藏红花素对急性心肌梗死大鼠心肌组织损伤和细胞凋亡的影响及机制

Effects and mechanism of crocin on myocardial tissue injury and apoptosis in rats with acute myocardial infarction

目的探索藏红花素(crocin)对急性心肌梗死大鼠心肌组织损伤和细胞凋亡的影响及作用机制。方法40只SD大鼠随机分为假手术(Sham)组、急性心肌梗死(AMI)组、AMI+crocin组、右美托咪定(Dex)组,每组各10只。三苯基氯化四氮唑(TTC)染色计算各组大鼠心肌梗死面积;超声心动图获得胸骨旁长轴切面M型记录,计算左室射血分数(LVEF)和左室缩短分数(LVFS);TUNEL染色检测心肌组织凋亡;酶联免疫吸附法(ELISA)检测各组大鼠血清中乳酸脱氢酶(LDH)、肌酸激酶(CK)和Caspase-3活性;实时荧光定量PCR(qRT-PCR)检测组织miR-146a-5p表达。设置H9c2细胞为对照组(control)、缺氧组(hypoxia)、hypoxia+crocin组、hypoxia+crocin+inhibitor组和Dex组,qRT-PCR检测细胞miR-146a-5p表达;流式细胞术和TUNEL染色检测细胞凋亡;蛋白印迹法(WB)检测Cleaved caspase-3表达;试剂盒检测大鼠细胞上清液CK和LDH活性。结果与Sham组比较,AMI组大鼠心室壁薄,心室舒缩行为异常,心肌梗死面积明显增大,LVEF、LVFS和相对miR-146a-5p表达明显下降,细胞凋亡率、CK、LDH和Caspase-3水平明显升高(P<0.05);与AMI组相比,AMI+crocin组和Dex组大鼠心肌组织增厚,心肌舒缩较正常,心肌梗死面积明显减小,LVEF、LVFS和相对miR-146a-5p表达均明显上升,细胞凋亡率、CK、LDH和Caspase-3水平明显下降(P<0.05)。与control组相比,hypoxia组细胞相对miR-146a-5p表达明显降低,细胞凋亡率、Cleaved caspase-3表达、LDH和CK水平明显上升(P<0.05);与hypoxia组相比,hypoxia+crocin组和Dex组细胞miR-146a-5p相对表达水平明显上升,细胞凋亡率、Cleaved caspase-3表达、LDH和CK水平明显下降(P<0.05)。与hypoxia+crocin组细胞相比,hypoxia+crocin+inhibitor组上述氧化应激指标明显上升(P<0.05)。结论藏红花素可能通过上调miR-146a-5p减轻急性心肌梗死大鼠的心肌组织损伤和细胞凋亡。

Objective To explore effects and mechanism of crocin on myocardial tissue injury and apoptosis in rats with acute myocardial infarction. Method A total of 40 SD rats were divided into sham operation(Sham) group,acute myocardial infarction(AMI) group, AMI+crocin group and Dexmedetomidine(Dex) group, 10 rats in each group. The area of myocardial infarction was calculated by triphenyl tetrazolium chloride(TTC) staining. The Mtype records of parasternal long-axis cross-section were obtained by echocardiography to calculate left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS). The apoptosis of myocardial tissues was detected by TUNEL staining. The activities of serum lactate dehydrogenase(LDH), creatine kinase(CK) and Caspase-3 were detected by enzyme linked immunosorbent assay. The expression of tissue miR-146a-5p was detected by fluorescent quantitative real-time PCR(qRT-PCR). H9c2 cells were divided into control group(control), hypoxia group(hypoxia), hypoxia+crocin group, hypoxia+crocin+inhibitor group and Dex group. The expression of miR-146a-5p in cells was detected by qRT-PCR. The apoptosis was detected by flow cytometry and TUNEL staining. The expression of Cleaved caspase-3 was detected by Western blot(WB). The activities of CK and LDH in cells supernatant were detected by kits. Results Compared with Sham group, ventricular walls were thin in AMI group, ventricular diastolic and systolic behaviors were abnormal, area of myocardial infarction was significantly increased, LVEF, LVFS and relative expression of miR-146a-5p were significantly decreased,apoptosis rate, CK, LDH and Caspase-3 level were significantly increased(P<0.05). Compared with AMI group,myocardial tissues were thickened in AMI+crocin group and Dex group, myocardial diastolic and systolic function were normal, area of myocardial infarction was significantly reduced, LVEF, LVFS and relative expression of miR-146a-5p were significantly increased, apoptosis rate, CK, LDH and Caspase-3 level were significantly decreased(P<0.05). Compared with control group, relative expression of miR-146a-5p was significantly decreased in Hypoxia group, apoptosis rate, expression of Cleaved caspase-3, LDH and CK levels were significantly increased(P<0.05). Compared with hypoxia group, relative expression of miR-146a-5p was significantly increased in hypoxia+crocin group and Dex group, apoptosis rate, expression of Cleaved caspase-3, LDH and CK levels were significantly decreased(P<0.05). Compared with hypoxia+crocin group, the above-mentioned oxidative stress indexes were significantly increased in hypoxia+crocin+inhibitor group(P<0.05). Conclusion Crocin may reduce myocardial tissue damage and apoptosis in rats with acute myocardial infarction by up-regulating miR-146a-5p.