自噬作为一种新的机制见解探讨丹芪片防治急性心肌梗死后心力衰竭。

Autophagy as a novel insight into mechanism of Danqi pill against post-acute myocardial infarction heart failure.

机构信息

College of Chinese Medicine, Beijing University of Chinese Medicine, Beijing, 100029, China.

School of Life Sciences, Beijing University of Chinese Medicine, Beijing, 100029, China.

出版信息

J Ethnopharmacol. 2021 Feb 10;266:113404. doi: 10.1016/j.jep.2020.113404. Epub 2020 Oct 5.

DOI:10.1016/j.jep.2020.113404

PMID:32976970

Abstract

ETHNOPHARMACOLOGICAL RELEVANCE

Danqi Pill, composed of the root of Salvia miltiorrhiza Bunge and the root of Panax notoginseng, is effective in the clinical treatment of myocardial ischemia in coronary heart diseases. A number of studies have shown that autophagy plays an essential role in cardiac function and energy metabolism, and disordered autophagy is associated with the progression of heart failure. However, the effect and mechanism of Danqi pill on autophagy have not been reported yet.

AIM OF THE STUDY

This study aims to elucidate whether Danqi pill restores autophagy to protect against HF and its potential mechanism.

MATERIALS AND METHODS

Left anterior descending ligation was established to induce an HF rat model, HO-stimulated H9C2 cells model was conducted to clarify the effects and potential mechanism of Danqi pill. In vivo, Danqi pill (1.5 g/kg) were orally administered for four weeks and Fenofibrate (10 mg/kg) was selected as a positive group. In vitro, Danqi pill (10-200 μg/mL) was pre-cultured for 24 h and co-cultured with HO stimulation for 4 h. Importantly, transmission electron microscopy and fluorescence GPF-mRFP-LC3 reporter system were combined to monitor autophagy flux. Furtherly, we utilized Compound C, a specific AMPK inhibitor, to validate whether the autophagy was mediated by AMPK-TSC2-mTOR pathway.

RESULTS

Danqi pill significantly improved cardiac function and myocardial injury in HF rats. Intriguingly, Danqi pill potently regulated autophagy mainly by promoting the formation of autophagosomes in vivo. Further results demonstrated that expressions of p-AMPK (P < 0.001) and p-TSC2 (P < 0.001) in cardiac tissue were upregulated by Danqi pill, accompanied with downregulation of p-mTOR (P < 0.01) and p-ULK1(P < 0.01). In parallel with the vivo experiment, in vitro study indicated that Danqi pill dramatically restored autophagy flux and regulated expressions of critical autophagy-related molecules. Finally, utilization of Compound C abrogated the effects of Danqi pill on autophagy flux and the expressions of p-TSC2 (P < 0.05), p-mTOR (P < 0.01) and p-ULK1 (P < 0.05).

CONCLUSION

Danqi pill could improve cardiac function and protect against cardiomyocytes injury by restoring autophagy via regulating the AMPK-TSC2-mTOR signaling pathway.

摘要

民族药理学相关性

丹七片由丹参和三七根组成，在冠心病心肌缺血的临床治疗中有效。多项研究表明，自噬在心脏功能和能量代谢中起着至关重要的作用，而自噬紊乱与心力衰竭的进展有关。然而，丹七片对自噬的作用及其机制尚未报道。

研究目的

本研究旨在阐明丹七片是否通过恢复自噬来防治心力衰竭及其潜在机制。

材料和方法

通过结扎左前降支建立心力衰竭大鼠模型，通过 HO 刺激 H9C2 细胞模型阐明丹七片的作用及潜在机制。体内实验中，丹七片（1.5 g/kg）连续灌胃四周，以非诺贝特（10 mg/kg）为阳性组。体外实验中，丹七片（10-200 μg/mL）预培养 24 h 后与 HO 刺激共培养 4 h。重要的是，采用透射电镜和 GFP-mRFP-LC3 报告系统联合监测自噬流。进一步采用 Compound C，一种特异性的 AMPK 抑制剂，验证自噬是否通过 AMPK-TSC2-mTOR 通路介导。

结果

丹七片显著改善心力衰竭大鼠的心功能和心肌损伤。有趣的是，丹七片在体内强力调节自噬，主要通过促进自噬体的形成。进一步的结果表明，丹七片可上调心肌组织中磷酸化 AMPK（P < 0.001）和磷酸化 TSC2（P < 0.001）的表达，同时下调磷酸化 mTOR（P < 0.01）和磷酸化 ULK1（P < 0.01）的表达。与体内实验一致，体外实验表明，丹七片可显著恢复自噬流，并调节关键自噬相关分子的表达。最后，使用 Compound C 可消除丹七片对自噬流和磷酸化 TSC2（P < 0.05）、磷酸化 mTOR（P < 0.01）和磷酸化 ULK1（P < 0.05）表达的影响。