摘要
目的探究抑制S-腺苷同型半胱氨酸水解酶(SAHH)活性,升高S-腺苷同型半胱氨酸(SAH)水平,对大鼠主动脉血管平滑肌细胞(RAVSMCs)衰老的影响。方法培养RAVSMCs细胞,随机分为Scrambled组、SAHH siRNA组、Control组、腺苷二醛(ADA)组,Scrambled组转染对照RNA(5 nmol/L),SAHH siRNA组转染SAHH siRNA(5 nmol/L),Control组在平滑肌细胞培养基内加入二甲基亚砜(DMSO),ADA组在平滑肌细胞培养基内加入SAHH抑制剂ADA(30μmol/L),干预时间24 h。采用质谱检测RAVSMCs内SAH水平,之后进行相关衰老指标的检测,使用衰老相关β-半乳糖苷酶(SA-β-gal)染色方法检测细胞衰老的情况;通过Western blot检测细胞中p16和p21的蛋白表达水平;采用EdU-488方法检测细胞的增殖能力。结果与Scrambled组比较,SAHH siRNA组SAH水平、SA-β-gal阳性率、p16蛋白、p21蛋白表达水平显著升高,细胞增殖能力显著下降,差异均有统计学意义(t=7.604、16.750、2.906、5.735、4.236,P均<0.05);与Control组比较,ADA组SAH水平、SA-β-gal阳性率、p16蛋白、p21蛋白表达水平显著升高,细胞增殖能力显著下降,差异均有统计学意义(t=27.910、34.080、3.359、4.769、16.670,P均<0.05)。结论抑制SAHH活性,升高SAH水平会诱导RAVSMCs衰老。
Objective To explore the effects of inhibiting S-adenosyl-homocysteine hydrolase(SAHH)activity and increasing the levels of S-adenosyl-homocysteine(SAH)on the rat aortic vascular smooth muscle cells(RAVSMCs)senescence.Methods RAVSMCs were cultured and randomly divided into Scrambled group,SAHH siRNA group,Control group and adenosine dialdehyde(ADA)group.The Scrambled group was transfected with control RNA(5 nmol/L),the SAHH siRNA group was transfected with SAHH siRNA(5 nmol/L),and the Control group was treated with dimethyl sulfoxide(DMSO)was added to the smooth muscle cell medium,and the SAHH inhibitor ADA(30μmol/L)was added to the smooth muscle cell medium in the ADA group for 24 h.Mass spectrometry was used to detect SAH levels in RAVSMCs,and the relevant aging indicators were detected;cell senescence was detected by senescence associated beta galactosidase(SA-β-gal)staining;the protein expression levels of p16 and p21 in cells were detected by Western blot;cell proliferation was detected by EdU-488 methods.Results Compared with the Scrambled group,the SAH level,SA-β-gal positive rate,p16 protein and p21 protein expression levels in the SAHH siRNA group were significantly increased,and the cell proliferation ability was significantly decreased,and the differences were statistically significant(t=7.604,16.750,2.906,5.735,4.236,all P<0.05);compared with the Control group,the SAH level,SA-β-gal positive rate,p16 protein and p21protein expression levels in the ADA group and the SAHH siRNA group were significantly increased,and the cell proliferation ability was significantly decreased,and the differences were statistically significant(t=27.910,34.080,3.359,4.769,16.670,all P<0.05).Conclusion Inhibition of SAHH activity could promote senescence in RAVSMCs.
作者
米佳欣
凌文华
MI Jia⁃xin;LING Wen⁃hua(School of Public Health,Sun Yat⁃sen University,Guangzhou,Guangdong 510080,China)
出处
《热带医学杂志》
CAS
2022年第6期741-745,894,共6页
Journal of Tropical Medicine
基金
国家自然科学基金(81973022)
