基于p38MAPK/SERCA2α通路观察羟基红花黄色素A对大鼠心肌缺血/再灌注损伤的保护作用

Protective effect of hydroxysafflor yellow A on myocardial ischemia/reperfusion injury in rats based on p38MAPK/SERCA2α pathway

目的:基于p38丝裂原活化蛋白激酶/肌质网Ca^(2+)-ATP酶2α(p38MAPK/SERCA2α)通路观察羟基红花黄色素A(HSYA)对大鼠心肌缺血/再灌注损伤(MIRI)的保护作用。方法:120只SD雄性大鼠随机分为假手术组(Sham组)、缺血再灌注组(I/R组)、HSYA 5 mg/kg组、HSYA 10 mg/kg组、HSYA 20 mg/kg组及p38MAPK信号通路抑制剂组(SB203580组)。5、10、20 mg/kg HSYA组大鼠分别灌胃给予对应剂量HSYA,SB203580组给予100µg/kg SB203580,末次灌胃结束后制作MIRI模型,Sham组仅切开左胸暴露心脏不结扎。缺氧4 h、复氧12 h建立H9C2心肌细胞OGD/R损伤模型并分为6组:对照组(Control组)、缺氧复氧模型组(OGD/R组)、5、10、20µmol/L HSYA组及SB203580组,Control组、OGD/R组不进行药物干预。TTC染色测定大鼠心肌梗死面积;超声心动图检测大鼠心脏功能;HE染色观察大鼠心肌组织病理学改变;Annexin V-FITC/PI双染法检测大鼠心肌组织和H9C2细胞凋亡;检测大鼠血清氧化应激及心肌酶指标;ELISA检测大鼠心肌组织和H9C2细胞炎症因子含量;CCK8及EdU染色检测H9C2细胞增殖能力;qRT-PCR检测心肌组织和H9C2细胞p38MAPK、SERCA2αmRNA表达;Western blot检测大鼠心肌组织和H9C2细胞p38MAPK、p-p38MAPK、SERCA2α、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)的蛋白表达。结果:10µmol/L或10 mg/kg HSYA能够提高左室射血分数(LVEF)、左室短轴缩短率(LVFS)、Bcl-2、SERCA2α水平,降低肌酸激酶(CK)、乳酸脱氢酶(LDH)、天冬氨酸转氨酶(AST)、超氧化物歧化酶(SOD)活力,降低Bax、肌钙蛋白Ⅰ(cTnⅠ)、丙二醛(MDA)、TNF-α、IL-1β、IL-6、p-p38MAPK/p38MAPK水平,促进心肌细胞增殖,抑制细胞凋亡,减少心肌梗死面积,改善心肌损伤。结论:HSYA能够抑制心肌细胞凋亡、氧化应激及炎症,从而减轻MIRI,其机制可能与p38MAPK/SERCA2α信号通路有关。

Objective:To explore protective effect of hydroxysafflor yellow A(HSYA)on myocardial ischemia/reperfusion injury(MIRI)of rats based on p38 mitogen-activated protein kinase/sarcoplasmic reticulum Ca^(2+)-ATPase 2α(p38MAPK/SERCA2α)pathway.Methods:A total of 120 male SD rats were randomly divided into sham operation group(Sham group),ischemia-reperfusion group(I/R group),HSYA 5 mg/kg group,HSYA 10 mg/kg group,HSYA 20 mg/kg group and p38MAPK signal pathway inhibitor group(SB203580 group).Rats in 5,10,20 mg/kg HSYA groups were intragastrically treated with corresponding doses of HSYA,SB203580 group was given 100µg/kg SB203580.MIRI models were made after the last intragastric administration,only left chest was cut open in Sham group to expose heart without ligation.OGD/R injury model of H9C2 cardiomyocytes was established by hypoxia for 4 h and reoxygenation for 12 h and divided into 6 groups:Control group,hypoxia-reoxygenation model group(OGD/R group),5µmol/L,10µmol/L and 20µmol/L HSYA groups and SB203580 group,there was no drug intervention in Control group and OGD/R group.Area of myocardial infarction was measured by TTC staining,cardiac function was detected by echocardiography,histopathological changes of myocardial tissues were observed by HE staining,apoptosis of myocardial tissues and H9C2 cells were detected by Annexin V-FITC/PI double staining,sesum indexes of oxidative stress and myocardial enzymes in rats were detected,contents of inflammatory factors of myocardial tissues and H9C2 cells were detected by ELISA,and proliferation ability of H9C2 cells was detected by CCK8 and EdU staining.p38MAPK and SERCA2αmRNA expressions in myocardial tissues and H9C2 cells were detected by qRT-PCR,and protein expressions of p38MAPK,p-p38MAPK,SERCA2α,B cell lymphoma-2(Bcl-2)and Bcl-2 associated X(Bax)in rat myo-cardial tissues and H9C2 cells were detected by Western blot.Results:10µmol/L or 10 mg/kg HSYA increased left ventricular ejec tion fraction(LVEF),left ventricular short axis shortening rate(LVFS),Bcl-2,SERCA2αlevels,and reduced creatine kinase(CK),lactate dehydrogenase(LDH),aspartame acid transaminase(AST),superoxide dismutase(SOD)activity,reduced Bax,tro-poninⅠ(cTnⅠ),malondialdehyde(MDA),TNF-α,IL-1β,IL-6,p-p38MAPK/p38MAPK levels,promoted proliferation of myocar-dial cells,inhibited cardiomyocytes apoptosis,reduced area of myocardial infarction,and improved myocardial damage.Conclusion:HSYA can inhibit cardiomyocytes apoptosis,oxidative stress and inflammation,thereby reducing MIRI,whose mechanism may be re-lated to p38MAPK/SERCA2αsignaling pathway.