摘要
目的探讨Ras同源基因家族成员A(RhoA)/Rho相关卷曲螺旋蛋白激酶(ROCK)通路在缺氧复氧(H/R)诱导的人心肌AC16细胞损伤中的作用及其机制。方法将体外培养的AC16细胞分为对照组(正常培养)、H/R组(构建H/R模型)、H/R+NC-siRNA组(转染NC-siRNA后构建H/R模型)和H/R+RhoA-siRNA组(转染RhoA-siRNA后构建H/R模型),采用MTT法检测AC16细胞存活率,流式细胞术检测AC16细胞凋亡率,比色法检测AC16细胞乳酸脱氢酶(LDH)活性和Caspase-3活性,ELISA检测AC16细胞上清液中白细胞介素6(IL-6)、白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)水平,试剂盒检测AC16细胞超氧化物歧化酶(SOD)和丙二醛(MDA)水平,实时荧光定量PCR检测AC16细胞中RhoA、ROCK1、ROCK2、核因子κB(NF-κB)p65及IkB激酶(IKK)的mRNA表达水平,Western blot检测AC16细胞中RhoA、ROCK1、ROCK2、NF-κB p65、磷酸化NF-κB(p-NF-κB)p65及IKK的蛋白表达水平。结果与对照组比较,H/R组细胞存活率、SOD水平显著降低,细胞凋亡率、LDH活性、Caspase-3活性、MDA水平和细胞上清液中IL-6、IL-1β、TNF-α水平以及细胞中RhoA、ROCK1、ROCK2、NF-κB p65、p-NF-κB p65、IKK mRNA与蛋白表达水平均显著升高(P<0.05);H/R+NC-siRNA组和H/R组之间上述各指标差异均无统计学意义(P>0.05);与H/R+NC-siRNA组比较,H/R+RhoA-siRNA组细胞存活率、SOD水平显著升高,细胞凋亡率、LDH活性、Caspase-3活性、MDA水平和细胞上清液中IL-6、IL-1β、TNF-α水平以及细胞中RhoA、ROCK1、ROCK2、NF-κB p65、p-NF-κB p65、IKK mRNA与蛋白表达水平均显著降低(P<0.05)。结论靶向抑制RhoA/ROCK通路可通过降低炎症反应、氧化应激和细胞凋亡减轻H/R诱导的心肌细胞损伤。
Aim To investigate the role and mechanism of Ras homolog gene family member A(RhoA)/Rho-associated coiled-coil forming protein kinase(ROCK)pathway in hypoxia/reoxygenation(H/R)-induced injury in human cardiac AC16 cells.Methods AC16 cells cultured in vitro were divided into control group(normal culture),H/R group(construction of H/R model),H/R+NC-siRNA group(H/R model was constructed after transfection of NC-siRNA)and H/R+RhoA-siRNA group(H/R model was established after transfection of RhoA-siRNA),MTT assay was used to detect the survival rate of AC16 cells,the apoptosis rate of AC16 cells was detected by flow cytometry,the activities of lactate dehydrogenase(LDH)and Caspase-3 in AC16 cells were detected by colorimetry,the levels of interleukin-6(IL-6),interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in the supernatant of AC16 cells were detected by ELISA,the levels of superoxide dismutase(SOD)and malondialdehyde(MDA)in AC16 cells were detected by kit,the mRNA expression levels of RhoA,ROCK1,ROCK2,nuclear factor-κB(NF-κB)p65 and IKB kinase(IKK)in AC16 cells were detected by real-time fluorescence quantitative PCR,the protein expression levels of RhoA,ROCK1,ROCK2,NF-κB p65,phosphorylated NF-κB(p-NF-κB)p65 and IKK in AC16 cells were detected by Western blot.Results Com-pared with those in the control group,the cell survival rate and SOD level in H/R group were significantly lower,apoptosis rate,LDH activity,Caspase-3 activity,MDA level,IL-6,IL-1βand TNF-αlevels in cell supernatant,RhoA,ROCK1,ROCK2,NF-κB p65,p-NF-κB p65,IKK mRNA and protein expression levels were significantly higher(P<0.05).There was no significant difference in the above indexes between H/R+NC-siRNA group and H/R group(P>0.05).Compared with those in H/R+NC-siRNA group,the cell survival rate and SOD level in H/R+RhoA-siRNA group were sig-nificantly higher,apoptosis rate,LDH activity,Caspase-3 activity,MDA level,IL-6,IL-1βand TNF-αlevels in cell su-pernatant,RhoA,ROCK1,ROCK2,NF-κB p65,p-NF-κB p65,IKK mRNA and protein expressio
作者
朱振侠
杨晓霞
冯斐斐
ZHU Zhenxia;YANG Xiaoxia;FENG Feifei(Department of Internal Medicine,Daping Coal Mine Hospital of Zhengzhou Coul Industry(Group)Co.,Ltd.,Zhengzhou,Henan 450000,China)
出处
《中国动脉硬化杂志》
CAS
2021年第12期1021-1027,共7页
Chinese Journal of Arteriosclerosis
基金
河南省科技攻关项目(182102310166)。
