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目的观察脂多糖(lipopolysaccharides,LPS)诱导足细胞(podocyte,PC)瞬时受体电位阳离子通道蛋白6(transient receptor potential cation channel 6,TRPC6)表达及胞内Ca2+浓度的变化,探讨地塞米松(dexamethasone,DEX)对TRPC6及Ca2+变化的影响。方法体外分化培养的永生化小鼠PC随机分为:1对照组;2LPS组;3DEX组;4LPS+DEX组。Fura-2/AM负载检测细胞内Ca2+浓度,Annexin V-FITC/PI标记流式细胞测定PC的凋亡百分率,免疫荧光染色及半定量分析骨架蛋白F-肌动蛋白分布及数量,Western blotting检测足细胞TRPC6、nephrin及desmin蛋白的表达。结果1对照组、DEX组、LPS组、LPS+DEX组PC Ca2+荧光强度变化分别为15.6±6.1、17.8±6.5、116.2±15.4、57.2±13.6;凋亡百分率分别为(0.44±0.24)%、(0.45±0.18)%、(2.28±0.36)%、(1.78±0.33)%;F-肌动蛋白平均荧光强度分别为925±136、924±148、217±44.4、634±71.9;TRPC6蛋白的表达分别为0.33±0.09、0.36±0.08、0.81±0.13、0.61±0.12;nephrin表达为1.14±0.15、1.10±0.15、0.39±0.11、0.68±0.10;desmin表达为0.32±0.05、0.38±0.04、1.01±0.15、0.84±0.14。2与对照组及DEX组相比,LPS组足细胞Ca2+浓度增加,凋亡百分率增加,F-肌动蛋白减少,TRPC6、desmin表达增加,nephrin表达降低。3与LPS组相比,LPS+DEX组足细胞Ca2+浓度及凋亡百分率降低,F-肌动蛋白增加,TRPC6、desmin表达下调,nephrin表达上调。结论 DEX拮抗LPS引起足细胞TRPC6高表达及细胞内Ca2+水平增加,对足细胞有保护作用。
Objective To observe the changes of transient receptor potential cation channel 6 (TRPC6) expression and intracellular Ca2 + concentration induced by lipopolysaccharides (LPS) in rats and to investigate the effect of dexamethasone , DEX) on TRPC6 and Ca2 + changes. Methods Immortalized mouse PC differentiated in vitro were randomly divided into 1 control group, 2 LPS group, 3DEX group and 4 LPS + DEX group. Fura-2 / AM load detection of intracellular Ca2 concentration, Annexin V-FITC / PI flow cytometry PC apoptosis percentage, immunofluorescence staining and semi-quantitative analysis of skeletal protein F-actin distribution and quantity, Western blotting detection Podocyte TRPC6, nephrin and desmin protein expression. Results The changes of the fluorescence intensity of PC Ca2 + in control group, DEX group, LPS group and LPS + DEX group were 15.6 ± 6.1, 17.8 ± 6.5, 116.2 ± 15.4 and 57.2 ± 13.6, respectively; the percentages of apoptosis were (0.44 ± 0.24)%, (0.45 ± 0.18)%, (2.28 ± 0.36)%, (1.78 ± 0.33)%, respectively. The average fluorescence intensity of F-actin was 925 ± 136,924 ± 148,217 ± 44.4,634 ± 71.9, The expression of nephrin was 1.14 ± 0.15, 1.10 ± 0.15, 0.39 ± 0.11, 0.68 ± 0.10 respectively; the expression of desmin was 0.32 ± 0.05, 0.38 ± 0.04, 1.01 ± 0.15,0.84 ± 0.14.2 Compared with the control group and the DEX group, the podocyte Ca2 + concentration increased, the percentage of apoptosis increased, F-actin decreased, the expression of TRPC6, desmin increased and the expression of nephrin decreased in LPS group. Compared with LPS group, the Ca2 + concentration and apoptosis percentage of podocytes in LPS + DEX group decreased, F-actin increased, the expression of TRPC6, desmin was down-regulated and the expression of nephrin was up-regulated. Conclusion DEX antagonizes LPS-induced high expression of TRPC6 in podocytes and an increase in intracellular Ca2 + level, which has a protective effect on podocytes.