肝纤维化通过抑制坏死性凋亡抵抗D-氨基半乳糖/脂多糖诱导的致死性损伤  

Liver fibrosis inhibits lethal injury through D-galactosamine/lipopolysaccharide-induced necroptosis

作　　者：李璐[1] 白丽[2] 郑素军[3] 陈煜[2] 段钟平[2] Li Lu;Bai Li;Zheng Sujun;Chen Yu;Duan Zhongping(Department of Infectious Diseases,Peking University Third Hospital,Beijing 100191,China;Beijing Municipal Key Laboratory of Liver Failure and Artificial Liver Treatment Research The Fourth Department of Hepatology,Beijing YouAn Hospital,Capital Medical University,Beijing 100069,China;The First Department of Hepatology,Beijing YouAn Hospital,Capital Medical University,Beijing 100069,China)

机构地区：[1]北京大学第三医院感染疾病科,北京100191 [2]首都医科大学附属北京佑安医院肝病中心四科肝衰竭与人工肝治疗研究北京市重点实验室,北京100069 [3]首都医科大学附属北京佑安医院肝病中心一科,北京100069

出　　处：《中华肝脏病杂志》2022年第4期413-418,共6页Chinese Journal of Hepatology

基　　金：大数据精准医疗高精尖创新中心(PXM2021_014226_000026);国家重点研发计划(2017YFA0103000);首都卫生发展科研专项项目(2021-1G-2181);北京市医院管理局临床医学发展专项(ZYLX201806,ZYLX202125);北京市医院管理局消化内科学科协同发展中心项目(XXZ0503);北京市自然科学基金(7202068,7222093,7222094)。

摘　　要：目的以坏死性凋亡为切入点,探讨肝纤维化抵抗D-氨基半乳糖/脂多糖(D-GalN/LPS)诱导致死性损伤的新机制。方法建立四氯化碳(CCl4)诱导的肝纤维化小鼠模型,于纤维化6周时以致死剂量的D-GalN/LPS进行攻击,以同样处理的正常小鼠作为对照。即实验共分为4组:对照组(Control)、急性损伤(D-GalN/LPS)组、肝纤维化(Fib)组、肝纤维化+急性攻击(Fib+D-GalN/LPS)组。定量PCR和免疫荧光法分析坏死性凋亡(necroptosis)关键信号分子受体相互作用蛋白(RIPK)1、RIPK3、混合谱系激酶结构域蛋白(MLKL)和/或P-MLKL在各组中的表达。给予正常小鼠以靶向necroptosis关键信号分子的抑制剂,再给予急性攻击,根据转氨酶水平及肝组织学的变化来评估抑制necroptosis对D-GalN/LPS触发急性肝损害的影响。建立肝纤维化自发消融模型,再给予急性攻击,免疫组织化学法分析和比较各组小鼠肝组织中necroptosis关键信号分子的表达。组间差异的比较采用t检验或方差分析。结果定量PCR和免疫荧光检测结果显示,D-GalN/LPS攻击触发了RIPK1、RIPK3、MLKL和/或P-MLKL的明显上调。抑制necroptosis信号的关键分子可明显减轻D-GalN/LPS诱导的肝损害,表现为血清丙氨酸转氨酶和天冬氨酸转氨酶水平的显著降低以及肝组织学的明显改善。纤维化肝脏中necroptosis信号分子的表达明显受到抑制。肝纤维化对D-GalN/LPS触发necroptosis的抑制随着纤维消融而逐渐削弱。结论肝纤维化可能通过抑制D-GalN/LPS触发的necroptosis而保护小鼠抵抗急性致死性损伤的攻击。Objective To explore the new mechanism of liver fibrosis through D-galactosamine/lipopolysaccharide(D-GalN/LPS)-induced necroptosis as an entry point to inhibit lethal injury.Methods The carbon tetrachloride(CCl4)-induced mouse model of liver fibrosis was established.At 6 weeks of fibrosis,the mice were challenged with a lethal dose of D-GalN/LPS,and the normal mice treated with the same treatment were used as the control.The experiment was divided into four groups:control group(Control),acute injury group(D-GalN/LPS),liver fibrosis group(Fib),and liver fibrosis+acute challenge group(Fib+D-GalN/LPS).Quantitative PCR and immunofluorescence were used to analyze the expression of necroptosis key signal molecules RIPK1,RIPK3,MLKL and/or P-MLKL in each group.Normal mice were treated with inhibitors targeting key signaling molecules of necroptosis,and then given an acute challenge.The inhibitory effect of D-GalN/LPS-induced-necroptosis on acute liver injury was evaluated according to the changes in transaminase levels and liver histology.Liver fibrosis spontaneous ablation model was established,and then acute challenge was given.Necroptosis key signal molecules expression was analyzed in liver tissue of mice in each group and compared by immunohistochemistry.The differences between groups were compared with t-test or analysis of variance.Results Quantitative PCR and immunofluorescence assays result showed that D-GalN/LPS-induced significant upregulation of RIPK1,RIPK3,MLKL and/or P-MLKL.Necroptosis key signal molecules inhibition had significantly reduced D-GalN/LPS-induced liver injury,as manifested by markedly reduced serum ALT and AST levels with improvement in liver histology.Necroptosis signaling molecules expression was significantly inhibited in fibrotic livers even under acute challenge conditions.Additionally,liver fibrosis with gradual attenuation of fibrotic ablation had inhibited D-GalN/LPS-induced necroptosis.Conclusion Liver fibrosis may protect mice from acute lethal challenge injury by inhibiting D-GalN

关 键 词：肝纤维化 损伤抵抗 坏死性凋亡 发病机制 

分 类 号：R575.2[医药卫生—消化系统]