高强度间歇运动联合司美格鲁肽对db/db小鼠心肌糖自噬和心功能的影响

关键词：糖尿病心肌病  高强度间歇运动  司美格鲁肽  糖自噬  糖原累积  心肌保护

Keywords:diabetic cardiomyopathy  high-intensity intermittent exercise  semaglutide  glycophagy  glycogen accumulation  myocardial protection

摘要：

      摘要 目的：探究高强度间歇运动（high-intensity interval training，HIIT）联合司美格鲁肽（semaglutide）对db/db小鼠心肌糖自噬和心功能的影响。 方法：40只雄性db/db小鼠随机分为糖尿病对照组（DC组，n=10）、糖尿病运动组（DE组，n=10）、糖尿病药物组（DM组，n=10）和联合干预组（EM组，n=10）。DE组与EM组进行8周HIIT运动，DM组与EM组每天以0.05mg/kg小鼠体重进行颈部皮下注射。定期测量小鼠体重及空腹血糖，干预结束后用超声心动图检测心功能。苏木精-伊红染色观察心肌形态；Masson染色观察心肌纤维化；糖原染色观察糖原累积；WGA染色观察心肌细胞肥大；透射电镜观察糖自噬情况；Western Blot法检测心肌组织中IRS-1、P-PI3K、PI3K、p-AKT、AKT、p-GSK3β、GSK3β、p-AMPK、AMPK、p-mTOR、mTOR、LC3Ⅱ/Ⅰ、TFEB、GABARAPL1蛋白的表达。 结果：与DC组相比，HIIT和司美格鲁肽组及联合干预组均能不同程度减轻db/db小鼠体重和空腹血糖，增强心功能，改善心肌形态和纤维化，减少心肌糖原累积和心肌细胞肥大，心肌细胞横截面积减少（P＜0.05），透射电镜观察到心肌存在自噬体以及含有不同数量糖原颗粒的自噬泡（P＜0.05）；与DC组相比，DE组自噬负调节因子p-mTOR/mTOR比值下降，LC3Ⅱ/Ⅰ、TFEB、GABARAPL1蛋白表达增加（P＜0.05）；DM组AKT、GSK3β磷酸化水平显著上升，TFEB、GABARAPL1蛋白表达增加（P＜0.05）；EM组IRS-1、LC3Ⅱ/Ⅰ、TFEB、GABARAPL1蛋白表达增加，PI3K、AKT、GSK3β、AMPK磷酸化水平显著上升，自噬负调节因子p-mTOR/mTOR比值下降（P＜0.05）；与单一治疗组相比，联合干预组AMPK磷酸化显著增加，LC3Ⅱ/Ⅰ蛋白表达增加（P＜0.05）。 结论：HIIT和司美格鲁肽均能改善db/db小鼠心肌纤维化、糖原累积和心功能，联合干预组治疗效果最显著，可能与激活AMPK/mTOR通路上调心肌细胞糖自噬，改善心脏糖原累积，减少心肌损伤有关。

Abstract:

      Abstract Objective： To explore the effects of high-intensity interval training （HIIT） and semaglutide on cardiomyopathy and glycophagy in db/db mice. Method： Forty male db/db mice were randomly divided into diabetic control group （DC， n=10）， diabetic + exercise group （DE， n=10）， diabetic + semaglutide group （DM， n=10） and diabetic + exercise + semaglutide group （EM， n=10）. DE mice and EM mice were conducted a 8-week HIIT treadmill protocol， DM mice and EM mice received daily subcutaneous neck injections of semaglutide at 0.05mg/kg. Body weight and fasting blood glucose were measured regularly. Echocardiography was used to detect cardiac function at the end of the intervention. Myocardial morphology was evaluated by H&E staining; The H&E staining was used to observe the myocardial morphological changes； fibrosis by Masson staining; glycogen accumulation by periodic acid-Schiff staining; and cardiomyocyte hypertrophy by wheat germ agglutinin (WGA) staining. The glycophagy was observed by transmission electron microscopy. Western Blot quantified the IRS-1， P-PI3K， PI3K， p-AKT， AKT， p-GSK3β， GSK3β， p-AMPK， AMPK， p-mTOR， mTOR， LC3Ⅱ/Ⅰ， TFEB and GABARAPL1 in myocardial tissue. Result： Compared with DC group， HIIT and/or semaglutide intervention reduced body weight and fasting glucose, improved cardiac function and myocardial morphology, decreased fibrosis and cardiomyocyte hypertrophy (reduced cross-sectional area, P<0.05), and lowered glycogen accumulation. Autophagosomes and autophagy vacuoles containing different amounts of glycogen granule were observed by transmission electron microscopy. Compared with DC group， the protein level of p-mTOR/mTOR in DE mice was significantly decreased， and the protein levels of LC3Ⅱ/Ⅰ， TFEB and GABARAPL1 in DE mice were significantly increased （P<0.05）. The phosphorylation levels of AKT and GSK3β in DM mice was significantly increased， and the protein level of TFEB and GABARAPL1 in DM mice was significantly increased （P<0.05）. The protein levels of IRS-1， LC3Ⅱ/Ⅰ， TFEB and GABAPARAL1 in EM mice significantly increased， the phosphorylation levels of PI3K， AKT， GSK3β and AMPK were significantly increased. The p-mTOR/mTOR ratio of autophagy negative regulator in EM mice was decreased （P<0.05）. Compared with single treatment group， the AMPK phosphorylation and LC3Ⅱ/Ⅰ protein expression in EM group were significantly increased （P<0.05）. Conclusion： HIIT and semaglutide can improve myocardial fibrosis， glycogen accumulation and cardiac function in db/db mice， and the combined intervention group yields the most pronounced effects， which may be related to AMPK/mTOR pathway activation, enhanced cardiac glycophagy, and reduced myocardial injury.