Mechanisms and network pharmacological analysis of Yangyin Fuzheng Jiedu prescription in the treatment of hepatocellular carcinoma.
Mechanisms and network pharmacological analysis of Yangyin Fuzheng Jiedu prescription in the treatment of hepatocellular carcinoma.
复制标题
养阴扶正解毒方治疗肝癌的作用机制及网络药理学分析
作者：
筛选养阴扶正解毒方的关键药物，并应用网络药理学方法研究其抗肝癌作用及其可能机制。建立H22荷瘤小鼠模型。30只雄性BALB/c小鼠随机分为5组。给小鼠连续灌胃YFJP拆方或生理盐水14天。在治疗期间每2天称重小鼠，并通过拍照观察肿瘤的外观。计算肿瘤抑制率、脾脏指数和胸腺指数。采用苏木精-伊红染色和免疫组化染色观察组织学变化和肿瘤浸润淋巴细胞。通过末端脱氧核苷酸转移酶介导的dUTP缺口末端标记染色测定细胞凋亡。使用流式细胞术分析CD 8 + T细胞的比例和程序性细胞死亡蛋白1（PD-1）、T细胞免疫球蛋白结构域和粘蛋白结构域-3（Tim-3）以及具有IG和ITIM结构域的T细胞免疫受体（TIGIT）的表达。使用Milliplex® MAP小鼠高灵敏度T细胞组试剂盒检测血清细胞因子的产生。从相应的数据库中获得关键药物的活性成分和HCC相关靶蛋白。通过靶点定位筛选HCC治疗的推定靶点，并通过构建组分-靶点网络筛选潜在活性组分。从STRING数据库中获得可能的相互作用靶点，构建蛋白质-蛋白质相互作用网络。基于潜在靶标进行基因本体论（GO）和基因和基因组途径富集分析的京都百科全书。构建基因-基因内部网络和组分-靶点-通路网络并进行分析，筛选关键靶点。使用蛋白质印迹法评估荷瘤小鼠模型中关键靶标的蛋白质表达。通过分子对接验证了关键靶点和化合物的结合活性。在YFJP的三个拆方中，扶正方（FZP）在治疗H22荷瘤小鼠期间显示出显著的抗肿瘤作用，并抑制体重减轻。FZP可提高H22荷瘤小鼠的免疫器官指数、脾脏和外周血中CD 8+和CD 3 + T细胞水平。FZP还降低了CD 8 + T细胞中PD-1、TIGIT和TIM 3的表达以及IL-10、IL-4、IL-6和IL-1β的产生。网络药理学和实验验证表明，FZP治疗HCC的关键靶点为PIK 3CA、TP 53、MAPK 1、MAPK 3和EGFR。基于HCC相关信号通路（包括癌症中的PIK 3-Akt信号通路、PD-L1表达和PD-1检查点通路）评估对HCC的治疗效果。GO富集分析表明，FZP通过膜筏、膜微区等细胞成分正向调节细胞表面转移酶和激酶的分子功能，抑制细胞死亡和程序性细胞死亡。FZP是YFJP抗肝癌和免疫调节作用的关键拆方。FZP通过HCC相关靶点、途径和生物学过程缓解T细胞耗竭并改善免疫抑制微环境。通过体内实验筛选YFJP的关键药物，探讨其抗肿瘤作用机制。通过网络药理学、GO和KEGG富集分析、分子对接等方法进一步探讨关键药物的潜在作用机制。
To identify the key drugs of Yangyin Fuzheng Jiedu prescription (YFJP) and investigate their therapeutic effects against hepatocellular carcinoma (HCC) and the potential mechanism using network pharmacology. The H22 tumor‐bearing mouse model was established. Thirty male BALB/c mice were divided randomly into five groups. The mice were orally treated with either disassembled prescriptions of YFJP or saline solution continuously for 14 days. The mice were weighed every 2 days during treatment and the appearance of tumors was observed by photographing. The tumor inhibition rate and the spleen and thymus indexes were calculated. Hematoxylin and eosin and immunohistochemical staining were performed to observe the histological changes and tumor‐infiltrating lymphocytes. Cell apoptosis was determined by terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling staining. The proportion of CD8+ T cells and the expression of programmed cell death protein 1 (PD‐1), T cell immunoglobulin domain and mucin domain‐3 (Tim‐3), and T cell immunoreceptor with Ig and ITIM domains (TIGIT) were analyzed using flow cytometry. The production of serum cytokines was detected using the Milliplex® MAP mouse high sensitivity T cell panel kit. The active components of the key drugs and HCC‐related target proteins were obtained from the corresponding databases. The putative targets for HCC treatment were screened by target mapping, and potential active components were screened by constructing a component‐target network. The interactive targets of putative targets were obtained from the STRING database to construct the protein–protein interaction network. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes pathway enrichment analyses were performed based on potential targets. The gene–gene inner and component‐target‐pathway networks were constructed and analyzed to screen the key targets. Western blotting was used to evaluate the protein expression of the key targets in the tumor‐bearing mouse model. The binding activity of the key targets and compounds was verified by molecular docking. Among the three disassembled prescriptions of YFJP, the Fuzheng prescription (FZP) showed significant antitumor effects and inhibited weight loss during the treatment of H22 tumor‐bearing mice. FZP increased the immune organ index and the levels of CD8+ and CD3+ T cells in the spleen and peripheral blood of H22 tumor‐bearing mice. FZP also reduced the expression of PD‐1, TIGIT, and TIM3 in CD8+ T cells and the production of IL‐10, IL‐4, IL‐6, and IL‐1β. Network pharmacology and experimental validation showed that the key targets of FZP in the treatment of HCC were PIK3CA, TP53, MAPK1, MAPK3, and EGFR. The therapeutic effect on HCC was evaluated based on HCC‐related signaling pathways, including the PIK3‐Akt signaling pathway, PD‐L1 expression, and PD‐1 checkpoint pathway in cancer. GO enrichment analysis indicated that FZP positively regulated the molecular functions of transferases and kinases on the cell surface through membrane raft, membrane microarea, and other cell components to inhibit cell death and programmed cell death. FZP was found to be the key disassembled prescription of YFJP that exerted antitumor and immunoregulatory effects against HCC. FZP alleviated T cell exhaustion and improved the immunosuppressive microenvironment via HCC‐related targets, pathways, and biological processes. In vivo experiments were conducted to screen the key drugs of YFJP and explore its anti‐tumor mechanism. Network pharmacology, GO and KEGG enrichment analysis, molecular docking were conducted to further explore the potential mechanisms of the key drugs.