TNF-α单克隆抗体治疗大鼠肝肺综合征作用机制的研究
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摘要
目的:
肝肺综合症(hepatopulmonary syndrome,HPS)是指在肝硬化或门脉高压基础上发生的呼吸功能障碍,通常包括肝硬化、肺内血管扩张和动脉低氧血症三联症。肺内毛细血管弥漫性扩张是HPS的基本病理改变,其发生与扩血管物质一氧化氮(nitric oxide,NO)异常增多密切相关。NO是肺血管扩张的重要介质,同时能够刺激血管内皮细胞增生和毛细血管向周围组织生长,致使肺泡间隔增宽和肺泡容量减小,最终导致PaO2下降、P(A-a)O2增大。NO由一氧化氮合酶(nitricoxide synthase,NOS)催化L-精氨酸生成。目前认为HPS时,内皮素(endothelin,ET)通过与内皮上ETB受体结合使内皮型NOS(endothelial NOS, eNOS)活化,促进NO合成;而血管内的巨噬细胞活化或聚集可以引起诱导型NOS(inducible NOS, iNOS)增加,促进NO合成。
磷脂酰肌醇-3激酶( phosphatidylinositol 3 kinase , PI3K )家族是一类特异性地催化磷酯酰肌醇(phosphatidylinositol, PI) 3位羟基磷酸化,并产生具有第二信使作用的肌醇脂物质的激酶。PI3K是由两部分组成的异源性二聚体,包括调节亚单位p85和催化亚单位p110。p85亚单位包含两个SH2功能域和一个SH3功能域。当细胞被生长因子刺激后,p85亚单位的SH2功能域与酪氨酸磷酸化受体或连接蛋白结合,进而将p85·p110复合体结合于细胞膜内侧以转导信息。
Akt又称蛋白激酶B( protein kinase B, PKB),是一种丝氨酸/苏氨酸蛋白激酶,在细胞内信号转导系统中位于PI3K的下游,是癌基因v-akt编码产物的同系物。目前发现至少存在3种Akt家族成员: Aktl/PKBα、Akt2/PKBβ、Akt3/PKBγ,尽管它们由不同基因编码产生,但是它们密切相关,相互间同源性> 80 %,但在不同组织中的表达各不相同。Akt主要通过依赖PI3K方式活化。其活化都依靠两个位点的磷酸化:一个是活性区的Thr308位点,另一个是羧基端疏水区Ser473。只有当两个位点全部磷酸化后,Akt才能够完全被激活。活化的Akt通过磷酸化作用激活或抑制其下游一系列底物如NF-κB、Bad、caspase9和GSK-3等改变,从而调节细胞的增殖、分化、凋亡以及迁移。
研究发现,细菌易位(bacterial translocation,BT)之所以与HPS发生有关,归因于超量生产的TNF-α。有研究报道,在HPS模型中,血液单核细胞可在肺血管内黏附、聚积,并于肺血管内皮分化成肺血管内巨噬细胞( pulmonary intravascular macrophages, PIM) ,PIM可被内毒素(LPS)活化,导致大量细胞因子如TNF-α、NO、IL-1、IL-6和IL-8等炎症介质释放入血液。其中,TNF-α是具有代表性的炎性介质,能够诱导局部释放单核细胞趋化因子,并刺激巨噬细胞和内皮细胞表达粘附因子,进一步加剧肺血管内巨噬细胞的大量聚集、粘附。TNF-α还能够以依赖PI3K/Akt信号通路的方式进一步激活NF-κB,活化的NF-κB参与了包括iNOS和TNF-α在内的众多炎症因子的转录。其结果是:一方面,巨噬细胞内iNOS过度表达,导致NO的过度生成;另一方面,新生成的TNF-α则又能进一步增强NF-κB的作用,形成级联正反馈。最终使NO过量生成,导致HPS的发生。
HPS在肝硬化中发病率为10～20%,预后不良。目前由于HPS的发病机理仍然未明,故药物治疗的效果并不满意。目前,仅肝移植是唯一肯定有效的治疗HPS方法。所以,研究HPS的肝肺综合征发病机制,寻找有效的治疗方法至关重要。
由于近年来TNF-α单克隆抗体用于治疗Crohn’s病、类风湿性关节炎和强直性脊柱炎等疾病取得了良好效果,所以我们推断应用TNF-αMcAb阻断TNF-α效应,对HPS的治疗应该有一定价值。而且,目前还没有关于应用TNF-αMcAb治疗HPS的研究,为此,我们采用多种实验方法,应用TNF-αMcAb治疗胆总管结扎(common bile duct ligation, CBDL)致肝肺综合征大鼠,观察TNF-αMcAb干预后对PI3K和Akt表达的影响,讨论TNF-αMcAb对HPS治疗作用,以及HPS的发生过程中的信号转导机制。
方法:
采用胆总管结扎CBDL致大鼠肝肺综合征模型。将60只雄性Sprague Dawley大鼠随机分为假手术组(sham组)、胆总管结扎组(CBDL组,分为胆总管结扎1周、2周、3周、4周、5周组五个亚组)及胆总管结扎+TNF-αMcAb干预组(CBDL+TNF-αMcAb,分为胆总管结扎1周后TNF-αMcAb治疗1周、2周、3周、4周组四个亚组),共10个亚组,每组各6只雄性SD大鼠。干预组与术后1周开始腹腔内注射TNF-αMcAb(0.1g/kg/2d), CBDL组对应注射相当剂量的生理盐水。各组动物在规定时间取材(sham组和CBDL组第五周动物同时取材),取静脉血测肝功能,TNF-α和NO的含量;取动脉血测定肺泡-动脉氧分压差(P(A-a)O2);取肝、肺组织进行HE染色和Masson三色观察以确定模型建立情况,肝硬化形成情况及不同组间的病理学变化差异;采用免疫组织化学法(SP法)检测PI3K、p-Akt在肺组织中的表达定位及表达程度;应用RT-PCR和Western blot方法分别检测PI3K、Akt在肺组织中mRNA和蛋白的表达。结果:
1肝脏及肺脏病理改变
1.1肝脏病理(HE及Masson染色):sham组大鼠肝小叶结构完整,肝板排列整齐,仅在门管区可见少量结缔组织。CBDL组大鼠肝脏小胆管广泛增生,胆管扩张,纤维组织增生,假小叶形成。TNF-αMcAb组大鼠肝脏小叶结构变化不明显,小胆管仍有增生,门管区胶原纤维轻微增多,未见假小叶形成。
1.2肺脏病理(HE染色):sham组大鼠肺脏毛细血管形态正常,肺泡壁薄厚一致,肺泡腔规则。CBDL组大鼠肺脏大鼠肺泡毛细血管增生、扩张,伴肺泡间隔增宽、容量减小,可见巨噬细胞。TNF-αMcAb组大鼠肺脏毛细血管轻度扩张,肺泡间隔轻度增宽。
2肝功能检测:
血浆ALT在CBDL术后造模组1周(177.00±56.32 U/L)较sham组(56.33±9.29 U/L)明显增加,有统计学意义(P<0.05)。之后逐渐下降,但均明显高于sham组(P<0.05)。TNF-αMcAb组干预2周即较同周CBDL组明显下降,差异有统计学意义(P<0.05),之后,呈下降趋势,至治疗4周(63.00±7.55 U/L)时与CBDL组(87.67±17.50 U/L)比较差异明显(P<0.05),虽仍高于sham组,但是差异无统计学意义(P>0.05)。
3血浆TNF-α检测:
CBDL术后1周血浆TNF-α水平(1.60±0.07 ng/ml)即明显升高,之后呈上升趋势,3周时血浆TNF-α水平(3.18±0.54 ng/ml)达高峰,4周时有所回落,5周又稍有回升,但均明显高于sham组(0.75±0.14 ng/ml)(P<0.05),差异有统计学意义。TNF-αMcAb干预治疗后1周即开始明显降低,之后继续下降,至干预治疗后4周(1.21±0.13 ng/ml)与CBDL组比较(2.43±0.43)明显降低((P<0.05),虽仍高于sham组,但差异无统计学意义(P>0.05)。
4动脉血气分析检测:
CBDL造模2周时P(A-a)O2(17.65±2.67 mmHg)开始增大,之后呈上升趋势,5周时达到(51.67±8.49 mmHg),明显高于sham组(6.70±3.20 mmHg),有统计学意义(P<0.05)。TNF-αMcAb干预治疗后2周较CBDL术后同周组开始下降,之后呈下降趋势,至治疗4周(10.5±8.51 mmHg)的P(A-a)O2与同周CBDL组(51.8±8.49 mmHg)比较,有明显差异(P<0.05)。虽仍高于sham组,但是差异无统计学意义(P>0.05)。
5 NO检测结果:
CBDL术后1周NO开始上升,与sham组比较有明显差异(P<0.05)。至3周末(95.67±15.41 umol/l)达到高峰后,之后呈下降趋势,但均明显高于sham组(36.19±1.59 umol/l)。TNF-αMcAb干预治疗后1周NO的量即开始明显低于同周CBDL组(74.38±2.67)水平,有统计学意义(P<0.05),之后继续下降,至干预治疗后3周(45.47±9.58 umol/l)、4周(44.42±9.27 umol/l)与CBDL组3周(72.22±6.11 umol/l)和4周(64.31±1.75 umol/l)比较差异明显,虽仍高于sham组,但已无明显差异(P>0.05)。
6肺组织PI3K的表达
6.1 RT-PCR扩增检测: PI3K mRNA的表达在CBDL组的第二周即达一个高峰(1.75±0.15),此后在第三周出现一个低谷(1.07±0.19),随后又上升至(1.26±0.16),第五周又降低至(0.98±0.11),但均高于sham组(0.28±0.08),差异有统计学意义(P<0.05)。TNF-αMcAb干预治疗组在干预一周(0.34±0.14)开始有明显的抑制,差异具有统计学意义(P<0.05),随后呈逐渐上升趋势,但是均较同时间点的CBDL组表达低,差异明显(P<0.05)。干预四周后与sham组比较,差异仍有统计学意义(P<0.05)。
6.2免疫组织化学检结果显示,①CBDL组:大鼠肺组织内PI3K蛋白的阳性表达较多,可见细胞胞浆呈棕褐色细颗粒,阳性表达多集中在肺内巨噬细胞胞浆中,于二周时达高峰(0.31±0.10),此后,CBDL组在三周时有短暂的下降,四周、五周又见回升,表达均高于sham组,差异有统计学意义(P<0.05)。②TNF-αMcAb组:干预治疗一周(0.19±0.03)后,与CBDL相比,PI3K蛋白表达即被明显抑制(P<0.05),此后,在各时间点的阳性表达均比同时间点的CBDL组弱,基本维持在上述的水平,但是仍明显高于sham组,差异有统计学意义(P<0.05)。
6.3 Western Blot分析: PI3K在85KD的位置出现杂交带。蛋白表达变化趋势同上述免疫组化检测结果。
7肺组织Akt和p-Akt的表达
7.1 RT-PCR扩增检测:①CBDL组Akt mRNA的表达呈逐渐升高的趋势,至第三周达峰值(2.07±0.18),与sham(0.58±0.06)组比较存在明显差异(P<0.05),随后呈下降趋势,但仍高于sham组。②TNF-αMcAb干预治疗组Akt mRNA的表达趋势与CBDL组基本一致,在治疗的前三周与相应的CBDL组比较,其表达即被明显抑制,表达较少,干预二周时差异性即具有统计学意义(P<0.05),但随着CBDL组的表达的逐渐减少,至第四周(0.64±0.06)与CBDL组比较无明显差异(P>0.05),但是与sham组的差异仍明显(P<0.05)。
7.2免疫组织化学分析p-Akt:p-Akt蛋白的阳性反应为黄色颗粒,主要定位在肺巨噬细胞的胞浆。①CBDL组p-Akt蛋白阳性表达在术后四周达峰值(0.31±0.03),与sham组(0.09±0.03),阳性细胞数目和表达强度均较高,差异有统计学意义(P<0.05)。②TNF-αMcAb干预治疗组各时间点的蛋白的阳性表达较CBDL组均有明显的减少,至治疗的第四周(0.20±0.03)明显低于同周CBDL组(0.28±0.03),差异有统计学意义(P<0.05),仍高于sham组,差异有统计学意义(P<0.05)。
7.3 Western Blot分析Akt:①CBDL组Akt蛋白表达呈逐渐上升的趋势,于术后第三周达一个高峰值(2.45±0.23),此后下降到(1.74±0.18),五周时稍有回升(1.92±0.10),均高于sham(1.08±0.20),差异有统计学意义(P<0.05)。②TNF-αMcAb组治疗1周(1.79±0.04)后与同时间的CBDL组相比较,即被明显的抑制,此后一直在此水平波动,前三周一直明显低于手术组,但是第四周(1.80±0.21)较CBDL组(1.92±0.10)比较差异不明显(P>0.05),但是仍高于sham组,差异亦有统计学意义(P<0.05)。
7.4 Western Blot分析p-Akt:p-Akt蛋白表达结果与免疫组化结果一致。8血浆中TNF-α和NO的相关性分析
相关系数r为0.719,相关系数的概率值为P =0.005<0.05,差异有统计学意义,说明肝肺综合征大鼠的血浆中TNF-α水平与NO水平有相关关系,并且为正相关,即随着血浆TNF-α的增加NO的水平也增加。
结论:
1 HPS的形成和发展过程中,肝脏功能的改变是肺功能变化的基础;
2 TNF-α可能是HPS的发生、发展过程中一个重要的因子;
3 HPS的发生过程中伴随PI3K、Akt表达明显增加,p-Akt的水平升高,存在PI3K/Akt信号转导通路的活化,说明PI3K/Akt的信号转导与HPS发生有关;
4 TNF-αMcAb可特异性结合血浆中的TNF-α,从而降低血浆中TNF-α水平,一定程度上抑制PI3K、Akt蛋白的表达和激活,使NO释放减少,改善了HPS严重程度。
Objective: The hepatopulmonary syndrome (HPS) is characterized by a defect in arterial oxygenation induced by pulmonary vascular dilatation in the setting of liver diseased or portal hypertension, accompanying with respiratory dysfunction.The basal pathological alteration is intrapulmonary vasodilatation, which is often related to the nitric oxide (NO) overproduction.
NO is an important mediator for intrapulmonary vasodilatation. It could stimulate the hyperplasia endothelialis and peripherad growth of micrangium, which lead to widened alveolar septum and diminished alveolar volume.The end-results are descending of partial pressure of oxygen in artery (PaO2) and widened of alveolar-arterial PO2 difference (P(A-a)O2). NO is produced by NO synthases (NOS) with the substrate of L-arginine. Now, it is considered that endothelin (ET) could bind to ETB receptor in endodermis, which leading to endothelial NOS (eNOS) activation, and following NO production. Unlike eNOS, inducible NOS (iNOS) activation dues to pulmonary macrophage activation or aggregation may increase NO production.
Phosphatidylinositol 3 kinase (PI3K) family is characterized by catalyzing the phosphorylation of phosphatidylinositol (PI) at the 3-OH position of the inositol phospholipids, and producing phosphatidylinositoltriphosphate (PIP3) that is the intracellular second messenger. This enzyme is composed of two subunits: an 85-kilodalton regulatory subunit and a 110-kilodalton catalytic subunit.The p85 subunit includes two SH2 domains and one SH3 domain. Having been activated by growth factor receptors, SH2 domains would directly bind to phosphotyrosine residues of growth factor receptors or adaptors, and then,the p85-p110 complex is brought to the membrane for next message transduction.
Akt, also known as protein kinase B (PKB), which is identified as the human homologues for the viral oncogene v-akt, is a serine/threonine kinase downstream target of PI3K. To date, three members of this PKB family have been isolated, named Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ. Although they are products of different genes, they are closely related to each other, with up to 80% of amino acid homology. Nevertheless, their expression is different in distinct tissues. Activation of three isoforms is in a similar way that phosphorylation of two sites, one in the activation domain and another in the COOH-terminal hydrophobic motif, and both of them are necessary for full activity. Activated Akt could activate or restrain a series of substrates through phosphorylation, for example, NF-κB, Bad, caspase9 and GSK-3 and so on, to adjust cellular proliferation, differentiate, apoptosis and migration.
Bacterium translocation (BT) leads to overproduction of TNF-α, the latter is an important factor in the development of HPS. It has been identified that the monocytes in circulating could stick to and accumulate in pulmonary vascular endodermis.Where they can differentiate into pulmonary intravascular cmacrophages (PIM), which could be activated by LPS. Activation of PIM could release a great of mediators of inflammation, for example, TNF-α, NO, IL-1, IL-6 and IL-8 etc. Especially, TNF-αcould induce local release of monocyte chemotactic factors and expression of macrophage and endothelium adhesion molecules. In addition, TNF-αcould activate NF-κB through PI3K/Akt signal pathway. NF-κB participates in the transcribing process of numerous inflammatory factors, including iNOS and TNF-αexpression by macrophage. So that iNOS induces NO overproduction and TNF-αand NF-κB that could activate each other in circles. Then the cascade reaction is triggered, which promotes the development of HPS.
The prevalence of HPS in the setting of cirrhosis ranges between 10% - 20%, with prognosis mala. Owing to unknown pathogeny of HPS, there is still no effective medical therapy to reverse this condition. Liver transplantation is the only effective method among choices of treatment though mortality is comparatively high. So it is important to define its pathogeny, and look for effective therapeutic approach.
For the past years, TNF-αMcAb has been used for treatment of Crohn’s disease, rheumatoid arthritis and ankylosing spondylitis etc, and shows well effect. So we conclude that TNF-αMcAb should be effective on HPS through combining to TNF-α. Up to now, there is few people have done the similar study. Therefore, we carried out this study. The aim of the present study was to evaluate if TNF-αMcAb influences experimental HPS when administered after CBDL and to define the mechanisms about the development of HPS.
Metheds: Male Sprague-Dawley rats, weighing 250±25g underwent sham operation (6 rats), CBDL (30 rats, divided into 5 subgroups: 1week、2week、3week、4week and 5week after ligation), CBDL+TNF-αMcAb (24 rats, divided into 4 subgroups: treating 1week, 2week, 3week, and 4week after 1 week of ligation). CBDL+TNF-αMcAb ( 0.1g/kg/2d ) was given by intraperitoneal injection for 1 week, 2week, 3week and 4 week , respectively, beginning 1 week after CBDL. At the same time, Rats in CBDL group were given NS for the same dose. Rat samples were gotten at different timepoints, as follow 1 wk, 2 wk, 3 wk, 4 wk, 5 wk after CBDL (Sham group were gotten at the fifth week). Hepatopulmonary syndrome was assessed by measurements of alveolar-arterial PO2 difference (P(A-a)O2). The level of NO and TNF-αwas measured. Liver histopathological changes were evaluated by hematoxylin and eosin staining and Masson’s trichrome methods. Lung histopathological changes were evaluated by hematoxylin and eosin staining. Immunohistochemistry, Western Blot and RT-PCR were employed to investigate the expression and the change of PI3K, Akt and p-Akt in rats’lung.
Results:
1. Histopathological changes of liver and lung:
1.1 Liver:①Control group: Hematoxylin and eosin staining showed that there were normal hepatic lobules, and hepatic plate to line in order. Masson showed that there was a little connective tissue in portal area.②CBDL group: Hematoxylin and eosin staining and Masson’s trichrome methods showed that the rats of CBDL group were with extensive biliary ducts accrementition and dilation, even complete cirrhotic nodule;③TNF-αMcAb treatment group: Hematoxylin and eosin staining showed that there was also biliary ducts accrementition, but not significant difference compared with the sham group in liver. Masson showed that there was increasing collagen fiber in portal areas, but not complete cirrhotic nodule.
1.2 Lung:①Control group: micrangiums of lung were eumorphism, alveolus wall and alveolus space were regular;②CBDL group: Hematoxylin and eosin staining showed that there was widened interalveolar septum, and obviously inflammatory reaction in lung interstitium. Macrophages were found. Capillary density increased, which showed that there was vasodilatation in lung tissue;③TNF-αMcAb treament group: Hematoxylin and eosin staining showed that there was a gentle vasodilatation and widened interalveolar septum.
2. Hepatic function check:①CBDL group: ALT in plasma obviously increased after CBDL for 1 week(177.00±56.32 U/L). Compared with the control group(56.33±9.29 U/L), which showed that there existed obvious difference(P<0.05). Following, the level of ALT in plasma fell off, but it was notablely elevated compared with that of control group.②TNF-αMcAb treatment group: Having treated with TNF-αMcAb for 2week, the level of ALT in plasma decreased obviously compared with that of CBDL group (P<0.05). Following, the downtrend continued, up to 4 week (63.00±7.55), it was still higher than that of control group, but the difference was not significant (P>0.05) .
3. Measurement of plasma TNF-α:①CBDL group: 1 week(1.60±0.07 ng/ml) after CBDL, the level of TNF-αin plasma increased significantly. Then, it kept the rising tendency. At the third week(3.18±0.54 ng/ml)after CBDL, it reached the peak. Between the third week and the fifth week after CBDL, it had a transient drop. Following, it returned to increase. But, it was at a higher level compared with that of control group (P<0.05).②TNF-αMcAb treament group: Compared with the CBDL group, the level of TNF-αin plasma decreased obviously after one week treatment. Then, the level of TNF-αkept downturn. Until the fourth week(1.21±0.13 ng/ml) after the CBDL, the level of TNF-αin plasma was also much lower than the CBDL group(2.43±0.43)(P<0.05),but compared with that of control group, there was no significant difference (P>0.05).
4. The blood gas analysis:①CBDL group: two weeks(17.65±2.67 mmHg) after the CBDL, P(A-a)O2 began to increase. Compared with that of control group(6.70±3.20)mmHg, which showed that there existed obvious difference(P<0.05). Later, it kept increasing.②TNF-αMcAb treatment group: The P(A-a)O2 increasing of the treatment group was restrained after 2 weeks treatment, then it kept increasing. After treatment for 4 week(10.5±8.51 mmHg),to contrast that of CBDL group(51.8±8.49 mmHg),there was significant difference(P<0.05). Compared with that of control group, the level of the P(A-a)O2 was also higher , but no significant difference (P>0.05).
5. Measurement of plasma NO:①CBDL group: One week after CBDL, the level of NO in plasma increased significantly. Then, it remained elevated tendency. It reached peak at the third week(95.67±15.41 umol/l) after CBDL. Then it had a transient drop. But, it was at a higher level compared with that of control group(36.19±1.59 umol/l)(P<0.05).②TNF-αMcAb: Compared with that of CBDL group, the level of TNF-αin plasma decreased obviously after one week treatment. Then, the level of TNF-αkept decreasing. Until the third week(45.47±9.58)and 4 week(44.42±9.27)after the CBDL, the level of NO in plasma was obviously lower than CBDL that of group at the same time( 72.22±6.11 umol/l , 64.31±1.75 umol/l ), and there was statistical significance. The level of NO in plasma was also higher than that of control group, but the difference was not significant (P>0.05).
6. Measurement of the expression of PI3K in lung
6.1 RT-PCR showed as follow:①CBDL group:The expression of PI3K mRNA peaked at the second week(1.75±0.15)after CBDL,and there was a transient descent at the third week(1.07±0.19),then a ascensus at fourth week (1.26±0.16)and another descent at the fifth week(0.98±0.11).Compared with the expression of PI3K mRNA of sham group(0.28±0.08),there was significant difference.②TNF-αMcAb treatment group: Compaerd with CBDL group, the expression of PI3K mRNA was obviously depressed at the second week (0.34±0.14).Then, the level of the PI3K mRNA kept ascending, but it’s aways lower than that of sham group, and the difference was significant(P<0.05).
6.2 Immunohistochemistry showed as follow:①CBDL group: The positive protein expression of PI3K was higher than that in the control group, the cell kytoplasm is brown, and the positive expression localized in the kytoplasm of phagocyte.The second week(0.31±0.10), expression of PI3K protein was up to peak,which was higher than that of control group(0.06±0.01)(P<0.05). Next, it had a temporary descent.Then, it returned to increase. Compared with that of control group, there was significant difference (P<0.05).②TNF-αMcAb treatment group:After 1 week treament ( 0.15±0.03, the protein expression of PI3K was obviously restrained, compared with that of CBDL group, there was significant difference (P<0.05). Following, it kept on this level. Compared with that of control group, there was significant difference (P<0.05).
6.2 Western blot showed as follow: The result was similar with IHC.
7 Measurement of the expression of Akt and p-Akt in lung
7.1 RT-PCR of Akt showed as follow:①CBDL group: Expression of Akt mRNA showed gradually elevated tendency. At the third week, it reached peak (2.07±0.18). Next, it kept descending. But compared with sham group (0.58±0.06), the expression of Akt mRNA was higher. And the difference was significant.②TNF-αMcAb treatment group: The tendency of the expression of Akt mRNA was similar with that of CBDL group, but level of the expression of Akt mRNA was obviously depressed, and there was significant difference (P<0.05). At the third week, the difference was not significant (P>0.05).But compared with that of sham group, there was significant difference (P<0.05).
7.2 Immunohistochemistry of p-Akt showed as follow:①CBDL group: The positive protein expression of p-Akt was higher than that in the control group, the cell kytoplasm is brown, and the positive expression localized in the kytoplasm of phagocyte. After CBDL, protein expression of p-Akt was uo to peak on the fourth weeek(0.31±0.03. Compared with that of control group(0.09±0.03, there was significant difference (P<0.05).②TNF-αMcAb treatment group: compared with that CBDL group, the expression of p-Akt decreased. After treatment for 4 weeks(0.20±0.03, it was much lower than CBDL group(P<0.05), but still higher than that of control group(P<0.05).
7.3 Western blot of Akt showed as follow:①CBDL group: After CBDL, the expression of Akt kept increasing. At the third week(2.45±0.23, it was up to peak. Following, its expression was significantly restrained, but there was significant difference between it and the control group(1.08±0.20)(P<0.05).②TNF-αMcAb treatment group: For 1 week treatment, compared with that of CBDL group, the expression of Akt was visiblely inhibited. Following, it kept on this level. Compared with that of control group, there was significant difference (P<0.05).
7.4 Western blot of p-Akt showed as follow: The result was similar with IHC.
8 Analysis of correlation of TNF-αand NO in plasma
Coefficient correlation was 0.719, and probability value of coefficient correlation was 0.005<0.05, there was statistical significance, which showed correlativity between TNF-αand NO in plasma of rats with HPS. The direct correlation indicated that NO would increase along with TNF-α.
Conclusions:
1 Liver function changes were the foundation of the lung function changes in the forming process of HPS;
2 TNF-αmay probably be an important essential cause of the development of HPS;
3 Development of HPS accompanies by increasing expression of PI3K and Akt protein. Activation of Akt increasing showes activation of PI3K/Akt signalling pathway, which suggested that PI3K/Akt was concerned with HPS happenning;
4 TNF-αMcAb may combine with TNF-αin the plasma, and decrease the expression of TNF-α, partly inhibited expression and activation of PI3K and Akt protein, and the release of NO, then improve HPS.
肝肺综合症(hepatopulmonary syndrome,HPS)是指在肝硬化或门脉高压基础上发生的呼吸功能障碍,通常包括肝硬化、肺内血管扩张和动脉低氧血症三联症。肺内毛细血管弥漫性扩张是HPS的基本病理改变,其发生与扩血管物质一氧化氮(nitric oxide,NO)异常增多密切相关。NO是肺血管扩张的重要介质,同时能够刺激血管内皮细胞增生和毛细血管向周围组织生长,致使肺泡间隔增宽和肺泡容量减小,最终导致PaO2下降、P(A-a)O2增大。NO由一氧化氮合酶(nitricoxide synthase,NOS)催化L-精氨酸生成。目前认为HPS时,内皮素(endothelin,ET)通过与内皮上ETB受体结合使内皮型NOS(endothelial NOS, eNOS)活化,促进NO合成;而血管内的巨噬细胞活化或聚集可以引起诱导型NOS(inducible NOS, iNOS)增加,促进NO合成。
磷脂酰肌醇-3激酶( phosphatidylinositol 3 kinase , PI3K )家族是一类特异性地催化磷酯酰肌醇(phosphatidylinositol, PI) 3位羟基磷酸化,并产生具有第二信使作用的肌醇脂物质的激酶。PI3K是由两部分组成的异源性二聚体,包括调节亚单位p85和催化亚单位p110。p85亚单位包含两个SH2功能域和一个SH3功能域。当细胞被生长因子刺激后,p85亚单位的SH2功能域与酪氨酸磷酸化受体或连接蛋白结合,进而将p85·p110复合体结合于细胞膜内侧以转导信息。
Akt又称蛋白激酶B( protein kinase B, PKB),是一种丝氨酸/苏氨酸蛋白激酶,在细胞内信号转导系统中位于PI3K的下游,是癌基因v-akt编码产物的同系物。目前发现至少存在3种Akt家族成员: Aktl/PKBα、Akt2/PKBβ、Akt3/PKBγ,尽管它们由不同基因编码产生,但是它们密切相关,相互间同源性> 80 %,但在不同组织中的表达各不相同。Akt主要通过依赖PI3K方式活化。其活化都依靠两个位点的磷酸化:一个是活性区的Thr308位点,另一个是羧基端疏水区Ser473。只有当两个位点全部磷酸化后,Akt才能够完全被激活。活化的Akt通过磷酸化作用激活或抑制其下游一系列底物如NF-κB、Bad、caspase9和GSK-3等改变,从而调节细胞的增殖、分化、凋亡以及迁移。
研究发现,细菌易位(bacterial translocation,BT)之所以与HPS发生有关,归因于超量生产的TNF-α。有研究报道,在HPS模型中,血液单核细胞可在肺血管内黏附、聚积,并于肺血管内皮分化成肺血管内巨噬细胞( pulmonary intravascular macrophages, PIM) ,PIM可被内毒素(LPS)活化,导致大量细胞因子如TNF-α、NO、IL-1、IL-6和IL-8等炎症介质释放入血液。其中,TNF-α是具有代表性的炎性介质,能够诱导局部释放单核细胞趋化因子,并刺激巨噬细胞和内皮细胞表达粘附因子,进一步加剧肺血管内巨噬细胞的大量聚集、粘附。TNF-α还能够以依赖PI3K/Akt信号通路的方式进一步激活NF-κB,活化的NF-κB参与了包括iNOS和TNF-α在内的众多炎症因子的转录。其结果是:一方面,巨噬细胞内iNOS过度表达,导致NO的过度生成;另一方面,新生成的TNF-α则又能进一步增强NF-κB的作用,形成级联正反馈。最终使NO过量生成,导致HPS的发生。
HPS在肝硬化中发病率为10～20%,预后不良。目前由于HPS的发病机理仍然未明,故药物治疗的效果并不满意。目前,仅肝移植是唯一肯定有效的治疗HPS方法。所以,研究HPS的肝肺综合征发病机制,寻找有效的治疗方法至关重要。
由于近年来TNF-α单克隆抗体用于治疗Crohn’s病、类风湿性关节炎和强直性脊柱炎等疾病取得了良好效果,所以我们推断应用TNF-αMcAb阻断TNF-α效应,对HPS的治疗应该有一定价值。而且,目前还没有关于应用TNF-αMcAb治疗HPS的研究,为此,我们采用多种实验方法,应用TNF-αMcAb治疗胆总管结扎(common bile duct ligation, CBDL)致肝肺综合征大鼠,观察TNF-αMcAb干预后对PI3K和Akt表达的影响,讨论TNF-αMcAb对HPS治疗作用,以及HPS的发生过程中的信号转导机制。
方法:
采用胆总管结扎CBDL致大鼠肝肺综合征模型。将60只雄性Sprague Dawley大鼠随机分为假手术组(sham组)、胆总管结扎组(CBDL组,分为胆总管结扎1周、2周、3周、4周、5周组五个亚组)及胆总管结扎+TNF-αMcAb干预组(CBDL+TNF-αMcAb,分为胆总管结扎1周后TNF-αMcAb治疗1周、2周、3周、4周组四个亚组),共10个亚组,每组各6只雄性SD大鼠。干预组与术后1周开始腹腔内注射TNF-αMcAb(0.1g/kg/2d), CBDL组对应注射相当剂量的生理盐水。各组动物在规定时间取材(sham组和CBDL组第五周动物同时取材),取静脉血测肝功能,TNF-α和NO的含量;取动脉血测定肺泡-动脉氧分压差(P(A-a)O2);取肝、肺组织进行HE染色和Masson三色观察以确定模型建立情况,肝硬化形成情况及不同组间的病理学变化差异;采用免疫组织化学法(SP法)检测PI3K、p-Akt在肺组织中的表达定位及表达程度;应用RT-PCR和Western blot方法分别检测PI3K、Akt在肺组织中mRNA和蛋白的表达。结果:
1肝脏及肺脏病理改变
1.1肝脏病理(HE及Masson染色):sham组大鼠肝小叶结构完整,肝板排列整齐,仅在门管区可见少量结缔组织。CBDL组大鼠肝脏小胆管广泛增生,胆管扩张,纤维组织增生,假小叶形成。TNF-αMcAb组大鼠肝脏小叶结构变化不明显,小胆管仍有增生,门管区胶原纤维轻微增多,未见假小叶形成。
1.2肺脏病理(HE染色):sham组大鼠肺脏毛细血管形态正常,肺泡壁薄厚一致,肺泡腔规则。CBDL组大鼠肺脏大鼠肺泡毛细血管增生、扩张,伴肺泡间隔增宽、容量减小,可见巨噬细胞。TNF-αMcAb组大鼠肺脏毛细血管轻度扩张,肺泡间隔轻度增宽。
2肝功能检测:
血浆ALT在CBDL术后造模组1周(177.00±56.32 U/L)较sham组(56.33±9.29 U/L)明显增加,有统计学意义(P<0.05)。之后逐渐下降,但均明显高于sham组(P<0.05)。TNF-αMcAb组干预2周即较同周CBDL组明显下降,差异有统计学意义(P<0.05),之后,呈下降趋势,至治疗4周(63.00±7.55 U/L)时与CBDL组(87.67±17.50 U/L)比较差异明显(P<0.05),虽仍高于sham组,但是差异无统计学意义(P>0.05)。
3血浆TNF-α检测:
CBDL术后1周血浆TNF-α水平(1.60±0.07 ng/ml)即明显升高,之后呈上升趋势,3周时血浆TNF-α水平(3.18±0.54 ng/ml)达高峰,4周时有所回落,5周又稍有回升,但均明显高于sham组(0.75±0.14 ng/ml)(P<0.05),差异有统计学意义。TNF-αMcAb干预治疗后1周即开始明显降低,之后继续下降,至干预治疗后4周(1.21±0.13 ng/ml)与CBDL组比较(2.43±0.43)明显降低((P<0.05),虽仍高于sham组,但差异无统计学意义(P>0.05)。
4动脉血气分析检测:
CBDL造模2周时P(A-a)O2(17.65±2.67 mmHg)开始增大,之后呈上升趋势,5周时达到(51.67±8.49 mmHg),明显高于sham组(6.70±3.20 mmHg),有统计学意义(P<0.05)。TNF-αMcAb干预治疗后2周较CBDL术后同周组开始下降,之后呈下降趋势,至治疗4周(10.5±8.51 mmHg)的P(A-a)O2与同周CBDL组(51.8±8.49 mmHg)比较,有明显差异(P<0.05)。虽仍高于sham组,但是差异无统计学意义(P>0.05)。
5 NO检测结果:
CBDL术后1周NO开始上升,与sham组比较有明显差异(P<0.05)。至3周末(95.67±15.41 umol/l)达到高峰后,之后呈下降趋势,但均明显高于sham组(36.19±1.59 umol/l)。TNF-αMcAb干预治疗后1周NO的量即开始明显低于同周CBDL组(74.38±2.67)水平,有统计学意义(P<0.05),之后继续下降,至干预治疗后3周(45.47±9.58 umol/l)、4周(44.42±9.27 umol/l)与CBDL组3周(72.22±6.11 umol/l)和4周(64.31±1.75 umol/l)比较差异明显,虽仍高于sham组,但已无明显差异(P>0.05)。
6肺组织PI3K的表达
6.1 RT-PCR扩增检测: PI3K mRNA的表达在CBDL组的第二周即达一个高峰(1.75±0.15),此后在第三周出现一个低谷(1.07±0.19),随后又上升至(1.26±0.16),第五周又降低至(0.98±0.11),但均高于sham组(0.28±0.08),差异有统计学意义(P<0.05)。TNF-αMcAb干预治疗组在干预一周(0.34±0.14)开始有明显的抑制,差异具有统计学意义(P<0.05),随后呈逐渐上升趋势,但是均较同时间点的CBDL组表达低,差异明显(P<0.05)。干预四周后与sham组比较,差异仍有统计学意义(P<0.05)。
6.2免疫组织化学检结果显示,①CBDL组:大鼠肺组织内PI3K蛋白的阳性表达较多,可见细胞胞浆呈棕褐色细颗粒,阳性表达多集中在肺内巨噬细胞胞浆中,于二周时达高峰(0.31±0.10),此后,CBDL组在三周时有短暂的下降,四周、五周又见回升,表达均高于sham组,差异有统计学意义(P<0.05)。②TNF-αMcAb组:干预治疗一周(0.19±0.03)后,与CBDL相比,PI3K蛋白表达即被明显抑制(P<0.05),此后,在各时间点的阳性表达均比同时间点的CBDL组弱,基本维持在上述的水平,但是仍明显高于sham组,差异有统计学意义(P<0.05)。
6.3 Western Blot分析: PI3K在85KD的位置出现杂交带。蛋白表达变化趋势同上述免疫组化检测结果。
7肺组织Akt和p-Akt的表达
7.1 RT-PCR扩增检测:①CBDL组Akt mRNA的表达呈逐渐升高的趋势,至第三周达峰值(2.07±0.18),与sham(0.58±0.06)组比较存在明显差异(P<0.05),随后呈下降趋势,但仍高于sham组。②TNF-αMcAb干预治疗组Akt mRNA的表达趋势与CBDL组基本一致,在治疗的前三周与相应的CBDL组比较,其表达即被明显抑制,表达较少,干预二周时差异性即具有统计学意义(P<0.05),但随着CBDL组的表达的逐渐减少,至第四周(0.64±0.06)与CBDL组比较无明显差异(P>0.05),但是与sham组的差异仍明显(P<0.05)。
7.2免疫组织化学分析p-Akt:p-Akt蛋白的阳性反应为黄色颗粒,主要定位在肺巨噬细胞的胞浆。①CBDL组p-Akt蛋白阳性表达在术后四周达峰值(0.31±0.03),与sham组(0.09±0.03),阳性细胞数目和表达强度均较高,差异有统计学意义(P<0.05)。②TNF-αMcAb干预治疗组各时间点的蛋白的阳性表达较CBDL组均有明显的减少,至治疗的第四周(0.20±0.03)明显低于同周CBDL组(0.28±0.03),差异有统计学意义(P<0.05),仍高于sham组,差异有统计学意义(P<0.05)。
7.3 Western Blot分析Akt:①CBDL组Akt蛋白表达呈逐渐上升的趋势,于术后第三周达一个高峰值(2.45±0.23),此后下降到(1.74±0.18),五周时稍有回升(1.92±0.10),均高于sham(1.08±0.20),差异有统计学意义(P<0.05)。②TNF-αMcAb组治疗1周(1.79±0.04)后与同时间的CBDL组相比较,即被明显的抑制,此后一直在此水平波动,前三周一直明显低于手术组,但是第四周(1.80±0.21)较CBDL组(1.92±0.10)比较差异不明显(P>0.05),但是仍高于sham组,差异亦有统计学意义(P<0.05)。
7.4 Western Blot分析p-Akt:p-Akt蛋白表达结果与免疫组化结果一致。8血浆中TNF-α和NO的相关性分析
相关系数r为0.719,相关系数的概率值为P =0.005<0.05,差异有统计学意义,说明肝肺综合征大鼠的血浆中TNF-α水平与NO水平有相关关系,并且为正相关,即随着血浆TNF-α的增加NO的水平也增加。
结论:
1 HPS的形成和发展过程中,肝脏功能的改变是肺功能变化的基础;
2 TNF-α可能是HPS的发生、发展过程中一个重要的因子;
3 HPS的发生过程中伴随PI3K、Akt表达明显增加,p-Akt的水平升高,存在PI3K/Akt信号转导通路的活化,说明PI3K/Akt的信号转导与HPS发生有关;
4 TNF-αMcAb可特异性结合血浆中的TNF-α,从而降低血浆中TNF-α水平,一定程度上抑制PI3K、Akt蛋白的表达和激活,使NO释放减少,改善了HPS严重程度。
Objective: The hepatopulmonary syndrome (HPS) is characterized by a defect in arterial oxygenation induced by pulmonary vascular dilatation in the setting of liver diseased or portal hypertension, accompanying with respiratory dysfunction.The basal pathological alteration is intrapulmonary vasodilatation, which is often related to the nitric oxide (NO) overproduction.
NO is an important mediator for intrapulmonary vasodilatation. It could stimulate the hyperplasia endothelialis and peripherad growth of micrangium, which lead to widened alveolar septum and diminished alveolar volume.The end-results are descending of partial pressure of oxygen in artery (PaO2) and widened of alveolar-arterial PO2 difference (P(A-a)O2). NO is produced by NO synthases (NOS) with the substrate of L-arginine. Now, it is considered that endothelin (ET) could bind to ETB receptor in endodermis, which leading to endothelial NOS (eNOS) activation, and following NO production. Unlike eNOS, inducible NOS (iNOS) activation dues to pulmonary macrophage activation or aggregation may increase NO production.
Phosphatidylinositol 3 kinase (PI3K) family is characterized by catalyzing the phosphorylation of phosphatidylinositol (PI) at the 3-OH position of the inositol phospholipids, and producing phosphatidylinositoltriphosphate (PIP3) that is the intracellular second messenger. This enzyme is composed of two subunits: an 85-kilodalton regulatory subunit and a 110-kilodalton catalytic subunit.The p85 subunit includes two SH2 domains and one SH3 domain. Having been activated by growth factor receptors, SH2 domains would directly bind to phosphotyrosine residues of growth factor receptors or adaptors, and then,the p85-p110 complex is brought to the membrane for next message transduction.
Akt, also known as protein kinase B (PKB), which is identified as the human homologues for the viral oncogene v-akt, is a serine/threonine kinase downstream target of PI3K. To date, three members of this PKB family have been isolated, named Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ. Although they are products of different genes, they are closely related to each other, with up to 80% of amino acid homology. Nevertheless, their expression is different in distinct tissues. Activation of three isoforms is in a similar way that phosphorylation of two sites, one in the activation domain and another in the COOH-terminal hydrophobic motif, and both of them are necessary for full activity. Activated Akt could activate or restrain a series of substrates through phosphorylation, for example, NF-κB, Bad, caspase9 and GSK-3 and so on, to adjust cellular proliferation, differentiate, apoptosis and migration.
Bacterium translocation (BT) leads to overproduction of TNF-α, the latter is an important factor in the development of HPS. It has been identified that the monocytes in circulating could stick to and accumulate in pulmonary vascular endodermis.Where they can differentiate into pulmonary intravascular cmacrophages (PIM), which could be activated by LPS. Activation of PIM could release a great of mediators of inflammation, for example, TNF-α, NO, IL-1, IL-6 and IL-8 etc. Especially, TNF-αcould induce local release of monocyte chemotactic factors and expression of macrophage and endothelium adhesion molecules. In addition, TNF-αcould activate NF-κB through PI3K/Akt signal pathway. NF-κB participates in the transcribing process of numerous inflammatory factors, including iNOS and TNF-αexpression by macrophage. So that iNOS induces NO overproduction and TNF-αand NF-κB that could activate each other in circles. Then the cascade reaction is triggered, which promotes the development of HPS.
The prevalence of HPS in the setting of cirrhosis ranges between 10% - 20%, with prognosis mala. Owing to unknown pathogeny of HPS, there is still no effective medical therapy to reverse this condition. Liver transplantation is the only effective method among choices of treatment though mortality is comparatively high. So it is important to define its pathogeny, and look for effective therapeutic approach.
For the past years, TNF-αMcAb has been used for treatment of Crohn’s disease, rheumatoid arthritis and ankylosing spondylitis etc, and shows well effect. So we conclude that TNF-αMcAb should be effective on HPS through combining to TNF-α. Up to now, there is few people have done the similar study. Therefore, we carried out this study. The aim of the present study was to evaluate if TNF-αMcAb influences experimental HPS when administered after CBDL and to define the mechanisms about the development of HPS.
Metheds: Male Sprague-Dawley rats, weighing 250±25g underwent sham operation (6 rats), CBDL (30 rats, divided into 5 subgroups: 1week、2week、3week、4week and 5week after ligation), CBDL+TNF-αMcAb (24 rats, divided into 4 subgroups: treating 1week, 2week, 3week, and 4week after 1 week of ligation). CBDL+TNF-αMcAb ( 0.1g/kg/2d ) was given by intraperitoneal injection for 1 week, 2week, 3week and 4 week , respectively, beginning 1 week after CBDL. At the same time, Rats in CBDL group were given NS for the same dose. Rat samples were gotten at different timepoints, as follow 1 wk, 2 wk, 3 wk, 4 wk, 5 wk after CBDL (Sham group were gotten at the fifth week). Hepatopulmonary syndrome was assessed by measurements of alveolar-arterial PO2 difference (P(A-a)O2). The level of NO and TNF-αwas measured. Liver histopathological changes were evaluated by hematoxylin and eosin staining and Masson’s trichrome methods. Lung histopathological changes were evaluated by hematoxylin and eosin staining. Immunohistochemistry, Western Blot and RT-PCR were employed to investigate the expression and the change of PI3K, Akt and p-Akt in rats’lung.
Results:
1. Histopathological changes of liver and lung:
1.1 Liver:①Control group: Hematoxylin and eosin staining showed that there were normal hepatic lobules, and hepatic plate to line in order. Masson showed that there was a little connective tissue in portal area.②CBDL group: Hematoxylin and eosin staining and Masson’s trichrome methods showed that the rats of CBDL group were with extensive biliary ducts accrementition and dilation, even complete cirrhotic nodule;③TNF-αMcAb treatment group: Hematoxylin and eosin staining showed that there was also biliary ducts accrementition, but not significant difference compared with the sham group in liver. Masson showed that there was increasing collagen fiber in portal areas, but not complete cirrhotic nodule.
1.2 Lung:①Control group: micrangiums of lung were eumorphism, alveolus wall and alveolus space were regular;②CBDL group: Hematoxylin and eosin staining showed that there was widened interalveolar septum, and obviously inflammatory reaction in lung interstitium. Macrophages were found. Capillary density increased, which showed that there was vasodilatation in lung tissue;③TNF-αMcAb treament group: Hematoxylin and eosin staining showed that there was a gentle vasodilatation and widened interalveolar septum.
2. Hepatic function check:①CBDL group: ALT in plasma obviously increased after CBDL for 1 week(177.00±56.32 U/L). Compared with the control group(56.33±9.29 U/L), which showed that there existed obvious difference(P<0.05). Following, the level of ALT in plasma fell off, but it was notablely elevated compared with that of control group.②TNF-αMcAb treatment group: Having treated with TNF-αMcAb for 2week, the level of ALT in plasma decreased obviously compared with that of CBDL group (P<0.05). Following, the downtrend continued, up to 4 week (63.00±7.55), it was still higher than that of control group, but the difference was not significant (P>0.05) .
3. Measurement of plasma TNF-α:①CBDL group: 1 week(1.60±0.07 ng/ml) after CBDL, the level of TNF-αin plasma increased significantly. Then, it kept the rising tendency. At the third week(3.18±0.54 ng/ml)after CBDL, it reached the peak. Between the third week and the fifth week after CBDL, it had a transient drop. Following, it returned to increase. But, it was at a higher level compared with that of control group (P<0.05).②TNF-αMcAb treament group: Compared with the CBDL group, the level of TNF-αin plasma decreased obviously after one week treatment. Then, the level of TNF-αkept downturn. Until the fourth week(1.21±0.13 ng/ml) after the CBDL, the level of TNF-αin plasma was also much lower than the CBDL group(2.43±0.43)(P<0.05),but compared with that of control group, there was no significant difference (P>0.05).
4. The blood gas analysis:①CBDL group: two weeks(17.65±2.67 mmHg) after the CBDL, P(A-a)O2 began to increase. Compared with that of control group(6.70±3.20)mmHg, which showed that there existed obvious difference(P<0.05). Later, it kept increasing.②TNF-αMcAb treatment group: The P(A-a)O2 increasing of the treatment group was restrained after 2 weeks treatment, then it kept increasing. After treatment for 4 week(10.5±8.51 mmHg),to contrast that of CBDL group(51.8±8.49 mmHg),there was significant difference(P<0.05). Compared with that of control group, the level of the P(A-a)O2 was also higher , but no significant difference (P>0.05).
5. Measurement of plasma NO:①CBDL group: One week after CBDL, the level of NO in plasma increased significantly. Then, it remained elevated tendency. It reached peak at the third week(95.67±15.41 umol/l) after CBDL. Then it had a transient drop. But, it was at a higher level compared with that of control group(36.19±1.59 umol/l)(P<0.05).②TNF-αMcAb: Compared with that of CBDL group, the level of TNF-αin plasma decreased obviously after one week treatment. Then, the level of TNF-αkept decreasing. Until the third week(45.47±9.58)and 4 week(44.42±9.27)after the CBDL, the level of NO in plasma was obviously lower than CBDL that of group at the same time( 72.22±6.11 umol/l , 64.31±1.75 umol/l ), and there was statistical significance. The level of NO in plasma was also higher than that of control group, but the difference was not significant (P>0.05).
6. Measurement of the expression of PI3K in lung
6.1 RT-PCR showed as follow:①CBDL group:The expression of PI3K mRNA peaked at the second week(1.75±0.15)after CBDL,and there was a transient descent at the third week(1.07±0.19),then a ascensus at fourth week (1.26±0.16)and another descent at the fifth week(0.98±0.11).Compared with the expression of PI3K mRNA of sham group(0.28±0.08),there was significant difference.②TNF-αMcAb treatment group: Compaerd with CBDL group, the expression of PI3K mRNA was obviously depressed at the second week (0.34±0.14).Then, the level of the PI3K mRNA kept ascending, but it’s aways lower than that of sham group, and the difference was significant(P<0.05).
6.2 Immunohistochemistry showed as follow:①CBDL group: The positive protein expression of PI3K was higher than that in the control group, the cell kytoplasm is brown, and the positive expression localized in the kytoplasm of phagocyte.The second week(0.31±0.10), expression of PI3K protein was up to peak,which was higher than that of control group(0.06±0.01)(P<0.05). Next, it had a temporary descent.Then, it returned to increase. Compared with that of control group, there was significant difference (P<0.05).②TNF-αMcAb treatment group:After 1 week treament ( 0.15±0.03, the protein expression of PI3K was obviously restrained, compared with that of CBDL group, there was significant difference (P<0.05). Following, it kept on this level. Compared with that of control group, there was significant difference (P<0.05).
6.2 Western blot showed as follow: The result was similar with IHC.
7 Measurement of the expression of Akt and p-Akt in lung
7.1 RT-PCR of Akt showed as follow:①CBDL group: Expression of Akt mRNA showed gradually elevated tendency. At the third week, it reached peak (2.07±0.18). Next, it kept descending. But compared with sham group (0.58±0.06), the expression of Akt mRNA was higher. And the difference was significant.②TNF-αMcAb treatment group: The tendency of the expression of Akt mRNA was similar with that of CBDL group, but level of the expression of Akt mRNA was obviously depressed, and there was significant difference (P<0.05). At the third week, the difference was not significant (P>0.05).But compared with that of sham group, there was significant difference (P<0.05).
7.2 Immunohistochemistry of p-Akt showed as follow:①CBDL group: The positive protein expression of p-Akt was higher than that in the control group, the cell kytoplasm is brown, and the positive expression localized in the kytoplasm of phagocyte. After CBDL, protein expression of p-Akt was uo to peak on the fourth weeek(0.31±0.03. Compared with that of control group(0.09±0.03, there was significant difference (P<0.05).②TNF-αMcAb treatment group: compared with that CBDL group, the expression of p-Akt decreased. After treatment for 4 weeks(0.20±0.03, it was much lower than CBDL group(P<0.05), but still higher than that of control group(P<0.05).
7.3 Western blot of Akt showed as follow:①CBDL group: After CBDL, the expression of Akt kept increasing. At the third week(2.45±0.23, it was up to peak. Following, its expression was significantly restrained, but there was significant difference between it and the control group(1.08±0.20)(P<0.05).②TNF-αMcAb treatment group: For 1 week treatment, compared with that of CBDL group, the expression of Akt was visiblely inhibited. Following, it kept on this level. Compared with that of control group, there was significant difference (P<0.05).
7.4 Western blot of p-Akt showed as follow: The result was similar with IHC.
8 Analysis of correlation of TNF-αand NO in plasma
Coefficient correlation was 0.719, and probability value of coefficient correlation was 0.005<0.05, there was statistical significance, which showed correlativity between TNF-αand NO in plasma of rats with HPS. The direct correlation indicated that NO would increase along with TNF-α.
Conclusions:
1 Liver function changes were the foundation of the lung function changes in the forming process of HPS;
2 TNF-αmay probably be an important essential cause of the development of HPS;
3 Development of HPS accompanies by increasing expression of PI3K and Akt protein. Activation of Akt increasing showes activation of PI3K/Akt signalling pathway, which suggested that PI3K/Akt was concerned with HPS happenning;
4 TNF-αMcAb may combine with TNF-αin the plasma, and decrease the expression of TNF-α, partly inhibited expression and activation of PI3K and Akt protein, and the release of NO, then improve HPS.
引文
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