益肾清热化湿方调控NF-κB介导的炎症通路改善大鼠慢性肾炎

Experimental Study on Yishen Qingre Huashi Decoction Improving Chronic Nephritis in Rats by Regulating NF-κB-Mediated Inflammatory Pathway

关键词:  益肾清热化湿方  NF-κB通路  炎症通路  慢性肾炎  腺嘌呤

Key words:  Yishen Qingre Huashi Decoction  NF-κB pathway  inflammatory pathway  chronic nephritis  adenine

摘要:

目的 明确益肾清热化湿方对慢性肾炎的调控作用及分子机制。方法 采用临床数据挖掘、动物实验验证与分子机制解析递进式研究策略：利用GEO数据库GSE35487数据集（5例健康人及25例肾炎患者）筛选差异基因并进行通路富集分析；再通过HERB数据库获取肾炎与益肾清热化湿方的交集靶标基因并进行富集分析；然后构建腺嘌呤诱导慢性肾炎大鼠模型，设正常组、模型组、阳性药黄葵胶囊组及益肾清热化湿方低/高剂量组，干预后检测生理、尿液及病理指标；最后采用ELISA检测血清炎症因子，RT-qPCR验证核心基因表达，MOE软件进行分子对接筛选活性成分。结果 生信分析显示NF-κB与TNF等炎症通路为肾炎核心通路，FOS、JUN与GDF-15等基因是其关键调控及方剂靶标基因。动物实验证实，方剂可剂量依赖性降低大鼠肾质量及肾体比，减少尿蛋白排泄，升高尿肌酐水平，缓解肾小球炎细胞浸润、肾小管坏死及纤维化程度；同时显著下调血清TNF-α、IL-1β、IL-6及肾脏中FOS、JUN、GDF-15表达。分子对接显示β-胡萝卜素与辣椒素等成分可与FOS及JUN稳定结合。结论 益肾清热化湿方通过靶向NF-κB通路及调控下游FOS、JUN与GDF-15等关键基因，协同发挥抗炎、抗纤维化及肾保护作用，为该方剂在慢性肾炎治疗中的临床应用提供了实验依据。

Abstract:

OBJECTIVE To clarify the regulatory effect and underlying molecular mechanism of Yishen Qingre Huashi Decoction (YSQRHS) on chronic nephritis. METHODS A progressive strategy combining clinical data mining, animal experiment verification and molecular mechanism analysis was adopted. The GSE35487 dataset from the GEO database (5 healthy individuals and 25 nephritis patients) was used to identify differential expressed genes (DEGs) and conduct pathway enrichment analysis. The intersection target genes between nephritis and YSQRHS were retrieved from the HERB database. An adenine-induced chronic nephritis rat model was established, consisting of the normal group, model group, Huangkui Capsule group (positive control), and low/high-dose YSQRHSD groups. After the treatments, physiological, urinary and pathological indicators were detected. ELISA was used to detect serum inflammatory factors, while RT-qPCR was employed to verify the expression levels of core genes. Additionally, molecular docking was performed using MOE software to screen potential active components of YSQRHS. RESULTS Bioinformatics analysis revealed that inflammatory pathways such as NF-κB and TNF were the core pathways involved in nephritis, with FOS, JUN, and GDF-15 identified as key DEGs and target genes of YSQRHS. Animal experiments confirmed that YSQRHS could dose-dependently reduce kidney weight and kidney-to-body weight ratio, decrease urinary protein excretion, increase urinary creatinine level, and alleviate glomerular inflammatory cell infiltration, renal tubular necrosis and fibrosis. Meanwhile, it significantly downregulated the serum levels of TNF-α, IL-1β, IL-6 as well as the renal levels of FOS, JUN, GDF-15. Molecular docking results demonstrated that β-carotene, capsaicin, and other active components could bind to FOS and JUN (binding affinity < -5 kcal/mol, RMSD < 2 ?). CONCLUSION Yishen Qingre Huashi Decoction exerts synergistic anti-inflammatory, anti-fibrotic and renoprotective effects targeting the NF-κB pathway and its downstream key genes (FOS, JUN and GDF-15), providing experimental evidence for its clinical application in the treatment of chronic nephritis.