敲除MLKL可以显著缓解肝实质细胞的损伤和程序性坏死，并显著的抑制肝星状细胞的活化

纤维化是一种世界范围内高发病率与高死亡率的疾病，是肝损伤发展成严重肝病的重要阶段，影响着全球数百万人的生命健康。然而，肝纤维化尚无有效的治疗药物上市。肝纤维化的发病机制复杂，但肝实质细胞的异常损伤及损伤后的死亡一直是公认的起始诱因。肝实质细胞的异常损伤和死亡会激活肝脏内的炎症反应，并进一步诱导肝星状细胞的增殖、活化和细胞外基质累积，最终进展为肝纤维化。在此项研究中，我们首先分析了肝纤维化临床病人的肝活检样本以及肝纤维化小鼠的组织样品，检测到MLKL在肝纤维化状态下高表达，并且MLKL的表达水平与肝纤维化程度呈强相关性。为详细探究MLKL在肝纤维化发生中的作用，我们构建了MLKL敲除（Mlkl-/-）的小鼠，并在四氯化碳和胆管结扎手术诱导的纤维化模型上，发现Mlkl-/-小鼠肝脏损伤、肝脏炎症以及肝纤维化程度明显减轻。肝纤维化的发生是一种多细胞参与的过程，涉及肝实质细胞和非实质肝细胞（包括巨噬细胞、肝星状细胞等）的共同作用。为详细阐明MLKL作用的细胞类型及机制，我们分别分离了这些关键细胞进行分析。发现敲除MLKL可以显著缓解肝实质细胞的损伤和程序性坏死，并显著的抑制肝星状细胞的活化，但不影响巨噬细胞的分化和功能。最后，针对此发现，我们设计了靶向肝实质细胞MLKL的基因疗法（AAV8-TBG-shMlkl），并证实AAV8-TBG-shMlkl可以有效治疗四氯化碳诱导的小鼠肝纤维化。综上所述，该项研究发现，MLKL与肝纤维化的发生密切相关，敲除MLKL可以显著减轻四氯化碳及胆管结扎诱导的小鼠肝纤维化的发生，并揭示了MLKL作用的细胞类型及机制。最终，建立了靶向肝实质细胞MLKL以治疗肝纤维化的新方法。该研究结果为抗纤维化药物的研发提供了新靶点和方法。

Fibrosis is a disease with high global morbidity and mortality, representing a critical stage in the progression of liver injury to severe liver disease, affecting the health and lives of millions of people worldwide. However, no effective therapeutic drugs have been approved for hepatic fibrosis. The pathogenesis of hepatic fibrosis is complex, but abnormal injury and subsequent death of hepatocytes (liver parenchymal cells) have long been recognized as the initiating triggers. Abnormal injury and death of hepatocytes activate intrahepatic inflammatory responses, further inducing the proliferation, activation, and extracellular matrix accumulation of hepatic stellate cells, ultimately leading to the progression of hepatic fibrosis.

In this study, we first analyzed liver biopsy samples from clinical hepatic fibrosis patients and tissue samples from hepatic fibrosis mouse models, and detected that MLKL is highly upregulated in the fibrotic state, with its expression level strongly correlated with the severity of hepatic fibrosis. To thoroughly investigate the role of MLKL in the development of hepatic fibrosis, we generated Mlkl knockout (Mlkl-/-) mice. On fibrosis models induced by carbon tetrachloride and bile duct ligation, we found that Mlkl-/- mice exhibited significantly alleviated liver injury, hepatic inflammation, and hepatic fibrosis.

The development of hepatic fibrosis is a multicellular process involving the combined actions of hepatocytes and non-parenchymal liver cells (including macrophages, hepatic stellate cells, etc.). To clarify the specific cell types and mechanisms through which MLKL exerts its effects, we isolated and analyzed these key cell populations separately. We found that knockout of MLKL significantly alleviated hepatocyte injury and necroptosis, and markedly inhibited the activation of hepatic stellate cells, but did not affect the differentiation and function of macrophages.

Based on these findings, we designed a hepatocyte-targeted MLKL gene therapy (AAV8-TBG-shMlkl), and confirmed that AAV8-TBG-shMlkl could effectively treat carbon tetrachloride-induced hepatic fibrosis in mice.

In summary, this study demonstrated that MLKL is closely associated with the development of hepatic fibrosis. Knockout of MLKL significantly attenuated the development of hepatic fibrosis induced by carbon tetrachloride and bile duct ligation in mice, and revealed the specific cell types and underlying mechanisms of MLKL's actions. Ultimately, we established a novel therapeutic strategy targeting MLKL in hepatocytes for hepatic fibrosis. The findings of this study provide new targets and approaches for the development of anti-fibrotic drugs.