氧化槐定碱体内体外通过AKT/mTOR通路调控自噬抑制HBV诱发肝纤维化

Oxysophoridine Regulates Autophagy through the AKT/mTOR Pathway and Inhibits HBV-Induced Liver Fibrosis

目的观察氧化槐定碱抑制HBV诱发肝纤维化的机制。方法采用细胞间非接触式的共培养体系进行培养,同时建立HBV诱发肝纤维化的动物模型,四甲基偶氮唑蓝(MTT)法检测LX-2细胞抑制率,qRT-PCR法检测α-SMA、collagenⅠmRNA水平,Western blot检测细胞纤维化、自噬、AKT/mTOR信号通路相关蛋白表达水平,运用AKT-siRNA、雷帕霉素、自噬抑制剂(3-MA)探讨自噬对肝纤维化的影响。结果体外实验显示:0.5-32 mg·L^(-1)氧化槐定碱抑制LX-2细胞的增殖(P<0.05);与对照组比较,氧化槐定碱4、8、16、32 mg·L^(-1)组α-SMA、collagenⅠmRNA表达水平降低,Beclin-1、LC3Ⅱ/LC3Ⅰ水平上调,p-AKT/AKT、p-mTOR/mTOR水平下调(P<0.05);与氧化槐定碱16 mg·L^(-1)组比较,氧化槐定碱16 mg·L^(-1)+AKT-siRNA组抑制率、LC3Ⅱ/LC3Ⅰ明显增加,p-AKT/AKT、collagenⅠ明显降低(P<0.05);与氧化槐定碱16 mg·L^(-1)组比较,氧化槐定碱16 mg·L^(-1)+3-MA组抑制率、LC3Ⅱ/LC3Ⅰ明显降低,p-AKT/AKT、collagenⅠ明显增加(P<0.05)。体内实验显示:与氧化槐定碱组比较,氧化槐定碱+雷帕霉素组ALT、AST、collagenⅠ、p-mTOR/mTOR水平明显下调,LC3Ⅱ/LC3Ⅰ水平明显上调;氧化槐定碱+3-MA组LC3Ⅱ/LC3Ⅰ水平明显下调,collagenⅠ、p-mTOR/mTOR明显上调(均P<0.05)。结论氧化槐定碱能调控肝细胞自噬,上调细胞纤维化水平,其机制可能与调控AKT/mTOR信号通路有关。

Objective To observe the mechanism of oxysophoridine inhibiting HBV-induced liver fibrosis.Methods A non-contact co-cultivation system between cells was used for culture. Simultaneously, an animal model of HBVinduced liver fibrosis was established. The inhibition rate of LX-2 cells was detected by the tetramethylazazole blue(MTT) method. The levels of α-SMA and collagen Ⅰ mRNA were detected by qRT-PCR, Protein expression levels of cell fibrosis, autophagy, and AKT/mTOR signaling pathway was detected by Western blot. And AKT-siRNA, rapamycin and autophagy inhibitor(3-MA) was used to explore the effect of autophagy on liver fibrosis.Results In vitro experiments showed 0.5-32 mg·L^(-1)oxysophoridine inhibited the proliferation of LX-2 cells(P<0.05). Compared with the control group, the expression level of α-SMA and collagen Ⅰ mRNA decreased, the levels of Beclin-1, LC3 Ⅱ/LC3 Ⅰ were upregulated, and the levels of p-AKT/AKT, p-mTOR/mTOR were down-regulated in oxysophoridine 4, 8, 16, 32 mg·L^(-1)groups(P<0.05). Compared with oxysophoridine 16 mg·L^(-1)group, the inhibition rate and LC3 Ⅱ/LC3 Ⅰ was significantly increased, and collagen Ⅰ,p-AKT/AKT was significantly decreased in oxysophoridine 16 mg·L^(-1)+AKT-siRNA group(P<0.05). Compared with oxysophoridine 16 mg·L^(-1)group, the inhibition rate and LC3 Ⅱ/LC3 Ⅰ decreased significantly, and collagen Ⅰ and p-AKT/AKT increased significantly in oxysophoridine 16 mg·L^(-1)+3-MA group(P<0.05). Compared with the oxysophoridine group, the ALT, AST, collagen Ⅰ, and p-mTOR/mTOR levels in the oxysophoridine + rapamycin group were significantly down-regulated, and the LC3 Ⅱ/LC3 Ⅰ levels were significantly up-regulated. The levels of LC3 Ⅱ/LC3 Ⅰ in the oxysophoridine +3-MA group were significantly down-regulated, and collagen Ⅰ and p-mTOR/mTOR were significantly up-regulated in vivo experiments(all P<0.05).Conclusion Oxysophoridine can regulate hepatocyte autophagy and down-regulate the level of cell fibrosis. The mechanism may be related to the regulation of AKT/mTOR signaling pathway.