摘要
目的:探讨氢分子对血管平滑肌细胞钙化的缓解作用及其机制。方法:将大鼠胸主动脉平滑肌细胞株A7r5分为对照组、钙化组和氢干预钙化组。对照组细胞正常培养,钙化组细胞在钙化培养液(10 mmol/Lβ-磷酸甘油+1.5 mmol/L CaCl_(2))中培养,氢干预钙化组细胞在钙化培养液中并置于氢培养箱培养。诱导12 d后,茜素红染色检测钙盐沉积;β-半乳糖苷酶染色检测细胞衰老;二氢乙啶荧光探针检测细胞内活性氧(reactive oxygen species,ROS)水平;Western blot法检测α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、Runt相关转录因子2(Runt-related transcription factor 2,RUNX2)、平滑肌蛋白22α(smooth muscle protein-22α,SM-22α)、p21、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、磷酸化mTOR(phosphorylated mTOR,p-mTOR)、AMP活化蛋白激酶(AMP-activated protein kinase,AMPK)和磷酸化AMPK(phosphorylated AMPK,p-AMPK)的蛋白水平。结果:与对照组相比,钙化组A7r5细胞钙盐沉积和衰老明显,细胞内ROS水平显著升高(P<0.05),α-SMA、SM-22α和pAMPK蛋白水平显著降低(P<0.05),RUNX2、p21和p-mTOR蛋白水平显著升高(P<0.05);与钙化组相比,氢干预钙化组细胞钙盐沉积和衰老得到明显缓解,ROS水平显著降低(P<0.05),α-SMA、SM-22α和p-AMPK蛋白水平显著升高(P<0.05),RUNX2、p21和p-mTOR蛋白水平显著降低(P<0.05)。结论:氢分子可通过激活AMPK/mTOR信号通路,抑制氧化应激,抵抗细胞衰老,从而减轻高磷诱导的血管平滑肌细胞钙化。
AIM:To investigate the mitigating effect of hydrogen molecules on the calcification of vascular smooth muscle cells(VSMCs)and its mechanism.METHODS:Rat thoracic aortic smooth muscle cell line A7r5 was divided into control group,calcification group and hydrogen intervention+calcification group.The VSMCs were cultured in normal medium(control group)or calcification medium(calcification group).For the hydrogen intervention+calcification group,the cells cultured in calcification medium were put in a hydrogen incubator.After 12 d of incubation,Alizarin red staining was used to detect calcium salt deposition.β-Galactosidase staining was used to detect cellular senescence.Dihydroethidium fluorescent probe was used to detect intracellular reactive oxygen species(ROS)level.Western blot was used to detect the protein levels ofα-smooth muscle actin(α-SMA),Runt-related transcription factor 2(RUNX2),smooth muscle protein-22α(SM-22α),p21,mammalian target of rapamycin(mTOR),phosphorylated mTOR(p-mTOR),AMPactivated protein kinase(AMPK)and phosphorylated AMPK(p-AMPK).RESULTS:Compared with control group,the calcium salt deposition and cellular aging were observed in calcification group,and the ROS level was significantly in‐creased(P<0.05).The protein levels ofα-SMA,SM-22αand p-AMPK were significantly decreased(P<0.05).The pro‐tein levels of RUNX2,p21 and p-mTOR were significantly increased(P<0.05).Compared with calcification group,the calcium salt deposition and cellular aging were significantly alleviated after hydrogen intervention.The ROS level was sig‐nificantly reduced(P<0.05),the protein levels ofα-SMA,SM-22αand p-AMPK were significantly increased(P<0.05),and the protein levels of RUNX2,p21 and p-mTOR werer significantly decreased(P<0.05).CONCLUSION:Hydrogen molecules can inhibit oxidative stress and resist cellular aging by activating AMPK/mTOR signaling pathway,thereby attenuating high phosphorus-induced calcification of VSMCs.
作者
杨超
薛俊莉
陶鸽如
许昕鑫
宋国华
YANG Chao;XUE Junli;TAO Geru;XU Xinxin;SONG Guohua(School of Clinical and Basic Medicine,Shandong First Medical University&Shandong Academy of Medical Science,Taian 271000,China;The Second Affiliated Hospital,Shandong First Medical University&Shandong Academy of Medi-cal Science,Taian 271000,China.)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2023年第6期982-987,共6页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81873517,No.81670422)
山东省青年泰山学者项目(No.tsqn20161045)。
