PERK-ATF4-CHOP信号通路在褪黑素抵抗血管紧张素Ⅱ诱导乳鼠心肌细胞肥厚中的作用

Role of PERK-ATF4-CHOP pathway in melatonin attenuates cardiomyocyte hypertrophy induced by angiotensin Ⅱ

目的初步探讨内质网应激PERK-ATF4-CHOP通路在褪黑素抵抗血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导的心肌细胞肥厚中的作用。方法将培养的心肌细胞随机分为对照组、AngⅡ处理组、褪黑素组。采用乳酸脱氢酶(lactate dehydrogenase,LDH)法检测细胞活性,原位切口末端标记法(TUNEL)检测细胞凋亡率,Western blot检测蛋白激酶R样内质网激酶(protein kinase r-like endoplasmic reticulum kinase,PERK)、活性转录因子(Activating Transcription Factor,ATF)4、C/EBP同源蛋白(C/EBP homologous protein,CHOP)的表达水平,实时荧光定量聚合酶链反应(quantitative real-time polymerase chain reaction,qRT-PCR)检测心肌肥厚相关标识物心房钠尿肽(atrial natriuretic peptide,ANP)、脑钠尿肽(brain natriuretic peptide,BNP)和心肌β-肌球蛋白重链(β-myosin heavy chain,β-MHC)mRNA表达量的变化;免疫荧光检测心肌细胞横截面积。结果与对照组相比,AngⅡ能够刺激心肌细胞上调ANP、BNP和β-MHC表达(P<0.05),刺激LDH释放(P<0.05)、增加细胞凋亡比率和心肌细胞横截面积(P<0.05);与AngⅡ组相比,褪黑素可以浓度依赖性显著抑制AngⅡ诱导的ANP、BNP和β-MHC的上调和LDH的积累,抑制心肌细胞的横截面积增加,降低细胞凋亡,并抑制诱导的PERK-ATF4-CHOP通路的激活(P<0.05);与对照组相比,AngⅡ显著增加PERK、ATF4和CHOP的表达水平(P<0.05),但给予褪黑素后,激活的PERK-ATF4-CHOP则被明显抑制(P<0.05)。结论褪黑素可能通过抑制内质网应激PERK-ATF4-CHOP信号通路激活,改善AngⅡ诱导的乳鼠心肌细胞肥厚,为未来褪黑素的进一步临床研究拓展新思路。

AIM To explore the role of the PERK-ATF4-CHOP pathway in melatonin attenuated cardiomyocyte hypertrophy induced by angiotensin II （AngII）. METHODS Cells were divided randomly into three groups： the blank control group, the AngII-stimulated group, the melatonin group. Lactate dehydrogenase （LDH） release was used to test cell viability. TUNEL staining was used to detect the apoptosis. Western blot method was used to detect the expression of PERK, ATF4 and CHOP. Real time-PCR was used to detect gene expression of ANP, BNP and β-MHC. Immunostaining was used to observe the cell morphology by α-actinin. RESULTS Compared with those in the control group, AngII pretreatment significantly augmented the expression of ANP, BNP and β-MHC, up-regulated LDH activity and apoptosis （P〈0.05）, and increased cell cross-sectional area of myocardial cells （P〈0.05）. Melatonin treatment resulted in reduction of expression of ANP, BNP and β-MHC （P〈0.05）, down-regulation of cardiomyocyte LDH activity and apoptosis （P〈0.05）, and the inhibition of cardiomyocyte hypertrophy （P〈0.05）. Moreover, Western blot analysis revealed that AngII exhibited a marked augmentation of the protein of ERK, ATF4 and CHOP （P〈0.05）, whereas melatonin suppressed the activation of ERK, ATF4 and CHOP stimulated by AngII （P〈0.05）. CONCLUSION SThe present study suggests that melatonin attenuates cardiomyocyte hypertrophy induced by AngII through the endoplasmic reticulum stress PERK-ATF4-CHOP signaling pathway. These results contribute to providing new therapeutic targets for future clinical treatment with melatonin.