白细胞介素27在肾小球足细胞损伤的作用及他克莫司对其的影响

The role of interleukin-27 in glomerular podocyte injury and the effect of Tacrolimus on glomerular podocyte injury

目的研究嘌呤霉素(PAN)致肾小球足细胞损伤中白细胞介素27(IL-27)mRNA和蛋白表达的变化,探讨他克莫司(FK506)在肾小球足细胞损伤中的保护作用机制。方法体外培养小鼠肾小球足细胞,分为3组:对照组、PAN组和FK506组。3组足细胞经处理8 h、24 h、48 h后在显微镜下观察细胞形态,应用酶联免疫吸附试验(ELISA)观察IL-27水平的变化,并收集培养的足细胞行实时荧光定量PCR(qPCR)检测IL-27 mRNA表达的变化,采用Western blot检测IL-27蛋白表达的变化。结果1.足细胞形态:在各个时间点(8 h、24 h、48 h),PAN组细胞体积较对照组缩小,形态改变;FK506组较PAN组体积偏大并饱满,趋于正常。2.IL-27的水平:在各个时间点(8 h、24 h、48 h),PAN组[(110.00±3.52)ng/L、(302.00±6.23)ng/L、(397.00±8.92)ng/L]均高于对照组[(90.00±5.12)ng/L、(85.00±4.21)ng/L、(88.00±4.20)ng/L],差异均有统计学意义(均P<0.05);FK506组[(96.00±4.17)ng/L、(107.00±4.86)ng/L、(112.00±6.24)ng/L]均低于PAN组,差异均有统计学意义(均P<0.05)。3.IL-27 mRNA表达量:在各个时间点(8 h、24 h、48 h),PAN组(1.25±0.11、1.57±0.08、1.73±0.13)均高于对照组(1.02±0.02、1.10±0.04、0.96±0.02),差异均有统计学意义(均P<0.05);FK506组8 h时(1.10±0.06)无明显升高,24 h和48 h时(1.21±0.04、1.30±0.09)表达量稍有升高;与PAN组比较,FK506组均低于PAN组,差异均有统计学意义(均P<0.05)。4.IL-27蛋白的表达量:在各个时间点(8 h、24 h、48 h),PAN组(0.94±0.04、1.56±0.07、1.63±0.04)均高于对照组(0.83±0.04、0.85±0.03、0.83±0.05),差异均有统计学意义(均P<0.05);FK506组(0.84±0.05、0.89±0.04、0.91±0.06)与PAN组比较,8 h无明显差异,24 h后IL-27蛋白表达下降,差异有统计学意义(P<0.05),48 h后IL-27蛋白表达下降更为明显,差异有统计学意义(P<0.05)。结论IL-27参与肾脏疾病的发病,FK506通过降低IL-27的表达,抑制肾小球足细胞的损伤,为临床应用FK506治疗肾脏病提供一定依据。

Objective To investigate changes in mRNA and protein expression of interleukin-27(IL-27)in glomerular podocyte injury caused by Puromycinonucleoside(PAN),and to explore the mechanism of the protective effect of Tacrolimus(FK506)on glomerular podocyte injury.Methods Glomerular foot cells from mice were cultured in vitro and divided into 3 groups,which were the control group,PAN group and FK506 group.The morphology of 3 groups of foot cells was observed under a microscope after 8 h,24 h,and 48 h treatment.The changes in IL-27 concentrations were detected by analyzing the enzyme-linked immunosorbent assay(ELISA)method.The cultured foot cells were then collected.The changes of IL-27 mRNA expression were measured by real-time fluorescence quantitative PCR(qPCR),and the changes of IL-27 protein expression were detected by Western blot.Results(1)At each time point(8 h,24 h,48 h),the cells of the PAN group had smaller volume and different morphology than the cells of the control group,and the cells of the FK506 group was larger and fuller than the cells of the PAN group.(2)The concentrations of IL-27 in the PAN group[(110.00±3.52)ng/L,(302.00±6.23)ng/L,(397.00±8.92)ng/L]were significantly higher than those in the control group[(90.00±5.12)ng/L,(85.00±4.21)ng/L,(88.00±4.20)ng/L]and those in the FK506 group[(96.00±4.17)ng/L,(107.00±4.86)ng/L,(112.00±6.24)ng/L]at 8 h,24 h and 48 h(all P<0.05).(3)At each time point(8 h,24 h,48 h),the IL-27 mRNA expression of the PAN group(1.25±0.11,1.57±0.08,1.73±0.13)was significantly higher than that of the control group(1.02±0.02,1.10±0.04,0.96±0.02)(all P<0.05).Compared with the control group,the IL-27 mRNA expression of the FK506 group did not significantly increase at 8 h(1.10±0.06),and showed a slight increase at 24 h and 48 h(1.21±0.04,1.30±0.09).Compared with PAN group,HC506 group were all lower(all P<0.05).(4)At each time point(8 h,24 h,48 h),the expression of IL-27 protein in the PAN group(0.94±0.04,1.56±0.07,1.63±0.04)was significantly higher than that in the control group(0.83±0.04,0.85±0.03,0.83±0.05),there was significant difference(all P<0.05).Compared with the PAN group,the expression of IL-27 protein in the FK506 group(0.84±0.05,0.89±0.04,0.91±0.06)was not significantly different at 8 h,but decreased significantly at 24 h and 48 h,there was significant difference(P<0.05).Conclusions IL-27 is involved in the pathogenesis of kidney diseases.FK506 can prevent the glomerular podocyte injury by reducing the expression of IL-27.This study provides experimental basis for clinical application of FK506 in the treatment of kidney diseases.