四逆散联合黄芪(SNSQ)治疗肝纤维化(HF)疗效显著,经临床实践证实。然而,其药理机制尚不清楚。本研究利用网络药理学来确定分子对接的关键靶标和蛋白质。此外,进行了动物实验以验证网络药理学结果,为SNSQ治疗HF的机制提供了进一步的见解。从中药系统药理学(TCMSP)和中药百科全书(ETCM)数据库中筛选出有效的SNSQ化合物。这些有效化合物的分子式结构从PubChem数据库获得。概率大于0.6的部分靶蛋白来自SWISS数据库。从SUPERPRED数据库检索对应于这些靶蛋白的UniprotID。化合物的剩余靶蛋白基于UniprotID从Uniprot数据库获得。然后总结药物靶蛋白。从GeneCards和OMIM数据库中选择与HF相关的目标点。在维恩图中确定了常见的目标点,并将其导入Cytoscape3.9.1软件以构建“SNSQ有效化合物-目标途径-HF”网络。利用AutoDock软件对具有高值的化合物和靶蛋白进行分子对接。使用DAVID数据库对共同目标点进行GO功能富集和KEGG途径富集分析。建立HF大鼠模型,测定血清AST和ALT活性。Hyp测定试剂盒用于检测肝组织中的Hyp含量。对促炎因子(IL-1β,TNF-α,IL-6)和抗炎因子(IL-10,TGF-β1,IL-4)在大鼠血清和肝脏中的表达。IL-1β,TNF-α,选择IL-10和TGF-β1用于通过ELISA验证。Westernblotting和qRT-PCR用于评估相关蛋白的表达。即NFKB1、NF-κBp65、NF-κBp50、α-SMA、和Col-1在肝组织中。qRT-PCR还用于研究ECM合成和增殖相关基因的表达,HSC-T6细胞和大鼠肝组织中的细胞周期蛋白D1、TIMP1、COL1A1,以及抑制HSC-T6细胞和大鼠肝组织中ECM相关基因MMP13。共预测了16个有效化合物,用山奈酚,谷甾醇,和异鼠李素表现出高度的价值。KEGG富集分析显示,SNSQ的靶基因富集在多个病理通路,以NF-κB信号通路为主。分子对接模拟表明SNSQ的主要成分山奈酚之间有很强的亲和力,谷甾醇,异鼠李素和NFKB1。实验结果表明,AST显著降低,ALT,和SNSQ组中的Hyp级别。促炎因子(IL-1β,TNF-α)显著降低,而抗炎因子(IL-10、TGF-β1)显著升高。α-SMA和Col-1的蛋白表达和转录水平显著降低,而NFKB1,NF-κBp65和NF-κBp50的表达明显升高。HSC-T6细胞和年夜鼠肝组织中CyclinD1、TIMP1、COL1A1的mRNA表达量显著降低,而MMP13mRNA表达水平显著升高。用SNSQ治疗HF涉及多个靶点和途径,与NFKB1的过表达和NF-κB信号通路的激活密切相关。其机制与炎症反应的激活密切相关,HSC激活,和扩散。
Si Ni San combined with Astragalus (SNSQ) has demonstrated significant efficacy in the treatment of hepatic fibrosis (HF), as confirmed by clinical practice. However, its pharmacological mechanism remains unclear. This
study employs network pharmacology to identify key targets and proteins for molecular docking. Additionally, animal experiments were conducted to validate the network pharmacology results, providing further insights into the mechanism of SNSQ in treating HF. Effective compounds of SNSQ were screened from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) and Encyclopedia of Traditional Chinese Medicine (ETCM) databases. Molecular formula structures of these effective compounds were obtained from the PubChem database. Partial target proteins with a probability greater than 0.6 were sourced from the SWISS database. Uniprot IDs corresponding to these target proteins were retrieved from the SUPERPRED database. The remaining target proteins of the compounds were obtained from the Uniprot database based on the Uniprot IDs. The drug target proteins were then summarized. Target points related to HF were selected from the GeneCards and OMIM databases. Common target points were identified in the Venn diagram and imported into Cytoscape 3.9.1 software to construct the \"SNSQ-effective compound-target pathway-HF\" network. AutoDock software was used for molecular docking of compounds and target proteins with high-degree values. The common target points underwent GO function enrichment and KEGG pathway enrichment analysis using the DAVID database. An HF rat model was established, and serum AST and ALT activities were measured. The Hyp assay kit was utilized to detect the Hyp content in liver tissue. To the transcription levels of pro-inflammatory factors (IL-1β, TNF-α, IL-6) and anti-inflammatory factors (IL-10, TGF-β1, IL-4) in rat serum and liver.IL-1β, TNF-α, IL-10, and TGF-β1 were chosen for validation through ELISA. Western blotting and qRT-PCR were used to assess the expression of related proteins, namely NFKB1, NF-κBp65, NF-κBp50, α-SMA, and Col-1 in liver tissue. qRT-PCR was also employed to
study the expression of ECM synthesis and proliferation-related genes, including Cyclin D1, TIMP1, COL1A1 in HSC-T6 cells and rat liver tissue, as well as the inhibition of the ECM-related gene MMP13 in HSC-T6 cells and rat liver tissue. A total of 16 valid compounds were predicted, with kaempferol, sitosterol, and isorhamnetin exhibiting high-degree values. KEGG enrichment analysis revealed that the target genes of SNSQ were enriched in multiple pathological pathways, with the NF-Kappa B signaling pathway being predominant. Molecular docking simulations indicated strong affinities between SNSQ\'s primary components-kaempferol, sitosterol, isorhamnetin-and NFKB1. Experimental results demonstrated significant reductions in AST, ALT, and Hyp levels in the SNSQ group. Pro-inflammatory factors (IL-1β, TNF-ɑ) were markedly reduced, while anti-inflammatory factors (IL-10, TGF-β1) were substantially increased. The protein expression and transcription levels of α-SMA and Col-1 were significantly decreased, whereas those of NFKB1, NF-κBp65, and NF-κBp50 were notably elevated. mRNA expression levels of Cyclin D1, TIMP1, COL1A1 in HSC-T6 cells and rat liver tissue were significantly decreased, whereas MMP13 mRNA expression level was significantly increased. Treatment of HF with SNSQ involves multiple targets and pathways, with a close association with the overexpression of NFKB1 and activation of the NF-Kappa B signaling pathway. Its mechanism is closely linked to the activation of inflammatory responses, HSC activation, and proliferation.