目的:研究槲皮素是否通过调控miR-146抑制PI3K/Akt信号通路诱导肝星状细胞凋亡。
方法:用TGF-β和不同浓度(40、60和80μmol/L)槲皮素处理大鼠肝星状细胞(HSC-T6),CCK-8法和流式细胞术检测细胞增殖和凋亡的变化。RT-qPCR检测miR-146的表达和α-SMA的mRNA表达,胶原Ⅰ,处理细胞中的TRAF6、PI3K和Akt,和α-SMA的蛋白质表达,胶原Ⅰ,TRAF6,PI3K,使用蛋白质印迹检测Akt和p-Akt。免疫荧光法检测α-SMA和Ⅰ型胶原的蛋白表达。还使用Western印迹检查了用miR-146模拟物和抑制剂转染对细胞蛋白表达的影响。
结果:槲皮素剂量和时间依赖性地抑制HSC-T6细胞的增殖,并显着增加总细胞凋亡率(P<0.01)。TGF-β刺激的HSC-T6细胞显示α-SMA的mRNA和蛋白表达水平显著升高,胶原Ⅰ,TRAF6、PI3K和Akt(P<0.05),槲皮素显著下调(P<0.05)。槲皮素显著上调HSC-T6细胞miR-146的表达(P<0.01),转染miR-146模拟物显著降低了α-SMA的mRNA和蛋白表达水平,胶原蛋白Ⅰ,TRAF6、PI3K和Akt(P<0.05),和miR-146抑制剂产生相反的作用(P<0.05)。
结论:槲皮素通过上调miR-146抑制PI3K/Akt信号通路,抑制HSCs增殖并促进凋亡。
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-β and different concentrations (40, 60 and 80 μmol/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 of α-SMA, collagenⅠ, TRAF6, PI3K and Akt in the treated cells, and the protein expressions of α-SMA, collagenⅠ, TRAF6, PI3K, Akt and p-Akt were detected using Western blotting. Immunofluorescence assay was used to detect the protein expression of α-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 of α-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 of α-SMA, collagen Ⅰ, TRAF6, PI3K and Akt (P<0.05), and miR- 146 inhibitor produced the opposite effects (P<0.05).
CONCLUSIONS: Quercetin inhibits proliferation and promotes apoptosis of HSCs by upregulating miR-146 to inhibit the PI3K/Akt signaling pathway.