MCA17-1通过调控TGF-β1/Smad信号通路改善肝纤维化

Amelioration of Hepatic Fibrosis by MCA17-1 through TGF-β1/Smad Signaling Pathway

关键词:  MCA17-1  肝纤维化  体内外评价  TGF-β1/Smad信号通路

Key words:  MCA17-1  Liver fibrosis  In vitro and in vivo evaluation  TGF-β1/Smad signaling pathway

摘要:

目的 评价真菌来源的化合物MCA17-1抗肝纤维化作用，并探讨其可能的作用机制。方法 采用TGF-β1诱导LX-2细胞肝纤维化模型和CCl4诱导小鼠肝纤维化模型评价MCA17-1的抗纤维化作用。利用q-PCR和Western blot检测方法检测LX-2细胞和小鼠肝脏中α-SMA、COL1A1、Fibronectin 1基因转录水平和蛋白表达。细胞划痕实验评价MCA17-1对HSCs迁移的影响。肝脏指数、血清生化指标、H&E染色、Masson染色、天狼星红染色及免疫组化等评价MCA17-1体内抗肝纤维化作用。采用Western blot检测方法检测LX-2细胞和小鼠肝脏组织中TGF-β1、Smad 2/3、p-Smad 2和p-Smad 2/3蛋白表达变化。结果 与TGF-β1模型组相比，1μM和5μM 剂量的MCA17-1均降低LX-2细胞中α-SMA、COL1A1、Fibronectin 1基因转录水平和蛋白表达（P<0.001），削弱LX-2细胞的迁移能力（P<0.05）。与CCl4模型组相比，2.5mg?kg-1和5mg?kg-1剂量的MCA17-1均降低小鼠肝脏中α-SMA、COL1A1、Fibronectin 1基因转录水平和α-SMA、COL1A1蛋白表达（P<0.01），但对Fibronectin 1蛋白表达的影响无显著性差异（P>0.05）。与CCl4模型组相比，低剂量和高剂量的MCA17-1均降低小鼠血清中ALT和AST水平（P<0.05），改善CCl4导致的肝组织病理变化，减少胶原纤维的沉积（P<0.01），并降低TGF-β1及CCl4诱导肝纤维化模型中Smad2/3蛋白的磷酸化水平（P<0.05）。MCA17-1可以降低肝纤维化细胞模型中TGF-β1蛋白表达（P<0.001），但提高了纤维化小鼠肝脏中TGF-β1蛋白表达（P<0.001）。结论 MCA17-1可体外抑制TGF-β1诱导的HSC活化，并减轻CCl4诱导的小鼠肝损伤，改善肝纤维化，该作用可能与其特异性抑制TGF-β1/Smad信号通路有关，提示真菌来源的新型化合物MCA17-1有望成为肝纤维化治疗的一种新候选药物。

Abstract:

OBJECTIVE To evaluate the anti-hepatic fibrosis effect of the fungal-derived compound MCA17-1 and to explore its possible mechanism of action. METHODS TGF-β1-induced liver fibrosis model in LX-2 cells and CCl4-induced liver fibrosis model in mice were used to evaluate the antifibrotic effect of MCA17-1. The transcript levels and protein expression of α-SMA, COL1A1, and Fibronectin 1 genes were detected in LX-2 cells and mouse livers using q-PCR and Western blot. Cell scratch assay was performed to evaluate the effect of MCA17-1 on the migration of HSCs. Liver index, serum biochemical indexes, H&E staining, Masson staining, Sirius red staining as well as immunohistochemistry were used to evaluate the in vivo anti-hepatic fibrosis effect of MCA17-1. Western blot assay was used to detect the changes of TGF-β1, Smad 2/3, p-Smad 2 and p-Smad 2/3 protein expression in LX-2 cells and mouse liver tissues. RESULTS Compared with the TGF-β1 model group, both 1 μM and 5 μM doses of MCA17-1 significantly decreased the transcript levels and protein expression of α-SMA, COL1A1, and Fibronectin 1 (P<0.001) and impaired the migration ability of LX-2 cells (P<0.05). Compared with the CCl4 model group, both 2.5 mg·kg?1 and 5 mg·kg?1 doses of MCA17-1 significantly decreased the mRNA levels of α-SMA, COL1A1, and Fibronectin 1, as well as the protein expression of α-SMA and COL1A1 in mouse liver tissues (P<0.01); however, no significant effect was observed on Fibronectin 1 protein expression (P>0.05). Compared with the CCl4 model group, both low-dose and high-dose MCA17-1 significantly reduced serum ALT and AST levels (P<0.05), ameliorated CCl4-induced histopathological changes in the liver, decreased collagen fiber deposition (P<0.01), and suppressed the phosphorylation levels of Smad2/3 in both TGF-β1- and CCl4-induced liver fibrosis models (P<0.05). Additionally, MCA17-1 reduced TGF-β1 protein expression in the cellular model of liver fibrosis (P<0.001) but increased TGF-β1 protein expression in the livers of fibrotic mice (P<0.001) CONCLUSION MCA17-1 inhibited TGF-β1-induced HSC activation in vitro, ameliorated CCl4-induced hepatic injury, and attenuated hepatic fibrosis in mice. This effect may be related to its specific inhibition of the TGF-β1/Smad signaling pathway, suggesting that MCA17-1, a novel fungal-derived compound, may serve as a potential therapeutic candidate for the treatment of liver fibrosis.