他克莫司对嘌呤霉素损伤的足细胞中α-辅肌动蛋白-4表达的影响

Effect of Tacrolimus on the eXpression of α-actinin-4 in podocytes induced by Puromycin aminonucleoside

目的研究他克莫司( FK506)和嘌呤霉素( PAN)对肾小球足细胞凋亡和α-辅肌动蛋白-4(α-actinin-4)mRNA及蛋白表达的影响,探讨FK506对足细胞的保护作用.方法体外培养小鼠肾小球足细胞,设置对照组、PAN组和FK506组,处理8 h、24 h、48 h后观察细胞形态,检测细胞凋亡率,并收集细胞进行实时荧光定量-PCR、Western blot检测α-actinin-4的mRNA及蛋白表达情况.结果 PAN组各时间段胞体面积较对照组明显缩小,FK506组胞体面积较PAN组增大.8 h时各组间足细胞凋亡率无明显差异(均P>0. 05);24 h及48 h时PAN组足细胞凋亡率[(8. 21 ± 0. 41)%、(16. 32 ± 0. 17)%]较对照组[(4. 28 ± 0. 35)%、(6. 27 ± 0. 28)%]明显升高,差异均有统计学意义(均P<0. 05),FK506组足细胞凋亡率[(6. 26 ± 0. 24)%、(13. 32 ± 0. 24)%]均明显低于PAN组,差异均有统计学意义(均P<0. 05).8 h时各组α-actinin-4 mRNA和蛋白表达无明显差异(均P>0. 05),24 h、48 h 时PAN组mRNA(2. 42 ± 0. 21、3. 78 ± 0. 25)和蛋白(0. 77 ± 0. 04、1. 22 ± 0. 10)表达量较对照组mRNA(1. 50 ± 0. 22、2. 15 ± 0. 15)和蛋白(0. 44 ± 0. 03、0. 83 ± 0. 07)显著增高,差异均有统计学意义(均P<0. 01);FK506组mRNA(1. 65 ± 0. 24、1. 70 ± 0. 32)和蛋白(0. 52 ± 0. 05、0. 56 ± 0. 07)表达量均明显低于PAN组,差异均有统计学意义(均P<0. 05).结论 FK506可有效抑制PAN对足细胞的损伤作用,稳定α-actinin-4的表达,为临床应用FK506治疗肾小球疾病提供依据.

Objective To investigate the effects of Tacrolimus( FK506 )and Puromycin aminonucleoside ( PAN)on apoptosis and expression of α-actinin-4 mRNA and protein in mouse glomerular podocytes in order to ex-plore the protective effect of FK506 on podocytes. Methods Mouse glomerular podocytes were cultured in υitro,and the control group,PAN group and FK506 group were established. After 8 h,24 h and 48 h of treatment,the cell mor-phology was observed and the apoptosis rate was detected. The cells were collected by real-time PCR and Western blot was used to detect the mRNA and protein expression of α-actinin-4. Results The cell body area of the PAN group was significantly smaller than that of the control group,and the cell area of the FK506 group was larger than that of the PAN group. There was no significant difference in the rate of podocyte apoptosis between those groups at 8 h( all P>0. 05). At 24 h and 48 h,the apoptotic rate of podocytes in PAN group[(8. 21 ± 0. 41)%,(16. 32 ± 0. 17)%]were significantly higher than those in the control group[(4. 28 ± 0. 35)%,(6. 27 ± 0. 28)%],and the differences were significant(all P<0. 05). The apoptosis rate of podocytes in FK506 group[(6. 26 ± 0. 24)%,(13. 32 ± 0. 24)%] were significantly lower than those in PAN group,and the differences were significant(all P<0. 05). At 8 h,there was no significant difference in the expression of α-actinin -4 mRNA and protein( all P >0. 05 ). The expression of mRNA(2. 42 ± 0. 21,3. 78 ± 0. 25)and protein(0. 77 ± 0. 04,1. 22 ± 0. 10)in the PAN group was significantly higher than mRNA(1. 50 ± 0. 22,2. 15 ± 0. 15)and protein(0. 44 ± 0. 03,0. 83 ± 0. 07)in the control group at 24 h and 48 h,and the differences were significant(all P<0. 01). The expression of mRNA(1. 65 ± 0. 24,1. 70 ± 0. 32)and protein(0. 52 ± 0. 05,0. 56 ± 0. 07)in FK506 group was significantly lower than that of PAN group,and the differ-ences were significant(all P<0. 05). Conclusions FK506 can effectively inhibit the damage of PAN on podocytes and stabilize the expression of α-actinin-4,which provides a basis for the clinical application of FK506 in the treat-ment of glomerular diseases.