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抑制谷氨酰胺代谢减轻血管紧张素II诱导的心肌纤维化

王盼盼^1,2, 白浩淼³, 何思宇³, 夏子淇³, 刘美杰², 安冏², 周嘉恒², 李宸涵^1,2, 张伟^1,2, 张星², 王馨佩², 李嘉^2,*

¹西北大学生命科学学院，西安 710069；²中国人民解放军空军军医大学航空航天医学系，西安 710032；³中国人民解放军空军军医大学基础医学院学员队，西安 710032

摘要

本文旨在探讨心脏成纤维细胞(cardiac fibroblasts, CFs)谷氨酰胺(glutamine, Gln)代谢在高血压所致心肌纤维化中的作用及机制。用微渗透泵给予C57BL/6J小鼠血管紧张素II (angiotensin II, Ang II, 1.6 mg/kg per d)诱导心肌纤维化，用免疫组织化学法和Western blot检测心肌组织谷氨酰胺酶1 (glutaminase 1, GLS1)的表达。小鼠腹腔注射GLS1抑制剂BPTES (12.5 mg/kg)以抑制Gln代谢，用Masson染色法观察心肌纤维化程度，用RT-qPCR和Western blot检测心肌组织I型和III型胶原蛋白表达变化。Sprague-Dawley (SD)大鼠乳鼠CFs在有/无Ang II (0.4 μmol/L)刺激下接受Gln 4 mmol/L或BPTES (5 μmol/L)处理。用划痕实验和CCK-8法分别检测CFs迁移和增殖，用RT-qPCR和Western blot检测CFs中GLS1、I型和III型胶原蛋白表达变化。在Ang II和BPTES处理的条件下，给予CFs 2 mmol/L α-酮戊二酸(α-ketoglutarate, α-KG)处理，检测CFs迁移、增殖、I型和III型胶原蛋白表达变化。结果显示，Ang II诱导的小鼠血压、心脏质量和心肌纤维化均增加，同时心肌组织GLS1表达显著上调。在体外实验中，无论有/无Ang II刺激，Gln均可显著促进CFs增殖、迁移及GLS1、I型和III型胶原蛋白的表达，而BPTES显著降低CFs上述指标；补充α-KG可逆转BPTES对Ang II刺激下CFs的抑制作用。给小鼠腹腔注射BPTES可改善Ang II诱导的小鼠心肌纤维化。以上结果提示，Gln的分解代谢在Ang II所致心肌纤维化进程中发挥重要作用，靶向抑制Gln分解代谢可能是治疗心肌纤维化的新策略。

关键词： 谷氨酰胺代谢; 谷氨酰胺酶1; 心肌纤维化

Inhibition of glutaminolysis alleviates myocardial fibrosis induced by angiotensin II

WANG Pan-Pan^1,2, BAI Hao-Miao³, HE Si-Yu³, XIA Zi-Qi³, LIU Mei-Jie², AN Jiong², ZHOU Jia-Heng², LI Chen-Han^1,2, ZHANG Wei^1,2, ZHANG Xing², WANG Xin-Pei², LI Jia^2,*

¹College of Life Sciences, Northwest University, Xi’an 710069, China；²School of Aerospace Medicine, Air Force Medical University, Xi’an 710032, China；³Cadet Regiment, School of Basic Medical Sciences, Air Force Medical University, Xi’an 710032, China

Abstract

The present study was aimed to investigate the role and mechanism of glutaminolysis of cardiac fibroblasts (CFs) in hypertension-induced myocardial fibrosis. C57BL/6J mice were administered with a chronic infusion of angiotensin II (Ang II, 1.6 mg/kg per d) with a micro-osmotic pump to induce myocardial fibrosis. Masson staining was used to evaluate myocardial fibrosis. The mice were intraperitoneally injected with BPTES (12.5 mg/kg), a glutaminase 1 (GLS1)-specific inhibitor, to inhibit glutaminolysis simultaneously. Immunohistochemistry and Western blot were used to detect protein expression levels of GLS1, Collagen I and Collagen III in cardiac tissue. Neonatal Sprague-Dawley (SD) rat CFs were treated with 4 mmol/L glutamine (Gln) or BPTES (5 μmol/L) with or without Ang II (0.4 μmol/L) stimulation. The CFs were also treated with 2 mmol/L α-ketoglutarate (α-KG) under the stimulation of Ang II and BPTES. Wound healing test and CCK-8 were used to detect CFs migration and proliferation respectively. RT-qPCR and Western blot were used to detect mRNA and protein expression levels of GLS1, Collagen I and Collagen III. The results showed that blood pressure, heart weight and myocardial fibrosis were increased in Ang II-treated mice, and GLS1 expression in cardiac tissue was also significantly up-regulated. Gln significantly promoted the proliferation, migration, mRNA and protein expression of GLS1, Collagen I and Collagen III in the CFs with or without Ang II stimulation, whereas BPTES significantly decreased the above indices in the CFs. α-KG supplementation reversed the inhibitory effect of BPTES on the CFs under Ang II stimulation. Furthermore, in vivo intraperitoneal injection of BPTES alleviated cardiac fibrosis of Ang II-treated mice. In conclusion, glutaminolysis plays an important role in the process of cardiac fibrosis induced by Ang II. Targeted inhibition of glutaminolysis may be a new strategy for the treatment of myocardial fibrosis.

Key words: glutaminolysis; glutaminase 1; myocardial fibrosis

收稿日期：　　录用日期：

通讯作者：李嘉　　E-mail: jiali816@fmmu.edu.cn

DOI: 10.13294/j.aps.2023.0029

引用本文：

王盼盼, 白浩淼, 何思宇, 夏子淇, 刘美杰, 安冏, 周嘉恒, 李宸涵, 张伟, 张星, 王馨佩, 李嘉. 抑制谷氨酰胺代谢减轻血管紧张素II诱导的心肌纤维化[J]. 生理学报 2023; 75 (2): 179-187.

WANG Pan-Pan, BAI Hao-Miao, HE Si-Yu, XIA Zi-Qi, LIU Mei-Jie, AN Jiong, ZHOU Jia-Heng, LI Chen-Han, ZHANG Wei, ZHANG Xing, WANG Xin-Pei, LI Jia. Inhibition of glutaminolysis alleviates myocardial fibrosis induced by angiotensin II. Acta Physiol Sin 2023; 75 (2): 179-187 (in Chinese with English abstract).