PAX6通过MEK/ERK信号通路抑制肝星状细胞活化和增殖实验研究

PAX6 inhibiting activation and proliferation of hepatic stellate cells through MEK/ERK signaling pathway in vitro

目的探讨PAX6通过丝裂原活化蛋白激酶(MEK)/细胞外调节蛋白激酶(ERK)信号通路抑制肝星状细胞活化和增殖的效果。方法取LX2肝星状细胞,分为对照组、PAX6 inhibitor组和PAX6 mimics组。采用CCK-8试剂盒测定细胞增殖,采用油红O染色测定细胞分化水平,使用流式细胞仪分析细胞凋亡,采用RT-PCR法和蛋白印迹法测定细胞PAX6、MEK和ERK mRNA和蛋白表达水平。结果PAX6 inhibitor组细胞PAX6 mRNA水平、凋亡率和G1期分别为(1.49±0.23)、(2.70±0.85)%和(59.02±1.25)%,显著低于LX2肝星状细胞组【分别为(1.85±0.19)、(3.40±0.47)%和(64.66±1.41)%,P<0.05】,而细胞增殖、存活率、分化率、MEK和ERK mRNA和蛋白水平分别为(0.79±0.03)、(73.35±9.74)%、(49.37±4.24)%、(2.55±0.43)、(3.90±0.49)、(0.89±0.15)和(1.17±0.17),显著高于LX2肝星状细胞组【分别为(0.58±0.05)、(60.74±9.24)%、(29.35±4.47)%、(2.67±0.47)、(4.55±0.50)、(0.74±0.14)和(1.35±0.16),P<0.05】;PAX6 mimics组细胞PAX6 mRNA水平、凋亡率和G1期分别为(2.67±0.20)、(6.70±1.04)%和(66.38±1.35)%,显著高于LX2肝星状细胞组【分别为(1.85±0.19)、(3.40±0.47)%和(64.66±1.41)%,P<0.05】,而细胞增殖、存活率、分化率、MEK和ERK mRNA和蛋白水平分别为(0.40±0.04)、(52.24±5.57)%、(13.85±3.35)%、(1.94±0.53)、(1.45±0.42)、(0.53±0.15)和(0.53±0.16),显著低于LX2肝星状细胞组【分别为(0.58±0.05)、(60.74±9.24)%、(29.35±4.47)%、(2.67±0.47)、(4.55±0.50)、(0.74±0.14)和(1.35±0.16),P<0.05】。结论PAX6过表达可抑制肝星状细胞活化和增殖,促进其凋亡,其机制可能与PAX6过表达可抑制肝星状细胞MEK和ERK表达进而抑制了MEK/ERK通路的激活有关。

Objective The purpose of this experiment was to investigate the effect of PAX6 on inhibition of activation and proliferation of hepatic stellate cells(HSCs)by through MEK/ERK signaling pathway.Methods The LX2 HSCs were divided into control,PAX6 inhibitor and PAX6 mimics group and managed accordingly.The cell proliferation was determined by CCK-8 kit,the cell differentiation was detected by oil red O staining,the cell apoptosis was analyzed by flow cytometry,and PAX6,MEK and ERK mRNA and their protein expression were assayed by RT-PCR and Western blotting,respectively.Results The PAX6 mRNA level,apoptosis rate and G1 stage in PAX6 inhibitor-intervened group were(1.49±0.23),(2.70±0.85)%and(59.02±1.25)%,significantly lower than[(1.85±0.19),(3.40±0.47)% and(64.66±1.41)%,respectively,P<0.05]in LX2 HSCs,and the cell proliferation,survival rate,differentiation rate,MEK and ERK mRNA and their protein expression in PAX6 inhibitor-intervened group were(0.79±0.03),(73.35±9.74)%,(49.37±4.24)%,(2.55±0.43),(3.90±0.49),(0.89±0.15)and(1.17±0.17),significantly higher than[(0.58±0.05),(60.74±9.24)%,(29.35±4.47)%,(2.67±0.47),(4.55±0.50),(0.74±0.14)and(1.35±0.16),respectively,P<0.05]in the control;the PAX6 mRNA level,apoptosis rate and G1 stages in PAX6 mimics-intervened group were(2.67±0.20),(6.70±1.04)% and(66.38±1.35)%,significantly higher than[(1.85±0.19),(3.40±0.47)%and(64.66±1.41)%,respectively,P<0.05]in the control,and the cell proliferation,survival rate,differentiation rate,MEK and ERK mRNA and their protein expression in PAX6 mimics group were(0.40±0.04),(52.24±5.57)%,(13.85±3.35)%,(1.94±0.53),(1.45±0.42),(0.53±0.15)and(0.53±0.16),significantly lower than[(0.58±0.05),(60.74±9.24)%,(29.35±4.47)%,(2.67±0.47),(4.55±0.50),(0.74±0.14)and(1.35±0.16),respectively,P<0.05]in the control.Conclusion The PAX6 overexpression could inhibit the activation and proliferation of hepatic stellate cells,and promote cell apoptosis.The mechanism involved might be related to the inhibition of MEK and ERK expression and MEK/ERK pathway in hepatic stellate cells.