槲皮素诱导肝星状细胞凋亡:基于调控miR 146影响PI3K/Akt信号通路  

Quercetin induces hepatic stellate cell apoptosis by inhibiting the PI3K/Akt signaling pathway via upregulating miR-146

作　　者：高晓阳 赵晓璐 张春艳 颜羽昕 金蓉 马月宏 GAO Xiaoyang;ZHAO Xiaolu;ZHANG Chunyan;YAN Yuxin;JIN Rong;MA Yuehong(Department of Basic Medical Sciences,Inner Mongolia Medical University,Hohhot 010000,China)

机构地区：[1]内蒙古医科大学基础医学院,内蒙古自治区呼和浩特010000

出　　处：《南方医科大学学报》2023年第10期1725-1733,共9页Journal of Southern Medical University

基　　金：国家自然科学基金(81960759,81560706);内蒙古自治区自然科学基金(2019MS08010,2014MS0841);内蒙古自治区草原英才培养计划(No.内人社办发[2016]348号);内蒙古医科大学致远人才项目(2020-11);内蒙古人才开发基金(2022年-个人项目22056);内蒙古医科大学蒙药抗肝纤维化作用研究科技创新团队(2022-01);内蒙古医科大学重点项目(YKD2022ZD019)。

摘　　要：目的探讨槲皮素(Que)通过调控肝星状细胞内miR-146水平影响PI3K/Akt信号通路诱导细胞凋亡的作用机制。方法以大鼠肝星状细胞系(HSC-T6)为研究对象,设计实验分组:空白对照组、TGF-β组、TGF-β+Que-L(40μmol/L)、TGF-β+Que-M(60μmol/L)和TGF-β+Que-H(80μmol/L)为药物治疗部分;各组阴性对照组、miR-146模拟剂(mimic)和抑制剂(inhibitor)为细胞转染部分。CCK-8法检测不同浓度的Que对细胞抑制情况,选出低、中、高3个剂量浓度用于后续细胞实验;细胞凋亡流式细胞术检测Que对HSCs细胞凋亡的影响;RT-qPCR法检测HSC-T6细胞药物治疗以及细胞转染后miR-146、α-SMA、CollagenⅠ、TRAF6、PI3K和Akt mRNA表达情况;Westernblot法检测以上各组中α-SMA、CollagenⅠ、TRAF6、PI3K、Akt和p-Akt蛋白表达情况;免疫荧光实验检测HSC-T6细胞药物治疗中α-SMA、CollagenⅠ蛋白表达强弱。结果CCK-8实验结果显示,相同时间不同浓度时,与Control组相比,随着药物浓度的递增,细胞A450 nm值显著降低(P<0.05);相同浓度不同时间比较,发现药物浓度在40、60、80μmol/L时细胞A450 nm值间的差异最为显著。流式细胞术检测结果显示,Que各给药组总细胞凋亡率均显著升高(P<0.01)。Que对HSC-T6细胞的作用结果显示,与Control组相比,TGF-β组α-SMA、CollagenⅠ、TRAF6、PI3K和Akt的mRNA和蛋白表达水平显著升高(P<0.05);与TGF-β相比,Que各治疗组α-SMA、CollagenⅠ、TRAF6、PI3K、Akt的mRNA和蛋白表达水平显著下调(P<0.05)。免疫荧光实验显示Que能显著减弱TGF-β组中α-SMA、CollagenⅠ蛋白的荧光强度。Que显著上调HSC-T6细胞中miR-146表达(P<0.01);与各自阴性对照组相比,miR-146 mimic显著降低α-SMA、CollagenⅠ、TRAF6、PI3K、Akt的mRNA和蛋白表达水平(P<0.05),而miR-146 inhibitor使上述指标mRNA和蛋白表达水平显著逆转(P<0.05)。结论Que通过上调miR-146影响PI3K/Akt信号通路来抑制HSCs增殖,促进其凋亡。Objective To investigate whether quercetin induces apoptosis of hepatic stellate cells by regulating miR-146 to inhibit the PI3K/Akt signaling pathway.Methods Rat hepatic stellate cells(HSC-T6)were treated with TGF-βand different concentrations(40,60 and 80μmol/L)of quercetin,and the changes in cell proliferation and apoptosis were detected using CCK-8 assay and flow cytometry.RT-qPCR was used to detect the expression of miR-146 and mRNA expressions ofα-SMA,collagenⅠ,TRAF6,PI3K and Akt in the treated cells,and the protein expressions ofα-SMA,collagenⅠ,TRAF6,PI3K,Akt and p-Akt were detected using Western blotting.Immunofluorescence assay was used to detect the protein expression ofα-SMA and collagenⅠ.The effects of transfection with miR-146 mimic and inhibitor on the protein expressions of the cells were also examined using Western blotting.Results Treatment with quercetin dose-and time-dependently inhibited the proliferation of HSC-T6 cells and significantly increased the total cell apoptosis rate(P<0.01).TGF-β-stimulated HSC-T6 cells showed significantly increased mRNA and protein expression levels ofα-SMA,collagenⅠ,TRAF6,PI3K and Akt(P<0.05),which were significantly down-regulated by quercetin treatment(P<0.05).Quercetin significantly upregulated the expression of miR-146 in HSC-T6 cells(P<0.01),Transfection of the cells with miR-146 mimic significantly decreased the mRNA and protein expression levels ofα-SMA,collagenⅠ,TRAF6,PI3K and Akt(P<0.05),and miR-146 inhibitor produced the opposite effects(P<0.05).Conclusion Quercetin inhibits proliferation and promotes apoptosis of HSCs by upregulating miR-146 to inhibit the PI3K/Akt signaling pathway.

关 键 词：槲皮素 肝星状细胞 miR-146 PI3K/AKT信号通路 

分 类 号：R285[医药卫生—中药学]