中药药理与临床

目的:基于网络药理学结合实验验证，探讨补阳还五汤含药血清对高糖损伤血管内皮细胞的影响。方法:基于复方网络药理学从TCMSP数据库、化源网、Swisstarget prediction数据库获取补阳还五汤有效化学成分及靶点，通过GeneCards、OMIM、Drugbank数据库收集糖尿病血管并发症相关靶点，运用String数据库构建关键靶点PPI网络图。基于Metascape数据库进行GO和KEGG富集分析。使用Autodock和Pymol软件进行分子对接验证。建立高糖诱导的HUVECS细胞损伤模型，给予5%、10%补阳还五汤含药血清；采用CCK-8法检测HUVECs细胞增殖情况，TBA法测定MDA含量；黄嘌呤氧化酶法测定SOD活性；Annexin V-FITC/PI双标记法联合Hoechst 33258、JC-1检测细胞凋亡情况；ELISA法检测IL-6、TNF-ɑ炎性因子含量；Western Blot法检测VEGF、p-AKT和AKT蛋白的表达。结果:补阳还五汤治疗糖尿病血管并发症有效成分为69个，潜在靶点306个，涉及的主要有效成分为槲皮素、山柰酚、β-谷甾醇，关键靶点有血管内皮生长因子(VEGFA),细胞炎性因子TNF、IL6等；生物学过程涉及对激素的反应、细胞对氮化合物的反应及对细胞因子的反应等，细胞组成涉及膜筏、膜侧和受体复合物等；分子功能涉及激酶活性与结合、脂质结合和转录因子结合等过程，其发挥作用机制的通路可能与脂质和动脉粥样硬化，细胞凋亡与衰老，生长激素的合成分泌，胰岛素抵抗，糖尿病并发症中的AGE-RAGE信号通路及PI3K-AKT信号通路等相关。基于网络药理学结果佐以实验验证，结果表明与模型对照组比较，18.5 g/kg补阳还五汤10%含药血清组细胞存活率显著增加(P<0.01),18.5 g/kg补阳还五汤含药血清5%、10%组可降低受损内皮细胞MDA含量(P<0.05或P<0.01),显著提高SOD活力(P<0.01),显著抑制细胞凋亡，降低受损内皮细胞IL-6、TNF-ɑ含量(P<0.05或P<0.01),降低VEGF、p-AKT/AKT蛋白表达(P<0.01),改善高糖状态的血管内环境。结论:补阳还五汤能够改善高糖状态的血管内环境，治疗糖尿病血管并发症，机制可能与其保护高糖损伤内皮细胞，促进细胞增殖，抑制细胞凋亡及炎症因子生成，以及下调VEGF和p-AKT/AKT蛋白，调节调控PI3K/AKT信号通路有关。

Objective: To investigate the effects of Buyang Huanwu Decoction(补阳还五汤) containing serum on high glucose-induced vascular endothelial cell injury based on network pharmacology and experimental validation. Methods:Based on network pharmacology, the active chemical components and targets of Buyang Huanwu Decoction were obtained from TCMSP database, ChemSource and Swisstarget prediction databases, and diabetic vascular complications related targets were collected from GeneCards, OMIM and DrugBank databases.The PPI network of the key targets was constructed by using the String database. The GO and KEGG enrichment analysis were performed based on Metascape database. Molecular docking validation was performed using Autodock and Pymol software. A high glucose-induced HUVECS cell injury model was established, and 5% and 10% Buyang Huanwu Decoction-containing serum was given to the cells. CCK-8 assay was used to detect the cell proliferation of HUVECs, the TBA method was used to determine the MDA content, and the xanthine oxidase method was used to determine the SOD activity. Annexin V-FITC/PI double-labeling combined with Hoechst 33258 and JC-1 staining was used to detect apoptosis. ELISA was used for the detection of IL-6 and TNF-ɑ levels, and Western Blot was used to detect protein expressions of vascular endothelial growth factor(VEGF),p-Akt and Akt. Results:A total of 69 active ingredients and 306 potential targets were involved in the treatment of diabetic vascular complications with Buyang Huanwu Decoction. The main active ingredients involved quercetin, kaempferol, and β-sitosterol, and the key targets were vascular endothelial growth factor(VEGFA),cellular inflammatory factors, such as TNF and IL6,etc. The biological process involved the response to hormones, cellular response to nitrogen compounds, and cellular response to cytokines, etc.,and the cellular composition involved membrane rafts, membrane sides and receptor complexes, etc. The molecular function involved kinase activity and binding, lipid binding, transcription factor binding, etc. The pathways of its possible mechanism of action may be related to lipid and atherosclerosis, apoptosis and senescence, growth hormone synthesis and secretion, insulin resistance, AGE-RAGE signaling pathway in diabetes complications, PI3K-Akt signaling pathway, etc. Based on the results of network pharmacology and experimental verification, compared with the model control group, the cell survival rate of the 18.5 g/kg Buyang Huanwu Decoction-containing serum 10% group was significantly increased(P<0.01),and the MDA content of damaged endothelial cells was reduced(P<0.05 or P<0.01),while the SOD activity was increased(P<0.01) in the 18.5 g/kg Buyang Huanwu Decoction-containing serum 5% and 10% groups. Compared with the model group, in the 18.5 g/kg Buyang Huanwu Decoction-containing serum 5% and 10% groups, cell apoptosis was significantly inhibited, and the contents of IL-6 and TNF-ɑ were decreased in damaged endothelial cells. In addition, the protein expression of VEGF(P<0.01),and p-AKT/AKT(P<0.01) were down-regulated, and the intravascular environment under hyperglycemic state was improved. Conclusion:Buyang Huanwu Decoction can improve the intravascular environment under hyperglycemic state in the treatment diabetic vascular complications, which may be related to its ability of protecting the endothelial cells in high glucose state, promoting cell proliferation, inhibiting apoptosis and inflammatory factor production, down-regulating the protein expression of VEGF and p-Akt/Akt, and regulating PI3k/Akt signaling pathway.