急性肝衰竭大鼠血清TNF-α、IL-10水平的动态变化和TNF-α单克隆抗体护肝机制的探讨

Dynamic changes of serum tumor necrosis factor-α, interleukin-10 in rats with acute liver failure and the hepatoprotective mechanism of tumor necrosis factor-α monoclonal antibody

目的探讨急性肝衰竭大鼠血清肿瘤坏死因子(TNF)￣α、白介素(IL)￣10水平的动态变化及TNF￣α单克隆抗体(英利西单抗)对肝损伤保护作用的可能机制。方法 Wistar清洁级大鼠14只,雌雄各半,按随机数字表法随机分成对照组及治疗组各7只。每只大鼠给予皮下注射脂多糖(LPS)10μg/kg及腹腔注射D￣氨基半乳糖(D￣GalN)800 mg/kg建立急性肝衰竭模型,对照组注射LPS和D￣GalN后,皮下注射生理盐水1 ml/kg,治疗组皮下注射英利西单抗5 mg/kg。应用酶联免疫吸附测定(ELISA)方法检测两组大鼠给药后0、12、24、48、72 h的血清TNF￣α、IL￣10的水平。两组间TNF￣α和IL￣10的比较采用t检验,组内不同时间点的TNF￣α和IL￣10比较采用重复测量方差分析。结果对照组组内比较:注射药物后12 h血清TNF￣α水平达到高峰,24、48、72 h随时间延长呈下降趋势;血清IL￣10水平随时间延长逐渐升高,24 h后迅速上升,72 h达到高峰,72 h时间点与其它时间点比较差异均有统计学意义(F=257.31、227.815、89.276、9.984,均为P<0.05)。治疗组组内比较:血清TNF￣α变化趋势同对照组,在12 h达到高峰,24、48、72 h呈缓慢下降;血清IL￣10水平随时间延长而升高,在12、24 h迅速上升,在48、72 h上升趋势减缓,72 h时间点与其它时间点比较差异均有统计学意义(F=451.471、195.105、26.259、22.962,均为P<0.05)。治疗组与对照组比较:治疗组12、24、72 h时间点的血清TNF￣α水平较对照组明显下降(t=2.392、3.393、2.276,均为P<0.05);治疗组血清IL￣10水平在0、12、24 h时间点高于对照组,其中12 h时比较差异有统计学意义(t=-4.556,P=0.002),在48、72 h时间点低于对照组,其中72 h时间点比较差异有统计学意义(t=7.997,P<0.001)。结论 TNF￣α、IL￣10是肝急性衰竭发生发展过程中重要细胞因子,在急性肝衰竭早期产生大量的促炎细胞因子TNF￣α,晚期血清抗炎细胞因子IL￣10水平升高;英利西单抗可能通过拮抗TNF￣α的生物活性,下调TNF￣α的水平和早期上调IL￣10的水平来保护肝脏,避免肝损伤。

Objective To observe the dynamic changes of serum TNF-a, IL-IO of rats with acute liver failure and to explore the hepatoprotective efficacy of anti-tumor necrosis factor alpha monoelonal antibody （anti-TNF-a mAb, Infiximab）. Methods divided into control group （n=7） and treatment group Fourteen Wistar rats （7 males and 7 females） were （n=7） randomly. All acute liver failure models of rats was established by hypodermic injection with lipopolysaccharides （10 μg/kg） and intraperitoneal injection with D-Galactosamin （800 mg/kg）. Then the rats in the control group were injected with saline hypodermically （1 ml/kg）, and the rats in the treatment group were given infiximab injection （5 mg/kg）. The levels of serum TNF-a, IL-10 of 2 groups were measured by enzyme-linked immunosorbent assay（ELISA）at the 0, 12, 24, 48 and 72 h and were compared by independent sample t-test between 2 groups. The serum TNF-a, IL-10 levels at different time points in the same group were compared using general linear model-repeated analysis. Results The levels of serum TNF-a reached a peak at 12 h in the control group and gradually decreased at 24, 48 and 72 h. The levels of serum IL-10 rose gradually and significantly at 24 h and peaked at 72 h. The levels of IL-10 and INF-a at 72 h showed significant difference compared with other time points （F=257.31,227.815, 89.276, 9.984; all in P〈0.05）. The levels of serum TNF-a in the treatment group showed similar changes as the control group, reached a peak at 12 h and gradually decreased at 24, 48 and 72 h. The levels of serum IL-10 increased evidently at 12 and 24 h, but gradually at 48 and 72 h. The levels of IL-10 and TNF-a at 72 h showed significant difference compared with other time points（F=51.471, 195.105, 26.259, 22.962; all in P〈0.05）. Compared with the control group, the TNF-a levels in the treatment group at 12, 24 and 72 h were significantly lower （t=2.392, 3.393, 2.276; all in P〈0.05）, and the IL-10 levels were higher at 0, 12 and 24 h with significant difference at 12 h （t=-4.556, P=0.002）, but lower at 48 and 72 h with significant difference at 72 h （t=7.997, P〈0.001）. Conclusions Serum TNF-a and IL-10 play import roles in the progression of acute liver failure. A large amount of pro-inflammatory cytokines such as TNF-a are induced at the early phase of acute liver failure, and the anti-inflammatory cytokines as IL-10 are induced at the advanced phase. Anti-TNF-a mAb（Infiximab） may probably alleviate liver injury by antagonizing the TNF-a activity and increasing the expression of IL-10 at the early phase.