灵杆菌多糖抗内毒素作用及作用机制研究
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摘要
前言
灵杆菌多糖是从灵杆菌菌体中提取精制得到的脂多糖物质,目前国内注册的英文通用名为hemarisin,国内曾经称灵杆菌多糖为“灵杆菌素”,国外,尤其是前苏联或俄罗斯所报道的同类产品叫做prodigiozan。由于灵杆菌(Bacillus prodigiosus)是粘质沙雷氏菌(Serratia marcescens)的老式叫法,因此,灵杆菌多糖和prodigiozan都是从粘质沙雷氏菌或灵杆菌中提取的脂多糖类物质。灵杆菌多糖虽然也是属于细菌内毒素的范畴,但其毒性极低,具有免疫促进作用,在临床主要用于各种原因引起的白细胞减少症、乙型肝炎及急慢性盆腔炎等疾病的辅助治疗。
目的
在药物筛选过程中,我们发现灵杆菌多糖具有明显的抗内毒素作用,据此,本研究采用动物模型对灵杆菌多糖的抗内毒素作用进行确证,并从不同角度探讨灵杆菌多糖抗内毒索的作用机制,为理解灵杆菌多糖的临床用途提供新的视角,同时为灵杆菌多糖用于内毒素血症的辅助治疗提供前期实验依据。
研究内容
1.灵杆菌多糖对小鼠内毒素休克及内毒素血症模型的影响
1.1灵杆菌多糖对小鼠内毒素休克致死模型的保护作用
1.1.1材料与方法
先给KM小鼠(以下称：小鼠)腹腔注射灵杆菌多糖或生理盐水或阳性对照药物,0.5h后,每组动物均腹腔注射内毒素(铜绿假单胞菌脂多糖,LPS)60mg/kg,观察3d内动物死亡情况。
1.1.2结果
腹腔注射大剂量LPS(60mg/kg)后,小鼠出现明显的中毒反应,20h后陆续有动物死亡,48h后仍存活的动物逐渐恢复正常活动与摄食。灵杆菌多糖(100、50U/kg)预防性腹腔注射给药使动物存活率分别提高60%和45%(χ2=15.000,P=0.000；χ2=9.231,Bonferroni校正P=0.006)。
1.2灵杆菌多糖对内毒素血症模型小鼠炎症相关因子产生的抑制作用
1.2.1材料与方法
先给小鼠腹腔注射灵杆菌多糖或生理盐水或阳性对照药物,0.5h后,正常对照组动物腹腔注射等体积生理盐水,其余动物均腹腔注射LPS(10mg/kg),导致内毒素血症。3h后,小鼠摘眼球放血制备血清,ELISA方法测定血清中白介素-1p(IL-1p)、白介素-6(IL-6)、肿瘤坏死因子α(TNF α)、前列腺素E2(PGE2)的含量。Griess法检测一氧化氮(NO)的含量。
1.2.2结果
小鼠腹腔注射LPS后,其血清中IL-1β、IL-6、TNF α、PGE2、NO的含量明显升高,预先给予灵杆菌多糖(100,50U/kg)腹腔注射,然后再给LPS,其血清中各指标升高的幅度明显下降,灵杆菌多糖组各指标均数与LPS组相比,差异有统计学意义(IL-1β：P=0.007,P=0.023；IL-6：P=0.018,P=0.046,TNFα：P=0.001,P=0.019;PGE2：P=0.001,P=0.009：NO：P=0.012,P=0.039)。
1.3灵杆菌多糖对内毒素血症模型小鼠炎症相关基因mRNA表达的抑制作用
1.3.1材料与方法
动物分组及处理同1.2.1节,小鼠腹腔注射LPS(10mg/kg)3h后,脱颈椎处死,取出脾脏置液氮中保存,提取总RNA,实时荧光定量PCR法检测IL-1p、IL-6、TNFα、COX-2(环氧酶-2)、iNOS(诱导型一氧化氮合酶)mRNA的相对含量(2-△Ct,以GAPDH为内参)。
1.3.2结果
小鼠腹腔注射LPS后3h,脾脏中IL-1β、IL-6、TNFα、COX-2、iNOS mRNA的表达明显增多,提前0.5h腹腔注射灵杆菌多糖(100,50U/kg)可部分抑制由LPS诱导的上述各目的基因mRNA的表达,灵杆菌多糖组各参数均数与LPS组相比,剂量为100U/kg时,差异均有统计学意义(IL-1β：P=0.026；IL-6：P=0.015；TNF仅：P=0.029：COX-2：P=0.016；iNOS:P=0.020)；剂量为50U/kg时,差异有些有统计学意义(IL-1β：P=0.072；IL-6：P=0.035；TNF α：P=0.069；COX-2：P=0.034；iNOS:P=0.399)。
2.灵杆菌多糖对体外内毒素激活小鼠腹腔巨噬细胞产生炎症相关因子及表达炎症相关基因的影响
2.1灵杆菌多糖对内毒素激活小鼠腹腔巨噬细胞产生炎症相关因子的抑制作用
2.1.1材料与方法
LPS(10μg/ml)刺激小鼠腹腔巨噬细胞活化作为体外炎症模型。微量96孔板作为培养载体,ELISA法检测培养上清中IL-1β、IL-6、TNF-α和PGE2的含量；Griess法检测NO的含量。
2.1.2结果
在LPS刺激小鼠腹腔巨噬细胞16h后,培养上清中促炎细胞因子IL-1p、IL-6、TNF-a及培养24h后炎症因子或介质PGE2、NO的含量大幅增加,灵杆菌多糖(20、10U/mL)能部分抑制由LPS诱导的上述促炎细胞因子及炎症因子或介质的分泌,灵杆菌多糖组各指标均数与LPS组相比,差异有统计学意义(IL-1β：P=0.001,P=0.016；IL-6：P=0.001,P=0.016；TNF-α：P=0.027,P=0.046；PGE2：P=0.010,P=0.038；NO：P=0.000,P=0.001)
2.2灵杆菌多糖对内毒素激活小鼠腹腔巨噬细胞表达炎症相关基因mRNA的抑制作用
2.2.1材料与方法
LPS(10μg/ml)刺激小鼠腹腔巨噬细胞活化作为体外炎症模型。6孔板作为培养载体,实时荧光定量PCR法检测目的基因mRNA的相对含量(2-△Ct,以GAPDH为内参)。
2.2.2结果
经过LPS体外刺激16h后,小鼠腹腔巨噬细胞IL-1p、IL-6、TNF-α、COX-2、iNOS mRNA的表达明显增多,灵杆菌多糖(20、10U/mL)可部分抑制由LPS诱导的上述目的基因mRNA的表达,灵杆菌多糖组各指标均数与LPS组相比,差异有统计学意义(IL-1β：P=0.001,P=0.009；IL-6：P=0.000,P=0.000；TNF-a：P=0.000,P=0.000；COX-2：P=0.000,P=0.001；iNOS:P=0.011,P=0.021)
3.灵杆菌多糖单独给药对小鼠产生促炎细胞因子的影响
3.1灵杆菌多糖对正常小鼠血清促炎细胞因子水平的影响
3.1.1材料与方法
小鼠腹腔注射灵杆菌多糖或生理盐水或LPS,3h后,小鼠摘眼球放血制备血清,ELISA方法测定血清中IL-1β、IL-6、TNF α的含量。
3.1.2结果
正常小鼠血清细胞因子IL-1p、IL-6、TNF α的含量很低,灵杆菌多糖(100、50U/kg)腹腔注射给药使上述细胞因子的血清含量有提升趋势,但均数与生理盐水组相比,差异无统计学意义(IL-1β：P=0.377,P=0.658；IL-6：P=0.903,P=0.994：TNF a：P=0.369,P=0.997)
3.2灵杆菌多糖对正常小鼠腹腔巨噬细胞产生促炎细胞因子的影响
3.2.1材料与方法
小鼠腹腔巨噬细胞体外培养模型,微量96孔板作为培养载体,ELISA法检测培养上清中IL-1p、IL-6、TNF-α的含量。
3.2.2结果
在体外培养的条件下,灵杆菌多糖对小鼠腹腔巨噬细胞产生IL-1β、IL-6、TNF-α均有弱的促进作用,在浓度为20U/mL时,其均数与全培组相比,差异有统计学意义(P=0.007,P=0.002,P=0.003)。在浓度为10U/mL时,有些有统计学意义(P=0.043,P=0.536,P=0.016)。
4.灵杆菌多糖对内毒素所致小鼠肺损伤的保护作用
4.1灵杆菌多糖抑制内毒素诱导的肺氧化损伤作用
4.1.1材料与方法
除LPS为静脉给药外,动物分组及处理同1.2.1节,小鼠尾静脉注射LPS(30mg/kg)6h后,腹腔注射麻醉,开胸取出全肺,经后续处理后,测量肺湿重干重比(W/D)、髓过氧化物酶(MPO)活力、丙二醛(MDA)含量。
4.1.2结果
小鼠静脉注射LPS6h后,出现明显肺水肿现象,肺组织中MPO的活性及MDA的含量均明显增加。
预先腹腔注射灵杆菌多糖(100、50U/kg)可减轻由LPS引起的肺水肿现象,肺组织湿重与干重的比值下降,当灵杆菌多糖剂量为100U/kg时其均数与LPS组相比,差异有统计学意义(P=0.010),剂量为50U/kg时差异无统计学意义(P=0.060)。
预先腹腔注射灵杆菌多糖(100、50U/kg)可部分抑制由LPS引起的MPO活性及MDA含量的增加,其均数与内毒素模型组相比,差异有统计学意义(MPO：P=0.001,P=0.010；MDA：P=0.000,P=0.002)。
4.2灵杆菌多糖抑制内毒素诱导肺内促炎细胞因子产生的作用
4.2.1材料与方法
除LPS为静脉给药外,实验分组及给药同1.2.1节,小鼠均尾静脉注射LPS(30mg/kg),3h后,腹腔注射麻醉,开胸取出肺脏,经冷冻后制备肺组织匀浆,ELISA法测定上清中IL-1β、IL-6和TNF α的含量。
4.2.2结果
小鼠静脉注射LPS3h后,肺组织中IL-1β、IL-6和TNFα的含量明显增加。预先腹腔注射灵杆菌多糖(100、50U/kg)可部分抑制由LPS引起的IL-1β、IL-6和TNFα的增加,当灵杆菌多糖剂量为100U/kg时其均数与LPS组相比,差异均有统计学意义(P=0.029,P=0.010,P=0.006)；剂量为50U/kg时,差异有些有统计学意义(P=0.695,P=0.065,P=0.017)。
5.灵杆菌多糖对内毒素所致小鼠肝损伤的保护作用
5.1灵杆菌多糖对内毒素诱导肝细胞损伤的保护作用
5.1.1材料与方法
除LPS为静脉给药外,动物分组及处理同1.2.1节,小鼠均尾静脉注射LPS(30mg/kg),6h后,小鼠摘眼球取血,制备血清用于谷丙转氨酶(ALT)和谷草转氨酶(AST)检测。
5.1.2结果
小鼠静脉注射LPS6h后,血清中ALT和AST的活性明显升高,预先腹腔注射灵杆菌多糖(100、50U/kg)可部分抑制由内毒素引起的ALT和AST活性的升高,当灵杆菌多糖剂量为100U/kg时其均数与LPS组相比,差异均有统计学意义(P=0.022,P=0.026)；剂量为50U/kg时,AST的差异有统计学意义(P=0.031)。
5.2灵杆菌多糖对内毒素诱导肝组织促炎细胞因子mRNA表达的抑制作用
5.2.1材料与方法
实验分组及给药同1.2.1节,小鼠均腹腔注射LPS(30mg/kg),3h后,脱颈椎处死,取出肝脏置液氮中保存,提取总RNA,实时荧光定量PCR法检测IL-1p、IL-6、TNF αmRNA的含量。5.2.2结果
小鼠腹腔注射细菌LPS后3h,肝脏中IL-1β、IL-6、TNF αmRNA的表达明显增多,提前0.5h腹腔注射灵杆菌多糖(100,50U/kg)可部分抑制由LPS诱导的IL-1p、IL-6、TNFαmRNA的表达,当灵杆菌多糖剂量为100U/kg时其均数与LPS组相比,差异均有统计学意义(P=0.020,P=0.012,P=0.014)；剂量为50U/kg时,IL-6和TNFα mRNA的差异有统计学意义(P=0.043,P=0.020)。
统计学处理
应用SPSS11.5统计软件进行统计分析。存活率以百分数表示,先采用R×C表资料的卡方检验对各组动物存活率差异进行多组间的总体检验,然后采用四格表资料的卡方检验进行组间存活率的两两比较。定量数据结果以均数±标准差表示,采用单向方差分析法分析,先进行方差齐性检验,若方差齐用LSD方法多重比较；若方差不齐,采用经Welch法校正方差分析的F和P值,用Dunnett's T3法多重比较。显著性水准取α=0.05,以P<0.05时,组间差异判断为具有统计学意义。
结论
(1)灵杆菌多糖预防性腹腔给药可减少内毒素致小鼠死亡的百分率。
(2)灵杆菌多糖预防性腹腔给药可部分抑制由内毒素所致小鼠血清IL-1β、IL-6、TNFα、PGE2、NO含量的升高。
(3)灵杆菌多糖预防性腹腔给药可部分抑制由内毒素所致小鼠脾脏IL-1pIL-6、TNFa、COX-2、iNOSmRNA的表达。
(4)灵杆菌多糖体外可部分抑制内毒素刺激小鼠腹腔巨噬细胞产生IL-1p、IL-6、TNF-a、PGE2、NO。
(5)灵杆菌多糖体外可部分抑制内毒素刺激小鼠腹腔巨噬细胞IL-1p、IL-6、TNF-α、COX-2、iNOS mRNA的表达。
(6)灵杆菌多糖体外可轻微刺激小鼠腹腔巨噬细胞产生IL-1p、IL-6、TNF-a,其作用明显弱于内毒素。
(7)灵杆菌多糖预防性腹腔给药对内毒素所致小鼠肺损伤有一定的保护作用。
(8)灵杆菌多糖预防性腹腔给药对内毒素所致小鼠肝损伤有一定的保护作用。
(9)由于慢性偷腔炎和乙型肝炎时均伴有局部内毒素浓度的升高,灵杆菌多糖临床上对上述病症有良好疗效的机制可能与拮抗内毒素有关。
(10)灵杆菌多糖与内毒素都属于细菌脂多糖,而细菌脂多糖作用的主要受体为TLR4(Toll-like receptor4)。灵杆菌多糖刺激作用弱,没有明显的毒性；而内毒素刺激作用强,有明显的毒性反应；提示灵杆菌多糖为部分激动剂,而内毒素为完全激动剂,两者同时存在时前者对后者的毒性作用有拮抗作用,不过,其确切机制有待进一步的研究。
INTRODUCTION
Bacterium prodigiosum polysaccharides or Hemarisin, an English generic name registred in China, refers to a group of polysaccharides isolated from a bacterium Bacillus prodigiosus. It used to be named as Ling Gan Jun Su in Chinese. In foreign countries, especially in the former Soviet Union or Russia, a similar product to hemarisin is called prodigiozan.Because Bacillus prodigiosus is the old name for Serratia marcescens, therefore, both hemarisin and prodigiozan are lipopolysaccharides isolated from Serratia marcescens. In essence, hemarisin belongs to the category of bacterial endotoxin, but its toxicity is very low. It has immune stimulant effect and is mainly used in the adjuvant treatment of leucopenia caused by different factors, hepatitis B, and acute or chronic pelvic inflammatory diseases, etc.
OBJECTIVES
Hemarisin was found to be effective against endotoxin in the process of screening for anti-endotoxin compounds. Inspired by the finding, the present study was carried out to confirm the anti-endotoxin effectiveness by using animal models and to explore the underlying mechanism from different aspects in an effort to provide new information for understanding the polysaccharides'clinical applications and at the same time to lay a foundation experimentally for the polysaccharides used in adjuvant treatment of endotoxemia.
RESEARCH CONTENTS
1. Effect of hemarisin on murine models of endotoxin shock and endotoxemia
1.1Protective effect of hemarisin on murine lethal model of endotoxin shock
1.1.1Materials and Methods
KM mice (hereinafter referred to as:mice) were intraperitoneally (ip) injected with hemarisin or saline or positive control drug at first. After0.5h, each animal was ip injected with endotoxin (Pseudomonas aeruginosa lipopolysaccharides, LPS) in a dose of60mg/kg. The animal was under observation for3d and deaths were recorded.
1.1.2Results
Injected ip with large dose of LPS (60mg/kg), mice exhibited obvious toxic response and some became to die after20h. Surviving animal gradually came back to normal activities and feeding after48h. Hemarisin (100,50U/kg) with ip administration made animal survival rates increase by60%and45%respectively (χ2=15.000, P=0.000;χ2=9.231, Bonferroni correction P=0.006)
1.2Inhibitory effect of hemarisin on inflammatory factors in murine model of endotoxemia
1.2.1Materials and Methods
Mice were ip injected with hemarisin or saline or positive control drug at first. After0.5h, animal in normal control group was ip injected equal volume saline and the rest was ip injected LPS in a dose of10mg/kg in order to cause endotoxemia.3h later, blood samples, from which serum samples were prepared, were collected from the eye vein by removing the eyeballs quickly. ELISA method was used to determine the amounts of serum interleukin-1β(IL-1β), interleukin-6(IL-6), tumor necrosis factor a (TNF a), prostaglandin E2(PGE2) and Griess method for the detection of nitric oxide (NO) content.
1.2.2Results
The mouse serum levels of IL-1β, IL-6, TNF a, PGE2and NO were dramatically increased after ip injection of LPS. Prophylactic administration of hemarisin (100,50U/kg) by ip injection to the animal who were then followed by LPS ip injection, the extent of increase in the above mentioned parameters was attenuated markedly compared to LPS injection alone. The differences of means for the parameters between hemarisin groups and LPS group were statistically significant (IL-1β:P=0.007, P=0.023; IL-6:P=0.018, P=0.046; TNF a:P=0.001, P=0.019; PGE2:P=0.001, P=0.009; NO:P=0.012, P=0.039)1.3Inhibitory effect of hemarisin on mRNA expression of inflammatory factors gene in murine model of endotoxemia
1.3.1Materials and Methods
Animal groups and treatment were the same as described in section1.2.1. Mice were sacrificed by cervical dislocation after receiving LPS (10mg/kg) ip injection for3h. Then spleens were removed and stored in liquid nitrogen for RNA isolation. Real time fluorescence quantitative PCR (Polymerase Chain Reaction) was applied to evaluate the mRNA expressions of IL-1β, IL-6, TNF α, COX-2(Cyclooxygenase-2) and iNOS (Inducible Nitric Oxide Synthase). The content of mRNA was expressed as2-ΔCt using GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) as internal reference.
1.3.2Results
After ip injection of LPS to the mice for3h, the mRNA expressions of IL-1β, IL-6, TNF a, COX-2and iNOS in spleen increased dramatically. Hemarisin (100,50U/kg) ip injected to the mice0.5h ahead of LPS injection partially inhibited expressions of the genes of interest induced by LPS. The differences of means for the examined parameters between hemarisin group and LPS group were statistically significant when hemarisin was in the dose of100U/kg (IL-1β:P=0.026; IL-6: P=0.015; TNF a:P=0.029; COX-2:P=0.016; iNOS:P=0.020). However, some of the differences were statistically significant when hemarisin was in the dose of50U/kg (IL-1β:P=0.072; IL-6:P=0.035; TNF a:P=0.069; COX-2:P=0.034; iNOS:P=0.399)
2. Effect of hemarisin on production and gene expression of inflammatory factors in endotoxin activated murine peritoneal macrophages in vitro
2.1Inhibitory effect of hemarisin on production of inflammatory factors in endotoxin activated murine peritoneal macrophages
2.1.1Materials and Methods
Murine peritoneal macrophages activated by LPS (10μg/ml) were used as an in vitro inflammatory model.96well microtitters were applied for cell culture. ELISA method was used to determine the amounts of IL-1β, IL-6, TNF a and PGE2, and Griess method for the detection of NO content in culture supernatants.
2.1.2Results
After LPS stimulation of murine peritoneal macrophages for16h, the amounts of proinflammatory cytokines IL-1β,IL-6and TNF-a, and inflammatory factors or mediators PGE2and NO in the supernatants increased dramatically. Hemarisin (20and10U/mL) partially inhibited the macrophages production of the proinflammatory cytokines and inflammatory factors or mediators induced by LPS. The differences of means for the examined parameters between hemarisin groups and LPS group were statistically significant (IL-1β:P=0.001, P=0.016; IL-6: P=0.001, P=0.016; TNF-a:P=0.027, P=0.046; PGE2:P=0.010, P=0.038; NO: P=0.000, P=0.001)
2.2Inhibitory effect of hemarisin on gene expression of inflammatory factors mRNA in endotoxin activated murine peritoneal macrophages
2.2.1Materials and Methods
Murine peritoneal macrophages activated by LPS (10μg/ml) were used as an in vitro inflammatory model.6well plates were applied for cell culture. Real time fluorescence quantitative PCR was applied to evaluate the mRNA expressions of genes of interest. The content of mRNA was expressed as2-ΔCt using GAPDH as internal reference.
2.2.2Results
After LPS stimulation of murine peritoneal macrophages for16h, the mRNA expressions of IL-1β,IL-6, TNF-a,COX-2and iNOS markedly increased. Hemarisin (20and10U/mL) partially inhibited the expressions of the genes of interest induced by LPS. The differences of means for the examined parameters between hemarisin groups and LPS group were statistically significant (IL-1β:P=0.001, P=0.009; IL-6: P=0.000, P=0.000; TNF-α:P=0.000, P=0.000; COX-2:P=0.000, P=0.001; iNOS: P=0.011,P=0.021)
3. Effect of hemarisin administered alone on production of proinflammatory cytokines in mice
3.1Effect of hemarisin on the serum levels of proinflammatory cytokines in mice
3.1.1Materials and Methods
Mice were ip injected with hemarisin or saline or LPS.3h later, blood samples, from which serum samples were prepared, were collected from the eye vein by removing the eyeballs quickly. ELISA method was used to determine the amounts of serum IL-1β, IL-6and TNF a.
3.1.2Results
The levels of the cytokines IL-1β, IL-6and TNF a in normal mice were very low. Peritoneal injection of hemarisin (100,50U/kg) to the animal resulted in an increase trend in the cytokines serum levels, but the differences of means for the examined parameters between hemarisin groups and saline control group were not statistically significant (IL-1β:P=0.377, P=0.658; IL-6:P=0.903, P=0.994; TNF a:P=0.369, P=0.997)
3.2Effect of hemarisin on production of inflammatory cytokines by normal murine peritoneal macrophages
3.2.1Materials and Methods
Normal murine peritoneal macrophages were cultured as an in vitro model.96well microtitter was applied for cell culture. ELISA method was used to determine the amounts of IL-1β, IL-6and TNF in culture supernatants.
3.2.2Results
Hemarisin exerted weak stimulating effect on production of IL-1β, IL-6and TNF-a by murine peritoneal macrophages in in vitro culture situation. The differences of means for the examined parameters between hemarisin group and culture medium control group were statistically significant when hemarisin was at the concentration of20U/mL(P=0.007, P=0.002, P=0.003). However, only some of the differences were statistically significant when hemarisin was at the concentration of10U/mL (P=0.043, P=0.536, P=0.016)
4. Protective effects of hemarisin on endotoxin-induced lung injury in mice
4.1Hemarisin inhibits endotoxin-induced pulmonary oxidative damage in mice
4.1.1Materials and Methods
Except for LPS administered by intravenous route, animal groups and treatment were the same as described in section1.2.1. Mice were injected LPS (30mg/kg) through tail vein.6h later, the animal were anesthetized by intraperitoneal injection and lungs were removed by thoracotomy. Followed by various treatments, lung wet weight and dry weight ratio (W/D) was measured, myeloperoxidase (MPO) activity assessed, and malondialdehyde (MDA) content determined.
4.1.2Results
Significant pulmonary edema appeared after mice receiving LPS (30mg/kg) intravenous injection for6h. Lung tissue MPO activity and MDA content were increased dramatically.
Prophylactic ip administration of hemarisin (100,50U/kg) reduced the pulmonary edema caused by LPS with a decrease in lung wet weight and dry weight ratio (W/D). The difference of means for the ratio between hemarisin group and LPS group was statistically significant when hemarisin was in the dose of100U/kg (P=0.010), while no statistical significance was observed in the dose of50U/kg (P=0.060)
Prophylactic ip administration of hemarisin (100,50U/kg) partially suppressed the increases in MPO activity and MDA content caused by LPS. The differences of means for the examined parameters between hemarisin group and LPS group were statistically significant (MPO:P=0.001, P=0.010; MDA:P=0.000, P=0.002)4.2Hemarisin inhibits endotoxin-induced increase in pulmonary proinflammatory cytokines
4.2.1Materials and Methods
Except for LPS administered by intravenous route, animal groups and treatment were the same as described in section1.2.1. Mice were injected LPS (30mg/kg) through tail vein.3h later, the animal were anesthetized by intraperitoneal injection and lungs were removed by thoracotomy and frozen immediately. Pulmonary tissue homogenates were made in ice bath. ELISA method was used to determine the amounts of IL-1β, IL-6and TNFa in the homogenates.
4.2.2Results
The amounts of IL-1β, IL-6and TNF a in murine pulmonary tissue were dramatically increased after iv injection of LPS for3h. Prophylactic administration of hemarisin (100and50U/kg) by ip injection to the animal partially inhibited the increase in the proinflammatory cytokines induced by LPS. The differences of means for the examined parameters between hemarisin group and LPS group were statistically significant when hemarisin was in the dose of100U/kg (P=0.029, P=0.010, P=0.006). However, some of the differences were statistically significant when hemarisin was in the dose of50U/kg (P=0.695, P=0.065, P=0.017)
5. Protective effects of hemarisin on endotoxin-induced liver injury in mice
5.1Protective effects of hemarisin on endotoxin-induced hepatocyte injury
5.1.1Materials and Methods
Except for LPS administered by intravenous route, animal groups and treatment were the same as described in section1.2.1. Mice were injected LPS (30mg/kg) through tail vein.6h later, blood samples, from which serum samples were prepared, were collected from the eye vein by removing the eyeballs quickly. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum samples were assessed.
5.1.2Results
The mouse serum levels of ALT and AST were dramatically increased after iv injection of LPS for6h. Prophylactic administration of hemarisin (100,50U/kg) by ip injection to the animal that were then followed by LPS ip injection partially inhibited the increase in the examined parameters.The differences of means for the examined parameters between hemarisin group and LPS group were statistically significant when hemarisin was in the dose of100U/kg (P=0.022, P=0.026). However, only the difference of AST was statistically significant when hemarisin was in the dose of50U/kg (P=0.031)
5.2Hemarisin inhibits endotoxin-induced increase in proinflammatory cytokines mRNA expression in liver tissue
5.2.1Materials and Methods
Animal groups and treatment were the same as described in section1.2.1. Mice were sacrificed by cervical dislocation after receiving LPS (30mg/kg) iv injection for3h. Then livers were removed and stored in liquid nitrogen for RNA isolation. Real time fluorescence quantitative PCR was applied to evaluate the mRNA expressions of IL-1β, IL-6and TNF a.
5.2.2Results
After ip injection of LPS to the mice for3h, the mRNA expressions of IL-1β, IL-6and TNF a in the liver increased dramatically. Hemarisin (100,50U/kg) ip injected to the mice0.5h ahead of LPS injection partially inhibited expressions of the genes of IL-1β, IL-6and TNF a induced by LPS. The differences of means for the examined parameters between hemarisin group and LPS group were statistically significant when hemarisin was in the dose of100U/kg (P=0.020, P=0.012, P=0.014). However, only the means differences of IL-6and TNF a were statistically significant when hemarisin was in the dose of50U/kg (P=0.043, P=0.020)
STATISTICAL ANALYSIS
SPSS11.5statistical software was applied for statistical analysis. The survival rate was expressed as percentage. The R X C crosstabs chi-square test was used for overall test of animal survival differences among experimental groups at first, and then the four rows crosstabs chi-square test was performed for two by two comparisons. Continuous variables were described as as Mean±SD and analyzed by One-way ANOVA (Analysis of Variance). Test of homogeneity of variances was carried out prior to ANOVA. If the variance was of homogeneity, LSD method was used for multiple comparison, otherwise, Dunnett's T3method was adopted for multiple comparison, using F and P values by Welch correction analysis of variance. Significance level was set at α=0.05and P<0.05was considered to be statistical significance.
CONCLUSSI ON
(1) Prophylactic ip administration of hemarisin can reduce endotoxin-induced mouse death.
(2) Prophylactic ip administration of hemarisin can partially suppress endotoxin-induced increases in murine serum IL-1β, IL-6,TNF a, PGE2and NO.
(3) Prophylactic ip administration of hemarisin can partially suppress endotoxin-induced increases in mRNA expressions of IL-1β, IL-6, TNF a, COX-2and iNOS in murine liver.
(4) Hemarisin can partially inhibit endotoxin-induced production of IL-1β, IL-6, TNF a, PGE2and NO by murine peritoneal macrophages in vitro.
(5) Hemarisin can partially inhibit endotoxin-induced increases in mRNA expressions of IL-1β, IL-6, TNF a, COX-2and iNOS in murine peritoneal macrophages in vitro.
(6) Hemarisin can slightly stimulate murine peritoneal macrophages to produce IL-1β, IL-6and TNF-a in vitro, but it is much weaker than endotoxin in potency.
(7) Prophylactic ip administration of hemarisin can protect mice from endotoxin-induced lung injury in some degree.
(8) Prophylactic ip administration of hemarisin can protect mice from endotoxin-induced liver injury in some degree.
(9) Considering chronic pelvic inflammatory disease and hepatitis B are accompanied with increase in local endotoxin concentration, it is conceivable that the good efficacy of hemarisin in the treatment of above mentioned clinical diseases may be associated with the anti-endotoxin mechanism of the medicine.
(10) Both hemarisin and endotoxin belong to bacterial lipopolysaccharide, and the main receptor for bacterial lipopolysaccharide is TLR4(Toll-like receptor4). Hemarisin has weak stimulating effect and no obvious toxicity, while endotoxin has strong stimulating action and obvious toxicity. It is indicated that hemarisin act as a partial agonist, and endotoxin a full agonist. The former may exert an antagonist effect against the toxicity of the latter when they are present at the same time. However, the exact mechanism needs further study.
灵杆菌多糖是从灵杆菌菌体中提取精制得到的脂多糖物质,目前国内注册的英文通用名为hemarisin,国内曾经称灵杆菌多糖为“灵杆菌素”,国外,尤其是前苏联或俄罗斯所报道的同类产品叫做prodigiozan。由于灵杆菌(Bacillus prodigiosus)是粘质沙雷氏菌(Serratia marcescens)的老式叫法,因此,灵杆菌多糖和prodigiozan都是从粘质沙雷氏菌或灵杆菌中提取的脂多糖类物质。灵杆菌多糖虽然也是属于细菌内毒素的范畴,但其毒性极低,具有免疫促进作用,在临床主要用于各种原因引起的白细胞减少症、乙型肝炎及急慢性盆腔炎等疾病的辅助治疗。
目的
在药物筛选过程中,我们发现灵杆菌多糖具有明显的抗内毒素作用,据此,本研究采用动物模型对灵杆菌多糖的抗内毒素作用进行确证,并从不同角度探讨灵杆菌多糖抗内毒索的作用机制,为理解灵杆菌多糖的临床用途提供新的视角,同时为灵杆菌多糖用于内毒素血症的辅助治疗提供前期实验依据。
研究内容
1.灵杆菌多糖对小鼠内毒素休克及内毒素血症模型的影响
1.1灵杆菌多糖对小鼠内毒素休克致死模型的保护作用
1.1.1材料与方法
先给KM小鼠(以下称：小鼠)腹腔注射灵杆菌多糖或生理盐水或阳性对照药物,0.5h后,每组动物均腹腔注射内毒素(铜绿假单胞菌脂多糖,LPS)60mg/kg,观察3d内动物死亡情况。
1.1.2结果
腹腔注射大剂量LPS(60mg/kg)后,小鼠出现明显的中毒反应,20h后陆续有动物死亡,48h后仍存活的动物逐渐恢复正常活动与摄食。灵杆菌多糖(100、50U/kg)预防性腹腔注射给药使动物存活率分别提高60%和45%(χ2=15.000,P=0.000；χ2=9.231,Bonferroni校正P=0.006)。
1.2灵杆菌多糖对内毒素血症模型小鼠炎症相关因子产生的抑制作用
1.2.1材料与方法
先给小鼠腹腔注射灵杆菌多糖或生理盐水或阳性对照药物,0.5h后,正常对照组动物腹腔注射等体积生理盐水,其余动物均腹腔注射LPS(10mg/kg),导致内毒素血症。3h后,小鼠摘眼球放血制备血清,ELISA方法测定血清中白介素-1p(IL-1p)、白介素-6(IL-6)、肿瘤坏死因子α(TNF α)、前列腺素E2(PGE2)的含量。Griess法检测一氧化氮(NO)的含量。
1.2.2结果
小鼠腹腔注射LPS后,其血清中IL-1β、IL-6、TNF α、PGE2、NO的含量明显升高,预先给予灵杆菌多糖(100,50U/kg)腹腔注射,然后再给LPS,其血清中各指标升高的幅度明显下降,灵杆菌多糖组各指标均数与LPS组相比,差异有统计学意义(IL-1β：P=0.007,P=0.023；IL-6：P=0.018,P=0.046,TNFα：P=0.001,P=0.019;PGE2：P=0.001,P=0.009：NO：P=0.012,P=0.039)。
1.3灵杆菌多糖对内毒素血症模型小鼠炎症相关基因mRNA表达的抑制作用
1.3.1材料与方法
动物分组及处理同1.2.1节,小鼠腹腔注射LPS(10mg/kg)3h后,脱颈椎处死,取出脾脏置液氮中保存,提取总RNA,实时荧光定量PCR法检测IL-1p、IL-6、TNFα、COX-2(环氧酶-2)、iNOS(诱导型一氧化氮合酶)mRNA的相对含量(2-△Ct,以GAPDH为内参)。
1.3.2结果
小鼠腹腔注射LPS后3h,脾脏中IL-1β、IL-6、TNFα、COX-2、iNOS mRNA的表达明显增多,提前0.5h腹腔注射灵杆菌多糖(100,50U/kg)可部分抑制由LPS诱导的上述各目的基因mRNA的表达,灵杆菌多糖组各参数均数与LPS组相比,剂量为100U/kg时,差异均有统计学意义(IL-1β：P=0.026；IL-6：P=0.015；TNF仅：P=0.029：COX-2：P=0.016；iNOS:P=0.020)；剂量为50U/kg时,差异有些有统计学意义(IL-1β：P=0.072；IL-6：P=0.035；TNF α：P=0.069；COX-2：P=0.034；iNOS:P=0.399)。
2.灵杆菌多糖对体外内毒素激活小鼠腹腔巨噬细胞产生炎症相关因子及表达炎症相关基因的影响
2.1灵杆菌多糖对内毒素激活小鼠腹腔巨噬细胞产生炎症相关因子的抑制作用
2.1.1材料与方法
LPS(10μg/ml)刺激小鼠腹腔巨噬细胞活化作为体外炎症模型。微量96孔板作为培养载体,ELISA法检测培养上清中IL-1β、IL-6、TNF-α和PGE2的含量；Griess法检测NO的含量。
2.1.2结果
在LPS刺激小鼠腹腔巨噬细胞16h后,培养上清中促炎细胞因子IL-1p、IL-6、TNF-a及培养24h后炎症因子或介质PGE2、NO的含量大幅增加,灵杆菌多糖(20、10U/mL)能部分抑制由LPS诱导的上述促炎细胞因子及炎症因子或介质的分泌,灵杆菌多糖组各指标均数与LPS组相比,差异有统计学意义(IL-1β：P=0.001,P=0.016；IL-6：P=0.001,P=0.016；TNF-α：P=0.027,P=0.046；PGE2：P=0.010,P=0.038；NO：P=0.000,P=0.001)
2.2灵杆菌多糖对内毒素激活小鼠腹腔巨噬细胞表达炎症相关基因mRNA的抑制作用
2.2.1材料与方法
LPS(10μg/ml)刺激小鼠腹腔巨噬细胞活化作为体外炎症模型。6孔板作为培养载体,实时荧光定量PCR法检测目的基因mRNA的相对含量(2-△Ct,以GAPDH为内参)。
2.2.2结果
经过LPS体外刺激16h后,小鼠腹腔巨噬细胞IL-1p、IL-6、TNF-α、COX-2、iNOS mRNA的表达明显增多,灵杆菌多糖(20、10U/mL)可部分抑制由LPS诱导的上述目的基因mRNA的表达,灵杆菌多糖组各指标均数与LPS组相比,差异有统计学意义(IL-1β：P=0.001,P=0.009；IL-6：P=0.000,P=0.000；TNF-a：P=0.000,P=0.000；COX-2：P=0.000,P=0.001；iNOS:P=0.011,P=0.021)
3.灵杆菌多糖单独给药对小鼠产生促炎细胞因子的影响
3.1灵杆菌多糖对正常小鼠血清促炎细胞因子水平的影响
3.1.1材料与方法
小鼠腹腔注射灵杆菌多糖或生理盐水或LPS,3h后,小鼠摘眼球放血制备血清,ELISA方法测定血清中IL-1β、IL-6、TNF α的含量。
3.1.2结果
正常小鼠血清细胞因子IL-1p、IL-6、TNF α的含量很低,灵杆菌多糖(100、50U/kg)腹腔注射给药使上述细胞因子的血清含量有提升趋势,但均数与生理盐水组相比,差异无统计学意义(IL-1β：P=0.377,P=0.658；IL-6：P=0.903,P=0.994：TNF a：P=0.369,P=0.997)
3.2灵杆菌多糖对正常小鼠腹腔巨噬细胞产生促炎细胞因子的影响
3.2.1材料与方法
小鼠腹腔巨噬细胞体外培养模型,微量96孔板作为培养载体,ELISA法检测培养上清中IL-1p、IL-6、TNF-α的含量。
3.2.2结果
在体外培养的条件下,灵杆菌多糖对小鼠腹腔巨噬细胞产生IL-1β、IL-6、TNF-α均有弱的促进作用,在浓度为20U/mL时,其均数与全培组相比,差异有统计学意义(P=0.007,P=0.002,P=0.003)。在浓度为10U/mL时,有些有统计学意义(P=0.043,P=0.536,P=0.016)。
4.灵杆菌多糖对内毒素所致小鼠肺损伤的保护作用
4.1灵杆菌多糖抑制内毒素诱导的肺氧化损伤作用
4.1.1材料与方法
除LPS为静脉给药外,动物分组及处理同1.2.1节,小鼠尾静脉注射LPS(30mg/kg)6h后,腹腔注射麻醉,开胸取出全肺,经后续处理后,测量肺湿重干重比(W/D)、髓过氧化物酶(MPO)活力、丙二醛(MDA)含量。
4.1.2结果
小鼠静脉注射LPS6h后,出现明显肺水肿现象,肺组织中MPO的活性及MDA的含量均明显增加。
预先腹腔注射灵杆菌多糖(100、50U/kg)可减轻由LPS引起的肺水肿现象,肺组织湿重与干重的比值下降,当灵杆菌多糖剂量为100U/kg时其均数与LPS组相比,差异有统计学意义(P=0.010),剂量为50U/kg时差异无统计学意义(P=0.060)。
预先腹腔注射灵杆菌多糖(100、50U/kg)可部分抑制由LPS引起的MPO活性及MDA含量的增加,其均数与内毒素模型组相比,差异有统计学意义(MPO：P=0.001,P=0.010；MDA：P=0.000,P=0.002)。
4.2灵杆菌多糖抑制内毒素诱导肺内促炎细胞因子产生的作用
4.2.1材料与方法
除LPS为静脉给药外,实验分组及给药同1.2.1节,小鼠均尾静脉注射LPS(30mg/kg),3h后,腹腔注射麻醉,开胸取出肺脏,经冷冻后制备肺组织匀浆,ELISA法测定上清中IL-1β、IL-6和TNF α的含量。
4.2.2结果
小鼠静脉注射LPS3h后,肺组织中IL-1β、IL-6和TNFα的含量明显增加。预先腹腔注射灵杆菌多糖(100、50U/kg)可部分抑制由LPS引起的IL-1β、IL-6和TNFα的增加,当灵杆菌多糖剂量为100U/kg时其均数与LPS组相比,差异均有统计学意义(P=0.029,P=0.010,P=0.006)；剂量为50U/kg时,差异有些有统计学意义(P=0.695,P=0.065,P=0.017)。
5.灵杆菌多糖对内毒素所致小鼠肝损伤的保护作用
5.1灵杆菌多糖对内毒素诱导肝细胞损伤的保护作用
5.1.1材料与方法
除LPS为静脉给药外,动物分组及处理同1.2.1节,小鼠均尾静脉注射LPS(30mg/kg),6h后,小鼠摘眼球取血,制备血清用于谷丙转氨酶(ALT)和谷草转氨酶(AST)检测。
5.1.2结果
小鼠静脉注射LPS6h后,血清中ALT和AST的活性明显升高,预先腹腔注射灵杆菌多糖(100、50U/kg)可部分抑制由内毒素引起的ALT和AST活性的升高,当灵杆菌多糖剂量为100U/kg时其均数与LPS组相比,差异均有统计学意义(P=0.022,P=0.026)；剂量为50U/kg时,AST的差异有统计学意义(P=0.031)。
5.2灵杆菌多糖对内毒素诱导肝组织促炎细胞因子mRNA表达的抑制作用
5.2.1材料与方法
实验分组及给药同1.2.1节,小鼠均腹腔注射LPS(30mg/kg),3h后,脱颈椎处死,取出肝脏置液氮中保存,提取总RNA,实时荧光定量PCR法检测IL-1p、IL-6、TNF αmRNA的含量。5.2.2结果
小鼠腹腔注射细菌LPS后3h,肝脏中IL-1β、IL-6、TNF αmRNA的表达明显增多,提前0.5h腹腔注射灵杆菌多糖(100,50U/kg)可部分抑制由LPS诱导的IL-1p、IL-6、TNFαmRNA的表达,当灵杆菌多糖剂量为100U/kg时其均数与LPS组相比,差异均有统计学意义(P=0.020,P=0.012,P=0.014)；剂量为50U/kg时,IL-6和TNFα mRNA的差异有统计学意义(P=0.043,P=0.020)。
统计学处理
应用SPSS11.5统计软件进行统计分析。存活率以百分数表示,先采用R×C表资料的卡方检验对各组动物存活率差异进行多组间的总体检验,然后采用四格表资料的卡方检验进行组间存活率的两两比较。定量数据结果以均数±标准差表示,采用单向方差分析法分析,先进行方差齐性检验,若方差齐用LSD方法多重比较；若方差不齐,采用经Welch法校正方差分析的F和P值,用Dunnett's T3法多重比较。显著性水准取α=0.05,以P<0.05时,组间差异判断为具有统计学意义。
结论
(1)灵杆菌多糖预防性腹腔给药可减少内毒素致小鼠死亡的百分率。
(2)灵杆菌多糖预防性腹腔给药可部分抑制由内毒素所致小鼠血清IL-1β、IL-6、TNFα、PGE2、NO含量的升高。
(3)灵杆菌多糖预防性腹腔给药可部分抑制由内毒素所致小鼠脾脏IL-1pIL-6、TNFa、COX-2、iNOSmRNA的表达。
(4)灵杆菌多糖体外可部分抑制内毒素刺激小鼠腹腔巨噬细胞产生IL-1p、IL-6、TNF-a、PGE2、NO。
(5)灵杆菌多糖体外可部分抑制内毒素刺激小鼠腹腔巨噬细胞IL-1p、IL-6、TNF-α、COX-2、iNOS mRNA的表达。
(6)灵杆菌多糖体外可轻微刺激小鼠腹腔巨噬细胞产生IL-1p、IL-6、TNF-a,其作用明显弱于内毒素。
(7)灵杆菌多糖预防性腹腔给药对内毒素所致小鼠肺损伤有一定的保护作用。
(8)灵杆菌多糖预防性腹腔给药对内毒素所致小鼠肝损伤有一定的保护作用。
(9)由于慢性偷腔炎和乙型肝炎时均伴有局部内毒素浓度的升高,灵杆菌多糖临床上对上述病症有良好疗效的机制可能与拮抗内毒素有关。
(10)灵杆菌多糖与内毒素都属于细菌脂多糖,而细菌脂多糖作用的主要受体为TLR4(Toll-like receptor4)。灵杆菌多糖刺激作用弱,没有明显的毒性；而内毒素刺激作用强,有明显的毒性反应；提示灵杆菌多糖为部分激动剂,而内毒素为完全激动剂,两者同时存在时前者对后者的毒性作用有拮抗作用,不过,其确切机制有待进一步的研究。
INTRODUCTION
Bacterium prodigiosum polysaccharides or Hemarisin, an English generic name registred in China, refers to a group of polysaccharides isolated from a bacterium Bacillus prodigiosus. It used to be named as Ling Gan Jun Su in Chinese. In foreign countries, especially in the former Soviet Union or Russia, a similar product to hemarisin is called prodigiozan.Because Bacillus prodigiosus is the old name for Serratia marcescens, therefore, both hemarisin and prodigiozan are lipopolysaccharides isolated from Serratia marcescens. In essence, hemarisin belongs to the category of bacterial endotoxin, but its toxicity is very low. It has immune stimulant effect and is mainly used in the adjuvant treatment of leucopenia caused by different factors, hepatitis B, and acute or chronic pelvic inflammatory diseases, etc.
OBJECTIVES
Hemarisin was found to be effective against endotoxin in the process of screening for anti-endotoxin compounds. Inspired by the finding, the present study was carried out to confirm the anti-endotoxin effectiveness by using animal models and to explore the underlying mechanism from different aspects in an effort to provide new information for understanding the polysaccharides'clinical applications and at the same time to lay a foundation experimentally for the polysaccharides used in adjuvant treatment of endotoxemia.
RESEARCH CONTENTS
1. Effect of hemarisin on murine models of endotoxin shock and endotoxemia
1.1Protective effect of hemarisin on murine lethal model of endotoxin shock
1.1.1Materials and Methods
KM mice (hereinafter referred to as:mice) were intraperitoneally (ip) injected with hemarisin or saline or positive control drug at first. After0.5h, each animal was ip injected with endotoxin (Pseudomonas aeruginosa lipopolysaccharides, LPS) in a dose of60mg/kg. The animal was under observation for3d and deaths were recorded.
1.1.2Results
Injected ip with large dose of LPS (60mg/kg), mice exhibited obvious toxic response and some became to die after20h. Surviving animal gradually came back to normal activities and feeding after48h. Hemarisin (100,50U/kg) with ip administration made animal survival rates increase by60%and45%respectively (χ2=15.000, P=0.000;χ2=9.231, Bonferroni correction P=0.006)
1.2Inhibitory effect of hemarisin on inflammatory factors in murine model of endotoxemia
1.2.1Materials and Methods
Mice were ip injected with hemarisin or saline or positive control drug at first. After0.5h, animal in normal control group was ip injected equal volume saline and the rest was ip injected LPS in a dose of10mg/kg in order to cause endotoxemia.3h later, blood samples, from which serum samples were prepared, were collected from the eye vein by removing the eyeballs quickly. ELISA method was used to determine the amounts of serum interleukin-1β(IL-1β), interleukin-6(IL-6), tumor necrosis factor a (TNF a), prostaglandin E2(PGE2) and Griess method for the detection of nitric oxide (NO) content.
1.2.2Results
The mouse serum levels of IL-1β, IL-6, TNF a, PGE2and NO were dramatically increased after ip injection of LPS. Prophylactic administration of hemarisin (100,50U/kg) by ip injection to the animal who were then followed by LPS ip injection, the extent of increase in the above mentioned parameters was attenuated markedly compared to LPS injection alone. The differences of means for the parameters between hemarisin groups and LPS group were statistically significant (IL-1β:P=0.007, P=0.023; IL-6:P=0.018, P=0.046; TNF a:P=0.001, P=0.019; PGE2:P=0.001, P=0.009; NO:P=0.012, P=0.039)1.3Inhibitory effect of hemarisin on mRNA expression of inflammatory factors gene in murine model of endotoxemia
1.3.1Materials and Methods
Animal groups and treatment were the same as described in section1.2.1. Mice were sacrificed by cervical dislocation after receiving LPS (10mg/kg) ip injection for3h. Then spleens were removed and stored in liquid nitrogen for RNA isolation. Real time fluorescence quantitative PCR (Polymerase Chain Reaction) was applied to evaluate the mRNA expressions of IL-1β, IL-6, TNF α, COX-2(Cyclooxygenase-2) and iNOS (Inducible Nitric Oxide Synthase). The content of mRNA was expressed as2-ΔCt using GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) as internal reference.
1.3.2Results
After ip injection of LPS to the mice for3h, the mRNA expressions of IL-1β, IL-6, TNF a, COX-2and iNOS in spleen increased dramatically. Hemarisin (100,50U/kg) ip injected to the mice0.5h ahead of LPS injection partially inhibited expressions of the genes of interest induced by LPS. The differences of means for the examined parameters between hemarisin group and LPS group were statistically significant when hemarisin was in the dose of100U/kg (IL-1β:P=0.026; IL-6: P=0.015; TNF a:P=0.029; COX-2:P=0.016; iNOS:P=0.020). However, some of the differences were statistically significant when hemarisin was in the dose of50U/kg (IL-1β:P=0.072; IL-6:P=0.035; TNF a:P=0.069; COX-2:P=0.034; iNOS:P=0.399)
2. Effect of hemarisin on production and gene expression of inflammatory factors in endotoxin activated murine peritoneal macrophages in vitro
2.1Inhibitory effect of hemarisin on production of inflammatory factors in endotoxin activated murine peritoneal macrophages
2.1.1Materials and Methods
Murine peritoneal macrophages activated by LPS (10μg/ml) were used as an in vitro inflammatory model.96well microtitters were applied for cell culture. ELISA method was used to determine the amounts of IL-1β, IL-6, TNF a and PGE2, and Griess method for the detection of NO content in culture supernatants.
2.1.2Results
After LPS stimulation of murine peritoneal macrophages for16h, the amounts of proinflammatory cytokines IL-1β,IL-6and TNF-a, and inflammatory factors or mediators PGE2and NO in the supernatants increased dramatically. Hemarisin (20and10U/mL) partially inhibited the macrophages production of the proinflammatory cytokines and inflammatory factors or mediators induced by LPS. The differences of means for the examined parameters between hemarisin groups and LPS group were statistically significant (IL-1β:P=0.001, P=0.016; IL-6: P=0.001, P=0.016; TNF-a:P=0.027, P=0.046; PGE2:P=0.010, P=0.038; NO: P=0.000, P=0.001)
2.2Inhibitory effect of hemarisin on gene expression of inflammatory factors mRNA in endotoxin activated murine peritoneal macrophages
2.2.1Materials and Methods
Murine peritoneal macrophages activated by LPS (10μg/ml) were used as an in vitro inflammatory model.6well plates were applied for cell culture. Real time fluorescence quantitative PCR was applied to evaluate the mRNA expressions of genes of interest. The content of mRNA was expressed as2-ΔCt using GAPDH as internal reference.
2.2.2Results
After LPS stimulation of murine peritoneal macrophages for16h, the mRNA expressions of IL-1β,IL-6, TNF-a,COX-2and iNOS markedly increased. Hemarisin (20and10U/mL) partially inhibited the expressions of the genes of interest induced by LPS. The differences of means for the examined parameters between hemarisin groups and LPS group were statistically significant (IL-1β:P=0.001, P=0.009; IL-6: P=0.000, P=0.000; TNF-α:P=0.000, P=0.000; COX-2:P=0.000, P=0.001; iNOS: P=0.011,P=0.021)
3. Effect of hemarisin administered alone on production of proinflammatory cytokines in mice
3.1Effect of hemarisin on the serum levels of proinflammatory cytokines in mice
3.1.1Materials and Methods
Mice were ip injected with hemarisin or saline or LPS.3h later, blood samples, from which serum samples were prepared, were collected from the eye vein by removing the eyeballs quickly. ELISA method was used to determine the amounts of serum IL-1β, IL-6and TNF a.
3.1.2Results
The levels of the cytokines IL-1β, IL-6and TNF a in normal mice were very low. Peritoneal injection of hemarisin (100,50U/kg) to the animal resulted in an increase trend in the cytokines serum levels, but the differences of means for the examined parameters between hemarisin groups and saline control group were not statistically significant (IL-1β:P=0.377, P=0.658; IL-6:P=0.903, P=0.994; TNF a:P=0.369, P=0.997)
3.2Effect of hemarisin on production of inflammatory cytokines by normal murine peritoneal macrophages
3.2.1Materials and Methods
Normal murine peritoneal macrophages were cultured as an in vitro model.96well microtitter was applied for cell culture. ELISA method was used to determine the amounts of IL-1β, IL-6and TNF in culture supernatants.
3.2.2Results
Hemarisin exerted weak stimulating effect on production of IL-1β, IL-6and TNF-a by murine peritoneal macrophages in in vitro culture situation. The differences of means for the examined parameters between hemarisin group and culture medium control group were statistically significant when hemarisin was at the concentration of20U/mL(P=0.007, P=0.002, P=0.003). However, only some of the differences were statistically significant when hemarisin was at the concentration of10U/mL (P=0.043, P=0.536, P=0.016)
4. Protective effects of hemarisin on endotoxin-induced lung injury in mice
4.1Hemarisin inhibits endotoxin-induced pulmonary oxidative damage in mice
4.1.1Materials and Methods
Except for LPS administered by intravenous route, animal groups and treatment were the same as described in section1.2.1. Mice were injected LPS (30mg/kg) through tail vein.6h later, the animal were anesthetized by intraperitoneal injection and lungs were removed by thoracotomy. Followed by various treatments, lung wet weight and dry weight ratio (W/D) was measured, myeloperoxidase (MPO) activity assessed, and malondialdehyde (MDA) content determined.
4.1.2Results
Significant pulmonary edema appeared after mice receiving LPS (30mg/kg) intravenous injection for6h. Lung tissue MPO activity and MDA content were increased dramatically.
Prophylactic ip administration of hemarisin (100,50U/kg) reduced the pulmonary edema caused by LPS with a decrease in lung wet weight and dry weight ratio (W/D). The difference of means for the ratio between hemarisin group and LPS group was statistically significant when hemarisin was in the dose of100U/kg (P=0.010), while no statistical significance was observed in the dose of50U/kg (P=0.060)
Prophylactic ip administration of hemarisin (100,50U/kg) partially suppressed the increases in MPO activity and MDA content caused by LPS. The differences of means for the examined parameters between hemarisin group and LPS group were statistically significant (MPO:P=0.001, P=0.010; MDA:P=0.000, P=0.002)4.2Hemarisin inhibits endotoxin-induced increase in pulmonary proinflammatory cytokines
4.2.1Materials and Methods
Except for LPS administered by intravenous route, animal groups and treatment were the same as described in section1.2.1. Mice were injected LPS (30mg/kg) through tail vein.3h later, the animal were anesthetized by intraperitoneal injection and lungs were removed by thoracotomy and frozen immediately. Pulmonary tissue homogenates were made in ice bath. ELISA method was used to determine the amounts of IL-1β, IL-6and TNFa in the homogenates.
4.2.2Results
The amounts of IL-1β, IL-6and TNF a in murine pulmonary tissue were dramatically increased after iv injection of LPS for3h. Prophylactic administration of hemarisin (100and50U/kg) by ip injection to the animal partially inhibited the increase in the proinflammatory cytokines induced by LPS. The differences of means for the examined parameters between hemarisin group and LPS group were statistically significant when hemarisin was in the dose of100U/kg (P=0.029, P=0.010, P=0.006). However, some of the differences were statistically significant when hemarisin was in the dose of50U/kg (P=0.695, P=0.065, P=0.017)
5. Protective effects of hemarisin on endotoxin-induced liver injury in mice
5.1Protective effects of hemarisin on endotoxin-induced hepatocyte injury
5.1.1Materials and Methods
Except for LPS administered by intravenous route, animal groups and treatment were the same as described in section1.2.1. Mice were injected LPS (30mg/kg) through tail vein.6h later, blood samples, from which serum samples were prepared, were collected from the eye vein by removing the eyeballs quickly. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum samples were assessed.
5.1.2Results
The mouse serum levels of ALT and AST were dramatically increased after iv injection of LPS for6h. Prophylactic administration of hemarisin (100,50U/kg) by ip injection to the animal that were then followed by LPS ip injection partially inhibited the increase in the examined parameters.The differences of means for the examined parameters between hemarisin group and LPS group were statistically significant when hemarisin was in the dose of100U/kg (P=0.022, P=0.026). However, only the difference of AST was statistically significant when hemarisin was in the dose of50U/kg (P=0.031)
5.2Hemarisin inhibits endotoxin-induced increase in proinflammatory cytokines mRNA expression in liver tissue
5.2.1Materials and Methods
Animal groups and treatment were the same as described in section1.2.1. Mice were sacrificed by cervical dislocation after receiving LPS (30mg/kg) iv injection for3h. Then livers were removed and stored in liquid nitrogen for RNA isolation. Real time fluorescence quantitative PCR was applied to evaluate the mRNA expressions of IL-1β, IL-6and TNF a.
5.2.2Results
After ip injection of LPS to the mice for3h, the mRNA expressions of IL-1β, IL-6and TNF a in the liver increased dramatically. Hemarisin (100,50U/kg) ip injected to the mice0.5h ahead of LPS injection partially inhibited expressions of the genes of IL-1β, IL-6and TNF a induced by LPS. The differences of means for the examined parameters between hemarisin group and LPS group were statistically significant when hemarisin was in the dose of100U/kg (P=0.020, P=0.012, P=0.014). However, only the means differences of IL-6and TNF a were statistically significant when hemarisin was in the dose of50U/kg (P=0.043, P=0.020)
STATISTICAL ANALYSIS
SPSS11.5statistical software was applied for statistical analysis. The survival rate was expressed as percentage. The R X C crosstabs chi-square test was used for overall test of animal survival differences among experimental groups at first, and then the four rows crosstabs chi-square test was performed for two by two comparisons. Continuous variables were described as as Mean±SD and analyzed by One-way ANOVA (Analysis of Variance). Test of homogeneity of variances was carried out prior to ANOVA. If the variance was of homogeneity, LSD method was used for multiple comparison, otherwise, Dunnett's T3method was adopted for multiple comparison, using F and P values by Welch correction analysis of variance. Significance level was set at α=0.05and P<0.05was considered to be statistical significance.
CONCLUSSI ON
(1) Prophylactic ip administration of hemarisin can reduce endotoxin-induced mouse death.
(2) Prophylactic ip administration of hemarisin can partially suppress endotoxin-induced increases in murine serum IL-1β, IL-6,TNF a, PGE2and NO.
(3) Prophylactic ip administration of hemarisin can partially suppress endotoxin-induced increases in mRNA expressions of IL-1β, IL-6, TNF a, COX-2and iNOS in murine liver.
(4) Hemarisin can partially inhibit endotoxin-induced production of IL-1β, IL-6, TNF a, PGE2and NO by murine peritoneal macrophages in vitro.
(5) Hemarisin can partially inhibit endotoxin-induced increases in mRNA expressions of IL-1β, IL-6, TNF a, COX-2and iNOS in murine peritoneal macrophages in vitro.
(6) Hemarisin can slightly stimulate murine peritoneal macrophages to produce IL-1β, IL-6and TNF-a in vitro, but it is much weaker than endotoxin in potency.
(7) Prophylactic ip administration of hemarisin can protect mice from endotoxin-induced lung injury in some degree.
(8) Prophylactic ip administration of hemarisin can protect mice from endotoxin-induced liver injury in some degree.
(9) Considering chronic pelvic inflammatory disease and hepatitis B are accompanied with increase in local endotoxin concentration, it is conceivable that the good efficacy of hemarisin in the treatment of above mentioned clinical diseases may be associated with the anti-endotoxin mechanism of the medicine.
(10) Both hemarisin and endotoxin belong to bacterial lipopolysaccharide, and the main receptor for bacterial lipopolysaccharide is TLR4(Toll-like receptor4). Hemarisin has weak stimulating effect and no obvious toxicity, while endotoxin has strong stimulating action and obvious toxicity. It is indicated that hemarisin act as a partial agonist, and endotoxin a full agonist. The former may exert an antagonist effect against the toxicity of the latter when they are present at the same time. However, the exact mechanism needs further study.
引文
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