Pannexin1/P2X7介导的铁死亡在糖尿病心肌损伤中的作用及机制

Role and mechanism of Pannexin1/P2X7⁃mediated ferroptosis in diabetic myocardial injury

摘要

目的：探讨Pannexin1（Panx1）/P2X7介导的铁死亡在糖尿病心肌损伤中的作用及其潜在分子机制。方法：选取18只6~8周龄雄性C57BL/6小鼠，采用随机数字表法分为假手术组（C组）、糖尿病组（DM组）及糖尿病+Panx1抑制剂甘珀酸组（DM+CBX组），每组6只。糖尿病小鼠予高脂饲料喂养8周后，单次腹腔注射链脲佐菌素（STZ，100 mg·kg^-1）建立糖尿病模型。DM+CBX组小鼠于取材前24 h腹腔注射CBX（20 mg·kg^-1）。采用HE染色观察心肌组织病理改变，天狼星红染色评估心肌纤维化程度，普鲁士蓝染色检测心肌铁沉积情况。酶联免疫吸附试验检测血清亚铁离子（Fe²⁺）、还原型谷胱甘肽（GSH）、乳酸脱氢酶（LDH）及肌钙蛋白I（cTn⁃I）水平。透射电镜观察心肌细胞线粒体超微结构变化。Western Blot检测心肌组织Panx1、P2X7受体及铁死亡相关蛋白GPX4、SLC7A11、TFR1的表达水平。结果：与C组相比，DM组小鼠血清cTn⁃I和LDH水平显著升高（P<0.05），心肌纤维排列紊乱，间质胶原沉积增多，铁染阳性信号明显增强；血清Fe²⁺含量显著升高而GSH含量明显下降（P<0.05）；线粒体呈现体积缩小、嵴断裂及膜结构破坏等典型铁死亡超微结构特征；心肌组织Panx1、P2X7、TFR1蛋白表达显著上调，而SLC7A11、GPX4表达显著下调（均P<0.05）。经CBX干预后，DM+CBX组心肌损伤标志物水平下降，铁沉积减少，氧化应激水平下降，线粒体结构相对完整；Panx1、P2X7、TFR1表达下调，SLC7A11、GPX4表达上调（均P<0.05）。结论：Panx1/P2X7信号轴在糖尿病心肌损伤中过度激活，通过促进铁离子积聚、破坏氧化还原平衡及诱导线粒体损伤，最终导致心肌细胞铁死亡。抑制Panx1可有效减轻糖尿病诱导的心肌铁死亡和组织损伤，为糖尿病心肌病的防治提供了新的分子靶点。

Abstract

Objective: To investigate the role of Pannexin1 (Panx1)/P2X7⁃mediated ferroptosis in diabetic myocardial injury and to elucidate its underlying molecular mechanisms. Methods: Eighteen male C57BL/6 mice (aged 6⁃8 weeks) were randomly divided to a sham group (C), a diabetes group (DM), and a diabetes mellitus plus Panx1 inhibitor group (DM+CBX) (n=6 per group). DM was induced by feeding a high⁃fat diet for 8 weeks followed by a single intraperitoneal injection of streptozotocin (STZ, 100 mg·kg^-1). Mice in the DM+CBX group received carbenoxolone (CBX, 20 mg·kg^-1, i.p.) 24 h before tissue collection. Hematoxylin⁃eosin (HE) staining was used to evaluate myocardial histopathology. Sirius Red staining was used to assess myocardial fibrosis. Prussian blue staining was used to detect cardiac iron deposition. Serum ferrous iron (Fe²⁺), reduced glutathione (GSH), lactate dehydrogenase (LDH), and cardiac troponin I (cTn⁃I) were measured by ELISA. Transmission electron microscopy (TEM) was used to examine mitochondrial ultrastructure. Western Blot was performed to assess myocardial expression of Panx1, the P2X7 receptor, and ferroptosis⁃related proteins including GPX4, SLC7A11, and TFR1. Results: Compared with the C group, the DM group showed significantly increased serum cTn⁃I and LDH levels (P<0.05), as well as disordered myocardial fiber arrangement with increased interstitial collagen deposition and enhanced iron⁃positive signals on Prussian blue staining. Serum Fe²⁺ was significantly elevated, whereas GSH was reduced (P<0.05). TEM revealed classic ferroptosis⁃associated mitochondrial features, including mitochondrial shrinkage, cristae disruption, and membrane rupture. Myocardial Panx1, P2X7, and TFR1 protein levels were up⁃regulated, whereas SLC7A11 and GPX4 were down⁃regulated (allP<0.05). Following CBX treatment, the DM+CBX group exhibited lower levels of myocardial injury markers, reduced iron deposition, attenuated oxidative stress, and relatively preserved mitochondrial structure. Additionally, Panx1, P2X7, and TFR1 expression was decreased, while SLC7A11 and GPX4 expression were increased (allP<0.05). Conclusion: Pharmacological inhibition of Panx1 alleviated diabetes⁃induced myocardial ferroptosis and tissue injury, suggesting that Panx1 may serve as a potential molecular target for the prevention and treatment of diabetic cardiomyopathy.

关键词

糖尿病 / 心肌损伤 / Panx1 / P2X7 / 铁死亡 / 糖尿病心肌病

Key words

Diabetes / Myocardial Injury / Panx1 / P2X7 / Ferroptosis / Diabetic Cardiomyopathy

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基金资助

国家自然科学基金资助项目(81772049)