基于网络药理学的雷公藤抗肾纤维化作用及实验验证_刘馨月.pdf

基金项目:山西省卫生健康委员会青年项目(2023121)作者简介:刘馨月，女，1998 03 生，在读硕士，E-mail:liuxyue1163 com收稿日期:2022 11 22基于网络药理学的雷公藤抗肾纤维化作用及实验验证刘馨月1，田杰2，李赟2，周雪娇2，李维鹏1，李贤霖1，郑晓俊2*(1山西医科大学药学院，太原030001;2山西医科大学第一医院药剂科;*通讯作者，E-mail:zhengxiaojun0525163 com)摘要:目的运用网络药理学探究雷公藤中药缓解肾纤维化(renal fibrosis，F)的核心成分及相关信号通路，并进一步针对肾纤维化关键信号通路之一 Wnt 信号通路进行细胞实验验证。方法运用网络药理学筛选雷公藤核心成分的潜在靶点，构建“雷公藤核心成分 潜在靶点”及蛋白质互作网络分析图，进行京都基因与基因组百科全书(Kyoto encyclopedia of genesand genomes，KEGG)通路富集分析。将不同浓度的山柰酚(0，5，10，15，20 mol/L)、谷甾醇(0，5，10，15，20 mol/L)、雷公藤甲素(0，5，10，15，20 nmol/L)作用于人肾小管上皮细胞(human embryonic kidney epithelial cells，HKC)，通过 CCK-8 实验检测存活率，存活率最高的药物浓度作为后续实验的给药浓度。蛋白免疫印迹法及 q-PC 检测空白组、山柰酚组、谷甾醇组、雷公藤甲素组磷脂酶 D1(phospholipase D1，PLD-1)表达水平，筛选影响 Wnt 通路的核心成分。进一步将细胞分组:对照组、雷公藤甲素组、TGF-组、TGF-+雷公藤甲素组，观察各组细胞形态改变，蛋白免疫印迹法及 q-PC 法检测各组细胞 平滑肌肌动蛋白(-SMA)表达水平。结果网络药理学表明雷公藤通过 WNT/-catenin 信号通路发挥抗肾纤维化作用，主要核心成分为山柰酚、谷甾醇、雷公藤甲素。CCK-8 实验结果显示，与 0 mol/L 相比，山柰酚浓度15 mol/L 时，HKC 细胞存活率降低(P0 01);谷甾醇浓度15 mol/L 时，HKC 细胞存活率降低(P0 01);雷公藤甲素浓度10 nmol/L 时，HKC细胞存活率降低(P0 01)。故选择山柰酚 10 mol/L、谷甾醇 10 mol/L 和雷公藤甲素 5 nmol/进行后续实验研究。与空白组相比，雷公藤甲素组 PLD-1 蛋白及 mNA 表达量明显降低(P 0 01)，山柰酚组及 谷甾醇组 PLD-1 蛋白及 mNA表达量差异均无统计学意义(P0 05)。细胞形态学结果表明，与对照组相比，TGF-组梭形转化增多(P 0 01);与 TGF-组相比，TGF-+雷公藤甲素组梭形转化减少(P 0 01)。与对照组相比，TGF-组-SMA 蛋白及 mNA 表达升高(P 0 01);与 TGF-组相比，TGF-+雷公藤甲素组-SMA 蛋白及 mNA 表达明显降低(P0 01)。结论雷公藤中雷公藤甲素可以通过抑制 PLD-1 表达，调节 WNT/-catenin 信号通路发挥抗肾纤维化。关键词:雷公藤;肾纤维化;WNT/-catenin 信号通路;网络药理学;雷公藤甲素中图分类号:285文献标志码:A文章编号:1007 6611(2023)04 0535 09DOI:1013753/j issn1007 6611202304018Mechanism of Tripterygium wilfordii in treatment of renal fibrosis based on network pharmacology and experimental verificationLIU Xinyue1，TIAN Jie2，LI Yun2，ZHOU Xuejiao2，LI Weipeng1，LI Xianlin1，ZHENG Xiaojun2*(1School of Pharmacy，ShanxiMedical University，Taiyuan 030001，China;2Department of Pharmacy，First Hospital of Shanxi Medical University;*Correspondingauthor，E-mail:zhengxiaojun0525163 com)Abstract:ObjectiveTo investigate the core components and related signalling pathways of Tripterygium wilfordii in improvement ofrenal fibrosis(F)using network pharmacology，and further validate the Wnt signalling pathway，one of the key signalling pathways inrenal fibrosis in cellular experimentsMethodsThe database was used to screen the potential targets of core components of Tripterygiumwilfordii“Core components of Tripterygium wilfordii-potential target”network analysis and PPI network were constructed Kyoto ency-clopedia of genes and genomes(KEGG)pathway was used to complete enrichment analysis Human embryonic kidney epithelial cells(HKC)were respectively treated with different concentrations of kaempferol(0，5，10，15，20 mol/L)，-sitosterol(0，5，10，15，20 mol/L)and triptolide(0，5，10，15，20 nmol/L)to screen the drug concentration with the highest survival rate for subsequentexperiments The expression levels of phospholipase D1(PLD-1)were detected in blank group，kaempferol group，-sitosterol geoupand triptolide group by Western blot test and q-PC to screen the core components affecting the Wnt pathway Cells were further dividedinto control group，triptolide group，TGF-group，and TGF-+triptolide group Morphological changes of cells in each group wereobserved The expression level of-smooth muscle actin(-SMA)was detected by Western blotting and q-PCesultsNetworkpharmacology showed that Tripterygium wilfordii exerted anti-renal fibrosis effect through Wnt/-catenin signaling pathway，and themain core components of Tripterygium wilfordi were kaempferol，-sitosterol and triptolide CCK-8 assay showed that compared with0 mol/L，HKC cell survival was reduced at kaempferol concentration 15 mol/L(P 0 01)，-sitosterol concentration 15 mol/L535山西医科大学学报，2023 年 4 月，第 54 卷 第 4 期(P0 01)，and triptolide concentration 10 nmol/L(P0 01)Therefore，10 mol/L kaempferol，10 mol/L-sitosterol and 5nmol/L triptolide were selected for the subsequent experimental study Compared with blank group，PLD-1 protein and mNA expres-sion levels in triptolide group were significantly decreased(P0 01)Compared with blank group，PLD-1 protein and mNA expres-sion levels in kaempferol group and-sitosterol group showed no statistical difference(P0 05)Cytomorphological results showed anincrease in spindle cell transformation in TGF-group compared to control group(P0 01)and a decrease in spindle cell transforma-tion in TGF-+triptolide group compared to TGF-group(P0 01)The-SMA protein and mNA expression levels were higher inTGF-group than in control group(P0 01)，but lower in TGF-+triptolide group than in TGF-group(P0 01)ConclusionTriptolide in Tripterygium wilfordii can regulate the WNT/-catenin signaling pathway to play an anti-renal fibrosis role by inhibitingthe expression of PLD-1Key words:Tripterygium wilfordii;renal fibrosis;WNT signaling pathway;network pharmacology;Triptolide肾纤维化(renal fibrosis，F)是以大量成纤维细胞及肌成纤维细胞增生、细胞外基质堆积而导致肾小球硬化、肾小管间质纤维化为特征的病理过程。肾纤维化是慢性肾脏病(chronic kidney disease，CKD)进行性肾功能衰竭的原因1，CKD 已成为影响人类健康较为常见的一种危害。2019 年断面调查我国成年人 CKD 总体患病率达 16 4%2。近年治疗 CKD 主要通过肾脏移植及透析治疗等，这给患者及家庭带来较大的经济负担。中医药可通过多靶点调控来缓解 F，具有现代医学不可替代的优势3。Wnt/-catenin 信号通路是肾纤维化调控机