摘要
目的:观察抗衰老Klotho蛋白对大鼠乳鼠原代心肌细胞缺氧/复氧(H/R)损伤的作用并探讨其作用机制。方法:建立大鼠乳鼠心肌细胞H/R模型,并将心肌细胞分为正常对照组、H/R模型组和不同浓度(0.1μmol/L、1μmol/L和10μmol/L)Klotho作用H/R组。观察各组心肌细胞H/R前后搏动频率变化,利用MTT方法检测细胞存活率;测定各组心肌细胞H/R后LDH、CK、AST的漏出量及MDA含量、SOD活性;流式细胞术检测各组心肌细胞的凋亡率;real-time PCR检测各组心肌细胞中内质网应激标记及凋亡相关分子GRP78、CRT和CHOP和caspase-12 mRNA的表达情况;Western blot法检测心肌细胞内内质网应激凋亡蛋白CHOP和caspase-12蛋白表达及Akt磷酸化水平。结果:与正常对照组相比,H/R模型组中心肌细胞搏动频率和细胞存活率显著下降,细胞凋亡率显著升高(P<0.05);LDH、CK、AST和MDA含量升高而SOD活性显著降低(P<0.05);GRP78、CRT、CHOP和caspase-12 mRNA表达显著增高(P<0.05);CHOP和caspase-12蛋白表达随之增高而Akt的磷酸化水平显著降低(P<0.05)。与H/R模型组相比,抗衰老Klotho蛋白作用H/R心肌细胞后,心肌细胞搏动频率和细胞存活率显著升高,细胞凋亡率逐渐降低(P<0.05),LDH、CK、AST和MDA含量下降而SOD活性显著增高(P<0.05),GRP78、CRT、CHOP和caspase-12 mRNA的表达逐渐降低(P<0.05),CHOP和caspase-12蛋白表达也随之降低,而Akt磷酸化水平显著增加(P<0.05)。结论:抗衰老Klotho蛋白能够提升H/R损伤后心肌细胞的存活率,抑制细胞凋亡,通过抵抗氧化应激和过度内质网应激反应发挥作用,并与激活Akt磷酸化有关。
AIM：To study the effects of anti-aging Klotho protein on neonatal rat myocardial cells with hypo-xia/reoxygenation （ H/R） injury.METHODS：The cardiomyocytes of neonatal SD rats were cultured to establish hypoxia/reoxygenation model.The myocardial cells were divided into normal control group, H/R model group, different concentra-tions of Klotho protein （0.1μmol/L, 1μmol/L and 10μmol/L） pretreatment groups.The myocardial cells pulse frequen-cy was observed before and after H/R.The cell viability was measured by MTT assay.The leakages of LDH, CK and AST, the content of MDA and the activity of SOD were detected.The apoptotic rate of the myocardial cells was analyzed by flow cytometry.The mRNA expression of endoplasmic reticulum stress markers and apoptosis-related molecules GRP78, CRT, CHOP and caspase-12 was measured by real-time PCR.The protein levels of CHOP, caspase-12 and phosphorylated Akt in the myocardial cells were determined by Western blot.RESULTS： Compared with normal control group, the pulse fre-quency, cell viability rate and SOD activity of myocardial cells were significantly decreased, the cell apoptotic rate as well as the contents of LDH, CK, AST and MDA were increased in H/R model group.The mRNA expressions of GRP78, CRT, CHOP and caspase-12 as well as the protein levels of CHOP and caspase-12 were increased, whereas p-Akt level was decreased obviously.Compared with H/R model group, the pulse frequency, cell viability rate and SOD activity were in-creased significantly, the cell apoptotic rate as well as the contents of LDH, CK, AST and MDA were decreased in Klotho pretreated group.The mRNA expression of GRP78, CRT, CHOP and caspase-12 as well as the protein levels of CHOP and 〈br〉 caspase-12 were decreased, while p-Akt level increased significantly.CONCLUSION：Anti-aging Klotho protein improves the myocardial cell survival and inhibits the apoptosis by increasing the resistance of the cells to oxidative stress and exces-sive endoplasmic reticulum stress response, which is related with the activation of Akt phosphorylation in H/R-injured my-cardial cells.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2015年第6期980-987,共8页
Chinese Journal of Pathophysiology
基金
湖北省教育厅自然科学研究计划重点项目(No.D20091308)
作者简介
通讯作者Tel：0717-555730；E-mail：zhoujingqun-1@medmail．com．cn
