摘要
目的:观察补阳还五汤含药血清对高糖诱导小鼠足细胞损伤的影响,并探讨其潜在作用机制.方法:体外培养小鼠足细胞(MPC5),筛选补阳还五汤含药血清的最佳干预浓度与干预时间;将细胞分为正常组(5.5 mmol·L-1葡萄糖)、等渗组(5.5 mmol·L-1葡萄糖+24.5 mmol·L-1甘露醇)、高糖组(30mmol·L-1葡萄糖)、空白血清组(30 mmol·L-1葡萄糖+20％空白血清)、补阳还五汤含药血清组(30 mmol·L-1葡萄糖+10％补阳还五汤含药血清)、铁死亡抑制剂Fer-1组(30 mmol·L-1葡萄糖+Fer-11 µmol·L-1),干预24h.FerroOrange荧光探针检测细胞二价铁(Fe2+)水平;荧光染核(Hoechst)染色检测细胞内活性氧(ROS)水平;试剂盒检测细胞内微量还原型谷胱甘肽(GSH)、丙二醛(MDA)水平;酶联免疫吸附测定法(ELISA)检测细胞4-羟基壬烯醛(4-HNE)水平;蛋白免疫印迹法(Western blot)检测细胞结蛋白(Desmin)、足细胞裂孔膜蛋白(Nephrin)、足萼蛋白(Podocalyxin)、长链脂酰辅酶A合成酶4(ACSL4)、溶质载体家族7成员11(SLC7A11)、谷胱甘肽过氧化物酶4(GPX4)、核因子E2相关因子2(Nrf2)、血红素加氧酶-1(HO-1)蛋白表达情况;实时荧光定量聚合酶链式反应(Real-time PCR)检测细胞Desmin、Nephrin、Podocalyxin、ACSL4、SLC7A11、GPX4、Nrf2、HO-1 的 mRNA 表达水平.结果:与正常组比较,高糖组Desmin、ACSL4表达明显增加,Fe2+及 ROS水平升高,MDA、4-HNE含量增加,GSH 活性降低,Nephrin、Podocalyxin、SLC7A11、GPX4、Nrf2、HO-1蛋白及mRNA表达减少(P＜0.05,P＜0.01);等渗组上述结果无明显变化.与高糖组比较,补阳还五汤含药血清组与Fer-1组Desmin、ACSL4表达明显下降,Fe2+及ROS水平降低,MDA、4-HNE含量减少,GSH活性增加,Nephrin、Podocalyxin、SLC7 A11、GPX4、Nrf2、HO-1的蛋白及mRNA表达增加(P＜0.05,P＜0.01);空白血清组上述结果无明显变化.结论:补阳还五汤含药血清可减轻高糖诱导的足细胞损伤,其机制与调控Nrf2/HO-1通路,抑制铁死亡有关.
Abstract
Objective:To observe the effect of serum containing Buyang Huanwutang on podocyte injury induced by high glucose in mice,and to explore its potential mechanism.Methods:Mouse podocytes(MPC5)were cultured in vitro,and the optimal intervention concentration and time of serum containing Buyang Huanwutang were screened.Cells were divided into normal group(5.5 mmol·L-1 glucose),isotonic group(5.5 mmol·L-1 glucose+24.5 mmol·L-1 Mannitol),high glucose group(30 mmol·L-1 glucose),blank serum group(30 mmol·L-1 glucose+20％blank serum)and Buyang Huanwutang(30 mmol·L-1 glucose+10％serum containing).FerroOrange fluorescent probe was used to detect the level of iron(Fe2+)in cells.The level of reactive oxygen species(ROS)in cells was detected by Hoechst staining.The levels of glutathione(GSH)and malondialdehyde(MDA)in cells were detected by the kit.Enzyme-linked immunosorbent assay(ELISA)was used to determine the level of 4-hydroxynonenal(4-HNE).Western blot was used to detect Desmin,podocyte hole membrane protein(Nephrin),Podocalyxin,long-chain acyl-CoA synthetase 4(ACSL4),member 11 of solute carrier family 7(SLC7A11),glutathione peroxidase 4(GPX4),nuclear transcription factor E2-related factor 2(Nrf2)and heme oxygenase-1(HO-1).The mRNA expression levels of Desmin,Nephrin,Podocalyxin,ACSL4,SLC7A11,GPX4,Nrf2 and HO-1 were detected by real-time fluorescence quantitative polymerase chain reaction.Results:Compared with the normal control group,the expressions of Desmin and ACSL4,the levels of Fe2+and ROS,and the contents of MDA and 4-HNE in the high glucose group increased significantly,and the expressions of GSH,Nephrin,Podocalyxin,SLC7A11,GPX4,Nrf2 and HO-1 decreased(P＜0.05,P＜0.01).There was no significant change in the above results in isotonic group.Compared with the high glucose group,the expressions of Desmin and ACSL4,the levels of Fe2+and ROS,and the contents of MDA and 4-HNE in Buyang Huanwutang-containing serum group and Fer-1 group decreased significantly,and the expressions of GSH,Nephrin,Podocalyxin,SLC7A11,GPX4,Nrf2 and HO-1 increased(P＜0.05,P＜0.01).There was no significant change in the above results in blank serum group.Conclusion:Buyang Huanwutang medicated serum can alleviate podocyte injury induced by high glucose,and its mechanism is related to regulating Nrf2/HO-1 pathway and inhibiting ferroptosis.
国家科技期刊平台
NSTL
维普
万方数据