TMT quantitative proteomics and network pharmacology reveal the mechanism by which asiaticoside regulates the JAK2/STAT3 signaling pathway to inhibit peritoneal fibrosis
TMT quantitative proteomics and network pharmacology reveal the mechanism by which asiaticoside regulates the JAK2/STAT3 signaling pathway to inhibit peritoneal fibrosis
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DOI：
10.1016/j.jep.2023.116343
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发表时间：
2023-03-13
影响因子：
5.4
通讯作者：
Sheng, Meixiao
中科院分区：
文献类型：
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作者：
Sun, Jinyi;Tang, Lei;Sheng, Meixiao
民族药理学相关性：传统中药积雪草 (Centella asiatica (L.) Urb.) 已在临床中广泛用于治疗多种纤维化疾病。积雪草苷（ASI）作为一种重要的活性成分，在该领域备受关注。然而，ASI 对腹膜纤维化（PF）的影响仍不清楚。因此，我们评估了 ASI 对 PF 和间皮间质转化 (MMT) 的益处，并揭示了其潜在机制。研究目的：本研究的目的是利用蛋白质组学和网络药理学预测 ASI 对抗腹膜间皮细胞 (PMC) MMT 的潜在分子机制，并通过体内和体外研究对其进行证实。材料和方法：使用串联质量标签（TMT）技术对腹膜纤维化小鼠和正常小鼠的肠系膜差异表达的蛋白质进行定量分析。接下来，通过网络药理学分析筛选ASI抗PF的核心靶基因，并利用Cytoscape Version 3.7.2构建PPI和C-P-T网络。根据差异蛋白及核心靶基因的GO和KEGG富集分析结果，选择关联度较高的信号通路作为ASI抑制PMCs MMT的关键信号通路，进行进一步的分子对接分析和实验验证。结果：基于TMT的定量蛋白质组分析结果鉴定出5727个蛋白，其中70个蛋白下调，178个蛋白上调。其中，腹膜纤维化小鼠肠系膜中STAT1、STAT2和STAT3的水平显着低于对照组，表明STAT家族在腹膜纤维化的发病机制中发挥着重要作用。然后，通过网络药理学分析总共鉴定了 98 个 ASI-PF 相关靶点。 JAK2 是代表潜在治疗靶点的十大核心靶基因之一。 JAK/STAT 信号传导可能代表 ASI 介导 PF 效应的核心途径。分子对接研究表明，ASI 有可能与 JAK/STAT 信号通路中涉及的靶基因（例如 JAK2 和 STAT3）发生良好的相互作用。实验结果表明，ASI可以显着减轻葡萄糖酸氯己定（CG）引起的腹膜组织病理学变化，并增加JAK2和STAT3磷酸化水平。在TGF-β1刺激的HMrSV5细胞中，E-钙粘蛋白表达水平显着降低，而波形蛋白、p-JAK2、α-SMA和p-STAT3表达水平显着增加。 ASI抑制TGF-β1诱导的HMrSV5细胞MMT，降低JAK2/STAT3信号传导的激活，并增加p-STAT3的核转位，这与JAK2/STAT3通路抑制剂AG490的作用一致。结论：ASI可通过调节JAK2/STAT3信号通路抑制PMCs MMT，减轻PF。
Ethnopharmacological relevance: Traditional Chinese medicine, Centella asiatica (L.) Urb., has been extensively utilized in clinics to treat a variety of fibrotic disorders. Asiaticoside (ASI), as an important active ingredient, has attracted much attention in this field. However, the effect of ASI on peritoneal fibrosis (PF) is still unclear. Therefore, we evaluated the benefits of ASI for PF and mesothelial-mesenchymal transition (MMT) and revealed the underlying mechanisms. Aim of study: The objective of this investigation was to anticipate the potential molecular mechanism of ASI against peritoneal mesothelial cells (PMCs) MMT employing proteomics and network pharmacology, and to confirm it using in vivo and in vitro studies. Materials and methods: The mesentery of peritoneal fibrosis mice and normal mice were analyzed quantitatively for proteins that were differentially expressed using a technique tandem mass tag (TMT). Next, the core target genes of ASI against PF were screened through network pharmacology analysis, and PPI and C-P-T networks were constructed by Cytoscape Version 3.7.2. According to the findings of a GO and KEGG enrichment analysis of differential proteins and core target genes, the signaling pathway with a high correlation degree was selected as the key signaling pathway of ASI inhibiting the PMCs MMT for further molecular docking analysis and experi-mental verification. Results: TMT-based quantitative proteome analysis revealed the identification of 5727 proteins, of which 70 were downregulated and 178 were upregulated. Among them, the levels of STAT1, STAT2, and STAT3 in the mes-entery of mice with peritoneal fibrosis were considerably lower than in the control group, indicating a role for the STAT family in the pathogenesis of peritoneal fibrosis. Then, a total of 98 ASI-PF-related targets were identified by network pharmacology analysis. JAK2 is one of the top 10 core target genes representing a potential therapeutic target. JAK/STAT signaling may represent a core pathway mediating PF effects by ASI. Molecular docking studies showed that ASI had the potential to interact favorably with target genes involved in the JAK/ STAT signaling pathway, such as JAK2 and STAT3. The experimental results showed that ASI could significantly alleviate Chlorhexidine Gluconate (CG)-induced peritoneal histopathological changes and increase JAK2 and STAT3 phosphorylation levels. In TGF-beta 1-stimulated HMrSV5 cells, E-cadherin expression levels were dramati-cally reduced whereas Vimentin, p-JAK2, alpha-SMA, and p-STAT3 expression levels were considerably increased. ASI inhibited the TGF-beta 1-induced HMrSV5 cell MMT, decreased the activation of JAK2/STAT3 signaling, and increased the nuclear translocation of p-STAT3, which was consistent with the effect of the JAK2/STAT3 pathway inhibitor AG490. Conclusion: ASI can inhibit PMCs MMT and alleviate PF by regulating the JAK2/STAT3 signaling pathway.