摘要
目的探讨热休克蛋白22(heat shock protein 22, Hsp22)对苯肾上腺素(phenylephrine, PE)诱导的心肌细胞肥大反应的作用。方法在郑州大学附属第一医院心血管实验室分离培养原代大鼠心肌细胞,将培养的心肌细胞随机分为对照组、刺激组、1 μg/mL Hsp22的处理组、10 μg/mL Hsp22的处理组;采用PE刺激诱导心肌细胞肥大反应,用不同浓度的人重组Hsp22孵育心肌细胞,对每组细胞通过MTT法检测细胞存活率;通过α-肌动蛋白免疫荧光检测心肌细胞面积;采用DCFH-DA荧光探针检测活性氧的水平;TUNEL染色检测细胞凋亡数量;应用反转录-聚合酶链反应(RT-PCR)检测肥厚标志物的转录;免疫印迹检测信号通路的改变。使用SPSS 13.0进行统计分析,计量资料以均数±标准差(Mean±SD)表示,所有数据均使用单因素方差分析检测各组间的差异,组间两两比较采用LSD-t检验,以P<0.05为差异有统计学意义。结果不同浓度的Hsp22对心肌细胞活性没有影响(F=6.622,P>0.05);PE刺激明显增加心肌细胞面积(t=16.01,P<0.05),增加心肌肥厚标志物心房利钠肽(t=44.84,P<0.05),B型利钠肽(BNP)(t=16.85,P<0.05),肌球蛋白重链β(β-MHC)(t=41.53,P<0.05)的转录水平,增加心肌细胞氧化应激水平(t=43.61,P<0.05),增加心肌细胞凋亡数量(t=60.82,P<0.05)。不同浓度的Hsp22处理明显减小心肌细胞面积[PE+1 μg/mL Hsp22组:(3 872±212)μm2,t=4.018,P<0.05;PE+10 μg/mL Hsp22组:(2 563±287)μm2,t=10.80,P<0.05];减少肥厚标志物ANP、BNP、β-MHC的转录水平(均P<0.05),减少心肌细胞氧化应激水平(均P<0.05);减少心肌细胞凋亡数量[PE+1 μg/mL Hsp22组:(21.7±1.2)%,t=20.02,P<0.05;PE+10 μg/mL Hsp22组:(19.8±1.6)%,t=24.70,P<0.05]。免疫印迹结果显示,PE刺激组AMPKα的磷酸化减少(t=7.75,P<0.05),mTOR的磷酸化增加(t=25.22,P<0.05);不同浓度的Hsp22增加AMPKα的磷酸化(均P<0.05),减少mTOR的磷酸化(均P<0.05)。结论Hsp22通过激活AMPKα抑制心肌细胞肥大反应,H
Objective To investigate the effect of Hsp22 on phenylephrine-induced cardiomyocytes hypertrophy. Methods Primary rat myocardial cells were isolated and cultured in Department of Cardiology, the First Affiliated Hospital of Zhengzhou University. Cells were divided into four groups randomly: Control group, model group, treatment group with 1 μg/mL Hsp22, and treatment group with 10 μg/mL Hsp22. Phenylephrine stimuli was used to induce cardiomyocytes hypertrophy model. Cell viability was measured by 3-(4,5-Dimcthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cardiomyocytes surface area was evaluated by α-actin immunofluorescence staining. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the transcription level of hypertrophic markers. Reactive oxygen species level was detected by 2',7'-Dichlorodihydronuoresccin diacetate (DCFH-DA) fluorescent probe. Apoptosis was detected by TUNEL staining. Signal pathway protein expression was detected by Western blot. SPSS 13.0 was used for statistical analysis. Data were expressed as mean ± standard deviation. All data were analyzed by one-way ANOVA between groups. Comparisons between two groups were perfonned using LSD-r test. A P<0.05 was considered statistically significant. Results Different concentrations of Hsp22 had no effect on cardiomyocytes viability (F=6.622;P>0.05). Phenylephrine stimulation significantly increased cardiomyocytes area (t=10.80;P<0.05), increased the transcription level of hypertrophy markers atrial natriuretic peptide (t=37.72;P<0.05), type B natriuretic peptide (t=16.85;P<0.05), and myosin heavy chain beta (r=41.53;P<0.05). Different concentrations of Hsp22 significantly reduced cardiomyocytes area (PE+1 μg/mL Hsp22 t=4.018: P<0.05;PE+10 μg/mL Hsp22 t=10.80;P<0.05), reduced the transcription level of hypertrophic markers atrial natriuretic peptide (PE+1 μg/mL Hsp22 t=27.12, P<0.05;PE+10μg/mL Hsp22 t=37.72, P<0.05), type B natriuretic peptide (PE+1 μg/mL Hsp22 t=4.82, P<0.05;PE+10 μg/mL Hsp22 /=12.74
作者
肖莉丽
谷玉雷
高路
陈君
王小芳
李凌
Xiao Lili;Gu Yulei;Gao Li;Chen Jun;Wang Xiaofang;Li Ling(Department of Cardiology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052,China;Department of Emergency, the First Affiliated Hospital of Zhengzhou University, Zhengzhou450052, China)
出处
《中华急诊医学杂志》
CAS
CSCD
北大核心
2019年第3期344-349,共6页
Chinese Journal of Emergency Medicine
基金
河南省医学科技攻关项目( 201702063).
关键词
热休克蛋白22
苯肾上腺素
心肌细胞肥大
氧化应激
AMPKα
Heat shock protein 22
Phenylephrine
Cardiomyocytes hypertrophy
Oxidative stress
AMP-activated protein kinaseα
