火把花根片关键药效物质的“效益-风险”体外评价及其网络调控机制研究

In vitro "benefit-risk" evaluation and network regulation mechanism of Colquhounia Root Tablets and its key material basis

雷公藤属中药品种火把花根片对类风湿关节炎、糖尿病肾病和膜性肾病均具有良好的临床疗效,然而,肝脏损伤、恶心呕吐等副反应限制了其广泛应用。该研究旨在通过体外药效与毒性评价,整合生物分子网络分析,明确火把花根片及其潜在活性成分针对类风湿关节炎、糖尿病肾病和膜性肾病的效益与风险,并初步探究其“异病同治”的作用机制。首先,采用超高效液相色谱串联四极杆飞行时间质谱分析火把花根片入血成分,并考察火把花根片及其所含雷公藤甲素、雷公藤红素对人正常肝细胞L02、人类风湿成纤维样滑膜细胞MH7A、人肾小管上皮细胞HK-2和小鼠足细胞MPC-5活性的影响;采用酶联免疫吸附法考察各组炎症诱导性MH7A细胞和HK-2细胞模型中炎性因子的水平;采用蛋白免疫印迹法检测药物对MPC-5细胞模型中nephrin和podocin蛋白异常表达的干预作用,并开展免疫荧光检测考察对MPC-5细胞骨架蛋白表达水平的调节。接着,通过TCMIP v2.0数据库获得火把花根片入血成分候选靶点,利用GEO数据库收集3类疾病靶点,基于STRING数据库构建“疾病基因-药物靶标”互作网络,进行通路富集分析。最后,利用AutoDock Vina分子对接方法探究雷公藤甲素和雷公藤红素与关键通路中相应靶标的结合能力。结果表明,3种药物均可降低MH7A及HK-2细胞上清炎性因子浓度,增加MPC-5细胞标志性蛋白含量,其中,雷公藤甲素对于L02肝细胞毒性最强,雷公藤红素次之,火把花根片毒性最小。“疾病基因-药物靶标”关联网络挖掘结果表明,火把花根片可能通过调节PI3K/HIF1α/NOS2信号通路,干预3类疾病。分子对接结果显示其关键药效物质与PI3K、HIF1α、NOS2蛋白均具有较高亲和力。综上,该研究结果表明,火把花根片对于类风湿关节炎、糖尿病肾病、膜性肾病的疗效与雷公藤甲素及雷公藤红素相似,但细胞毒性均小于二者,即效益较高,而风险更小。此外,生物分子网络分析初步表明PI3K/HIF1α/NOS2信号通路可能是其“异病同治”的关键环节。

Colquhounia Root Tablets, prepared from Tripterygium, is effective for rheumatoid arthritis, diabetic nephropathy, and membranous nephropathy. However, the adverse reactions, such as liver injury, nausea, and vomiting, limit its application. This study aims to evaluate the advantages and risk of Colquhounia Root Tablets and its key active components in the treatment of rheumatoid arthritis, diabetic nephropathy, and membranous nephropathy and explore the potential mechanism in treating different diseases based on in vitro efficacy and toxicity assessment and biomolecular network analysis. First, the components of Colquhounia Root Tablets absorbed in blood were detected via ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry, and the influence of Colquhounia Root Tablets and its key components triptolide and celastrol on viability of human hepatocyte L02, human rheumatoid fibroblast-like synovial cell MH7A, human renal tubular epithelial cell HK-2, and mouse podocyte MPC-5 was detected by cell counting kit 8(CCK8) assay. Then the expression of inflammatory cytokines of MH7A and HK-2 cells was detected by enzyme-linked immunosorbent assay(ELISA). Moreover, the expression of nephrin and podocin in MPC-5 cells was measured by Western blot, and the expression of cytoskeletal protein by immunofluorence assay. Candidate targets of components from Colquhounia Root Tablets absorbed in blood were retrieved from TCMIP v2.0, and targets of the three diseases from GEO. The "disease-related genes-drug targets" network was constructed based on STRING, followed by pathway enrichment. Finally, molecular docking was performed by AutoDock Vina to explore the binding affinity of triptolide and celastrol with putative targets in the key signaling pathway. Results showed that Colquhounia Root Tablets, triptolide, and celastrol can obviously reduce the levels of inflammatory cytokines in supernatant of MH7A and HK-2 cells and enhance the expression of nephrin and podocin in MPC-5 cells. In addition, triptolide had the strongest toxicity to L02 cells, while Huobahuagen Tablets had the least toxicity to hepatocytes. Network analysis revealed that Colquhounia Root Tablets may intervene the three diseases through PI3 K/HIF1α/NOS signaling pathway. Both triptolide and celastrol had high binding affinities to corresponding targets in this signaling pathway. In conclusion, Colquhounia Root Tablets exerts similar effects on rheumatoid arthritis, diabetic nephropathy, and membranous nephropathy to triptolide and celastrol, but the toxicity was lower. PI3 K/HIF1α/NOS signaling pathway may be the common pathway of Colquhounia Root Tablets in the treatment of the three diseases.