NPLC0393调节内质网应激反应及相关蛋白减轻肝纤维化的作用机制研究

Hepatic fibrosis (liver fibrosis) is the excessive accumulation of extracellular matrix proteins including collagen that occurs in most types of chronic liver diseases. Advanced liver fibrosis leads to the pathogeneses of cirrhosis, liver failure, and portal hypertension, and increases the risk of hepatocellular carcinoma. Endoplasmic reticulum (ER) stress plays an important role in liver diseases, such as viral hepatitis, alcohol liver disease, drug induced hepatitis, nonalcoholic fatty liver diseases. Studies on investigation of liver fibrogenesis are helpful to develop targeted therapies to reverse the fibrotic response and to improve the outcomes of patients with chronic liver disease. NPLC0393 is a triterpene saponin extracted from Gynostemma pentaphyllum, which is widely used in the treatment of liver disease. Previous study showed that the natural product NPLC0393 could activate PP2Cα and terminate TGF signaling pathway. Meanwhile, NPLC0393 could inhibit collagen expression and significantly decrease the number of HSCs. Moreover, NPLC0393 also exhibited effective antifibrotic effects in liver fibrosis mice. These findings suggested that the natural product NPLC0393 might be used in antifibrotic drug development. However, the detailed mechanism of NPLC0393 to ameliorate liver fibrosis still needs to be further investigated. The endoplasmic reticulum (ER) is a central organelle that plays essential roles in cell survival and homeostasis. Disorders that affect ER function can lead to cell dysfunction and disease. Recent research indicated that mitochondria-associated membranes (MAM), a sub-region of the endoplasmic reticulum (ER) that facilitates crosstalk between the ER and mitochondria, played an important role in cellular processes including protein folding and lipid synthesis. Our label-free proteomics research on NPLC0393-treated mouse liver tissues indicated that NPLC0393 could regulate ER associated proteins involving protein folding and lipid metabolism to attenuated liver fibrosis. In this study, we will investigate the mechanisms of NPLC0393 on regulation of liver ER stress and endoplasmic reticulum associated proteins through quantitative proteomics approach.

肝纤维化是慢性肝病发展到肝硬化的中间环节，多种不良因素都可导致肝纤维化。最近研究显示内质网应激与肝纤维化的形成密切相关。研究开发抗肝纤维化药物，探讨其作用机制对肝纤维化防治具有十分重要的意义。NPLC0393是从传统中药绞股蓝中提取的化合物，研究表明，NPLC0393可增强PP2Cα酶活性，抑制TGFβ通路，减轻小鼠肝纤维化作用。我们研究发现，NPLC0393可通过调节内质网相关蛋白表达减轻肝纤维化。本项目拟探讨内质网应激与肝纤维化的相关性，并考察NPLC0393是否参与改善肝纤维化的内质网应激，并采用定量蛋白质组学技术研究内质网以及内质网线粒体间膜（mitochondrial associated ER membrane, MAM）蛋白变化情况，探索肝细胞中内质网特别是MAM生理功能及在肝纤维化发生发展中的作用，探讨NPLC0393调节内质网相关蛋白减轻肝纤维化的作用机理。

从绞股蓝中提取的三萜皂苷NPLC0393在体内外均具有抗纤维化活性。为了进一步探索其抗肝纤维化的分子机制，本研究通过蛋白质组学分析和生物学验证，证明甲硫氨酸腺苷转移酶2A（MAT2A）在四氯化碳（CCl4）诱导的纤维化小鼠模型中表达显著增加，而N-Myc下游调节基因2(NDRG2)表达显著降低。NPLC0393处理可以逆转肝纤维化中MAT2A的表达增加和NDRG2的表达降低。进一步的机制研究发现，TGF-β1可诱导HSCs p65磷酸化和NF-κB活化，从而促进MAT2A的mRNA转录和蛋白表达，降低S-腺苷蛋氨酸（SAM）浓度。体内和体外敲除MAT2A可减轻CCl4-和TGF-β1诱导的HSC活化，而体内过表达MAT2A可促进肝纤维化并消除NPLC0393的治疗作用。在活化肝星状细胞中下调NDRG2可以逆转NPLC0393的抗肝纤维化作用以及对TGF-β通路的抑制作用。此外，下调NDRG2还可以逆转NPLC0393对MAPK通路的抑制作用。本研究表明，NPLC0393可通过调节MAT2A和NDRG2的蛋白表达来有效减轻肝纤维化，为肝纤维化的治疗提供了潜在靶点。

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