基于AMPK/mTOR/ULK1信号通路探究黄连素对大鼠心肌纤维化的作用机制

摘要/Abstract

摘要： 目的探讨基于腺苷一磷酸活化蛋白激酶（AMPK）-哺乳动物雷帕霉素靶蛋白（mTOR）-Unc-51样激酶1（Ulk1）通路观察黄连素（BBR）改善心肌纤维化大鼠的心肌损伤的作用。方法选取健康6周龄55只雄性SD大鼠（n=11）,应用异丙肾上腺素（浓度0.5 mg·mL^-1,第1天5 mg·kg^-1,剩余20 d按照2.5 mg·kg^-1·d^-1）连续皮下注射21 d诱导大鼠心肌纤维化,模型制备后将大鼠分为模型组、BBR组、BBR+AMPK抑制剂组及美托洛尔组,另设1组空白对照组,各组大鼠连续给药28 d,最后一次给药12 h后麻醉处死动物并采集标本。通过观察实验过程中各组大鼠的活动度、精神状态,实验结束后通过HE染色观察大鼠心肌组织的病理形态变化,通过Masson染色观察各组大鼠心肌病理形态及纤维化程度,并计算计算心肌胶原容积分数。通过Western Blot法检测心肌组织中的AMPK、mTOR、ULK1、p-AMPK、p-mTOR、p-ULK1、Beclin1、Ⅰ型胶原蛋白(CollagenⅠ)的蛋白表达。结果一般情况：空白对照组毛色白、有光泽、行动敏捷;模型组毛色干枯、行动迟缓、精神萎靡、活动力差;与模型组相比,实验组症状均有不同程度改善。空白组大鼠心肌纤维呈束状排列,无典型的纤维化现象。模型组心肌纤维排列紊乱,出现大量胶原沉积,纤维化程度严重。BBR+AMPK抑制剂组、胶原纤维较模型组减少。BBR组、美托洛尔组较BBR+AMPK抑制剂组纤维化程度降低。模型组Beclin1蛋白、p-AMPK、p-ULK1显著降低,p-mTOR、Collagen Ⅰ显著升高。与模型组相比,BBR组及美托洛尔组Beclin1蛋白、p-AMPK、p-ULK1升高,p-mTOR、Collagen Ⅰ降低。与BBR组相比,BBR+AMPK抑制剂组Beclin1蛋白、p-AMPK、p-ULK1水平降低,p-mTOR、Collagen Ⅰ升高。差异均具有统计学意义（P<0.05）。结论 BBR可通过AMPK/mTOR/ULK1通路激活自噬来减轻异丙肾上腺素诱导的心肌纤维化大鼠心肌损伤,抑制心肌纤维化,发挥心肌保护功能。

关键词: 心肌纤维化, 黄连素, AMPK-mTOR-ULK1信号通路, 自噬

Abstract: Objective To investigate the effect of BBR on myocardial injury in rats with myocardial fibrosis based on AMPK - mammalian target of rapamycin (mTOR) -UNC-51-like kinase 1 (Ulk1) pathway. Methods Fifty-five healthy male SD rats (n = 11) aged 6 weeks were selected and treated with isoproterenol (concentration 0.5 mg·mL^-1, 5 mg·kg^-1 on the first day, 2.5 mg·kg^-1·d^-1 on the remaining 20 days) by subcutaneous injection for 21 days to induce myocardial fibrosis. After model preparation, the rats were divided into model group, BBR group, BBR+AMPK inhibitor group and metoprolol group, and a blank control group was set up. The rats in each group were given continuous administration for 28 days, and the animals were anesthetized 12 hours after the last administration, and specimens were collected. The activity and mental state of the rats in each group were observed during the experiment. After the experiment, the pathological morphological changes of the myocardium of the rats were observed by HE staining, and the pathological morphology and fibrosis degree of the myocardium of the rats were observed by Masson staining, and the myocardial collagen volume fraction was calculated. The protein expression of AMPK, mTOR, ULK1, p-AMPK, p-mTOR, p-ULK1, Beclin1 and CollagenⅠ in myocardial tissue was detected by Western Blot. Results The blank control group was white, glossy and quick in action; The model group had dry hair color, slow movement, listless spirit and poor activity. Compared with the model group, the symptoms of the experimental group were improved to different degrees. In the blank group, the myocardial fibers were arranged in bundles without typical fibrosis. In the model group, the myocardial fiber arrangement was disordered, a large amount of collagen deposition occurred, and the degree of fibrosis was serious. BBR +AMPK inhibitor group and collagen fiber decreased compared with model group. BBR group and metoprolol group had lower fibrosis degree than BBR+AMPK inhibitor group. In the model group, Beclin1 protein, p-AMPK and p-ULK1 were significantly decreased, while p-mTOR and Collagen Ⅰ were significantly increased. Compared with the model group, Beclin1 protein, p-AMPK and p-ULK1 were increased in BBR group and metoprolol group, while p-mTOR and Collagen I were decreased. Compared with the BBR+AMPK inhibitor group, Beclin1 protein, p-AMPK and p-ULK1 levels were decreased. p-mTOR and Collagen I were elevated. The differences were statistically significant (P<0.05). Conclusion BBR can activate autophagy through AMPK/mTOR/ULK1 pathway to alleviate myocardial damage induced by isoproterenol in rats with myocardial fibrosis, inhibit myocardial fibrosis, and exert myocardial protection function.

Key words: Myocardial fibrosis, Berberine, AMPK/mTOR/ULK1 signaling pathway, Autophagy

中图分类号:

R542.2

刘志强, 刘振, 孙经武. 基于AMPK/mTOR/ULK1信号通路探究黄连素对大鼠心肌纤维化的作用机制[J]. 承德医学院学报, 2025, 42(1): 7-11.

LIU Zhi-qiang, LIU Zhen, SUN Jing-wu. Mechanism of Berberine on Isoproterenol-induced Myocardial Fibrosis in Rats based on AMPK/mTOR/ULK1 Signaling Pathway[J]. Journal of Chengde Medical University, 2025, 42(1): 7-11.

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http://tougao.cdmc.edu.cn:8088/CN/Y2025/V42/I1/7

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