颅内动脉瘤组织中Septin4的表达及其通过调节自噬对人脑血管平滑肌细胞的作用研究

The expression of Septin4 in intracranial aneurysm tissue and its effect on human cerebral vascular smooth muscle cells by regulating autophagy

目的:探究Septin4在颅内动脉瘤中的表达及其对人脑血管平滑肌细胞(HBVSMCs)的作用及可能的作用机制。方法:收集颅内动脉瘤组织和正常颅动脉组织各32份,通过免疫组化(IHC)、逆转录-聚合酶链反应(RT-PCR)和蛋白质印迹法检测组织中Septin4的表达;免疫荧光染色(IF)检测平滑肌22α(SM22α)和平滑肌α肌动蛋白(αSMA)表达;分别将Septin4过表达载体(Septin4)、Septin4干扰序列(si-Septin4)及其阴性对照(NC和si-NC)转染HBVSMCs,RT-PCR和蛋白质印迹法检测HBVSMCs中Septin4的表达;蛋白质印迹法检测αSMA、SM22α、基质金属蛋白酶(MMP)2、MMP9、微管相关蛋白1轻链3(LC3)、sequestosome 1(p62)、磷酸化腺苷酸活化蛋白激酶(p-AMPK)、腺苷酸活化蛋白激酶(AMPK)、磷酸化哺乳动物类雷帕霉素靶蛋白(p-mTOR)和哺乳动物类雷帕霉素靶蛋白(mTOR)蛋白表达。结果:与正常颅动脉组织相比,颅内动脉瘤组织中Septin4阳性细胞数、mRNA表达、蛋白表达及SM22α和αSMA荧光强度明显降低(P<0.05)。与NC组相比,Septin4组细胞中Septin4 mRNA表达及Septin4、αSMA、SM22α、p62、p-mTOR/mTOR蛋白表达明显升高(P<0.05),MMP2、MMP9、LC3Ⅱ/LC3Ⅰ和p-AMPK/AMPK蛋白表达明显降低(P<0.05);与si-NC组相比,si-Septin4组细胞中Septin4 mRNA表达及Septin4、αSMA、SM22α、p62、p-mTOR/mTOR蛋白表达明显降低(P<0.05),MMP2、MMP9、LC3Ⅱ/LC3Ⅰ和p-AMPK/AMPK蛋白表达明显升高(P<0.05)。结论:Septin4可能通过调节AMPK/mTOR信号通路抑制自噬,促进HBVSMCs向收缩型表型转化,在颅内动脉瘤中发挥保护作用。

Objective:To explore the expression of Septin4 in intracranial aneurysms and its effect on human brain vascular smooth muscle cells(HBVSMCs)and its possible mechanism.Methods:32 samples of intracranial aneurysm tissue and normal cranial artery tissue were collected,and the expression of Septin4 in the tissue was detected by immunohistochemistry(IHC),reverse transcription-polymerase chain reaction(RT-PCR)and Western blot;immunofluorescence staining were used to detect smooth muscle 22α(SM22α)and smooth muscleαactin(ΑSMA)expression.Septin4 overexpression vector(Septin4),interference sequence(si-Septin4)and negative control(NC and si-NC)were transfected into HBVSMCs,respectively.RT-PCR and Western blot experiments were used to detect the expression of Septin4 in cells;Western blot was used to detect the expression ofαSMA,SM22α,matrix metalloproteinase 2(MMP2),matrix metalloproteinase 9(MMP9),Microtubule-associated protein 1 light chain 3(LC3),sequestosome 1(p62),Phosphorylated adenylate activated protein kinase(p-AMPK),adenosine monophosphate activated protein kinase(AMPK),Phosphorylated mammalian target of rapamycin(p-mTOR)and mammalian target of rapamycin(mTOR)protein.Results:Compared with the normal cranial artery tissue,the number of Septin4 positive cells,mRNA expression,protein expression and the fluorescence intensity of SM22αandαSMA in the intracranial aneurysm tissue were significantly reduced(P<0.05).Compared with the NC group,Septin4 mRNA expression,Septin4,αSMA,SM22α,p62 and p-mTOR/mTOR protein expression were significantly increased in Septin4 cells(P<0.05),MMP2,MMP9,LC3Ⅱ/LC3Ⅰand p-AMPK/AMPK protein expression was significantly reduced(P<0.05);compared with si-NC group,Septin4 mRNA expression,Septin4,αSMA,SM22α,p62 and p-mTOR/mTOR protein expression were significantly reduced in si-Septin4 group cells(P<0.05),MMP2,MMP9,LC3Ⅱ/LC3Ⅰand p-AMPK/AMPK protein expression were significantly increased(P<0.05).Conclusion:Septin4 may inhibit autophagy by regulating AMPK/mTOR signaling pathway,promote the transformation of HBVSMCs to contractile phenotype,and play a protective role in intracranial aneurysms.