p16^( INK4a)基因通过非周期依赖作用促进心脏成纤维细胞纤维化蛋白表达

p16^( INK4a) gene promotes cardiac fibroblast fibrosis protein expression through a cell cycle-independent effect

目的:探究p16^( INK4a)基因在转化生长因子-β1(TGF-β1)介导的促心肌纤维化中的作用及机制。方法:在人原代心脏成纤维细胞中转染p16^( INK4a)过表达腺病毒,TGF-β1刺激并检测胶原蛋白(Collagen)Ⅰ、α-平滑肌肌动蛋白(α-SMA)和骨膜蛋白(POSTN)蛋白表达水平。其次,TGF-β1刺激细胞后,分别加入细胞周期蛋白依赖性激酶4和6(CDK4/6)抑制剂Palbociclib和Ribociclib,模拟p16蛋白细胞周期作用,检测α-SMA和POSTN蛋白表达水平。最后,同以上方法转染p16^( INK4a)过表达腺病毒和TGF-β1刺激,使用Ravoxertinib阻断ERK信号通路,检测其对CollagenⅠ、α-SMA和POSTN表达水平的影响。结果:与空载腺病毒处理对照组比较,p16^( INK4a)过表达(p16-OE)组TGF-β1介导的Collagen I[(0.33±0.08)比(1.36±0.22)]、α-SMA[(0.62±0.22)比(1.43±0.45)]和POSTN[(0.67±0.04)比(1.69±0.50)]蛋白表达水平均显著升高,P<0.05或<0.01。与对照(NC)+二甲亚砜(DMSO)组比较,NC+Palbociclib组和NC+Ribociclib组α-SMA和POSTN蛋白表达水平均无显著变化,P均>0.05。与p16-OE+DMSO组比较,p16-OE+Ravoxertinib组Collagen I[(1.38±0.06)比(0.77±0.07)]、α-SMA[(1.09±0.03)比(0.70±0.13)]和POSTN[(1.34±0.11)比(0.69±0.06)]蛋白表达水平均显著降低,P均<0.01。结论:p16^( INK4a)基因可能通过ERK1磷酸化通路激活而非细胞周期依赖作用,促进心脏成纤维细胞分泌促纤维化蛋白,进而参与心肌纤维化病理过程。

Objective:To explore the role and mechanism of p16^( INK4a) gene in promoting myocardial fibrosis mediated by transforming growth factor(TGF)-β1.Methods:Human primary cardiac fibroblasts were transfected with p16^( INK4a) over-expressing adenovirus and stimulated with TGF-β1.Then expression levels of Collagen I,α-smooth muscle actin(α-SMA)and periostin(POSTN)protein were measured.Secondly,after TGF-β1 stimulation,cyclin-dependent kinase 4 and 6(CDK4/6)inhibitors Palbociclib and Ribociclib were respectively added to imitate the cell cycle effects of p16 protein,then expressions ofα-SMA and POSTN protein were measured.Finally,the p16^( INK4a) overexpressing adenovirus was transfected in the same way as above and stimulated by TGF-β1,and then Ravoxertinib was used to block the ERK signaling pathway,and its impact on expressions of CollagenⅠ,α-SMA and POSTN was detected.Results:Compared with empty adenovirus-treated control group,there were significant rise in expression levels of Collagen I[(0.33±0.08)vs.(1.36±0.22)],α-SMA[(0.62±0.22)vs.(1.43±0.45)]and POSTN protein[(0.67±0.04)vs.(1.69±0.50)]mediated by TGF-β1 in p16^( INK4a) over-expressing(p16-OE)group,P<0.05 or<0.01.Compared with control(NC)+dimethyl sulfoxide(DMSO)group,there were no significant changes in expressions ofα-SMA and POSTN protein in NC+Palbociclib group and NC+Ribociclib group,P>0.05 all.Compared with p16-OE+DMSO group,there were significant reductions in expressions of Collagen I[(1.38±0.06)vs.(0.77±0.07)],α-SMA[(1.09±0.03)vs.(0.70±0.13)]and POSTN protein[(1.34±0.11)vs.(0.69±0.06)]in p16-OE+Ravoxertinib group,P<0.01 all.Conclusion:p16^( INK4a) gene may promote cardiac fibroblasts to secrete pro-fibrotic proteins through ERK1 phosphorylation pathway instead of cell cycle-dependent effect,then participate in the pathological process of myocardial fibrosis.