中药药理与临床

目的:采用网络药理学-分子对接的方法，探讨黄芪桂枝五物汤预防运动性疲劳(Exercise-induced fatigue, EF)的作用机制，并进行动物实验验证分析。方法:通过网络药理学工具获得黄芪桂枝五物汤预防EF的有效成分、核心靶点和信号通路。构建EF大鼠模型，进行HE染色、ELISA、PCR法验证网络药理学预测的主要靶点及预防效果。结果:筛选出黄芪桂枝五物汤中43个活性成分和242个潜在靶点，运动性疲劳潜在靶点1 002个；药物和疾病交集靶点104个，包括AKT1、IL6、TNF、IL1B、PPARG、PTGS2等。KEGG富集分析结果包括AGE-RAGE信号通路、TNF信号通路、IL-17信号通路、HIF-1信号通路等；分子对接结果显示豆甾醇、刺芒柄花素及原阿片碱等活性成分与PTGS2、PTGS1、CHRM1等关键靶点表现出良好的对接活性，黄芪桂枝五物汤可能通过影响AKT1、IL-6、TNF、IL-1B、PPARG、PTGS2等靶蛋白的表达进而调控炎症反应和细胞凋亡。黄芪桂枝五物汤可以使大鼠肌纤维排列更紧密，增强大鼠四肢肌力、运动耐力、心脏供血能力，且缓解心脏过度代偿。与正常对照组比较，模型对照组游泳力竭时间与四肢抓力显著减少，血清中肌酸激酶(CK)、血乳酸(BLA)、白介素6(IL-6)、肿瘤坏死因子(TNF-α)、缺氧诱导因子(HIF-1α)的含量显著升高，血管内皮生长因子(VEGF)与促红细胞生成素(EPO)的含量显著降低(P<0.01),雌激素受体1(Esr1)表达下调，前列腺素内过氧化物合酶2(Ptgs2)、蛋白激酶1(Akt1)、过氧化物酶体增殖物激活受体γ(Pparg)和果寡糖(Fos)mRNA表达上调(P<0.05);与模型对照组比较，黄芪桂枝五物汤组游泳力竭时间与四肢抓力显著增加，血清中CK、BLA、IL-6、TNF-α、HIF-1α的含量显著减少；VEGF、EPO的含量显著增加(P<0.01);Esr1 mRNA表达上调，Ptgs2、Akt1、Pparg、Fos mRNA表达下调(P<0.05)。结论:黄芪桂枝五物汤中槲皮素、β-谷甾醇、山柰酚等化合物发挥核心作用，通过影响PTGS2、PTGS1、ADRB2等靶蛋白的表达并参与AGE-RAGE、TNF、IL-17信号传导途径，发挥预防运动性疲劳的作用。

Objective:To investigate the mechanism of action of Huangqi Guizhi Wuwu(黄芪桂枝五物) Decoction in the treatment of exercise-induced fatigue(EF) by using network pharmacology and molecular docking, and to validate and analyze it through animal experiments. Methods:The active ingredients, core targets and signaling pathways of Huangqi Guizhi Wuwu Decoction in the treatment of EF were obtained by network pharmacology tools. An EF rat model was constructed, and HE staining, ELISA,and PCR experiments were performed to verify the principal targets and therapeutic effects predicted by network pharmacology. Results:A total of 43 active ingredients and 242 potential targets in Huangqi Guizhi Wuwu Decoction were screened out, and 1002 potential targets for EF. A total of 104 targets was observed at the intersection of drug and disease, including AKT1,IL6,TNF,IL1B,PPARG,PTGS2,etc. The results of KEGG enrichment analysis showed that AGE-RAGE,TNF,IL-17,TNF,IL-17and HIF-1 signaling pathways were involved. The molecular docking results showed that the active ingredients, such as leguminol, prickly aristolochicin and proto-opiomelanocarpine hadgood binding activities with key targets, such as PTGS2,PTGS1,CHRM1,etc. Huangqi Guizhi Wuwu Decoction affected the expression of target proteins, such as AKT1,IL-6,TNF,IL-1B,PPARG,PTGS2,etc.,and thus regulated inflammatory response and apoptosis. Animal experiment results showed that Huangqi Guizhi Wuwu Decoction could induce rat muscle fibers more closely aligned, enhance rat limb muscle strength, exercise endurance, cardiac blood supply, and alleviate the cardiac overcompensation. Compared with the control group, the swimming exhaustion time and limb grip strength were significantly reduced in the model group. The serum levels of creatine kinase(CK),blood lactate(BLA),interleukin 6(IL-6),tumor necrosis factor-α(TNF-α),and hypoxia-inducible factor-1α(HIF-1α) were significantly elevated. The levels of vascular endothelial growth factor(VEGF) and erythropoietin(EPO) were significantly reduced(P<0.01). The mRNA expression of estrogen receptor 1(ESR1) was down regulated, and the mRNA expressions of prostaglandin endometriosis synthase 2(Ptgs2),threonine kinase 1(Akt1),peroxisome proliferator-activated receptor γ(Pparg),and fructooligosaccharides(Fos) were up regulated(P<0.05). Compared with the model group, the swimming exhaustion time and limb grip strength was significantly increased in the Huangqi Guizhi Wuwu Decoction group, the serum levels of BLA,CK,IL-6,TNF-α,and HIF-1α were significantly decreased, and the levels of VEGF and EPO were significantly increased(P<0.01). The mRNA expression of Esr1 was up regulated, while the mRNA expressions of Ptgs2,Akt1,Pparg,and Fos were down regulated(P<0.05). Conclusion:Quercetin, β-sitosterol, kaempferol and other compounds in Huangqi Guizhi Wuwu Decoction played a key role in the prevention and treatment of EF by affecting the expression of target proteins, such as PTGS2,PTGS1 and ADRB2,and participating in the signaling pathways, such as AGE-RAGE,TNF and IL-17 signaling pathways.