基于TLR4、NF-κB信号通路探讨当归拈痛汤治疗痛风性关节炎的作用机制

Mechanism of Danggui Niantong decoction for gouty arthritis based on TLR4 and NF-κB signaling pathway

目的通过网络药理学方法探讨当归拈痛汤改善痛风性关节炎(GA)的作用机制,并通过动物体内实验验证分析。方法通过TCMSP数据库筛选当归拈痛汤的活性成分,并检索对应靶点,在GeneCards、OMIM等数据库检索GA的相关靶点。利用韦恩图得到药物-疾病交集靶点,用Cytoscape 3.6.1软件分别构建“中药-活性成分-靶点”作用网络及PPI蛋白互作网络。用R语言对交集靶点进行GO和KEGG分析。结合网络药理学分析结果,采用注射尿酸钠晶体溶液诱导建立GA大鼠模型,验证当归拈痛汤改善GA可能的分子机制。结果确定当归拈痛汤治疗GA的活性成分315个,治疗靶点110个,PPI网络筛选得到20个核心靶点。GO分析表明靶点主要涉及DNA结合转录因子结合、磷酸酶结合和RNA聚合酶Ⅱ特异性DNA结合转录因子结合等多种生物过程;KEGG分析提示NF-κB、Toll样受体及TNF信号通路在当归拈痛汤治疗GA过程中起着关键作用。动物实验表明,与空白对照组相比,模型对照组关节肿胀度显著升高,血清中IL-1β、TNF-α、IL-13水平升高;血清UREA、CREA无明显差异;关节滑膜组织HE染色显示模型对照组滑膜细胞增生,局部细胞变性坏死,大量炎性细胞浸润;TLR4、NF-κB-p65蛋白表达明显上调。与模型对照组相比,各给药组关节肿胀度均有降低;血清中IL-1β、TNF-α水平均降低,IL-13差异无统计学意义;关节滑膜组织HE染色显示双氯芬酸钠组与当归拈痛汤组较模型对照组炎性细胞浸润明显减少。TLR4、NF-κB-p65蛋白表达双氯芬酸钠组、当归拈痛汤组较模型对照组明显降低。结论当归拈痛汤可能通过调节NF-κB、Toll样受体及TNF等信号通路上相关蛋白表达改善GA。

Objective To determine the mechanism underlying the therapeutic effect of Danggui Niantong decoction on gouty arthritis(GA)with network pharmacological methods and validate it via in vivo animal experiments.Methods The active components of Danggui Niantong decoction were screened in the TCMSP database,and their corresponding targets were identified.Relevant targets for GA were searched in GeneCards,OMIM,and other databases.Drug-disease intersection targets were obtained with a Wayne diagram,and an interaction network between traditional Chinese medicine active ingredients and their targets as well as a protein-protein interaction network were established with Cytoscape 3.6.1 software.GO and KEGG analysis was performed on intersection targets by R language.Based on the network pharmacological analysis,we established a rat model of GA by injecting sodium urate crystal solution to verify the possible molecular mechanism of the efficacy of Danggui Niantong decoction for GA.Results Totally 315 active ingredients and 110 therapeutic targets were identified.Additionally,a protein-protein interaction(PPI)network was established to select 20 core targets.Gene ontology(GO)analysis revealed that these targets primarily participated in various biological processes,including DNA binding transcription factor binding,phosphatase binding,and RNA polymeraseⅡspecific DNA binding transcription factor binding.Furthermore,KEGG analysis indicated that the NF-κB,Toll-like receptors,and TNF signaling pathways played pivotal roles in the therapeutic effect of Danggui Niantong decoction on GA.Animal experiments demonstrated significant joint swelling and elevated levels of serum IL-1β,TNF-α,and IL-13 in the model group as compared with those in the blank group.Histological examination with HE staining revealed synovial cell proliferation as well as degeneration and necrosis of local cells accompanied by infiltration from numerous inflammatory cells in the model group.Moreover,upregulation of TLR4 and NF-κB-p65 protein expression levels was significantly.Compared with the model group,each medication group demonstrated a reduction in the degree of joint swelling.Levels of IL-1β,TNF-αdecreased in the serum,while there was no significant difference in IL-13.HE staining revealed reduced inflammatory cell infiltration in both the diclofenac sodium group and Danggui Niantong decoction group as compared with the model group.Expression levels of TLR4 and NF-κB-p65 protein in the model group were significantly lower than both the sodium bifenate group and Danggui Niantong decoction groups.Conclusion Danggui Niantong decoction may modulate the expression of relatd proteins in signaling pathways to ameliorate GA,including NF-κB,Toll-like receptor,and TNF pathways.These findings provide a theoretical and practical foundation for the clinical application of Danggui Niantong decoction.