[关键词]
[摘要]
目的 采用网络药理学结合体外实验验证的方法,探讨补阳还五汤苷类组分治疗动脉粥样硬化(atherosclerosis,AS)的作用机制。方法 采用网络药理学方法对补阳还五汤苷类组分与阿托伐他汀治疗AS的靶点和通路进行预测;SD大鼠随机分为对照组、补阳还五汤(10.8 g/kg)组、苷类组分(0.64 g/kg)组和阿托伐他汀(0.9 mg/kg)组,各给药组ig相应药物,2次/d,连续7次,制备空白血清、补阳还五汤血清、苷类组分血清及阿托伐他汀血清。采用试剂盒考察含药血清对氧化型低密度脂蛋白(oxidized low density lipoprotein,ox-LDL)诱导的小鼠单核巨噬细胞RAW264.7脂质含量的影响;采用油红O染色考察含药血清对ox-LDL诱导的RAW264.7细胞泡沫细胞形成率的影响;采用ELISA试剂盒观察含药血清对ox-LDL诱导的RAW264.7细胞上清液肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素-6(interleukin-6,IL-6)水平的影响;采用Western blotting考察含药血清对ox-LDL诱导的RAW264.7细胞Janus蛋白酪氨酸激酶2(Janus kinase-2,JAK2)、磷酸化JAK2(p-JAK2)、信号转导和转录激活因子3(signal transducer and activator of transcription 3,STAT3)和p-STAT3蛋白表达的影响;采用qRT-PCR考察含药血清对ox-LDL诱导的RAW264.7细胞JAK2和STAT3 mRNA表达的影响。结果 与模型组比较,各给药组细胞内脂质沉积减少,RAW264.7细胞吞噬脂质受到抑制,泡沫化细胞数量减少,上清液中TNF-α和IL-6水平显著降低(P<0.01),细胞内总胆固醇(total cholesterol,TC)、游离胆固醇(free cholesterol,FC)和胆固醇酯(cholesteryl ester,CE)含量显著减少(P<0.01),JAK2/STAT3通路相关蛋白及mRNA表达水平显著降低(P<0.01)。结论 补阳还五汤苷类组分是治疗AS的主要药效物质,其作用机制可能与降低细胞内脂质沉积、抑制JAK2/STAT3信号通路、抑制炎性因子分泌有关。
[Key word]
[Abstract]
Objective To explore the mechanism of Buyang Huanwu Decoction (补阳还五汤) glycosides on treatment of atherosclerosis (AS) by network pharmacology combined with in vitro experiment. Methods Network pharmacology method was used to predict the target and pathway of Buyang Huanwu Decoction glycosides and atorvastatin in the treatment of AS. SD rats were randomly divided into control group, Buyang Huanwu Decoction (10.8 g/kg) group, glycoside component (0.64 g/kg) group and atorvastatin (0.9 mg/kg) group, rats in each administration group were ig corresponding drugs, twice one day for 7 d. The blank serum, Buyang Huanwu Decoction serum, glycoside component serum and atorvastatin serum were prepared. Kit was used to investigate the effect of drug-containing serum on lipid content of RAW264.7 cells induced by oxidized low-density lipoprotein (ox-LDL); Oil red O staining was used to investigate the effect of drug-containing serum on foam cell formation rate of RAW264.7 cells induced by ox-LDL; ELISA kit was used to investigate the effect of drug-containing serum on levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the supernatant of RAW264.7 cells induced by ox-LDL; Western blotting was used to investigate the effect of drug-containing serum on expressions of Janus protein tyrosine kinase 2 (JAK2), phosphorylated JAK2 (p-JAK2), signal transducer and activator of transcription 3 (STAT3) and p-STAT3 in RAW264.7 cells induced by ox-LDL; qRT-PCR was used to investigate the effect of drug-containing serum on JAK2 and STAT3 mRNA expressions in RAW264.7 cells induced by ox-LDL. Results Compared with model group, the intracellular lipid deposition of each administration group was reduced, the phagocytic lipid of RAW264.7 cells was inhibited, the number of foamed cells was reduced; Levels of TNF-α and IL-6 in the supernatant were significantly reduced (P < 0.01); Intracellular total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) content were significantly reduced (P < 0.01); Expressions of JAK2/STAT3 pathway related protein and mRNA were significantly reduced (P < 0.01). Conclusion Glycoside components of Buyang Huanwu Decoction are the main pharmacological substances for the treatment of AS, of which mechanism may be related to the reduction of intracellular lipid deposition, inhibition of JAK2/STAT3 signal pathway, and inhibition the secretion of inflammatory factors.
[中图分类号]
R285.5
[基金项目]
国家自然科学基金资助项目(81774032);湖南省自然科学杰出青年基金资助项目(2020JJ2024);湖南省自然科学青年基金资助项目(2018JJ3391);湖南省教育厅科学研究重点项目(19A374);湖南中医药大学研究生培养质量工程专项(2020CX62)