Li L, Bai L, Zheng Y, Chen Z P, 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.
Zhonghua Gan Zang Bing Za Zhi. 2022 Apr 20;30(4):413-418. doi: 10.3760/cma.j.cn501113-20201204-00639.
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. 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. 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. Liver fibrosis may protect mice from acute lethal challenge injury by inhibiting D-GalN/LPS-induced necroptosis.
以D-半乳糖胺/脂多糖（D-GalN/LPS）诱导的坏死性凋亡为切入点，探索肝纤维化的新机制，以抑制致死性损伤。建立四氯化碳（CCl4）诱导的小鼠肝纤维化模型。在纤维化6周时，用致死剂量的D-GalN/LPS对小鼠进行攻击，以接受相同处理的正常小鼠作为对照。实验分为四组：对照组（Control）、急性损伤组（D-GalN/LPS）、肝纤维化组（Fib）和肝纤维化+急性攻击组（Fib + D-GalN/LPS）。采用定量PCR和免疫荧光法分析各组坏死性凋亡关键信号分子RIPK1、RIPK3、MLKL和/或P-MLKL的表达。用针对坏死性凋亡关键信号分子的抑制剂处理正常小鼠，然后进行急性攻击。根据转氨酶水平和肝脏组织学变化评估D-GalN/LPS诱导的坏死性凋亡对急性肝损伤的抑制作用。建立肝纤维化自发消退模型，然后进行急性攻击。通过免疫组化分析各组小鼠肝组织中坏死性凋亡关键信号分子的表达并进行比较。组间差异采用t检验或方差分析。定量PCR和免疫荧光分析结果显示，D-GalN/LPS诱导RIPK1、RIPK3、MLKL和/或P-MLKL显著上调。抑制坏死性凋亡关键信号分子可显著减轻D-GalN/LPS诱导的肝损伤，表现为血清ALT和AST水平明显降低，肝脏组织学改善。即使在急性攻击条件下，纤维化肝脏中坏死性凋亡信号分子的表达也显著受到抑制。此外，随着纤维化逐渐消退的肝纤维化抑制了D-GalN/LPS诱导的坏死性凋亡。肝纤维化可能通过抑制D-GalN/LPS诱导的坏死性凋亡来保护小鼠免受急性致死性攻击损伤。
