摘要
目的探讨盐酸右美托咪对高糖诱导的心肌线粒体损伤与细胞凋亡的影响及作用机制。方法用葡萄糖浓度为33 mmol/L的培养基培养H9C2细胞作为高糖组,正常培养基培养的细胞作为正常对照组;用1μmol/L的盐酸右美托咪定预处理H9C2细胞1 h后用葡萄糖浓度为33 mmol/L的培养基培养作为盐酸右美托咪定+高糖组;将con小干扰RNA(si-con)、HIF-1α小干扰RNA(si-HIF-1α)质粒分别转染至H9C2细胞中用1μmol/L的盐酸右美托咪定预处理H9C2细胞1 h后用葡萄糖浓度为33 mmol/L的培养基培养作为盐酸右美托咪定+si-con+高糖组、盐酸右美托咪定+si-HIF-1α+高糖组。四甲基偶氮唑盐比色法(MTT)检测细胞活力;乳酸脱氢酶(LDH)试剂盒检测LDH活性;蛋白质印迹(Western Blot)法检测裂解半胱氨酸天冬氨酸蛋白酶-3(Cleaved caspase-3)、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、细胞色素c(Cyt-C)、缺氧诱导因子-1α(HIF-1α)蛋白表达水平;流式细胞术检测细胞凋亡;JC-1荧光标记法检测线粒体膜电位;2’,7’-二氯荧光黄双乙酸盐(DCFH-DA)荧光探针法检测活性氧(ROS)荧光强度;实时荧光定量PCR(RT-q PCR)检测细胞HIF-1αmRNA水平。结果与正常对照组相比,高糖组心肌细胞活力显著降低,LDH活性显著升高,Cleaved caspase-3、Bax表达水平显著升高,Bcl-2表达水平显著降低,细胞凋亡率显著升高,线粒体膜电位降低的细胞比率显著升高,Cyt-C蛋白表达水平显著升高,ROS荧光强度显著升高,HIF-1αmRNA和蛋白表达显著降低(P <0. 05)。与高糖组相比,盐酸右美托咪定+高糖组心肌细胞活力显著升高,LDH活性显著降低,Cleaved caspase-3、Bax表达水平显著降低,Bcl-2表达水平显著升高,细胞凋亡率显著降低,线粒体膜电位降低的细胞比率显著降低,Cyt-C蛋白表达水平显著降低,ROS荧光强度显著降低,HIF-1αmRNA和蛋白表达显著升高(P <0. 05)。与盐酸右美托咪定+si-con+高糖组相比,盐酸右美托咪定+si-HIF-1α+高糖组可逆转上述盐酸右美托咪定对高糖诱导的心肌细胞线粒体损伤与细胞凋亡的抑制作用。结论盐酸右美托咪定可促进心肌细胞存活,抑制LDH的漏出和ROS的产生,抑制细胞凋亡,稳定线粒体膜电位,减轻高糖对线粒体和细胞凋亡的影响,其机制可能与HIF-1α有关。
Objective To investigate the effect and mechanism of dexmedetomidine hydrochloride on high glucose-induced myocardial mitochondrial damage and apoptosis. Methods H9 C2 cells cultured in a medium with glucose concentration of 33 mmol/L were used as the high glucose group,cells cultured in normal medium as the normal control group. H9 C2 cells pretreated with 1 μmol/L dexmedetomidine hydrochloride for 1 h,and then cultured in a medium with glucose concentration of 33 mmol/L were used as dexmedetomidine hydrochloride + high glucose group;H9 C2 cells transfected by the plasmids of si-con and si-HIF-1α respectively,and pretreated with 1 μmol/L dexmedetomidine hydrochloride for 1 h,and then cultured in a medium with a glucose concentration of 33 mmol/L were used as dexmedetomidine hydrochloride + si-con + high glucose group,dexmedetomidine hydrochloride + si-HIF-1α + high glucose group. Cell viability was measured by methyl thiazolyl tetrazolium( MTT) method;LDH activity was detected by lactate dehydrogenase( LDH) kit;the expression of cleaved cysteine-containing aspartate-specific proteases-3( Cleaved caspase-3),B cell lymphoma/leukemia-2( Bcl-2),Bcl-2 associated X protein( Bax),cytochrome c( Cyt-C),hypoxia-inducible factor-1 alpha( HIF-1α) protein were detected by Western Blot;apoptosis was detected by flow cytometry;mitochondrial membrane potential was detected by JC-1 fluorescent labeling method;2’,7’-dichlorodihydrofluorescein diacetate( DCFH-DA)fluorescence probe was used to detect the fluorescence intensity of reactive oxygen species( ROS);real-time quantitative PCR( RT-qPCR) was used to detect the level of HIF-1α mRNA. Results Compared with normal control group,the myocardial cell viability of the high glucose group was significantly decreased,the LDH activity was significantly increased,the expressions of Cleaved caspase-3 and Bax were significantly increased,the expression of Bcl-2 was significantly decreased,and the apoptosis rate was significantly increased,the proportion of cells with decreased mitochondrial membrane potential was significantly increased,the expression of Cyt-C protein was significantly increased,the fluorescence intensity of ROS was significantly increased,and the expression of HIF-1α mRNA and protein was significantly decreased( P < 0. 05).Compared with high glucose group,the myocardial cell viability of dexmedetomidine hydrochloride + high glucose group was significantly increased,LDH activity was significantly decreased,Cleaved caspase-3 and Bax expressions were significantly decreased,and Bcl-2 expression was significantly increased,the apoptosis rate was significantly decreased,the proportion of cells with decreased mitochondrial membrane potential was significantly decreased,the expression of Cyt-C protein was significantly decreased,the fluorescence intensity of ROS was significantly decreased,and the expression of HIF-1α mRNA and protein was significantly increased( P < 0. 05). Compared with dexmedetomidine hydrochloride + si-con+ high sugar group,dexmedetomidine hydrochloride + si-HIF-1α + high sugar group can reverse the above-mentioned the inhibitory effect of dexmedetomidine hydrochloride on high glucose-induced mitochondrial damage and apoptosis in cardiomyocytes. Conclusion Dexmedetomidine hydrochloride can promote myocardial cell survival,inhibit LDH leakage and ROS production,inhibit cell apoptosis,stabilize mitochondrial membrane potential,and alleviate the effects of high glucose on mitochondrial damage and apoptosis. The mechanism may be related to HIF-1α.
作者
邹田田
杨龙
陈春玲
ZOU Tian-tian;YANG Long;CHEN Chun-ling(Department of Anesthesiology,The First Affiliated Hospital of Xinjiang Medical University,Urumqi Xinjiang 830054,China)
出处
《临床和实验医学杂志》
2020年第9期902-906,共5页
Journal of Clinical and Experimental Medicine
基金
国家自然科学基金联合基金项目(编号:U1603129)。
