基于网络药理学和实验验证揭示麻黄抗肾纤维化的作用

Unveiling the Anti-fibrotic Effect of Ephedrae Herba in Kidney Fibrosis Based on Network Pharmacology and Experimental Validation

关键词:  麻黄  肾纤维化  网络药理学  蛋白质组学  单侧输尿管梗阻模型

Key words:  Ephedrae Herba  renal fibrosis  network pharmacology  proteomics  unilateral ureteral obstruction model

摘要:

目的 本研究旨在筛选一种最具抗纤维化潜力的中药，并探究其抗肾纤维化的作用及机制。方法 通过整合OMIM、GeneCards、TTD及TCMSP数据库资源，系统筛选具有潜在抗纤维化作用的中药。随后，构建单侧输尿管梗阻(unilateral ureteral obstruction, UUO)小鼠模型，探究目标中药对肾纤维化的干预效果。通过原代肾成纤维细胞(primary renal fibroblasts, PRFs)模型，进一步验证目标中药的抗肾纤维化作用。接下来，采用串联质谱标签(tandem mass tag, TMT)定量蛋白质组学技术鉴定差异表达蛋白(differentially expressed proteins, DEPs)并对其进行生物信息学分析。通过构建PPI网络并结合文献挖掘，筛选关键靶点蛋白，最终通过细胞实验验证其功能。结果 网络药理学结果表明麻黄是最具潜力的抗纤维化中药；体内实验证实，麻黄(Ephedrae Herba, MH)能够有效改善肾脏病理损伤并抑制肾纤维化进程；体外实验进一步验证了其在细胞水平上的抗纤维化作用。通过TMT定量蛋白质组学技术分析，在检测到的4973个蛋白质中，共鉴定出65个显著差异表达蛋白，其中FN1、MDA5、MX1、ALB以及ISG15被确定为关键调控蛋白。细胞实验验证结果显示，经麻黄干预后，FN1、MDA5、MX1、ALB和ISG15的蛋白表达水平显著下调，这一结果与TMT定量蛋白质组学分析呈现的表达趋势一致。结论 本研究首次系统阐明了麻黄在体内外实验模型中均具有显著的抗肾纤维化作用。通过整合网络药理学分析、TMT定量蛋白质组学技术及实验验证，首次发现FN1、MDA5、MX1、ALB和ISG15可能是麻黄发挥抗肾纤维化作用的关键分子靶点。

Abstract:

OBJECTIVE This study aims to explore one of the most effective potential traditional Chinese medicine for anti-fibrosis, evaluate its effects and mechanisms in anti-renal fibrosis. METHODS OMIM, Gene Cards, TTD and TCMSP databases were used to explore potential anti-fibrosis Chinese herbal medicines. Unilateral ureteral obstruction (UUO) model was used to evaluate the effects of potential herbal on renal interstitial fibrosis. Next, the anti-renal fibrosis effect of herbal medicine on primary renal fibroblasts (PRFs) was examined, and the technique tandem mass tag (TMT) was used to detect differentially expressed proteins (DEPs) and bioinformatics analysis was performed. Then, the key proteins were selected by both probable protein-protein interactions (PPI) network and literature search, followed by the next step of cellular experimental validation. RESULTS Ephedrae Herba (MH) is the most potential herbal medicine for anti-fibrosis. MH alleviated renal pathological injury and renal fibrosis in vivo, and also inhibited renal fibrosis at the cellular level. TMT proteomics analysis identified 65 significant DEPs among 4973 proteins after MH treatment, with FN1, MDA5, MX1, ALB and ISG15 were obtained as key proteins. Cellular experiments substantiated that the protein expression levels of FN1，MDA5, MX1, ALB, and ISG15 were markedly diminished upon exposure to MH, corroborating the downward trend observed through TMT-based quantitative proteomics profiling. CONCLUSION Our study first revealed that MH attenuated renal fibrosis in vivo and vitro, and validated that its possible targets were FN1，MDA5, MX1, ALB and ISG15 through bioinformatics analysis, proteomics results and cellular experiments.