目的 探讨丁苯酞（3-N-丁基苯酞，NBP）减轻大鼠心肌缺血再灌注损伤（MIRI）的作用及机制。方法 综合网络药理学方法，预测NBP减轻大鼠心肌缺血再灌注损伤的潜在分子靶点与通路。继而结扎心脏的左前降支冠状动脉法构建SD大鼠MIRI模型，NBP（80 mg·kg^-1）预处理7 d，通过超声心动图量化心脏功能，TTC染色法评估心肌梗死面积，ELISA法检测心肌组织肌酸激酶-MB（CK-MB）、肌钙蛋白T（cTnI）、超氧化物歧化酶（SOD）、丙二醛（MDA）和乳酸脱氢酶（LDH）水平，并借助Western blotting法分析心肌组织分子靶点的蛋白表达差异。最后体外培养H9C2大鼠心肌细胞，细胞分为：对照组，正常培养；模型组，进行氧糖剥夺/再氧合（OGD/R）处理； NBP组，在NBP（100 μmol·L^-1）预处理4 h后进行OGD/R；NBP＋LY组，在NBP组的基础上额外给予PI3K特异性抑制剂LY294002 15 μmol·L^-1预处理1 h； FTS（RAS抑制剂Salirasib）组，在OGD/R的基础上以FTS（39.36 μmol·L^-1）预处理12 h； FTS＋NBP组，在FTS组的基础上以100 μmol·L^-1的NBP预处理4 h，通过免疫荧光和Western blotting法进一步验证与探索NBP的作用通路。结果 网络药理学结果表明，NBP发挥作用的信号通路可能包括：脂质和动脉粥样硬化、磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（Akt）信号通路、T细胞受体信号通路、FoxO信号通路、细胞凋亡、MAPK信号通路和Ras信号通路。体内实验显示，相较于对照组，NBP预处理显著减轻MIRI诱导的SD大鼠心肌炎症反应与氧化应激，缩小梗死率，增强射血功能，同时激活PI3K信号通路蛋白表达，抑制凋亡通路蛋白表达，差异均显著（P＜0.05、0.01、0.001）。体外实验证实，NBP预处理通过RAS/PI3K信号通路调控凋亡蛋白，并激活热休克蛋白A12A（HSPA12A）表达，LY294002和Salirasib可以部分抵消其作用，该调节方式可能呈单向性。体内外实验结果相互佐证。结论 NBP可有效缓解MIRI诱导的细胞凋亡，发挥心脏保护作用，机制可能与RAS/PI3K信号通路相关。

Objective The role and mechanism of 3-N-butylphthalide (NBP) in alleviating myocardial ischemia-reperfusion injury (MIRI) in rats were explored. Methods A comprehensive network pharmacology approach was used to predict the potential molecular targets and pathways of NBP in reducing MIRI in rats. Then, a MIRI model in SD rats was established by ligating the left anterior descending coronary artery. Rats were pretreated with NBP (80 mg·kg^-1) for seven days. Cardiac function was quantified by echocardiography, myocardial infarction area was evaluated by TTC staining, and levels of creatine kinase-MB (CK-MB), troponin T (cTnI), superoxide dismutase (SOD), malondialdehyde (MDA), and lactate dehydrogenase (LDH) in myocardial tissue were detected by ELISA. Western blotting was used to analyze the protein expression differences of molecular targets in myocardial tissue. In addition, H9C2 rat cardiomyocytes were cultured in vitro. The cells were divided into: control group, normal culture; model group, subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) treatment; NBP group, pretreated with NBP (100 μmol·L^-1) for 4 h before OGD/R; NBP + LY group, pretreated with PI3K-specific inhibitor LY294002 15 μmol·L^-1 for 1 h on the basis of the NBP group; FTS (RAS inhibitor Salirasib) group, pretreated with FTS (39.36 μmol·L^-1) for 12 h before OGD/R; FTS + NBP group, pretreated with 100 μmol·L^-1 NBP for 4 h on the basis of the FTS group. Immunofluorescence and Western blotting were used to further verify and explore the action pathways of NBP. Results Network pharmacology results indicated that the possible signaling pathways involved in the action of NBP might include: lipid and atherosclerosis, PI3K/Akt signaling pathway, T-cell receptor signaling pathway, FoxO signaling pathway, apoptosis, MAPK signaling pathway, and Ras signaling pathway. In vivo experiments showed that compared with the control group, NBP pretreatment significantly reduced myocardial inflammatory response and oxidative stress induced by MIRI in SD rats, reduced infarction rate, enhanced ejection function, and activated PI3K signaling pathway protein expression while inhibiting apoptosis pathway protein expression, with significant differences (P < 0.05, 0.01, 0.001). In vitro experiments confirmed that NBP pretreatment regulated apoptosis proteins through the RAS/PI3K signaling pathway and activated HSPA12A expression. LY294002 and Salirasib could partially cancel its effect, and this regulatory mode might be unidirectional. The results of in vivo and in vitro experiments were mutually supportive. Conclusion NBP can effectively alleviate MIRI-induced apoptosis and exert cardioprotective effects, and the mechanism may be related to the RAS/PI3K signaling pathway.

R285

山东省中医药科技项目（M-2023061）