石斛合剂含药血清对高糖诱导小鼠足细胞损伤的保护作用  

Dendrobii Caulis Mixture-containing Serum Protects Mice from Podocyte Injury Induced by High Glucose

作　　者：陈勇[1] 林晓晖[1] 张捷平[1] 郑燕芳[2] 庄舒婷 余文珍[1] CHEN Yong;LIN Xiaohui;ZHANG Jieping;ZHENG Yanfang;ZHUANG Shuting;YU Wenzhen(College of Integrative Medicine,Fujian University of Traditional Chinese Medicine(TCM),Fuzhou 350122,China;College of Pharmacy,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,China)

机构地区：[1]福建中医药大学中西医结合学院,福州350122 [2]福建中医药大学药学院,福州350122

出　　处：《中国实验方剂学杂志》2024年第5期65-71,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81973827);福建省自然科学基金项目(2021J01890,2021J01886)。

摘　　要：目的:探讨石斛合剂含药血清对高糖诱导的小鼠足细胞损伤的影响及作用机制。方法:体外培养小鼠肾小球足细胞(MPC5),筛选高糖造模浓度和时间及石斛合剂含药血清最佳给药浓度;将细胞分为正常组(5.5 mmol·L^(-1)葡萄糖+10%空白血清)、模型组(30 mmol·L^(-1)葡萄糖+10%空白血清)、石斛合剂含药血清组(30 mmol·L^(-1)葡萄糖+10%石斛合剂含药血清),铁死亡抑制剂(Fer-1)组(30 mmol·L^(-1)葡萄糖+10%空白血清+1μmol·L^(-1)Fer-1)。采用试剂盒检测细胞中Fe^(2+)、乳酸脱氢酶(LDH)水平;酶联免疫吸附测定法(ELISA)检测细胞谷胱甘肽(GSH)、过氧化脂质(LPO)含量;荧光探针检测活性氧(ROS)水平;实时荧光定量聚合酶链式反应(Real-time PCR)检测足细胞中肾母细胞瘤基因-1(WT-1)、结蛋白(Desmin)、长链脂酰辅酶A合成酶4(ACSL4)和谷胱甘肽过氧化物酶4(GPX4)m RNA表达水平;蛋白免疫印迹法(Western blot)检测足细胞中WT-1、Desmin、ACSL4和GPX4蛋白表达水平。结果:与空白组比较,30 mmol·L^(-1)高糖干预48 h可显著降低足细胞活力(P<0.01),10%石斛合剂含药血清改善高糖诱导的足细胞活力最为显著(P<0.01)。与正常组比较,模型组足细胞内Fe^(2+)、LDH、LPO、ROS水平及Desmin、ACSL4 m RNA和蛋白表达显著升高(P<0.01),GSH水平及WT-1、GPX4 m RNA和蛋白表达显著降低(P<0.01);与模型组比较,石斛合剂含药血清组足细胞内Fe^(2+)、LDH、LPO、ROS水平及Desmin、ACSL4 m RNA和蛋白表达明显降低(P<0.05,P<0.01),GSH水平及WT-1、GPX4 m RNA和蛋白表达明显升高(P<0.05,P<0.01)。结论:石斛合剂含药血清对高糖诱导的足细胞损伤具有保护作用,与其抑制铁死亡有关。Objective:To investigate the effect and mechanism of Dendrobium mixture(DMix)-containing serum on high glucose-induced podocyte injury in mice.Method:The MPC5 mouse glomerular podocytes were cultured in vitro,and the optimal glucose concentration for modeling,modeling time,and concentration of DMix-containing serum for administration were determined.The cells were classified into normal(5.5 mmol·L^(-1) glucose+10%blank serum),model(30 mmol·L^(-1) glucose+10%blank serum),DMix-containing serum(30 mmol·L^(-1) glucose+10%DMix-containing serum),ferroptosis inhibitor(Fer-1,30 mmol·L^(-1) glucose+10%blank serum+1μmol·L^(-1) Fer-1)groups.The corresponding kits were used to measure the levels of Fe^(2+)and lactate dehydrogenase(LDH)in cells.Enzyme-linked immunosorbent assay was employed to determine the content of glutathione(GSH)and lipid peroxide(LPO)in cells.Fluorescence probe was used to measure the reactive oxygen species(ROS)level.Real-time fluorescence quantitative polymerase chain reaction and Western blotting were employed to determine the mRNA and protein levels,respectively,of Wilms'tumor-1(WT-1),desmin,long chain acyl-CoA synthase 4(ACSL4),and glutathione peroxidase 4(GPX4)in podocytes.Result:Compared with the blank group,the intervention with 30 mmol·L^(-1) glucose for 48 h reduced podocyte viability(P<0.01),and the 10%DMix-containing serum showed the most significant improvement in podocyte viability(P<0.01).Compared with the normal group,the model group presented elevated levels of Fe^(2+),LDH,LPO,and ROS,lowered GSH level,up-regulated mRNA and protein levels of desmin and ACSL4,and down-regulated mRNA and protein levels of WT-1 and GPX4(P<0.01).Compared with the model group,the DMix-containing serum lowered the Fe^(2+),LDH,LPO,and ROS levels,elevated the GSH level,down-regulated the mRNA and protein levels of desmin and ACSL4,and up-regulated the mRNA and protein levels of WT-1 and GPX4 in podocytes(P<0.05,P<0.01).Conclusion:DMix-containing serum exerts a protective effect on high glucose-induced po

关 键 词：石斛合剂 糖尿病肾病 足细胞 铁死亡 

分 类 号：R2-0[医药卫生—中医学] R33[生物学—生物化学] R289R692Q592.4