摘要
目的探讨艾司氯胺酮对大鼠心肌缺血再灌注损伤(MIRI)及腺苷-磷酸活化蛋白激酶(AMPK)-哺乳动物雷帕霉素靶蛋白(mTOR)通路相关蛋白的影响。方法构建MIRI大鼠模型,将大鼠分为Control组、MIRI组、艾司氯胺酮低剂量组(KET-L组)、艾司氯胺酮高剂量组(KET-H组)、艾司氯胺酮高剂量+AMPK抑制剂Compound C组(KET-H+CC组)。采用HE染色观察心肌组织病理形态变化;采用酶联免疫吸附试验(ELISA)检测心肌损伤标志物肌酸激酶同工酶(CK-Mb)、肌钙蛋白I(cTnI)、肌钙蛋白T(cTnT)水平;采用透射电镜观察心肌细胞线粒体形态变化;采用免疫组化法检测自噬相关蛋白微管相关蛋白轻链3Ⅱ(LC3Ⅱ)、重组人自噬效应蛋白(Beclin-1)表达水平;采用原位末端标记法(TUNEL)染色检测心肌组织细胞凋亡情况;采用蛋白免疫印迹法(Western blot)检测B细胞淋巴瘤因子2相关X蛋白(Bax)、B细胞淋巴瘤因子2(Bcl-2)、AMPK、磷酸化的腺苷-磷酸活化蛋白激酶(p-AMPK)、mTOR、磷酸化雷帕霉素靶蛋白(p-mTOR)的表达。结果与Control组相比,MIRI组大鼠心肌纤维排列紊乱,心肌细胞肥大且结构模糊,细胞核固缩、核膜皱缩,炎症细胞浸润明显,线粒体结构紊乱,嵴断裂甚至消失,MIRI严重,CK-Mb、cTnI、cTnT、LC3Ⅱ、Beclin-1、Bax表达水平,以及细胞凋亡率、p-mTOR/mTOR升高(P<0.05),Bcl-2表达水平、p-AMPK/AMPK降低(P<0.05);与MIRI组相比,KET-L组、KET-H组心肌细胞损伤减轻,炎症细胞浸润减少,CK-Mb、cTnI、cTnT、LC3Ⅱ、Beclin-1、Bax表达水平,以及细胞凋亡率、p-mTOR/mTOR降低(P<0.05),Bcl-2表达水平、p-AMPK/AMPK升高(P<0.05),且KET-H组优于KET-L组(P<0.05);与KET-H组相比,KET-H+CC组心肌损伤加重,线粒体膜消失,嵴断裂或者消失,CK-Mb、cTnI、cTnT、LC3Ⅱ、Beclin-1、Bax表达水平,以及细胞凋亡率、p-mTOR/mTOR升高(P<0.05),Bcl-2表达水平、p-AMPK/AMPK降低(P<0.05)。结论艾司氯胺酮可能通过激活AMPK-mTOR通路降低线粒体自噬水平,减少心肌细胞凋亡,减轻心肌损伤,缓解大鼠MIRI。
Objective To investigate the effects of esketamine on myocardial ischemia-reperfusion injury(MIRI)and adenosine monophosphate-activated protein kinase(AMPK)-mammalian target of rapamycin(mTOR)pathway-related proteins in rats.Methods The MIRI rat model was constructed,and the rats were divided into the Control group,MIRI group,low-dose esketamine group(KET-L group),high-dose esketamine group(KET-H group),high-dose esketamine+AMPK inhibitor Compound C group(KET-H+CC group).The pathological changes of myocardial tissue were observed by HE staining.The levels of myocardial injury markers creatine kinase isoenzyme(CK-Mb),cardiac troponin I(cTnI)and cardiac troponin T(cTnT)were detected by enzyme-linked immunosorbent assay(ELISA).The morphological changes of myocardial cellular mitochondria were observed by transmission electron microscopy.The expression levels of autophagy-related protein microtubule-associated protein light chain 3Ⅱ(LC3Ⅱ)and recombinant human autophagy effector protein(Beclin-1)were detected by immunohistochemistry.The apoptosis of myocardial tissue cells was detected by the situ end labeling(TUNEL)staining.The expression levels of B-cell lymphoma factor 2-associated X protein(Bax),B-cell lymphoma factor 2(Bcl-2),AMPK,phosphorylated adenylate-activated protein kinase(p-AMPK),mTOR and phosphorylated target of rapamycin(p-mTOR)were detected by Western blot.Results Compared with the Control group,the myocardial fibers in the MIRI group were arranged in disorder,the myocardial cells were hypertrophic and the structure was blurred,the nucleus was pyknosis,the nuclear membrane was shrunk,the inflammatory cell infiltration was obvious,the mitochondrial structure was disordered,the ridge was broken or even disappeared,MIRI was serious,the expression levels of CK-Mb,cTnI,cTnT,LC3Ⅱ,Beclin-1 and Bax,apoptosis rate and p-mTOR/mTOR were increased(P<0.05),the expression levels of Bcl-2 and p-AMPK/AMPK were decreased(P<0.05).Compared with the MIRI group,the myocardial cell injury in the KET-L group and KET-H group was alleviated,the inflammatory cell infiltration was reduced,the expression level of CK-Mb,cTnI,cTnT,LC3Ⅱ,Beclin-1,Bax,apoptosis rate and p-mTOR/mTOR were decreased,and the expression level of Bcl-2 and p-AMPK/AMPK were increased(P<0.05),moreover the KET-H group was better than the KET-L group(P<0.05).Compared with the KET-H group,the myocardial injury in the KET-H+CC group was aggravated,the mitochondrial membrane disappeared,cristae ruptured or disappeared,the expression levels of CK-Mb,cTnI,cTnT,LC3Ⅱ,Beclin-1,and Bax,apoptosis rate and p-mTOR/mTOR were increased(P<0.05),the expression level of Bcl-2 and p-AMPK/AMPK were decreased(P<0.05).Conclusion Esketamine may reduce the level of mitophagy by activating the AMPK-mTOR pathway,reduce the cardiomyocyte apoptosis,reduce the myocardial injury and alleviate MIRI in rats.
作者
贺燕羽
阳仲琴
郭娜
马凤莲
周瑜
王婷
HE Yanyu;YANG Zhongqin;GUO Na;MA Fenglian;ZHOU Yu;WANG Ting(Department of Anesthesiology,Jianyang Municipal Hospital of Traditional Chinese Medicine,Jianyang,Sichuan 641400,China)
出处
《检验医学与临床》
CAS
2024年第14期2040-2046,共7页
Laboratory Medicine and Clinic
基金
四川省卫生健康委员会科研课题普及应用项目(20PJ0236)。
关键词
艾司氯胺酮
腺苷-磷酸活化蛋白激酶
雷帕霉素靶蛋白
心肌缺血再灌注损伤
线粒体自噬
凋亡
esketamine
adenosine monophosphate-activated protein kinase
mammalian target of rapamycin
myocardial ischemia-reperfusion injury
mitochondrial autophagy
apoptosis
