异土木香内酯通过miR-5195-3p/KLF5/N-cadherin对肝纤维化的作用机制

The mechanism of isoalantolactone on liver fibrosis through miR-5195-3p/KLF5/N-cadherin

目的探讨异土木香内酯(isoalantolactone,IAL)通过miR-5195-3p/KLF5/N-cadherin对肝纤维化的作用机制。方法将SD大鼠随机分为正常组、模型组、IAL低、中、高剂量(IAL组)、IAL+miR-5195-3p mimic组和奥贝胆酸组,每组20只。采用ELISA法检测炎症因子水平,HE染色和天狼星红染色观察病理学改变情况和纤维化分级、评分情况,比色法测定氧化和抗氧化酶水平;qRT-PCR和Western blotting检测肝脏组织各因子mRNA和相关蛋白表达。结果与正常组相比,模型组ALT、AST、IL-6、IL-β、TNF-α、MDA、KLF5 mRNA、N-cadherin mRNA水平及肝纤维化分级、Ishak评分明显升高(P均<0.05)。与模型组相比,IAL低、中、高剂量组和奥贝胆酸组的以上各项指标依次显著降低(P均<0.05)。与正常组相比,模型组的SOD、GSH-Px、miR-5195-3p水平明显降低(P均<0.05),与模型组相比,IAL低、中、高剂量组和奥贝胆酸组的以上指标水平依次显著升高(P均<0.05)。与正常组相比,模型组大鼠肝脏小叶炎症明显升高,并发生严重损伤,与模型组相比,IAL低、中、高剂量组和奥贝胆酸组肝脏病理状态依次减轻。荧光素酶报告发现,KLF5是miR-5195-3p的重要靶点。与IAL组相比,IAL+miR-5195-3p mimic组肝脏损伤明显减轻,纤维化程度明显改善。结论IAL可以明显改善肝功能及炎症因子水平,增强肝脏的抗氧化能力,减轻肝脏脂质过氧化损伤程度,对肝脏纤维化具有一定的防治疗作用,这可能与IAL通过提高miR-5195-3p表达,降低KLF5和N-cadherin水平有关。

Objective To investigate the mechanism of isoalantolactone(IAL)on liver fibrosis through miR-5195-3p/KLF5/N-cadherin.Methods SD rats were randomly divided into normal group,model group,IAL low,medium,and high dose(IAL group),IAL+miR-5195-3p mimic group and obeticolic acid group,with 20 rats in each group.ELISA was used to detect levels of inflammatory factors,HE staining and Siiued staining were used to observe pathological changes and fibrosis grading and scoring,and colorimetric methods were used to measure levels of oxidation and antioxidant enzymes.qRT-PCR and Western blotting were used to detect the mRNA and related protein expression of various factors in liver tissue.Results Compared with the normal group,the levels of ALT,AST,IL-6,IL-β,TNF-α,MDA,KLF5 mRNA,N-cadherin mRNA,fibrosis grade and Ishak score in the model group were significantly higher(all P<0.05).Compared with the model group,the levels of the above indicators in the low,middle,high dose of IAL and obecholic acid groups were successively lower(all P<0.05).Compared with the normal group,the levels of SOD,GSH-Px,miR-5195-3p in the model group were significantly lower(all P<0.05).Compared with the model group,the levels of the above indicators in the low,middle,high dose of IAL and Obecholic acid groups were successively higher(all P<0.05).Compared with the normal group,the inflammation of the liver lobules in the model group rats was significantly increased and severe damage occurred.Compared with the model group,the pathological state of the liver in the low,middle,high dose IAL,and obecholic acid groups decreased in sequence.Luciferase reported that KLF5 was an important target of miR-5195-3p.Compared with the IAL group,the IAL+miR-5195-3p mimic group significantly reduced liver damage and improved the degree of fibrosis.Conclusion IAL can significantly improve the liver function and the level of inflammatory factors,enhance the antioxidant capacity of the liver,reduce the degree of liver lipid peroxidation injury,and it has a certain preventive and therapeutic effect on liver fibrosis,which may be related to the increase of miR-5195-3p expression and the decrease of KLF5 and N-cadherin levels by IAL.