中和白细胞介素-6减轻小鼠急性肝损伤

Neutralization of interleukin-6 alleviates acute liver injury in mice

目的研究白细胞介素-6(IL-6)在急性肝损伤发生、发展过程中的作用。方法将12只无特定病原体C57BL/6雄性小鼠随机分为对照组和模型组,每组各6只,对照组和模型组分别尾静脉注射等体积磷酸盐缓冲液(PBS)和刀豆蛋白A(ConA)(按10 mg/kg注射)。6 h收取标本进行肝组织HE染色、转氨酶测定以确定模型诱导成功。采用荧光定量PCR(qPCR)筛选白细胞介素(IL)-6、IL-17、IL-1β及干扰素(IFN)γ、肿瘤坏死因子α的表达,酶联免疫吸附法测定IL-6和IFNγ表达情况。后分别设立急性肝损伤对照组和IL-6中和抗体组各3只,等体积PBS或IL-6中和抗体(100μg/只)提前30 min尾静脉注射,再尾静脉注射ConA(10 mg/kg),6 h取眼血和肝组织,对肝组织进行HE染色和血清丙氨酸转氨酶(ALT)测定。对数据比较采用独立样本均数的t检验。结果模型组ALT和天冬氨酸转氨酶(AST)显著高于对照组[ALT:(2618.99±188.08)U/L与(43.34±5.02)U/L,t=-13.69,P=0.001;AST:(942.48±150.44)U/L与(57.80±4.84)U/L,t=-5.878,P=0.01]。肝组织HE染色显示肝细胞索结构紊乱,肝细胞胞质淡染,大片坏死灶逐渐形成,伴淋巴细胞浸润,小鼠急性肝损伤模型成功建立。模型组IL-6、IFNγmRNA和蛋白水平与对照组相比,表达明显上调。模型组IL-6 mRNA表达量是对照组的73.7倍(t=-6.218,P<0.001),血清IL-6表达量也高于对照组[(18537.02±92.57)pg/ml与(9.51±1.01)pg/ml,t=-199.782,P<0.001]。IFNγmRNA是对照组的108.4倍(t=-4.413,P=0.003),模型组血清IFNγ浓度也高于对照组[(12068.30±288.43)pg/ml与(25.97±3.52)pg/ml,t=-41.748,P<0.001]。其中IL-6水平增加最明显,提示其可能参与肝损伤的发生、发展过程。尾静脉注射IL-6中和抗体,IL-6中和抗体组小鼠ALT水平较急性肝损伤对照组明显下调[(167.41±47.80)U/L与(1520.34±190.21)U/L,t=6.899,P=0.015]。肝组织HE染色显示阻断血清IL-6后,肝细胞坏死明显减轻,坏死灶数量减少。免疫组织化学结果显示IL-6中和抗体组活化的caspase3表达减少,肝细胞凋亡减少。结论中和IL-6可明显减轻ConA引起的急性肝损伤。

Objective To study the role of interleukin 6(IL-6)in the occurrence and development of acute liver injury.Methods Twelve C57BL/6 male mice without specific pathogens were randomly divided into a control group and an acute liver injury model group,with six mice in each group.Control and model group were injected with an equal volume(dosage of 10 mg/kg)of phosphate-buffered saline(PBS)and concanavalin A(ConA)into the tail vein,respectively.Samples were collected at 6 h for liver HE staining.Transaminase assay was used to determine the success of the induction model.The expression of IL-6,IL-17,IL-1β,interferon(IFN)γand tumor necrosis factorαwere screened by quantitative fluorescence PCR(qPCR).The expressional condition of IL-6 and IFNγwere measured by enzyme-linked immunosorbent assay(ELISA).Subsequently,three control groups and three IL-6 neutralizing antibody groups were established for acute liver injury,respectively.Equal volumes of PBS or IL-6 neutralizing antibody(100μg/body)were injected prior 30 minutes,followed by injection of ConA(10 mg/kg)into the tail vein.Blood sampled from eye and liver tissue were fetched at 6 h.Liver tissues were stained with HE and serum alanine aminotransferase(ALT)was determined.An independent sample T-test was used for data comparison.Results Alanine aminotransferase(ALT)and aspartate aminotransferase(AST)of the model group was significantly higher than control group[ALT:(2618.99±188.08)U/L and(43.34±5.02)U/L,t=-13.69,P=0.001;AST:(942.48±150.44)U/L and(57.80±4.84)U/L,t=-5.878,P=0.01].Liver HE staining showed that the structure of hepatocyte cord was disordered,the cytoplasm of hepatocyte was lightly stained,and large necrotic foci were gradually formed,accompanied by lymphocyte infiltration,and then a mouse model of acute liver injury was successfully established.Protein levels of IL-6 and IFN,and mRNA of the model group were significantly up-regulated,as compared to control group.IL-6 mRNA expression of the model group was increased 73.7 times that of the control group(t=-6.218,P<0.001),and the serum IL-6 expression level was also higher than that of the control group(18537.02±92.57)pg/ml(t=-199.782,P<0.001).IFNγmRNA was 108.4 times higher than that of the control the group(t=-4.413,P=0.003),and serum IFNγconcentration of the model group was also higher than the control group(12068.30±288.43)pg/ml(t=-41.748,P<0.001).Among them,IL-6 level was obviously increased,suggesting that it could participate in the occurrence and development of liver injury.IL-6 neutralizing antibody was injected into the tail vein.ALT level of IL-6 neutralizing antibody was significantly lower than acute liver injury control group[(167.41±47.80)U/L and(1520.34±190.21)U/L,t=6.899,P=0.015].Liver tissue HE staining showed that hepatocyte necrosis and the number of necrotic foci was significantly alleviated after blocking serum IL-6.Immunohistochemical results showed that the expression of activated caspase3 and hepatocyte apoptosis in the IL-6 neutralizing antibody group was decreased.Conclusion Neutralizing IL-6 can significantly reduce acute liver injury caused by concanavalin A.