他克莫司对嘌呤霉素损伤的肾小球足细胞重组人帕金森蛋白7表达的影响

Effect of tacrolimus on the expression of Park7 in glomerular podocytes injured by puromycin aminonucleoside

目的探讨嘌呤霉素(puromycin aminonucleoside,PAN)对小鼠肾小球足细胞(mouse podocyte clone 5,MPC-5)凋亡及重组人帕金森蛋白7(Parkinson's disease 7,Park7)表达的影响,以及他克莫司(tacrolimus,FK506)对MPC-5损伤的保护机制。方法体外培养MPC-5,分为空白对照组(对照组)、PAN组和FK506组。PAN组滴加PAN(浓度50 mg/L)构建MPC-5损伤模型,FK506组同时滴加PAN(浓度50 mg/L)和FK506(浓度5 mg/L)。分别于12、24、48 h在倒置显微镜下观察MPC-5的形态结构;采用流式细胞术检测各组细胞凋亡率;采用实时荧光定量PCR法检测Park7 mRNA的表达;采用Western blot、免疫荧光染色法检测Park7蛋白表达。结果对照组各时间点细胞体足突数量多,细胞之间连接紧密,呈正常形态;PAN组各时间点细胞体积较对照组明显缩小,细胞间连接松散,可见破碎细胞;FK506组各时间点细胞体积较PAN组增大,细胞之间的连接较紧密,形态好转。PAN组各时间点细胞凋亡率较对照组明显升高,FK506组各时间点细胞凋亡率较PAN组均下降(P<0.05)。PAN组各时间点的Park7 mRNA表达量均较对照组显著增高,FK506组各时间点Park7 mRNA表达量较PAN组均下降(P<0.05)。Western blot结果显示PAN组中各时间点Park7蛋白表达量均较对照组显著增高,FK506组各时间点Park7蛋白表达量较PAN组均下降(P<0.05)。免疫荧光染色显示PAN组Park7蛋白分布在细胞膜和细胞质中,较对照组明显增加,呈密集团状分布,荧光强度增加;FK506组Park7蛋白分布较PAN组明显降低。结论PAN作用于MPC-5可引起细胞形态结构损伤及凋亡,以及Park7 mRNA及其蛋白的表达上调;FK506可使MPC-5损伤模型的Park7 mRNA及其蛋白表达下调,维持细胞功能稳态,减轻蛋白尿,延缓肾小球硬化。

Objective To study the effect of puromycin aminonucleoside(PAN)on the apoptosis of mouse podocyte clone 5(MPC-5)and the expression of recombinant human Parkinson's disease 7(Park7)and to study the protective mechanism of tacrolimus(FK506)against MPC-5 injury.Methods MPC-5 cells were cultured in vitro and then divided into three groups:blank control(control),PAN,and FK506.The cells in the PAN group were added with PAN(with a concentration of 50 mg/L)to establish a model of MPC-5 injury,and those in the FK506 group were added with PAN(with a concentration of 50 mg/L)and FK506(with a concentration of 5 mg/L).An inverted microscope was used to observe the morphology and structure of MPC-5 cells at 12,24,and 48 hours after treatment.Flow cytometry was used to measure cell apoptosis rate.Quantitative real-time PCR was used to measure the mRNA expression of Park7.Western blot and immunofluorescent staining were used to measure the protein expression of Park7.Results The control group had a large number of foot processes of the cell body at all time points,with tight connections between cells and a normal morphology.Compared with the control group,the PAN group had a significantly smaller cell volume at all time points,with loose connections between cells and the presence of ruptured cells.Compared with the PAN group,the FK506 group had an increased cell volume at all time points,with tighter connections between cells and a better morphology.The PAN group had a significantly higher apoptosis rate than the control group at all time points.Compared with the PAN group,the FK506 group had a significant reduction in the apoptosis rate at all time points(P<0.01).The PAN group had a significantly higher mRNA expression level of Park7 than the control group at all time points.Compared with the PAN group,the FK506 group had a significant reduction in the mRNA expression level of Park7 at all time points(P<0.01).Western blot showed that the PAN group had a significantly higher protein expression level of Park7 than the control group at all time points.Compared with the PAN group,the FK506 group had a significant reduction in the protein expression level of Park7 at all time points(P<0.01).Immunofluorescent staining showed that in the PAN group,there was a significantly lower expression of Park7 protein in cell membrane and cytoplasm,with a dense cluster distribution and increased fluorescence intensity.Compared with the PAN group,the FK506 group had a significant improvement in the distribution of Park7 protein.Conclusions PAN can act on MPC-5 cells and cause morphological and structural damage and apoptosis of MPC-5 cells,as well as upregulated mRNA and protein expression of Park7.FK506 can downregulate the mRNA and protein expression of Park7 in the model of MPC 5 injury,maintain cellular homeostasis,reduce proteinuria,and delay glomerulosclerosis.