目的 基于单细胞与空间转录组学探究复方丹参滴丸改善小鼠心肌缺血再灌注损伤（myocardial ischemia-reperfusion injury，MIRI）的作用及其潜在机制。方法 C57BL/6小鼠随机分为假手术组、模型组和复方丹参滴丸低、中、高剂量（61.43、122.85、245.70 mg/kg）组，每组15只。给药干预7 d，于末次给药30 min后采用左前降支结扎-再灌流方法构建小鼠MIRI模型。术后24 h应用超声心动图评估心功能变化；苏木素-伊红（hematoxylin-eosin，HE）及Masson染色观察心肌组织病理学改变。对假手术组、模型组及复方丹参滴丸中剂量组小鼠心脏组织进行单细胞与空间转录组学测序。单细胞转录组学进行细胞类型鉴定、心肌细胞亚群分型、差异基因表达分析、拟时序分析、差异表达基因分析及富集分析。空间转录组学分析识别MIRI空间转录组学特征与损伤边界区的特征基因。采用Western blotting检测小鼠心肌组织中核受体亚家族3C组成员1（nuclear receptor subfamily 3 group C member 1，NR3C1）、丙酮酸脱氢酶激酶 4（pyruvate dehydrogenase kinase 4，PDK4）、长链脂酰辅酶A合成酶4（acyl-CoA synthetase long-chain family member 4，ACSL4）和谷胱甘肽过氧化物酶4（glutathione peroxidase 4，GPX4）的蛋白表达。结果 与假手术组比较，模型组小鼠左室射血分数（left ventricular ejection fraction，LVEF）下降（P＜0.05），病理提示心肌细胞大量坏死，胶原纤维增多。与模型组比较，复方丹参滴丸显著改善MIRI小鼠心功能（P＜0.05），心肌损伤减轻，胶原纤维减少。单细胞转录组学结果表明，复方丹参滴丸可增加心肌细胞比例，减少成纤维细胞及炎症细胞比例。空间转录组学揭示了MIRI空间转录组学特质和损伤边界区的关键基因。Western blotting结果显示，与假手术组比较，模型组小鼠心肌组织NR3C1、PDK4和ACSL4蛋白表达水平显著升高（P＜0.05），GPX4蛋白表达水平显著降低（P＜0.05）；与模型组比较，复方丹参滴丸组心肌组织NR3C1、PDK4和ACSL4蛋白表达水平显著降低（P＜0.05），GPX4蛋白表达水平显著升高（P＜0.05）。结论 复方丹参滴丸可能通过减轻铁死亡，调节心肌代谢，减轻小鼠MIRI。

Objective To investigate the mechanisms by which Compound Danshen Dropping Pills (复方丹参滴丸) ameliorate myocardial ischemia-reperfusion injury (MIRI) in mice using single-cell and spatial transcriptomics. Methods C57BL/6 mice were randomly divided into sham group, model group, Compound Danshen Dropping Pills low-, medium-, and high-dose (61.43, 122.85, 245.70 mg/kg) groups, with 15 mice in each group. Mice were continuously given drug intervention for 7 d, and a MIRI model was constructed using left anterior descending artery ligation reperfusion method 30 min after the last administration. Cardiac function was assessed by echocardiography 24 h after surgery, and myocardial histopathology was evaluated by hematoxylin-eosin (HE) and Masson staining. Hearts from sham group, model group, and Compound Danshen Dropping Pills medium-dose group were subjected to single-cell and spatial transcriptomic analyses. Single cell transcriptomics was used for cell type identification, cardiomyocyte subpopulation typing, differential gene expression analysis, pseudo temporal analysis, differential gene expression analysis and enrichment analysis. Spatial transcriptomics analysis was used to identify the spatial transcriptomic features of MIRI and the characteristic genes of injury boundary region. Western blotting was performed to detect the expression levels of nuclear receptor subfamily 3 group C member 1 (NR3C1), pyruvate dehydrogenase kinase 4 (PDK4), acyl-CoA synthetase long-chain family member 4 (ACSL4) and glutathione peroxidase 4 (GPX4) in myocardial tissue of mice. Results Compared with sham group, the left ventricular ejection fraction (LVEF) of mice in model group was decreased (P < 0.05), pathology showed significant necrosis of myocardial cells and increased collagen fibers. Compared with model group, Compound Danshen Dropping Pills significantly improved the cardiac function of MIRI mice (P < 0.05), reduced myocardial injury and decreased collagen fibers. The results of single-cell transcriptomics indicated that Compound Danshen Dropping Pills could increase the proportion of myocardial cells and reduce the proportion of fibroblasts and inflammatory cells. Spatial transcriptomics revealed key genes involved in the spatial transcriptomic characteristics and damage boundary regions of MIRI. Western blotting results showed that compared with sham group, the expression levels of NR3C1, PDK4 and ACSL4 proteins in myocardial tissue of mice in model group mice were significantly increased (P < 0.05), while the expression level of GPX4 protein was significantly decreased (P < 0.05). Compared with model group, the expression levels of NR3C1, PDK4 and ACSL4 proteins in myocardial tissue of mice in Compound Danshen Dropping Pills group were significantly reduced (P < 0.05), while the expression level of GPX4 protein was significantly increased (P < 0.05). Conclusion Compound Danshen Dropping Pills may alleviate MIRI in mice by modulating myocardial metabolism and attenuating ferroptosis.

R285.5

国家自然科学基金面上项目（82074222）；国家自然科学基金面上项目（82374252）；上海市医学创新研究专项-重大专项（23Y31920200）；上海市中医临床重点实验室项目（20DZ2272200）；苏州工业园区东方华夏心血管健康研究院力心中药科研创新基金2024（2024-CCATCM-052）