小分子干扰RNA沉默T细胞免疫球蛋白黏蛋白分子3表达对暴发性肝衰竭小鼠Kupffer细胞分泌细胞因子的影响

Small interfering RNA silencing T cell immunoglobulin mucin 3 affect cytokines in Kupffer cells from fulminant hepatic failure mice

目的探讨T细胞免疫球蛋白及黏蛋白家族3(Tim-3)的小分子干扰RNA(si RNA)质粒对暴发性肝衰竭小鼠肝Kupffer细胞活化的调节作用并探讨其相关机制。方法采用D-氨基半乳糖(D-Galactosamine,D-Gal N)和脂多糖(LPS)建立暴发性肝衰竭小鼠模型。将30只大鼠分成对照组及模型组,每组15只。分离所有小鼠Kupffer细胞,用特异性靶向Tim-3的si RNA片段沉默小鼠肝Kupffer细胞中的Tim-3,同时将模型组小鼠进一步分成空转染组、特异si RNA组及阴性si RNA组。采用Real time PCR和Western blot检测Tim-3的表达,通过酶联免疫吸附试验(ELISA)检测Tim-3沉默后Kupffer细胞中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)及IL-6的表达,并采用ELISA及Western blot检测核因子-κB(NF-κB)通路对炎性细胞因子表达的影响。结果模型组Tim-3 m RNA的表达显著高于对照组(48.3±6.8 vs.10.3±1.8,t=0.007,P<0.05),且特异si RNA组Tim-3 m RNA的表达(18.3±3.5)显著低于空转染组(58.3±7.5)及阴性si RNA组(57.3±7.1)的表达(F=118.5,P<0.05)。在转染后3、6、24 h,特异si RNA组中TNF-α(F=68.76、73.55、92.36,P均<0.05),IL-1β(F=32.1、86.5、112.3,P均<0.05)及IL-6(F=178.9、98.7、89.9,P均<0.05)的表达均显著高于空转染组及阴性si RNA组。同时,特异si RNA组的NF-κB蛋白的表达在转染3 h(F=48.9,P=0.020)、6 h(F=107.4,P=0.002)、24 h(F=148.9,P=0.001)均显着高于空转染组及阴性si RNA组。结论 Tim-3通过NF-κB蛋白表达参与了暴发性肝衰竭小鼠肝Kupffer细胞活化的调节。

Objective To investigate the effect of down-regulating T cell immunoglobulin mucin 3（Tim-3） in Kupffer cells activation from fulminant hepatic failure mice and the related mechanism. Methods The fulminant hepatic failure model was established by D-Galactosamine（D-Gal N） and lipopolysaccharide（LPS）. Thirty mice were randomly divided into the control group and model group, 15 mice in each group. Kupffer cells were isolated from all mice and transfected by small interfering RNA（si RNA） targeting Tim3, and the Kupffer cells in the model group further divided into the mock group, si RNA group and negative si RNA group. The expression of Tim-3 was detected by Real time PCR and Western blotting. The levels of tumor necrosis factor-alpha（TNF-α）, interleukin-1β（IL-1β） and IL-6 in Kupffer cells after Tim-3 si RNA interference were examined by enzyme-linked immunosorbent assay（ELISA）. The ELISA and Western blotting were used to analyze the nuclear factor kappa B（NF-κB） pathway effects on inflammatory cytokines. Results The expression of Tim-3 m RNA in the model group was much higher than that in the control group（48.3 ± 6.8 vs. 10.3 ± 1.8, t = 0.007, P 0.05）. The expression of Tim-3 m RNA in the si RNA group, mock group and negative si RNA group were（18.3 ± 3.5）,（58.3 ± 7.5） and（57.3 ± 7.1）, respectively. The expression of Tim-3 m RNA in the si RNA group was much lower than those in the mock group and negative si RNA group（F =118.5, P 0.05）. In the si RNA group, the levels of TNF-α（F = 68.76, 73.55, 92.36, all P 0.05）, IL-1β（F = 32.1, 86.5, 112.3, all P 0.05） and IL-6（F = 178.9, 98.7, 89.9, all P 0.05）increased obviously as compared with those in the mock group and negative si RNA group on 3,6, 24 h after transfection. Meanwhile, the NF-κB protein in the si RNA group（F = 48.9, 107.4,148.9, P = 0.020, 0.002, 0.001） were also much higher than those in the mock group and negative si RNA group on 3, 6, 24 h after transfection. Conclusion Tim-3 is involved in the regulation of macrophage activation through NF-κB pathway.