目的 通过UHPLC-MS及分子对接技术筛选参附注射液（Shenfu Injection，SFI）核心有效成分，并通过体内外实验验证SFI对慢性心力衰竭（chronic heart failure，CHF）心阳虚证大鼠微管蛋白翻译后修饰表达的影响。方法 应用UHPLC-MS鉴定SFI的主要成分，并对其与微管蛋白相关靶点进行分子对接。SD大鼠随机分为对照组、模型组、秋水仙碱（0.1 mg/kg）组和SFI低、高剂量（3、6 mL/kg）组，每组8只，采用sc异丙肾上腺素复制CHF心阳虚证模型，给予药物干预2周。采用超声心动图检测心功能；ELISA检测血清N末端B型利钠肽原（N-terminal pro-B-type natriuretic peptide，NT-proBNP）水平；苏木素-伊红（hematoxylin-eosin，HE）染色检测心肌组织病理变化；免疫荧光检测心肌组织微管蛋白乙酰化（acetylα-tubulin）、微管蛋白谷氨酸化（polyglut α-tubulin）和微管蛋白去酪氨酸化（detyr α-tubulin）表达；免疫组化检测心肌组织微管蛋白酪氨酸连接酶（tubulin tyrosine ligase，TTL）和组蛋白去乙酰化酶6（histone deacetylase 6，HDAC6）表达。以异丙肾上腺素处理H9c2心肌细胞建立细胞损伤模型，给予SFI干预后，检测微管蛋白修饰水平。结果 UHPLC-MS筛选获得SFI中12种主要有效成分，分子对接结果显示其与tubulin及修饰相关蛋白结合力较强。动物实验结果显示，与模型组比较，SFI能显著改善大鼠心功能（P＜0.01），降低血清中NT-proBNP水平（P＜0.01），减轻心肌间质纤维化程度，降低心肌组织微管蛋白α-tubulin表达及微管密度（P＜0.05、0.01），减少微管蛋白去酪氨酸化和谷氨酸化表达，增加乙酰化表达，下调HDAC6阳性表达（P＜0.01），上调TTL阳性表达（P＜0.01）。细胞实验结果与动物实验一致，进一步验证SFI能够改善微管蛋白翻译后修饰异常。结论 SFI能够有效降低微管蛋白去酪氨酸化、谷氨酸化水平，提高乙酰化水平，纠正微管蛋白翻译后修饰失衡，从而改善CHF心阳虚证大鼠心肌细胞微管网络异常，增强微管稳定性并改善心功能，为SFI治疗CHF心阳虚证的分子机制提供实验依据。

Objective To screen the core active components of Shenfu Injection (参附注射液, SFI) using UHPLC-MS and molecular docking techniques, and to verify the effects of SFI on expressions of post-translational modifications of tubulin in chronic heart failure of heart-yang deficiency syndrome through in vivo and in vitro experiments. Methods UHPLC-MS was applied to identify the main components of SFI, and molecular docking was performed to analyze their interactions with tubulin-related targets. SD rats were randomly divided into control group, model group, colchicine (0.1 mg/kg) group, SFI low- and high-dose (3, 6 mL/kg) groups, with eight rats in each group. The CHF heart-yang deficiency model was replicated using sc isoproterenol, and drug intervention was given for two weeks. Echocardiography was used to detect cardiac function. ELISA was used to detect N-terminal pro-B-type natriuretic peptide (NT-proBNP) level in serum. Hematoxylin-eosin (HE) staining was used to detect pathological changes in myocardial tissue. Immunofluorescence was used to detect the expressions of acetyl α-tubulin, polyglut α-tubulin and detyr α-tubulin in myocardial tissue. Immunohistochemistry was used to detect the expressions of tubulin tyrosine ligase (TTL) and histone deacetylase 6 (HDAC6) in myocardial tissue. H9c2 cardiomyocytes were treated with isoproterenol to establish a cell injury model, and after intervention with SFI, the level of microtubule protein modification was detected. Results A total of 12 main active ingredients in SFI were obtained through UHPLC-MS screening, and molecular docking results showed strong binding affinity with tubulin and modification related proteins. The animal experiment results showed that compared with model group, SFI could significantly improve cardiac function of rats (P < 0.01), reduce the level of NT-proBNP in serum (P < 0.01), alleviate the degree of myocardial interstitial fibrosis, reduce the expressions of microtubule protein α-tubulin and microtubule density in myocardial tissue (P < 0.05, 0.01), reduce the expressions of microtubule protein tyrosine and glutamate, increase acetylation expression, down-regulate HDAC6 positive expression (P < 0.01), and up-regulate TTL positive expression (P < 0.01). The results of cell experiments were consistent with animal experiments, further verifying that SFI could improve post-translational modifications of microtubule proteins. Conclusion SFI could effectively reduce the levels of tyrosinization and glutamatzation of microtubules, increase acetylation levels, correct the imbalance of post-translational modifications of microtubules, thereby improving the abnormal microtubule network in cardiac myocytes of rats with CHF heart-yang deficiency, enhancing microtubule stability and improving heart function. This provides experimental evidence for the molecular mechanism of SFI treatment for CHF heart-yang deficiency.

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国家自然科学基金资助项目（82305092）；国家自然科学基金资助项目（82574922）；湖南中医药大学杏林英才支持计划（2025）；湖南省科技创新计划资助（2024RC3199）；湖南中医药大学优秀青年项目（Z2023XJYQ03）