活血解毒Ⅰ号方抗动脉粥样硬化的网络药理学分析及实验验证

Network pharmacologic analysis and experimental verification of Huoxue Jiedu Ⅰ Prescription against atherosclerosis

目的 基于网络药理学探索活血解毒Ⅰ号方治疗动脉粥样硬化的物质基础和作用机制,并进行实验验证。方法 通过中药系统药理学数据库与分析平台检索活血解毒Ⅰ号方活性成分及其靶点,采用Uniprot蛋白质数据库标准化蛋白质靶点信息。通过在线人类孟德尔遗传、Gene Cards、Drug Bank数据库获取动脉粥样硬化相关靶点基因,筛选药物和疾病共同靶点,构建“成分-靶点”网络并筛选关键活性成分。通过STRING平台构建共同靶点蛋白质-蛋白质相互作用网络,筛选关键靶点基因。采用Matascape数据库进行GO和KEGG通路富集分析,构建“药物-靶点-通路”多维网络图。选用SPF级8周龄雄性ApoE小鼠30只,体重18~22 g,通过高脂饲料喂养12周建立动脉粥样硬化模型,按随机数字表法将其分为模型组、他汀组、活血解毒Ⅰ号方组,每组10只,另选相同周龄及遗传背景的雄性C57BL/6J小鼠10只作为正常对照组。他汀组予阿托伐他汀3.400 mg/(kg·d),活血解毒Ⅰ号方组予药液2.171 g/(kg·d),正常对照组、模型组予等量生理盐水,四组均灌胃12周。实验结束后采用比色法检测四组血脂指标,对主动脉行HE染色及斑块比例定量分析,Western blot检测四组主动脉组织中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、caspase-3和核因子-κBp65(NF-κBp65)蛋白表达水平。结果 共筛选出活血解毒Ⅰ号方活性成分25个,潜在靶点214个,与动脉粥样硬化疾病共同靶点118个。GO分析显示2 173个结果包括通路165条。活血解毒Ⅰ号方治疗动脉粥样硬化的关键活性成分包括槲皮素、黄芩素、β-谷甾醇、鞣花酸、小檗浸碱、非洲防己碱、小檗碱等,可调控TNF、AKT1、IL-6、VEGFA、TP53、IL-1B、JUN、CASP3、PTGS2、PPARG等相关靶点,并可能通过TNF、Toll样受体、NF-κB、HIF-1、PI3K-Akt、细胞凋亡、T细胞受体、MAPK、Foxo等信号通路对动脉粥样硬化的发生发展产生影响。实验结果显示,模型组血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)高于正常对照组,高密度脂蛋白胆固醇水平低于正常对照组,活血解毒Ⅰ号方组血清TC、TG、LDL-C低于模型组,差异有统计学意义(P <0.05)。主动脉根部HE染色结果显示,正常对照组主动脉根部无粥样斑块沉积,余各组主动脉根部管壁均可观察到不同程度的粥样斑块沉积。斑块比例定量分析显示,活血解毒Ⅰ号方组斑块面积占血管面积的比例低于模型组,差异有统计学意义(P<0.05)。模型组TNF-α、IL-6、caspase-3、NF-κBp65蛋白水平高于正常对照组,活血解毒Ⅰ号方组低于模型组,差异有统计学意义(P <0.05)。结论 本研究初步揭示了活血解毒Ⅰ号方治疗动脉粥样硬化的“多成分-多靶点-多通路”作用网络,实验验证其作用机制与抑制相关炎症通路及细胞凋亡有关,为活血解毒Ⅰ号方治疗动脉粥样硬化临床应用提供研究依据。

Objective To explore the material basis and mechanism of Huoxue Jiedu Ⅰ Prescription in the treatment of atherosclerosis based on network pharmacology, and to carry out experimental verification. Methods The active ingredients and targets of Huoxue Jiedu Ⅰ Prescription were retrieved through the traditional Chinese medicine systems pharmacology database and analysis platform, and the information of protein targets was standardized by using Uniprot protein database. Target genes related to atherosclerosis were obtained from online Mendelian inheritance in man,Gene Cards, and Drug Bank databases, and common targets of drugs and diseases were screened. The“ingredient-target”network was constructed and key active components were screened. The STRING platform was used to construct the common target protein-protein interaction network and screen the key target genes. Matascape database was used for enrichment analysis of GO and KEGG pathways, and a multidimensional network diagram of “drug-target-pathway”was constructed.Thirty SPF 8-week-old male ApoEmice, weighing 18-22 g, were fed with high-fat diet for 12 weeks to establish atherosclerosis model. They were divided into model group, statin group, and Huoxue Jiedu Ⅰ Prescription group according to the random number table method, with ten mice in each group. Ten male C57BL/6J mice of the same age and genetic background were selected as normal control group. Statin group was given 3.400 mg/(kg·d) of Atorvastatin, Huoxue Jiedu ⅠPrescription group was given 2.171 g/(kg·d) of drug liquid, normal control group and model group was given the same amount of normal saline. All the four groups were given gavage for 12 weeks. At the end of the experiment, colorimetric method was used to detect blood lipid indexes of the four groups, and HE staining and plaque proportion of aorta were quantitatively analyzed. The protein expressions of tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), caspase-3, and nuclear factor-κBp65(NF-κBp65) in aortic tissues of the four groups were detected by Western blot. Results A total of 25active ingredients of Huoxue Jiedu Ⅰ Prescription were screened, 214 potential targets were identified, and 118 targets were common with atherosclerotic diseases. GO analysis showed 2 173 results, including 165 pathways. The key active ingredients of Huoxue Jiedu Ⅰ Prescription in the treatment of atherosclerosis include quercetin, baicalin, β-sitosterol,ellagic acid, berberine, tetrandrine afrika, berberine, etc. It can regulate TNF, AKT1, IL-6, VEGFA, TP53, IL-1B, JUN,CASP3, PTGS2, PPARG, and other related targets. It may affect the occurrence and development of atherosclerosis through TNF, Toll-like receptor, NF-κB, HIF-1, PI3K-Akt, apoptosis, T-cell receptor, MAPK, Foxo, and other signaling pathways.The results of the experiment showed that serum total cholesterol(TC), triglyceride(TG), and low density lipoprotein cholesterol(LDL-C) in the model group were higher than those in the normal control group, and the level of high density lipoprotein cholesterol in the model group was lower than that in the normal control group, serum TC, TG, and LDL-C in the Huoxue Jiedu Ⅰ Prescription group were lower than those in the model group, and the differences were statistically significant(P < 0.05). The results of HE staining of aortic root showed that there was no atherosclerotic plaque deposition in the aortic root of the normal control group, and different degrees of atherosclerotic plaque deposition could be observed in the aortic root wall of the other three groups. Quantitative analysis of plaque proportion showed that the proportion of plaque area in the Huoxue Jiedu Ⅰ Prescription group was lower than that in the model group, and the difference was statistically significant(P < 0.05). The protein levels of TNF-α, IL-6, caspase-3, and NF-κBp65 in the model group were higher than those in the normal control group, while those in the Huoxue Jiedu Ⅰ Prescription group were lower than those in the model group, and the differences were statistically significant(P < 0.05). Conclusion This study preliminarily reveales the“multi-component, multi-target, multi-pathway”action network of Huoxue Jiedu Ⅰ Prescription in the treatment of atherosclerosis, and experimentally verifies that its mechanism of action is related to the inhibition of related inflammatory pathways and apoptosis, providing a research basis for the clinical application of Huoxue Jiedu Ⅰ Prescription in the treatment of atherosclerosis.