敲除PTEN改善心肌缺血再灌注大鼠心脏功能并抑制NLRP3介导的心肌细胞焦亡

Knockout of PTEN improves cardiac function and inhibits NLRP3-mediated cardiomyocyte pyroptosis in rats with myocardial ischemia-reperfusion

目的通过敲除第10号染色体缺失的磷酸酶和张力蛋白同源蛋白(PTEN),探索其对大鼠心脏功能和含pyrin结构域NOD样受体家族3(NLRP3)介导的心肌细胞焦亡(pyroptosis)的影响。方法建立大鼠心肌缺血/再灌注(I/R)损伤大鼠模型,分为假手术组(野生型健康大鼠)、野生型I/R组(野生型大鼠进行心肌I/R处理)、敲除PTEN(PTEN KO)的I/R组(PTEN KO大鼠进行心肌I/R处理)。反转录PCR检测PTEN mRNA水平,HE染色观察心肌病理损伤;超声心动图仪检测心率(HR)和左心室壁厚度(LVWT)并通过BL-420F生物机能实验系统记录左心室收缩压(LVSP)、左室射血分数(LVEF)、缩短分数(FS);ELISA检测血清肌酸激酶同工酶(CK-MB)、肌红蛋白(Mb)、心肌肌钙蛋白I(cTnI)的含量;Western blot法检测心脏组织NLRP3、ELAV样RNA结合蛋白1(ELAVL1)、胱天蛋白酶1(caspase-1)、白细胞介素1β(IL-1β)的蛋白表达,免疫组织化学染色法检测心肌组织中caspase-1的含量;原位末端转移酶标记技术(TUNEL)染色观察心肌组织的凋亡。结果与假手术组相比,野生型I/R组PTEN mRNA和蛋白水平显著增加,HR、LVSP、LVEF、FS和LVWT均显著降低,血清CK-MB、Mb、cTnI含量均显著升高,NLRP3、ELAVL1、caspase-1、IL-1β蛋白表达水平均显著升高,敲除PTEN后PTEN mRNA和蛋白水平显著降低。敲除PTEN心肌细胞病理损伤减轻,HR、LVSP、LVEF、FS和LVWT均显著升高,血清CK-MB、Mb、cTnI含量显著降低。NLRP3、ELAVL1、caspase-1、IL-1β蛋白水平均显著降低,凋亡心肌细胞数量显著减少。结论敲除PTEN减缓心肌病理损伤,抑制NLRP3介导的心肌细胞焦亡,表明敲除PTEN能减轻心肌I/R损伤。

Objective To investigate the effects of phosphate and tension homology deleted on chromsome ten(PTEN)knockout on rat heart function and pyroptosis of cardiomyocytes mediated by NLR family pyrin domain containing 3(NLRP3).Methods Rat models of myocardial ischemia/reperfusion(I/R)injury were established.The rats were divided into sham operation group(wild-type healthy rats),wild-type I/R group(wild-type healthy rats treated with myocardial I/R),and I/R group(PTEN KO rats treated with myocardial I/R).PTEN mRNA level was detected by reverse transcription PCR,and myocardial pathological damage was observed by HE staining.Heart rate(HR)and left ventricular wall thickness(LVWT)were measured by echocardiography,and left ventricular systolic blood pressure(LVSP),left ventricular ejection fraction(LVEF),and fraction shortening(FS)were recorded by BL-420F bioassay system.Serum creatine kinase isoenzyme(CK-MB),myoglobin(Mb)and cardiac troponin I(cTnI)were detected by ELISA.Western blot analysis was used to detect the protein expression of NLRP3,embryonic lethal,abnormal vision,Drosophila-like 1(ELAVL1),caspase-1(caspase-1),and IL-1βin heart tissues.Immunohistochemical staining was performed to detect the content of caspase-1 in cardiac tissues.Apoptosis of myocardial tissue was observed with TUNEL staining.Results Compared with the sham operation group,PTEN mRNA and protein levels in the wild-type I/R group significantly increased,HR,LVSP,LVEF,FS,and LVWT went down significantly,and serum CK-MB,Mb,and cTnI levels significantly increased,and NLRP3,ELAVL1,caspase-1,and IL-1βprotein expression levels went up significantly.After PTEN was knocked out,PTEN mRNA and protein levels were significantly reduced,the pathological damage of cardiomyocytes was alleviated,and HR,LVSP,LVEF,FS,and LVWT were significantly elevated,and serum CK-MB,Mb,and cTnI levels were significantly inhibited.NLRP3,ELAVL1,caspase-1,and IL-1βprotein levels and the number of apoptotic cardiomyocytes were significantly reduced after PTEN knockout.Conclusion Knockout of PTEN can alleviate the pathological damage of myocardium and inhibit nlrp3-mediated apoptosis of cardiomyocytes,indicating that knockout of PTEN can alleviate myocardial I/R damage.