1,25(OH)_2D_3通过调节miR-146a水平抑制大鼠肝纤维化的体内和体外观察

1,25(OH)_2D_3 inhibits hepatic fibrosis by regulating miR-146a levels in vitro and in vivo

目的体内体外观察1,25(OH)_2D_3通过调节mi R-146a水平抑制大鼠肝纤维化的作用机制。方法建立CCl_4诱导的大鼠肝纤维化模型,体外转染肝星状细胞(HSCs)mi R-146a模拟剂/抑制剂,观察1,25(OH)_2D_3处理对动物肝组织变化和HSC增殖和凋亡的影响。采用qPCR法检测肝组织mi R-146a水平,采用CCK8法检测细胞增殖,使用流式细胞术检测HSC凋亡。结果在干预8 w末,1,25(OH)_2D_3干预组大鼠肝纤维化程度明显减轻;1,25(OH)_2D_3干预组大鼠肝组织mi R-146a水平为(0.70±0.03),显著高于橄榄油组【(0.33±0.17,P<0.05)】;1,25(OH)_2D_3组细胞增殖率为58.8%,较DMSO组下降了15.9%,转染mi R-146a模拟剂组大鼠HSC增殖率为46.5%,较对照组下降了53.3%,转染mi R-146a抑制剂组HSC增殖率为132.8%,较对照物组升高了32.8%(P<0.05),1,25(OH)_2D_3干预组细胞凋亡率为12.6%,较DMSO组增加了5.2%,转染mi R-146a模拟剂组细胞凋亡率为16.8%,较对照组细胞凋亡率增加了8.2%,转染mi R-146a抑制剂组细胞凋亡率为6.3%,较对照组细胞凋亡率减少了2.2%(P<0.05),提示1,25(OH)_2D_3具有抑制HSC增殖、促进凋亡作用。结论 1,25(OH)_2D_3可能通过调节mi R-146a水平抑制HSC活化和抑制大鼠肝纤维化。

Objective To observe the inhibitory effect of 1,25(OH)2D3 on liver fibrosis in hepatic stellate cells (HSCs) and in rat model. Method The CCl4-induced liver fibrosis was made in rats,and the activation of HSCs were induced by 10 pmmol/L TGF-β1 incubation. The miR-146a mimic/inhibitor were transfected in HSCs and 1,25(OH)2D3 intervention were paralleled. The miR-146a levels in liver tissues were detected by qPCR,the proliferation of cells was measured by CCK8 and the apoptosis was detected by flow cytometry. Results At the end of 8 weeks,the hepatic fibrosis in 1,25(OH)2D3 intervention group was significantly milder than that in the control group;the miR-146a level in 1,25(OH)2D3 intervention group was significantly higher than in oil control group[(0.70±0.03) vs.(0.33±0.17),P<0.05];the proliferation rate in1,25(OH)2D3 group was 58.8%,15.91% of lower than in DMSO group,in miR-146a mimic transfected group was 46.5%,53.3% of lower than in control,in miR-146a inhibitor group was132.8%,32.8% of higher than in control(P<0.05);the apoptosis rate of HSCs in1,25(OH)2D3 group was 12.6%,5.2% of higher than in DMSO,in miR-146a mimic transfected group was16.8%,8.2% of higher than in control,and in miR-146a inhibitor transfected group was 6.3%,2.2% of lower than in control (P<0.05),indicating the inhibition of 1,25(OH)2D3 on proliferation and promotion on apoptosis of HSCs. Conclusion 1,25(OH)2D3 might modulate miR-146a levels to inhibit liver fibrosis.