基于多层次交互网络和体内实验探讨藤茶总黄酮抗痛风性关节炎的作用机制

Mechanism of total flavonoids from Ampelopsis grossedentata against gouty arthritis based on multi-level interactive network and in vivo experimental validation

基于网络药理学预测分析藤茶总黄酮(total flavonoids from Ampelopsis grossedentata,AGTF)防治痛风性关节炎(gouty arthritis, GA)的作用机制,并进行实验验证。利用相关数据库,结合文献资料,筛选出AGTF的主要活性成分、作用靶点及疾病靶点,并分别构建蛋白质相互作用(protein-protein interaction, PPI)网络和“药物-关键成分-潜在靶点-核心通路”网络,预测AGTF抗GA潜在的作用靶点和机制。通过建立高尿酸血症(hyperuricemia, HUA)合并GA大鼠模型,对大鼠的步态行为评分,观察踝关节肿胀度;检测大鼠血清中尿酸(uric acid, UA)水平和黄嘌呤氧化酶(xanthine oxidase, XOD)活性,以及白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)含量;采用免疫组化法测定大鼠踝关节滑膜组织中Toll样受体-4(toll-like receptor 4,TLR4)、髓性分化因子-88(myeloid differentiation factor 88,MyD88)和核因子-κB(nuclear factor-kappa B,NF-κB)的蛋白表达。网络药理学筛选出AGTF有效活性成分10个,AGTF抗GA的候选靶点73个;KEGG富集显著的信号通路86条,其中与AGTF抗GA密切相关的通路有TNF信号通路、NF-κB信号通路、Toll样受体信号通路、NOD样受体信号通路及嘌呤代谢等。动物实验结果显示,AGTF能有效改善GA大鼠异常步态行为、缓解踝关节炎症反应、降低踝关节肿胀度;同时,AGTF能显著降低UA水平,抑制XOD活性,减少TNF-α、IL-6和IL-1β含量,下调踝关节滑膜组织TLR4、MyD88和NF-κB的表达(P<0.05,P<0.01)。网络药理学预测与验证实验结果相吻合,均表明AGTF通过调控尿酸代谢、改善尿酸水平异常、减少炎症因子释放、调节免疫及炎症TLR4/MyD88/NF-κB通路等多途径综合发挥防治GA的药效作用。

The present study investigated the mechanism of total flavonoids from Ampelopsis grossedentata(AGTF) against gouty arthritis(GA) by network pharmacology and experimental validation. The main active ingredients and targets of AGTF, as well as disease targets, were screened out using relevant databases and literature data. The "protein-protein interaction"(PPI) network and "drug-ingredient-target-pathway" network were constructed, and the potential targets and mechanism of AGTF against GA were predicted. The hyperuricemia(HUA) combined with GA model was induced in rats. The gait behaviors of rats were scored, and ankle swelling degree was observed. The uric acid(UA) level and xanthine oxidase(XOD) activity in the rat serum were detected, and the levels of interleukin-1β(IL-1β), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) were measured. The protein expression of toll-like receptor 4(TLR4), myeloid differentiation factor 88(MyD88), and nuclear factor-kappa B(NF-κB) in the synovial tissues of the rat ankle joint was determined by immunohistochemistry. Ten active ingredients of AGTF and 73 candidate targets of AGTF against GA were screened out by network pharmacology. Eighty-six signaling pathways were enriched, including TNF signaling pathway, NF-κB signaling pathway, TLR signaling pathway, Nod-like receptor signaling pathway, and purine metabolism signaling pathway, which were closely related to AGTF against GA. Animal experimental results showed that AGTF could effectively improve the abnormal gait behaviors of GA rats, relieve ankle inflammation, and reduce ankle joint swelling. In addition, AGTF could significantly reduce UA level, inhibit XOD activity, decrease TNF-α, IL-6, and IL-1β content, and down-regulate the expression of TLR4, MyD88, and NF-κB in ankle synovial tissues(P<0.05, P<0.01). The results of network pharmacology and experimental validation are consistent, indicating that AGTF exerts its therapeutic effect on GA by regulating UA metabolism, improving abnormal UA level, reducing the release of inflammatory factors, and regulating immunity and the TLR4/MyD88/NF-κB inflammatory pathway.