23]Gura KM, Lee S, Valim C, et al. Safety and efficacy of a fish-oil-based fat emulsion in the treatment of parenteral nutrition-associated liver disease. Pediatrics, 2008,121(3):e678-686.
[24]Van Aerde JE, Duerksen DR, Gramlich L, et al. Intravenous fish oil emulsion attenuates total parenteral nutrition-induced cholestasis in newborn piglets. Pediatr Res,1999,45 (2):202-208.
[25]Araya J, Rodrigo R, Videla LA, et al. Increase in long-chain polyunsaturated fatty acid n-6/n-3 ratio in relation to hepatic steatosis in patients with non-alcoholic fatty liver disease. Clin Sci (Lond),2004,106 (6):635-643.
[26]Babcock TA, Novak T, Ong E,et al. Modulation of lipopolysaccharide-stimulated macrophage tumor necrosis factor-aproduction by co-3 fatty acid is associated with differential cyclooxygenase-2 protein expression and is independent of interleukin-10.J Surg Res,2002,107 (1):135-139.
[27]Novak TE, Babcock TA, Jho DH,et al. NF-κB inhibition by ω-3 fatty acids modulates LPS-stimulated macrophage TNF-a transcription. Am J Physiol,2003, 284(1):L84-L89.
[28]Zhao Y, Joshi-Barve S, Barve S,et al. Eicosapentaenoic acid prevents LPS-induced TNF-a expression by preventing NF-κB activation. J Am Coll Nutr, 2004,23 (1):71-78.
[29]Weldon SM, Mullen AC, Loscher CE, et al. Docosahexaenoic acid induces an anti-inflammatory profile in lipopolysaccharide-stimulated human THP-1 macrophages more effectively than eicosapentaenoic acid. J Nutr Biochem,2007,18 (4):250-258.
[30]Wang S, Wu D, Lamon-Fava S, et al. In vitro fatty acid enrichment of macrophages alters inflammatory response and net cholesterol accumulation. Br J Nutr,2009,102 (4):497-501.
[31]Ruan H, Pownall HJ, Lodish HF.Troglitazone antagonizes tumor necrosis factor-alpha-induced reprogramming of adipocyte gene expression by inhibiting the transcriptional regulatory functions of NF-kappaB. J Biol Chem,2003,278 (30):28181-28192.
[32]Hassan A, Ibrahim A, Mbodji K, et al.An a-linolenic acid-rich formula reduces oxidative stress and inflammation by regulating NF-κB in rats with TNBS-induced colitis,2010,140(10):1714-1721.
[1]Klein CJ, Revenis M, Kusenda C,et al. Parenteral nutrition-associated conjugated hyperbilirubinemia in hospitalized infants. J Am Diet Assoc.2010,110(11):1684-95.
[2]Dawes LG, Laut HC, Woodruff M. Decreased bile acid synthesis with total parenteral nutrition.Am J Surg,2007,194(5):623-627.
[3]Tang QY, Wang Y, Feng Y,et al. Factors derived from parenteral nutrition associated with cholestasis in 612 neonates. Zhonghua Er Ke Za Zhi,2007, 45(11):838-842.
[4]Hsieh MH, Pai W, Tseng HI, et al. Parenteral nutrition-associated cholestasis in premature babies:risk factors and predictors. Pediatr Neonatol.2009,50(5):202-207.
[5]Kelly DA. Intestinal failure-associated liver disease:what do we know today? Gastroenterology.2006,130(2):S70-77.
[6]Blau J, Sridhar S, Mathieson S, et al. Effects of protein/nonprotein caloric intake on parenteral nutrition associated cholestasis in premature infants weighing 600-1000 grams. JPEN J Parenter Enteral Nutr.2007,31(6):487-490.
[7]Arnold CJ, Miller GG, Zello GA. Parenteral nutrition-associated cholestasis in neonates:the role of aluminum. Nutr Rev.2003,61(9):306-310.
[8]Wright K, Ernst KD, Gaylord MS, et al.Increased incidence of parenteral nutrition-associated cholestasis with aminosyn PF compared to trophamine. J Perinatol.2003,23(6):444-450.
[9]Spencer AU, Yu S, Tracy TF,et al. Parenteral nutrition-associated cholestasis in neonates:multivariate analysis of the potential protective effect of taurine. JPEN J Parenter Enteral Nutr.2005,29(5):337-344.
[10]Calder PC. Immunomodulation by omega-3 fatty acids. Prostaglandins Leukot Essent Fatty Acids.2007,77(5-6):327-335.
[11]Kelly DA.Preventing parenteral nutrition liver disease. Early Hum Dev,2010, 86(11):683-687.
[12]Chung PH, Wong KK, Wong RM,et al. Clinical experience in managing pediatric patients with ultra-short bowel syndrome using omega-3 fatty acid.Eur J Pediatr Surg, 2010,20(2):139-142.
[13]Puder M, Valim C, Meisel JA, et al. Parenteral fish oil improves outcomes in patients with parenteral nutrition-associated liver injury. Ann Surg,2009, 250(3):395-402.
[14]Gura KM, Lee S, Valim C, Zhou J, et al. Safety and efficacy of a fish-oil-based fat emulsion in the treatment of parenteral nutrition-associated liver disease. Pediatrics,2008,121(3):e678-686.
[15]Sterescu A, Pencharz PB, et al. Changing the paradigm:omegaven for the treatment of liver failure in pediatric short bowel syndrome. Diamond IR, J Pediatr Gastroenterol Nutr.2009,48(2):209-215.
[16]Calder PC. n-3 polyunsaturated fatty acids, inflammation, and inflammatory diseases. Am J Clin Nutr,2006,83(6):1505S-1519S.
[17]Calder PC. Polyunsaturated fatty acids and inflammation. Prostaglandins Leukot Essent Fatty Acids.2006,75(3):197-202.
[18]Geyeregger R, Zeyda M, Zlabnger G J, et a 1. Polyunsaturated fatty acid s in terfere w ith form ation of the immunological synapse. J Leukoc Biol,2005,77(5): 680-688.
[19]Wallace K, Burt AD, Wright MC. Liver fibrosis. Biochem J.2008,411(1):1-18.
[20]Deng X, Luyendyk JP, Ganey PE. Inflammatory stress and idiosyncratic hepatotoxicity:hints from animal models. Pharmacol Rev.2009,61(3):262-282.
[21]Bellinghausen I, Knop J, Saloga J. The role of interleukin 10 in the regulation of allergic immune responses. Int Arch Allergy Immunol.2001,126(2):97-101.
[22]Ling GS, Cook HT, Botto M,et al. An essential protective role of IL-10 in the immunological mechanism underlying resistance vs. susceptibility to lupus induction by dendritic cells and dying cells. Rheumatology (Oxford),2011,50(10):1773-1784.
[23]Calder PC, Yaqoob P. Postgrad Med. Understanding omega-3 polyunsaturated fatty acids.2009,121(6):148-157.
[24]Vedin I, Cederholm T, Freund Levi Y,et al. Effects of docosahexaenoic acid-rich n-3 fatty acid supplementation on cytokine release from blood mononuclear leukocytes:the OmegAD study.Am J Clin Nutr,2008,87(6):1616-1622.
[25]Fetterman JW Jr, Zdanowicz MM. Therapeutic potential of n-3 polyunsaturated fatty acids in disease. Am J Health Syst Pharm,2009,66(13):1169-1179.
[26]Simopoulos AP. Omega-3 fatty acids in inflammation and autoimmune diseases. J Am Coll Nutr,2002,21(6):495-505.
[27]Micallef MA, Garg ML. Anti-inflammatory and cardioprotective effects of n-3 polyunsaturated fatty acids and plant sterols in hyperlipidemic individuals. Atherosclerosis.2009,204(2):476-482.
[28]Elenkov IJ, Chrousos GP. Stress hormones,proinflammatory and antiinflammatory cytokines, and autoimmunity.Ann N Y Acad Sci,2002, 966:290-303.
[29]Fallon EM, Le HD, Puder M. Prevention of parenteral nutrition-associated liver disease:role of omega-3 fish oil. Curr Opin Organ Transplant.2010,15(3):334-340.
[30]Aldridge C, Razzak A, Babcock TA, et al. Lipopolysaccharide-stimulated RAW 264.7 macrophage inducible nitric oxide synthase and nitric oxide production is decreased by an omega-3 fatty acid lipid emulsion. J Surg Res.2008,149(2):296-302.
[31]Babcock TA, Helton WS, Anwar KN,et al. Synergistic anti-inflammatory activity of omega-3 lipid and rofecoxib pretreatment on macrophage proinflammatory cytokine production occurs via divergent NF-kappaB activation. JPEN J Parenter Enteral Nutr.2004,28(4):232-240.
[32]Babcock TA, Novak T, Ong E,et al. Modulation of lipopolysaccharide-stimulated macrophage tumor necrosis factor-alpha production by omega-3 fatty acid is associated with differential cyclooxygenase-2 protein expression and is independent of interleukin-10. J Surg Res.2002,107(1):135-139.
[33]Babcock TA, Helton WS, Hong D,et al. Omega-3 fatty acid lipid emulsion reduces LPS-stimulated macrophage TNF-alpha production. Surg Infect (Larchmt). 2002,3(2):145-149.
[34]Babcock TA, Kurland A, Helton WS, et al. Inhibition of activator protein-1 transcription factor activation by omega-3 fatty acid modulation of mitogen-activated protein kinase signaling kinases. JPEN J Parenter Enteral Nutr.2003,27(3):176-181.
[35]Babcock TA, Novak T, Ong E,et al. Modulation of lipopolysaccharide-stimulated macrophage tumor necrosis factor-aproduction by co-3 fatty acid is associated with differential cyclooxygenase-2 protein expression and is independent of interleukin-10. J Surg Res,2002,107(1):135-139.
[36]Boutros C, Somasundar P, Razzak A,et al. Omega-3 fatty acids:investigations from cytokine regulation to pancreatic cancer gene suppression. Arch Surg,2010, 145(6):515-520.
[37]Zhao Y, Joshi-Barve S, Barve S,et al. Eicosapentaenoic acid prevents LPS-induced TNF-a expression by preventing NF-κB activation. J Am Coll Nutr 2004,23(1):71-78.
[38]Weldon SM, Mullen AC, Loscher CE,et al. Docosahexaenoic acid induces an anti-inflammatory profile in lipopolysaccharide-stimulated human THP-1 macrophages more effectively than eicosapentaenoic acid. J Nutr Biochem,2007, 18(4):250-258.
[39]Wei Hao, Olive Y. Wong, Xuelai Liu,et al.co-3 fatty acids suppress inflammatory cytokine production by macrophages and hepatocytes. Journal of Pediatric Surgery, 2010,45(12):2412-2418
[40]Schwerbrock NM, Karlsson EA, Shi Q,et al. Fish oil-fed mice have impaired resistance to influenza infection.J Nutr,2009,139(8):1588-1594.
[41]Wang MY, Wu MH, Hsieh DY,et al. Home parenteral nutrition support in adults: experience of a medical center in Asia. J Parenter Enteral Nutr,2007,31(4):306-310.
[42]Zhang Q, Yu JC, Kang WM,et al. Effect of ω-3 Fatty Acid on Gastrointestinal Motility after Abdominal Operation in Rats.Mediators inflamm,2011,2011:152137.
[43]Sijben JW, Calder PC. Differential immunomodulation with long-chain n-3 PUFA in health and chronic disease.Proc Nutr Soc,2007,66(2):237-259.
[44]Miles EA, Banerjee T, Dooper MWBW,et al. The influence of different combinations ofy-linolenic acid, stearidonic acid and EPA on immune function in healthy young male subjects. Br J Nutr,2004,91(6):893-903.
[45]Gorjao R, Azevedo-Martins AK, Rodrigues HG,et al. Comparative effects of DHA and EPA on cell function.Pharmacol Ther,2009,122(1):56-64.
[46]Chapkin RS, Kim W, Lupton JR, et al. Dietary docosahexaenoic and eicosapentaenoic acid:emerging mediators of inflammation. Prostaglandins Lenkot Essent Fatty Acids,2009,81 (2-3):187-191.
[47]Ramakers JD, Mensink RP, Schaart G,et al. Arachidonic acid but not eicosapentaenoic acid (EPA) and oleic acid activates NF-kappaB and elevates ICAM-1 expression in Caco-2 cells. Lipids,2007,42(8):687-698.
[48]Myhrstad MC, Retterstol K, Telle-Hansen VH,et al. Effect of marine n-3 fatty acids on circulating inflammatory markers in healthy subjects and subjects with cardiovascular risk factors.Infamm Res,2011,60(4):309-319.
[49]Kasama T, Strieter RM, Lukacs NW, et al. Regulation of neutrophil-derived chemokine expression by IL-10. J Immunol,1994,152(7):3559-3569.
[50]Petit-Bertron AF, Fitting C, Cavaillon JM, et al. Adherence influences monocyte responsiveness to interleukin-10. J Leukoc Biol,2003,73(1):145-154.
[51]Spight D, Zhao B, Haas M, et al. Immunoregulatory effects of regulated, lung-targeted expression of IL-10 in vivo. Am J Physiol Lung Cell Mol Physiol,2005, 288(2):L251-265.
[52]Wanidworanun C, Strober W. Predominant role of tumor necrosis factor-alpha in human monocyte IL-10 synthesis. J Immunol,1993,151(12):6853-6861.
[53]Sierra S, Lara-Villoslada F, Olivares M, et al.11-10 expression is involved in the regulation of the immune response by omega 3 fatty acids. Nutr Hosp.2004, 19(6):376-382.
[54]LeBlanc CJ, Horohov DW, Bauer JE, et al. Effects of dietary supplementation with fish oil on in vivo production of inflammatory mediators in clinically normal dogs.Am J Vet Res,2008,69(4):486-493.
[55]Sierra S, Lara-Villoslada F, Comalada M, et al. Dietary fish oil n-3 fatty acids increase regulatory cytokine production and exert anti-inflammatory effects in two murine models of inflammation. Lipids.2006,41(12):1115-1125.
[2]Cheung HM, Lam HS, Tam YH,et al.Rescue treatment of infants with intestinal failure and parenteral nutrition-associated cholestasis (PNAC) using a parenteral fish-oil-based lipid. Clin Nutr,2009,28(2):209-212.
[3]Le HD, de Meijer VE, Robinson EM,et al. Parenteral fish-oil-based lipid emulsion improves fatty acid profiles and lipids in parenteral nutrition-dependent children. Am J Clin Nutr,2011,94(3):749-758.
[4]Nehra D, Fallon EM, Puder M. The prevention and treatment of intestinal failure-associated liver disease in neonates and children. Surg Clin North Am,2011, 91(3):543-563.
[5]Andorsky DJ, Lund DP, Lillehei CW, et al. Nutritional and other postoperative management of neonates with short bowel syndrome correlates with clinical outcomes. J Pediatr,2001,139(1):27-33.
[6]Kaufman SS, Atkinson JB, Bianchi A, et al. Indications for pediatric intestinal transplantation:a position paper of the American Society of Transplantation. Pediatr Transplant,2001,5(2):80-87.
[7]Robinson DT, Ehrenkranz RA. Parenteral nutrition-associated cholestasis in small for gestational age infants.J Pediatr,2008,152(1):59-62.
[8]Venigalla S,Gourley GR.Neonatal cholestasis.Semin Perinatol,2004, 28(5):348-355.
[9]Najati N, Gharebaghi MM, Mortazavi F. Underlying etiologies of prolonged icterus in neonates. Pak J Biol Sci,2010,13(14):711-714.
[10]Emerick KM, Whitington PF. Molecular basis of neonatal cholestasis. Pediatr Clin North Am,2002,49(1):221-235.
[11]Kubota A, Yonekura T, Hoki M, et al. Total parenteral nutrition-associated intrahepatic cholestasis in infants:25 years'experience.J Pediatr Surg,2000, 35(7):1049-1051.
[12]Lourenco R, Camilo ME. Taurine:a conditionally essential amino acid in humans? An overview in health and disease. Nutr Hosp,2002,17(6):262-270.
[13]Kosterg A, Karpen S J. The role of inflammation in cholestasis:clinical and basic aspects. Semin Liver Disease,2010,30(2):186-194.
[14]Ghaemi S, Fesharaki RJ, Kelishadi R. Late onset jaundice and urinary tract infection in neonates. Indian J Pediatr,2007,74(2):139-141.
[15]Kelly DA. Liver complications of pediatric parenteral nutrition-epidemiology. Nutrition,1998,4(1):153-157.
[16]Alwayn IP, Gura K, Nose V, et al. Omega-3 fatty acid supplementation prevents hepatic steatosis in a murine model of nonalcoholic fatty liver disease. Pediatr Res, 2005,57(3):445-452.
[17]Gura KM, Lee S, Valim C,et al.Safety and efficacy of a fish-oil-based fat emulsion in the treatment of parenteral nutrition-associated liver disease. Pediatrics, 2008,121(3):e678-686.
[18]Puder M, Valim C, Meisel JA, et al. Parenteral fish oil improves outcomes in patients with parenteral nutrition-associated liver injury.Ann Surg,2009, 250(3):395-402.
[19]Mata Lopez P, Ortega RM. Omega-3 fatty acids in the prevention and control of cardiovascular disease. Eur J Clin Nutr,2003,57(l):S22-25.
[20]Leaf A, Kang JX, Xiao YF et al.Clinical prevention of sudden cardiac death by n-3 polyunsaturated fatty acids and mechanism of prevention of arrhythmias by n-3 fish oils. Circulation,2003,107 (21):2646-2652.
[21]Su KP, Huang SY, Chiu CC, et al. Omega-3 fatty acids in major depressive disorder. A preliminary double-blind,placebo-controlled trial. Eur Neuropsychopharmacol,2003,13 (4):267-271.
[22]Freeman MP. Omega-3 fatty acids in psychiatry:a review. Ann Clin Psychiatry, 2000,12 (3):159-165.
[23]Arvindakshan M, Ghate M, Ranjekar PK, et al. Supplementation with a combination of omega-3 fatty acids and antioxidants improves (vitamins E and C) the outcome of schizophrenia. Schizophr Res,2003,62 (3):195-204.
[24]Simopoulos AP. Omega-3 fatty acids in inflammation and autoimmune diseases. J Am Coll Nutr,2002,21 (6):495-505.
[25]Calder PC. Polyunsaturated fatty acids and inflammatory processes:New twists in an old tale. Biochimie,2009,91(6):791-795.
[26]Simopoulos AP. Human requirement for N-3 polyunsaturated fatty acids.Poult Sci,2000,79(7):961-970.
[27]Christensen JH. n-3 fatty acids and the risk of sudden cardiac death. Emphasis on heart rate variability. Dan Med Bull,2003,50(4):347-367.
[28]Reiner E, Tedeschi-Reiner E, Stajminger G. The role of omega-3 fatty acids from fish in prevention of cardiovascular diseases. Lijec Vjesn,2007,129(10-11):350-355.
[29]Jung UJ, Torrejon C, Tighe AP, et al. n-3 Fatty acids and cardiovascular disease: mechanisms underlying beneficial effects. Am J Clin Nutr,2008,87(6):2003S-2009S.
[30]沈杨,施小燕,吴爱.ω-3多不饱和脂肪酸对急性肺损伤患者血浆TNF-α、IL-6、I L-10的影响,海峡药学,2011,23(6)：247-250.
[31]Engler MM, Engler MB. Omega-3 fatty acids:role in cardiovascular health and disease. J Cardiovasc Nurs,2006,21(1):17-24.
[32]扶志敏,王正.鱼油脂肪乳剂对内毒素致兔急性肺损伤保护作用机制的研究.中国急救医学,2009,29(8)：715-719.
[33]胡翊群,丁磊,夏文权,等.白介素-6和白介素-8对单核细胞组织因子表达的影响.中国免疫学杂志,2000,16(9)：506-507.
[34]Bhatia M, He M, Zhang H, et al. Sepsis as a model of SIRS. Front Biosci,2009, l(14):4703-4711.
[35]Holub M, Cheng CW, et al. Mott S Neutrophils sequestered in the liver suppress the proinflammatory response of Kupffer cells to systemic bacterial infection. J Immunol,2009,183(5):3309-3316.
[36]Gregory SH, Cousens LP, van Rooijen N, et al. Complementary adhesion molecules promote neutrophil-Kupffer cell interaction and the elimination of bacteria taken up by the liver. J Immunol,2002,168(1):308-315.
[37]谢钢,李运景.全胃肠外营养长期应用引起的肝损害.药物不良反应杂志,2007,9(5)：335-338
[38]李丹,任亚娜,范华骅.巨噬细胞的分类及其调节性功能的差异.生命科学,2011,23(3)：149-254.
[39]Sabater J, Masclans JR, Sacanell J,et al. Effects of an omega-3 fatty acid-enriched lipid emulsion on eicosanoid synthesis in acute respiratory distress syndrome (ARDS):A prospective, randomized, double-blind, parallel group study. Nutr Metab (Lond),2011,8(1):22.
[40]Hughes DA, Pinder AC, Piper Z, et al. Fish oil supplementation inhibits the expression of major histocompatibility complex class Ⅱ molecules and adhesion molecules on human monocytes. Am J Clin Nutr,1996,63 (2):267-272.
[41]Kew S, Banerjee T, Minihane AM, et al. Lack of effect of foods enriched with plant-or marine-derived n-3 fatty acids on human immune function. Am J Clin Nutr, 2003,77(5):1287-1295.
[42]Miles EA, Wallace FA, Calder PC. Dietary fish oil reduces intercellular adhesion molecule 1 and scavenger receptor expression on murine macrophages. Atherosclerosis,2000,152 (1):43-50.
[43]Farkas S, Bolder U, Schlittenbauer T, et al. Conditioning of liver grafts with prostaglandins improves bile acid transport. Transplant Proc,2005,37(1):435-438.
[44]Babcock TA, Espat NJ, Helton WS, et al. NF-kappa B inhibition by omega -3 fatty acids modulates LPS-stimulated macrophage TNF-alpha transcription. Am J Physiol Lung Cell Mol Physiol,2003,284 (1):L84-89.
[45]Lo CJ, Chiu KC, Fu M, et al.Fish oil decreases macrophage tumor necrosis factor gene transcription by altering the NF kappa B activity. J Surg Res,1999,82 (2):216-221.
[46]Boutros C, Somasundar P, Razzak A, et al. Omega-3 fatty acids:investigations from cytokine regulation to pancreatic cancer gene suppression. Arch Surg,2010, 145(6):515-520.
[47]Ruan H, Pownall HJ, Lodish HF.Troglitazone antagonizes tumor necrosis factor-alpha-induced reprogramming of adipocyte gene expression by inhibiting the transcriptional regulatory functions of NF-kappaB. J Biol Chem,2003,278 (30):28181-28192
[48]Wall R, Ross RP, Fitzgerald GF, et al. Fatty acids from fish:the anti-inflammatory potential of long-chain omega-3 fatty acids. Nutr Rev,2010, 68(5):280-289.
[49]Rosso GL. Dietary n-6 and n-3 polyunsaturated fatty acids:from biochemistry to clinical implications in cardiovascular prevention.Biochem Pharmacol,2009, 77(6):937-946.
[1]Alwayn IP, Gura K, Nose V, et al. Omega-3 fatty acid supplementation prevents hepatic steatosis in a murine model of nonalcoholic fatty liver disease. Pediatr Res, 2005,57 (3):445-452.
[2]Calder PC, Grimble RF.Polyunsaturated fatty acids, inflammation and immunity. Eur J Clin Nutr,2002,56(3):S14-19.
[3]Grimm H, Mayer K, Mayser P, et al. Regulatory potential of n-3 fatty acids in immunological and inflammatory processes. Br J Nutr,2002, (87) 1:S59-67.
[4]Tsou SS, Chiu WC, Yeh CL, et al. Effects of omega-3 fatty acids on inflammatory mediators and splenocyte cytokine mRNA expressions in rats with polymicrobial sepsis. Nutrition,2008,24 (5):484-491.
[5]Diamond IR, Sterescu A, Pencharz PB, et al. Changing the paradigm:omegaven for the treatment of liver failure in pediatric short bowel syndrome. J Pediatr Gastroenterol Nutr,2009,48(2):209-215.
[6]Ekema G, Falchetti D, Boroni G, et al.Reversal of severe parenteral nutrition-associated liver disease in an infant with short bowel syndrome using parenteral fish oil (Omega-3 fatty acids). J Pediatr Surg,2008,43(6):1191-1195.
[7]Gura KM, Duggan CP, Collier SB, et al. Reversal of parenteral nutrition-associated liver disease in two infants with short bowel syndrome using parenteral fish oil:implications for future management. Pediatrics,2006, 118(1):e197-201.
[8]Chung PHY, Wong KKY, Wong RMS, et al. Clinical experience in managing pediatric patients with ultra short bowel syndrome using omega-3-fatty acid. Eur J Pediatr Surg,2010,20(2):139-142.
[9]Lang R, Patel D, Morris JJ,et al. Shaping gene expression in activated and resting primarymacrophages by IL-10. J Immunol,2002,169(5):2253-2263.
[10]Couper KN, Blount DG, Riley EM. IL-10:the master regulator of immunity to infection. J Immunol,2008,180 (9):5771-5777.
[11]Georgia Perona-Wright, Katja Mohrs,Frank M, et al. Systemic but not local infections elicit immunosuppressive IL-10production by Natural Killer cells.Cell Host Microbe,2009,6(6):503-512.
[12]Anker SD, von Haehling S. Inflammatory mediators in chronic heart failure:an overview. Heart,2004,90 (4):464-470.
[13]Moore K, Malefyt R, Coffman R, et al. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol,2001,19 (1):683-765.
[14]Bolger AP, Sharma R, von Haehling S, et al. Effect of interleukin-10 on the production of tumor necrosis factor-alpha by peripheral blood mononuclear cells from patients with chronic heart failure. Am J Cardiol,2002,90 (4):384-389.
[15]Denys A, Udalova IA, Smith C, et al. Evidence for a dual mechanism for IL-10 suppression of TNF-{alpha} production that does not involve inhibition of p38 mitogen-activated protein kinase or NF-{kappa}B in primary human macrophages. J Immunol,2002,168 (10):4837-4845.
[16]Kaur K, Sharma AK, Dhingra S, et al.Interplay of TNF-alpha and IL-10 in regulating oxidative stress in isolated adult cardiac myocytes. J Mol Cell Cardiol, 2006,41 (6):1023-1030.
[17]Nozaki N, Yamaguchi S, Yamaoka M, et al. Enhanced expression and shedding of tumor necrosis factor (TNF) receptors from mononuclear leukocytes in human heart failure. J Moll Cell Cardiol,1998, (30):2003-2012.
[18]Stumpf C, Lehner C, Yilmaz A,et al. Decrease of serum levels of the anti-inflammatory cytokine interleukin-10 in patients with advanced chronic heart failure. Clin Sci (Lond),2003,105(1):45-50.
[19]De Maria A, Fogli M, Mazza S,et al. Increased natural cytotoxicity receptor expression and relevant IL-10 production in NK cells fromchronically infected viremic HCV patients. Eur J Immunol,2007,37 (2):445-455.
[20]Maroof A, Beattie L, Zubairi S, et al.Posttranscriptional regulation of il-10gene expression allows natural killer cells to express immunoregulatory function. Immunity,2008,29 (2):295-305.
[21]Kelly DA. Liver complications of pediatric parenteral nutrition-epidemiology. Nutrition,1998,14(1):153-157.
[22]Katakura T, Miyazaki M, Kobayashi M, et al. CCL17 and IL-10 as effectors that enable alternatively activated macrophages to inhibit the generation of classically activated macrophages. J Immunol,2004,172(3):1407-1413.
[23]Gura KM, Lee S, Valim C, et al. Safety and efficacy of a fish-oil-based fat emulsion in the treatment of parenteral nutrition-associated liver disease. Pediatrics, 2008,121(3):e678-686.
[24]Van Aerde JE, Duerksen DR, Gramlich L, et al. Intravenous fish oil emulsion attenuates total parenteral nutrition-induced cholestasis in newborn piglets. Pediatr Res,1999,45 (2):202-208.
[25]Araya J, Rodrigo R, Videla LA, et al. Increase in long-chain polyunsaturated fatty acid n-6/n-3 ratio in relation to hepatic steatosis in patients with non-alcoholic fatty liver disease. Clin Sci (Lond),2004,106 (6):635-643.
[26]Babcock TA, Novak T, Ong E,et al. Modulation of lipopolysaccharide-stimulated macrophage tumor necrosis factor-aproduction by co-3 fatty acid is associated with differential cyclooxygenase-2 protein expression and is independent of interleukin-10.J Surg Res,2002,107 (1):135-139.
[27]Novak TE, Babcock TA, Jho DH,et al. NF-κB inhibition by ω-3 fatty acids modulates LPS-stimulated macrophage TNF-a transcription. Am J Physiol,2003, 284(1):L84-L89.
[28]Zhao Y, Joshi-Barve S, Barve S,et al. Eicosapentaenoic acid prevents LPS-induced TNF-a expression by preventing NF-κB activation. J Am Coll Nutr, 2004,23 (1):71-78.
[29]Weldon SM, Mullen AC, Loscher CE, et al. Docosahexaenoic acid induces an anti-inflammatory profile in lipopolysaccharide-stimulated human THP-1 macrophages more effectively than eicosapentaenoic acid. J Nutr Biochem,2007,18 (4):250-258.
[30]Wang S, Wu D, Lamon-Fava S, et al. In vitro fatty acid enrichment of macrophages alters inflammatory response and net cholesterol accumulation. Br J Nutr,2009,102 (4):497-501.
[31]Ruan H, Pownall HJ, Lodish HF.Troglitazone antagonizes tumor necrosis factor-alpha-induced reprogramming of adipocyte gene expression by inhibiting the transcriptional regulatory functions of NF-kappaB. J Biol Chem,2003,278 (30):28181-28192.
[32]Hassan A, Ibrahim A, Mbodji K, et al.An a-linolenic acid-rich formula reduces oxidative stress and inflammation by regulating NF-κB in rats with TNBS-induced colitis,2010,140(10):1714-1721.
[1]Klein CJ, Revenis M, Kusenda C,et al. Parenteral nutrition-associated conjugated hyperbilirubinemia in hospitalized infants. J Am Diet Assoc.2010,110(11):1684-95.
[2]Dawes LG, Laut HC, Woodruff M. Decreased bile acid synthesis with total parenteral nutrition.Am J Surg,2007,194(5):623-627.
[3]Tang QY, Wang Y, Feng Y,et al. Factors derived from parenteral nutrition associated with cholestasis in 612 neonates. Zhonghua Er Ke Za Zhi,2007, 45(11):838-842.
[4]Hsieh MH, Pai W, Tseng HI, et al. Parenteral nutrition-associated cholestasis in premature babies:risk factors and predictors. Pediatr Neonatol.2009,50(5):202-207.
[5]Kelly DA. Intestinal failure-associated liver disease:what do we know today? Gastroenterology.2006,130(2):S70-77.
[6]Blau J, Sridhar S, Mathieson S, et al. Effects of protein/nonprotein caloric intake on parenteral nutrition associated cholestasis in premature infants weighing 600-1000 grams. JPEN J Parenter Enteral Nutr.2007,31(6):487-490.
[7]Arnold CJ, Miller GG, Zello GA. Parenteral nutrition-associated cholestasis in neonates:the role of aluminum. Nutr Rev.2003,61(9):306-310.
[8]Wright K, Ernst KD, Gaylord MS, et al.Increased incidence of parenteral nutrition-associated cholestasis with aminosyn PF compared to trophamine. J Perinatol.2003,23(6):444-450.
[9]Spencer AU, Yu S, Tracy TF,et al. Parenteral nutrition-associated cholestasis in neonates:multivariate analysis of the potential protective effect of taurine. JPEN J Parenter Enteral Nutr.2005,29(5):337-344.
[10]Calder PC. Immunomodulation by omega-3 fatty acids. Prostaglandins Leukot Essent Fatty Acids.2007,77(5-6):327-335.
[11]Kelly DA.Preventing parenteral nutrition liver disease. Early Hum Dev,2010, 86(11):683-687.
[12]Chung PH, Wong KK, Wong RM,et al. Clinical experience in managing pediatric patients with ultra-short bowel syndrome using omega-3 fatty acid.Eur J Pediatr Surg, 2010,20(2):139-142.
[13]Puder M, Valim C, Meisel JA, et al. Parenteral fish oil improves outcomes in patients with parenteral nutrition-associated liver injury. Ann Surg,2009, 250(3):395-402.
[14]Gura KM, Lee S, Valim C, Zhou J, et al. Safety and efficacy of a fish-oil-based fat emulsion in the treatment of parenteral nutrition-associated liver disease. Pediatrics,2008,121(3):e678-686.
[15]Sterescu A, Pencharz PB, et al. Changing the paradigm:omegaven for the treatment of liver failure in pediatric short bowel syndrome. Diamond IR, J Pediatr Gastroenterol Nutr.2009,48(2):209-215.
[16]Calder PC. n-3 polyunsaturated fatty acids, inflammation, and inflammatory diseases. Am J Clin Nutr,2006,83(6):1505S-1519S.
[17]Calder PC. Polyunsaturated fatty acids and inflammation. Prostaglandins Leukot Essent Fatty Acids.2006,75(3):197-202.
[18]Geyeregger R, Zeyda M, Zlabnger G J, et a 1. Polyunsaturated fatty acid s in terfere w ith form ation of the immunological synapse. J Leukoc Biol,2005,77(5): 680-688.
[19]Wallace K, Burt AD, Wright MC. Liver fibrosis. Biochem J.2008,411(1):1-18.
[20]Deng X, Luyendyk JP, Ganey PE. Inflammatory stress and idiosyncratic hepatotoxicity:hints from animal models. Pharmacol Rev.2009,61(3):262-282.
[21]Bellinghausen I, Knop J, Saloga J. The role of interleukin 10 in the regulation of allergic immune responses. Int Arch Allergy Immunol.2001,126(2):97-101.
[22]Ling GS, Cook HT, Botto M,et al. An essential protective role of IL-10 in the immunological mechanism underlying resistance vs. susceptibility to lupus induction by dendritic cells and dying cells. Rheumatology (Oxford),2011,50(10):1773-1784.
[23]Calder PC, Yaqoob P. Postgrad Med. Understanding omega-3 polyunsaturated fatty acids.2009,121(6):148-157.
[24]Vedin I, Cederholm T, Freund Levi Y,et al. Effects of docosahexaenoic acid-rich n-3 fatty acid supplementation on cytokine release from blood mononuclear leukocytes:the OmegAD study.Am J Clin Nutr,2008,87(6):1616-1622.
[25]Fetterman JW Jr, Zdanowicz MM. Therapeutic potential of n-3 polyunsaturated fatty acids in disease. Am J Health Syst Pharm,2009,66(13):1169-1179.
[26]Simopoulos AP. Omega-3 fatty acids in inflammation and autoimmune diseases. J Am Coll Nutr,2002,21(6):495-505.
[27]Micallef MA, Garg ML. Anti-inflammatory and cardioprotective effects of n-3 polyunsaturated fatty acids and plant sterols in hyperlipidemic individuals. Atherosclerosis.2009,204(2):476-482.
[28]Elenkov IJ, Chrousos GP. Stress hormones,proinflammatory and antiinflammatory cytokines, and autoimmunity.Ann N Y Acad Sci,2002, 966:290-303.
[29]Fallon EM, Le HD, Puder M. Prevention of parenteral nutrition-associated liver disease:role of omega-3 fish oil. Curr Opin Organ Transplant.2010,15(3):334-340.
[30]Aldridge C, Razzak A, Babcock TA, et al. Lipopolysaccharide-stimulated RAW 264.7 macrophage inducible nitric oxide synthase and nitric oxide production is decreased by an omega-3 fatty acid lipid emulsion. J Surg Res.2008,149(2):296-302.
[31]Babcock TA, Helton WS, Anwar KN,et al. Synergistic anti-inflammatory activity of omega-3 lipid and rofecoxib pretreatment on macrophage proinflammatory cytokine production occurs via divergent NF-kappaB activation. JPEN J Parenter Enteral Nutr.2004,28(4):232-240.
[32]Babcock TA, Novak T, Ong E,et al. Modulation of lipopolysaccharide-stimulated macrophage tumor necrosis factor-alpha production by omega-3 fatty acid is associated with differential cyclooxygenase-2 protein expression and is independent of interleukin-10. J Surg Res.2002,107(1):135-139.
[33]Babcock TA, Helton WS, Hong D,et al. Omega-3 fatty acid lipid emulsion reduces LPS-stimulated macrophage TNF-alpha production. Surg Infect (Larchmt). 2002,3(2):145-149.
[34]Babcock TA, Kurland A, Helton WS, et al. Inhibition of activator protein-1 transcription factor activation by omega-3 fatty acid modulation of mitogen-activated protein kinase signaling kinases. JPEN J Parenter Enteral Nutr.2003,27(3):176-181.
[35]Babcock TA, Novak T, Ong E,et al. Modulation of lipopolysaccharide-stimulated macrophage tumor necrosis factor-aproduction by co-3 fatty acid is associated with differential cyclooxygenase-2 protein expression and is independent of interleukin-10. J Surg Res,2002,107(1):135-139.
[36]Boutros C, Somasundar P, Razzak A,et al. Omega-3 fatty acids:investigations from cytokine regulation to pancreatic cancer gene suppression. Arch Surg,2010, 145(6):515-520.
[37]Zhao Y, Joshi-Barve S, Barve S,et al. Eicosapentaenoic acid prevents LPS-induced TNF-a expression by preventing NF-κB activation. J Am Coll Nutr 2004,23(1):71-78.
[38]Weldon SM, Mullen AC, Loscher CE,et al. Docosahexaenoic acid induces an anti-inflammatory profile in lipopolysaccharide-stimulated human THP-1 macrophages more effectively than eicosapentaenoic acid. J Nutr Biochem,2007, 18(4):250-258.
[39]Wei Hao, Olive Y. Wong, Xuelai Liu,et al.co-3 fatty acids suppress inflammatory cytokine production by macrophages and hepatocytes. Journal of Pediatric Surgery, 2010,45(12):2412-2418
[40]Schwerbrock NM, Karlsson EA, Shi Q,et al. Fish oil-fed mice have impaired resistance to influenza infection.J Nutr,2009,139(8):1588-1594.
[41]Wang MY, Wu MH, Hsieh DY,et al. Home parenteral nutrition support in adults: experience of a medical center in Asia. J Parenter Enteral Nutr,2007,31(4):306-310.
[42]Zhang Q, Yu JC, Kang WM,et al. Effect of ω-3 Fatty Acid on Gastrointestinal Motility after Abdominal Operation in Rats.Mediators inflamm,2011,2011:152137.
[43]Sijben JW, Calder PC. Differential immunomodulation with long-chain n-3 PUFA in health and chronic disease.Proc Nutr Soc,2007,66(2):237-259.
[44]Miles EA, Banerjee T, Dooper MWBW,et al. The influence of different combinations ofy-linolenic acid, stearidonic acid and EPA on immune function in healthy young male subjects. Br J Nutr,2004,91(6):893-903.
[45]Gorjao R, Azevedo-Martins AK, Rodrigues HG,et al. Comparative effects of DHA and EPA on cell function.Pharmacol Ther,2009,122(1):56-64.
[46]Chapkin RS, Kim W, Lupton JR, et al. Dietary docosahexaenoic and eicosapentaenoic acid:emerging mediators of inflammation. Prostaglandins Lenkot Essent Fatty Acids,2009,81 (2-3):187-191.
[47]Ramakers JD, Mensink RP, Schaart G,et al. Arachidonic acid but not eicosapentaenoic acid (EPA) and oleic acid activates NF-kappaB and elevates ICAM-1 expression in Caco-2 cells. Lipids,2007,42(8):687-698.
[48]Myhrstad MC, Retterstol K, Telle-Hansen VH,et al. Effect of marine n-3 fatty acids on circulating inflammatory markers in healthy subjects and subjects with cardiovascular risk factors.Infamm Res,2011,60(4):309-319.
[49]Kasama T, Strieter RM, Lukacs NW, et al. Regulation of neutrophil-derived chemokine expression by IL-10. J Immunol,1994,152(7):3559-3569.
[50]Petit-Bertron AF, Fitting C, Cavaillon JM, et al. Adherence influences monocyte responsiveness to interleukin-10. J Leukoc Biol,2003,73(1):145-154.
[51]Spight D, Zhao B, Haas M, et al. Immunoregulatory effects of regulated, lung-targeted expression of IL-10 in vivo. Am J Physiol Lung Cell Mol Physiol,2005, 288(2):L251-265.
[52]Wanidworanun C, Strober W. Predominant role of tumor necrosis factor-alpha in human monocyte IL-10 synthesis. J Immunol,1993,151(12):6853-6861.
[53]Sierra S, Lara-Villoslada F, Olivares M, et al.11-10 expression is involved in the regulation of the immune response by omega 3 fatty acids. Nutr Hosp.2004, 19(6):376-382.
[54]LeBlanc CJ, Horohov DW, Bauer JE, et al. Effects of dietary supplementation with fish oil on in vivo production of inflammatory mediators in clinically normal dogs.Am J Vet Res,2008,69(4):486-493.
[55]Sierra S, Lara-Villoslada F, Comalada M, et al. Dietary fish oil n-3 fatty acids increase regulatory cytokine production and exert anti-inflammatory effects in two murine models of inflammation. Lipids.2006,41(12):1115-1125.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |