基于网络药理学和实验验证雷公藤红素治疗肾癌的作用及机制

Effect and mechanism of Celastrol in the treatment of renal carcinoma based on network pharmacology and experimental verification

目的运用网络药理学、分子对接技术及实验验证方法探讨雷公藤红素(CeT)治疗肾癌的作用与机制。方法应用中药系统药理学数据库与分析平台、PubChem、NCBI Gene、STRING数据库及GO功能和KEGG通路富集分析CeT治疗肾癌的关键靶点和通路,然后运用Autodock Vina软件进行分子对接验证。选取人肾透明细胞腺癌细胞系786-O细胞,将其分为对照组(正常培养)及0.2、0.4、0.8μmol/L CeT组(给予对应浓度的CeT),培养24 h后,采用免疫印迹法检测各组凋亡蛋白及相关通路蛋白水平。结果共筛选得到CeT靶点53个,疾病靶点8881个,相互映射得到45个共有靶点。蛋白质-蛋白质相互作用网络分析共有靶点得到Bax、Bcl-2等核心靶点。GO富集分析得到469个条目,KEGG通路富集分析确立了67条相关通路,主要包括细胞凋亡、磷脂酰肌醇3激酶(PI3K)/Akt激酶(Akt)通路、胱天蛋白酶9(caspase-9)、核因子κB(NF-κB)、P53等信号通路。分子对接结果显示,CeT与核心靶点结合活性良好。细胞实验结果显示,0.4、0.8μmol/L CeT组caspase-9、P53表达量高于对照组,Bcl-2、NF-κB表达量低于对照组,0.4μmol/L CeT组Bax表达量高于对照组,0.8μmol/L CeT组PI3K表达量低于对照组,0.2、0.4、0.8μmol/L CeT组p-Akt/Akt表达量低于对照组,差异有统计学意义(P<0.05)。0.8μmol/L CeT组NF-κB表达量低于0.2μmol/L CeT组,差异有统计学意义(P<0.05)。结论CeT可能通过PI3K/Akt通路、NF-κB和P53等信号通路影响Bax、Bcl-2等靶蛋白的表达,发挥多靶点和多途径治疗肾癌的作用。

Objective To investigate the effect and mechanism of Celastrol(CeT)in the treatment of renal carcinoma by network pharmacology,molecular docking techniques,and experimental verification methods.Methods The key targets and pathways of CeT in the treatment of renal carcinoma were analyzed by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform,PubChem,NCBI Gene,STRING databases,GO function,and KEGG pathway enrichment,and then Autodock Vina software was used for molecular docking verification.Human renal clear cell adenocarcinoma cell line 786-O cells were selected and divided into control group(normal culture),and 0.2,0.4,0.8μmol/L CeT group(given the corresponding concentration of CeT).After 24 h culture,apoptotic proteins and related pathway proteins in each group were detected by Western blot.Results A total of 53 CeT targets and 8881 disease targets were screened,and 45 common targets were obtained by mutual mapping.Core targets such as Bax and Bcl-2 were obtained by analyzing common targets of protein-protein interaction network.GO enrichment analysis obtained 469 items,KEGG pathway enrichment analysis identified 67 related pathways,including apoptosis,phosp-ha tidylinositol 3 kinase(PI3K)/Akt kinase(Akt)pathway,cysteine aspartic acid specific protease-9(caspase-9),nuclear factorκB(NF-κB),P53,and other signaling pathways.Molecular docking results showed that CeT had good binding activity with core targets.Cell experiment results showed that the expressions of caspase-9 and P53 in 0.4 and 0.8μmol/L CeT groups were higher than those in the control group,while the expressions of Bcl-2 and NF-κB were lower than those in the control group,the expression of Bax in 0.4μmol/L CeT group was higher than that in control group,expression of PI3K in 0.8μmol/L CeT group was lower than that in control group,the expressions of P-Akt/Akt in 0.2,0.4,and 0.8μmol/L CeT groups were lower than those in control group,and the differences were statistically significant(P<0.05).The expression of NF-κB in 0.8μmol/L CeT group was lower than that in 0.2μmol/L CeT group,and the differences were statistically significant(P<0.05).Conclusion CeT may affect the expression of target proteins such as Bax and Bcl-2 through PI3K/Akt pathway,NF-κB,and P53 signaling pathways,and play a role of multi-target and multi-pathway treatment for renal carcinoma.