自噬依赖的"p62/IκBα-Bcl-2/Beclin-1"在肝星状细胞凋亡及肝纤维化逆转中的作用

Apoptosis of activated hepatic stellate cells(HSCs) is the key to reversion of hepatic fibrosis. It has been observed in our preliminary experiment that reduced autophagy by the inhibitor of 3-MA fuelled the apoptosis of activated HSCs, associated with regulation of NF-κB signaling pathway. Therefore, we hypothesize in this study that the activation of autophagy regulates the reversion of heaptic fibrosis through “p62/IκBα-NF-kB-Bcl-2/Beclin-1” signaling pathway. In the present study, liver fibrosis was induced in Atg6+/- mice by using bile duct ligation (BDL) and dimethylnitrosamine (DMN). Autophagy and apoptosis of activated HSCs were detected in the liver in vivo, when hepatic fibrosis was fully developed or was in the process of reversion. Meanwhile, the protein levels of representative molecules in “p62/IκBα-NF-kB-Bcl-2/Beclin-1” signaling pathway were measured also. And the reversion of liver fibrosis was evaluated correspondingly to explore the correlation between the autophagy of activated HSCs and the reversion of hepatic fibrosis. In vitro, HSCs were isolated from fibrotic or healthy liver, and were cultured on plastic flasks to mimic their activation in vivo. HSCs were set at different levels of autophagy by using siRNA or lentivirus transfection. The apoptosis of the cells was detected then to explore the role of autophagy in regulation of apoptosis of activated HSCs. By using signal inhibitors, the roles of “p62/IκBα-NF-kB” axis were revealed in regulation of cellular apoptosis. And the negative regulation of “NFκB-Bcl-2/Beclin-1” axis to cellular apoptosis was also demonstrated in this study. The expected results in this study are hopefully evaluable for understanding the mechanisms in apoptosis of activated HSCs and reversion of hepatic fibrosis, and also for the therapeutics of liver fibrosis.

活化的肝星状细胞（HSC）凋亡是肝纤维化逆转的关键。前期研究发现抑制自噬能促进HSC的凋亡，且与NF-κB途径相关。本研究提出“自噬通过p62/IκBα-NF-κB-Bcl-2/Beclin-1通路抑制活化HSC凋亡与肝纤维化逆转”的研究假说。本研究拟通过体内实验：在Atg6部分敲除的自噬抑制小鼠，通过DMN及胆管结扎建立肝纤维化模型，在模型期及恢复期检测HSC自噬与凋亡，信号通路相关蛋白表达，及肝纤维化逆转情况；通过体外实验：分离培养模型与对照小鼠HSC，利用基因过表达或siRNA基因沉默、信号通路抑制等方法，在不同自噬水平检测活化HSC凋亡及相关信号通路分子，探讨自噬对活化HSC凋亡的调节作用，“p62/IκBα-NF-κB”轴对凋亡的调控机制，及“NF-κB-Bcl-2/Beclin-1”轴对自噬的负反馈机制。这对活化的HSC凋亡调控、肝纤维化逆转的机制研究及临床治疗有重要价值。

活化的肝星状细胞（HSC）凋亡是肝纤维化逆转的关键。本课题研究主要研究了以下内容：1）通过体内实验：在Atg6部分敲除的自噬抑制小鼠，通过DMN及胆管结扎建立肝纤维化模型，在模型期及恢复期检测HSC自噬与凋亡，信号通路相关蛋白表达，及肝纤维化逆转情况；2）通过体外实验：分离培养模型与对照小鼠HSC，利用基因过表达或siRNA基因沉默、信号通路抑制等方法，在不同自噬水平检测活化HSC凋亡及相关信号通路分子，探讨自噬对活化HSC凋亡的调节作用，“p62/IκBα-NF-κB”轴对凋亡的调控机制，及“NF-κB-Bcl-2/Beclin-1”轴对自噬的负反馈机制。通过本课题的研究，我们发现：1）自噬的水平影响肝纤维化生成和恢复中活化的HSC 凋亡，是关键的调节者；2）通过p62，自噬降解IκBα，促进NF-κB 通路的激活，维持Bcl-2 高表达，抑制活化的HSC 凋亡。3）通过药物筛选，证实新先导结构Y0309可通过上调P62，抑制自噬溶酶体的生成。