藤黄酸通过IGF-1/IRS-1通路抑制膀胱癌炎症和血管生成的机制研究  

Mechanism of gambogic acid in inhibition of bladder cancer cell inflammation and angiogenesis through downregulation of IGF-1/IRS-1 pathway activity

作　　者：梅玉献 陈莎莎 MEI Yu-xian;CHEN Sha-sha(Department of Urology,Wenling Hospital of Traditional Chinese Medicine,Wenling,Zhejiang 317500,China;Department of Traditional Chinese Medicine,Taizhou Cancer Hospital,Wenling,Zhejiang 317502,China)

机构地区：[1]浙江中医药大学附属温岭中医院泌尿外科,温岭317500 [2]浙江省台州市肿瘤医院中医科,温岭317502

出　　处：《浙江中西医结合杂志》2023年第7期610-616,共7页Zhejiang Journal of Integrated Traditional Chinese and Western Medicine

基　　金：2020年台州市第二批社会发展科技计划项目(No.20ywb140);温岭市2021年社会发展科技项目(No.2021S00099);浙江中医药大学2021年附属医院科研专项项目(No.2021FSYYZY13)。

摘　　要：目的探究藤黄酸(GA)通过胰岛素样生长因子1(IGF-1)/胰岛素受体底物蛋白1(IRS-1)通路对膀胱癌炎症和血管生成的影响。方法体外培养人膀胱癌细胞5637和人膀胱移行细胞癌细胞T24,细胞分三组,Control组、IGF-1组(100 ng/mL)、IGF-1(100 ng/mL)+GA(0.8μg/mL)组,CCK8法检测不同浓度梯度GA对膀胱癌细胞活性的影响。酶联免疫吸附测定法检测各组细胞炎性因子白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平。CCK8和划痕法分别测定GA处理的膀胱癌条件培养基(CM)对膀胱癌细胞增殖和迁移能力的影响。小管形成法测定GA处理的膀胱癌CM对膀胱癌细胞血管网络形成能力的影响。通过qRT-PCR和Western blot测定GA处理的膀胱癌细胞系CM对膀胱癌细胞细胞内血管内皮生长因子(VEGF)和血管内皮生长因子受体(VEGFR)转录和翻译水平,以及对IRS-1/NF-κB通路的调控影响。结果IGF+GA组人膀胱癌细胞5637和人膀胱移行细胞癌细胞T24的IL-1β、IL-6、TNF-α分泌量较IGF-1组均减少[IL-1β:(32.25±2.49)pg/mL比(69.85±6.34)pg/mL,(24.92±3.16)pg/mL比(63.99±4.41)pg/mL;IL-6:(21.87±3.89)pg/mL比(39.54±4.75)pg/mL,(22.44±2.51)pg/mL比(57.69±3.18)pg/mL;TNF-α:(192.08±5.20)pg/mL比(384.78±30.79)pg/mL,(217.03±18.82)pg/mL比(604.30±72.88)pg/mL;P<0.05]。同时,GA处理的CM能减弱HUVEC细胞的增殖、迁移能力和相应的血管网络形成能力。qRT-PCR结果显示,IGF-1+GA-CM组人膀胱癌细胞5637和人膀胱移行细胞癌细胞T24的VEGF、VEGFR mRNA相对表达量较IGF-1-CM组均降低[VEGF:(1.63±0.25)比(4.45±0.36),(1.57±0.26)比(4.16±0.42);VEGFR:(1.60±0.14)比(5.90±0.48),(1.88±0.18)比(6.20±0.46);P<0.05],Western blot验证了这一结果。结论GA可能通过抑制膀胱癌内IGF-1/IRS-1通路,减轻炎症反应,从而抑制血管生成。Objective To assess the effect of gambogic acid(GA)on regulation of insulin-like growth factor 1(IGF-1)/insulin receptor substrate protein 1(IRS-1)activity and inflammation and angiogenesis in bladder cancer cells in vitro.Methods Human bladder carcinoma(5637)and human bladder transitional cell carcinoma(T24)cell lines were cultured in vitro and divided into control,IGF-1(100 ng/mL),and IGF-1(100 ng/mL)+GA(0.8μg/mL)groups.The cell viability CCK8 assay was used to detect changes in tumor cell viability,while levels of inflammatory cytokines interleukin(IL)-1β,IL-6,and tumor necrosis factor-α(TNF-α)in these groups of cells were deter mined by using the enzyme-linked immunosorbent assay.The effects of conditioned medium(CM)on tumor cell proliferation and migration were assayed by using the CCK8 and wound healing assays,respectively.The effect of CM on bladder cancer cell vascular network formation ability was determined by tubulogenesis assay.qRT-PCR and Western blot were used to assess changes in vascular endothelial growth factor(VEGF),vascular endothelial growth factor receptor(VEGFR),and IRS-1/NF-κB mRNA and proteins,respectively.Results Levels of IL-1β,IL-6,and TNF-αsecretions in IGF+GA group of 5637 and T24 cells were decreased compared with IGF-1 group(IL-1β,32.25±2.49 vs.69.85±6.34 pg/mL and 24.92±3.16 vs.63.99±4.41 pg/mL;IL-6,21.87±3.89 vs.39.54±4.75 pg/mL and 22.44±2.51 vs.57.69±3.18 pg/mL;TNF-α,192.08±5.20 vs.384.78±30.79 pg/mL and 217.03±18.82 vs.604.30±72.88 pg/mL;all P<0.05).Meanwhile,tumor cell viability was reduced and HUVEC cell migration and formation of vascular network were also weakened after addition of GA-treated CM.In addition,qRT-PCR data showed that levels of VEGF and VEGFR mRNA in IGF-1+GA-treated 5637 and T24 cell CM were lower than those in IGF-1 only CM(VEGF,1.63±0.25 vs.4.45±0.36 and 1.57±0.26 vs.4.16±0.42;VEGFR,1.60±0.14 vs.5.90±0.48 and 1.88±0.18 vs.6.20±0.46;all P<0.05).Western blot data further confirm qRT-PCR data.Conclusion GA can reduce bladder cance

关 键 词：藤黄酸 IGF-1/IRS-1 膀胱癌 

分 类 号：R285[医药卫生—中药学]