TGF-β1/Smads-Bmal1正反馈回路促进肝星状细胞自噬的机制研究

Activation of hepatic stellate cells (HSC) is the key to the development of liver fibrosis. Autophagy can promote the activation and proliferation of HSC, which may be a new way to treat liver fibrosis. We treated HSC with autophagy inhibitor and profibrotic factor, and screened for genes involved in autophagy using expression profiling chips. It was found that the circadian clock gene Bmal1 may be a key trigger point for HSC autophagy: Overexpression/inhibition of Bmal1 promotes/inhibits autophagy, proliferation and activation of HSC; TGF-β1 promotes Bmal1 expression by activating Smad3, while Bmal1 positive feedback promotes phosphorylation of Smad3. This study will further investigate the regulation of Smad3 on the promoter region of Bmal1 by luciferase reporter assay, ChIP, etc; Co-IP assay for the interaction of Bmal1, TGF-β1 receptor and Smad3; injection of HSC-targeting Bmal1 overexpressing lentivirus and Bmal1-inhibiting adeno-associated virus interfered with liver fibrosis in mice; detection of Bmal1 expression in patients with chronic liver disease. This study will explore the molecular mechanism of liver fibrosis from the new perspective of TGF-β1/Smads-Bmal1 positive feedback loop, and provide a new theoretical basis for clinical treatment of liver fibrosis.

肝星状细胞（HSC）的活化是肝纤维化发生发展的关键。自噬能促进HSC的活化和增殖，可能是治疗肝纤维化的新途径。我们用自噬抑制剂和促纤维化因子处理HSC，利用表达谱芯片等筛选参与自噬的基因。发现生物钟基因Bmal1可能是HSC自噬的关键触发点：过表达/抑制Bmal1能促进/抑制HSC的自噬、增殖和活化；TGF-β1通过激活Smad3促进Bmal1表达，同时Bmal1正反馈促进Smad3磷酸化。本课题将进一步采用荧光素酶报告实验、ChIP等探索Smad3对Bmal1启动子区的调控；Co-IP实验检测Bmal1、TGF-β1受体、Smad3的相互作用；注射靶向HSC的Bmal1过表达慢病毒和抑制表达的腺相关病毒干预小鼠肝纤维化进程；检测慢性肝病患者体内Bmal1的表达。本研究将从TGF-β1/Smads-Bmal1正反馈回路这一新视角探索肝纤维化的分子机制，为临床治疗肝纤维化提供新的理论依据。

肝星状细胞（HSC）的活化是肝纤维化发生发展的关键。自噬能促进HSC的活化和增殖，可能是治疗肝纤维化的新途径。我们用自噬抑制剂和促纤维化因子处理HSC，利用表达谱芯片等筛选参与自噬的基因。发现生物钟基因Bmal1可能是HSC自噬的关键触发点。Bmal1在肝星状细胞活化过程中的表达明显升高。过表达/抑制Bmal1能促进/抑制HSC的自噬、增殖和活化；TGF-β1通过激活Smad3能够促进Bmal1表达，同时Bmal1正反馈促进Smad3磷酸化。进一步采用荧光素酶报告实验、ChIP等探索Smad3对Bmal1启动子区的调控，结果显示，Bmal1能够结合到Smad3的启动子区，而不是Smad2的启动子区。为了验证体外实验发现的结果，体内水平干预Bmal1的水平，通过注射靶向HSC的Bmal1过表达慢病毒和抑制表达的腺相关病毒干预小鼠肝纤维化进程；检测慢性肝病患者体内Bmal1的表达。结果发现，体内干预Bmal1的表达水平后，能够影响肝纤维化的进程，Bmal1能够促进I型胶原蛋白的表达水平及α-SMA的水平。本研究将从TGF-β1/Smads-Bmal1正反馈回路这一新视角探索肝纤维化的分子机制，为临床治疗肝纤维化提供新的理论依据。