五苓散对TGF-β1诱导HK-2细胞纤维化、炎症和氧化应激损伤的改善作用及机制  

Improvement effect and mechanism of Wuling San on TGF-β1-induced fibrosis,inflammation,and oxidative stress damage in HK-2 cells

作　　者：吴君[1] 荆雪宁[1] 孟繁伟[1] 孔晓妮[1] 苗久旺 张彩霞[1] 李海伦 韩芸[3] WU Jun;JING Xue-ning;MENG Fan-wei;KONG Xiao-ni;MIAO Jiu-wang;ZHANG Cai-xia;LI Hai-lun;HAN Yun(Shandong College of Traditional Chinese Medicine,Yantai 264199,China;Affiliated Huai'an Hospital of Xuzhou Medical University,Huaian 223002,China;Binzhou Medical College,Yantai 264003,China)

机构地区：[1]山东中医药高等专科学校,山东烟台264199 [2]徐州医科大学附属淮安医院,江苏淮安223002 [3]滨州医学院,山东烟台264003

出　　处：《中国中药杂志》2025年第5期1247-1254,共8页China Journal of Chinese Materia Medica

基　　金：山东省自然科学基金面上项目(ZR2020MH350);齐鲁卫生与健康领军人才培育工程项目;山东省中医药科技发展计划项目(Z-2022056);山东省中医药高层次人才培育项目;国家中医药综合改革示范区中医药科技共建项目(GZY-KJS-SD-2023-052);烟台市校地融合发展项目(2023XDRHXMXK08)。

摘　　要：观察五苓散对转化生长因子-β1(TGF-β1)诱导人肾小管上皮细胞(HK-2)细胞纤维化、炎症和氧化应激的作用及其抗氧化应激损伤的机制。体外培养HK-2细胞,将细胞分为对照组、TGF-β1模型组、五苓散含药血清低剂量组(2.5%)、五苓散含药血清中剂量组(5.0%)和五苓散含药血清高剂量组(10.0%),除对照组外,其余各组采用TGF-β1造模。采用CCK-8分析不同浓度五苓散在含有或者不含有TGF-β1刺激HK-2细胞活性的影响。qPCR法检测纤维化关键分子肌动蛋白α2(Acta2)、Ⅰ型胶原α1(Col1α1)、Ⅲ型胶原α1(Col3α1)、金属肽酶抑制因子1(Timp1)和纤连蛋白1(Fn1)基因的表达;ELISA法检测炎性细胞因子肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、白细胞介素-8(IL-8)和白细胞介素-4(IL-4)的水平。采用谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)、过氧化氢酶(CAT)和超氧化物歧化酶(SOD)生化试剂盒分析五苓散对TGF-β1诱导HK-2细胞氧化应激损伤的作用,并通过qPCR和免疫荧光法分析核因子E2相关因子2(Nrf2)、血红素加氧酶1(HO-1)和NAD(P)H:醌氧化还原酶-1(NQO1)的表达。CCK-8结果显示,五苓散最佳给药质量分数分别为2.5%、5.0%、10.0%;与对照组比较,TGF-β1模型组纤维化关键分子(Acta2、Col1α1、Col3α1、Timp1、Fn1)和炎性细胞因子(TNF-α、IL-1β、IL-6、IL-8和IL-4)的水平显著升高;与TGF-β1模型组比较,五苓散给药能够剂量依赖性地抑制纤维化关键分子和炎性细胞因子的水平;五苓散能够显著增加TGF-β1刺激HK-2细胞中GSH-Px、CAT和SOD的活性,并且显著抑制MDA的水平。此外,与对照组比较,TGF-β1模型组Nrf2、HO-1和NQO1基因和蛋白的表达显著降低;五苓散干预后,能够显著增加Nrf2、HO-1和NQO1基因和蛋白的表达。相关性分析显示,在TGF-β1刺激的HK-2细胞模型中抗氧化应激酶(GSH-Px、CAT和SOD)和Nrf2信号与纤维化关键分子和炎性细胞因�This study investigated the effect of Wuling San on transforming growth factor-β1(TGF-β1)-induced fibrosis,inflammation,and oxidative stress in human renal tubular epithelial cells(HK-2)and its mechanism of antioxidant stress injury.HK-2 cells were cultured in vitro and divided into a control group,a TGF-β1 model group,and three treatment groups receiving Wuling San-containing serum at low(2.5%),medium(5.0%),and high(10.0%)doses.TGF-β1 was used to establish the model in all groups except the control group.CCK-8 was used to analyze the effect of different concentrations of Wuling San on the activity of HK-2 cells with or without TGF-β1 stimulation.The expression of key fibrosis molecules,including actin alpha 2(Acta2),collagen type Ⅰ alpha 1 chain(Col1α1),collagen typeⅢalpha 1 chain(Col3α1),TIMP metallopeptidase inhibitor 1(Timp1),and fibronectin 1(Fn1),was detected using qPCR.The expression levels of inflammatory cytokines,including tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6),interleukin-8(IL-8),and interleukin-4(IL-4),were measured using ELISA kits.Glutathione peroxidase(GSH-Px),malondialdehyde(MDA),catalase(CAT),and superoxide dismutase(SOD)biochemical kits were used to analyze the effect of Wuling San on TGF-β1-induced oxidative stress injury in HK-2 cells,and the expression of nuclear factor E2-related factor 2(Nrf2),heme oxygenase 1(HO-1),and NAD(P)H quinone oxidoreductase 1(NQO1)was analyzed by qPCR and immunofluorescence.The CCK-8 results indicated that the optimal administration concentrations of Wuling San were 2.5%,5.0%,and 10.0%.Compared with the control group,the TGF-β1 model group showed significantly increased levels of key fibrosis molecules(Acta2,Col1α1,Col3α1,Timp1,and Fn1)and inflammatory cytokines(TNF-α,IL-1β,IL-6,IL-8,and IL-4).In contrast,the Wuling San administration groups were able to dose-dependently inhibit the expression levels of key fibrosis molecules and inflammatory cytokines compared with the TGF-β1 model group.Wuling San significa

关 键 词：五苓散 氧化应激 炎症 纤维化 NRF2 

分 类 号：R285.5[医药卫生—中药学]