地塞米松拮抗脂多糖对TRPC6表达及足细胞损伤的影响

Effect of Dexamethasone Reverses Lipopolysaccharides on TRPC6 Expression and Podocyte Injury

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 Ca^2＋ concentration in LPS-induced podocytes, and to explore the effect of dexamethasone （DEX） on the change of TRPC6 and Ca^2＋ . Methods Immortalized mouse podocytes in vitro were randomly divided into control group, LPS group, DEX group and LPS ＋ DEX group. Intracellular Ca^2＋ concentration was measured by Fura-2/AM loading. The percentage of podocyte apoptosis was detected by flow cytometry with annexin V-FITC/PI double staining. Cytoskel- etal protein of F-actin immunofluoresence staining and semi - quantitative analysis was performed. The expression of TR PC6, nephrin and desmin proteins were detected by western blotting. Results Fluorescence intensity change of Ca^2 ＋ in podocytes was 15.6 ±6.1 in control group, 17.8 ±6.5 in DEX group, 116.2 ±15.4 in LPS group, and 57.2 ±13.6 in LPS ＋ DEX group respectively. The percentage of apoptosis podocytes was （0.44 ±0.24） % in control group, （0.45 ±0.18） % in DEX group, （2.28 ±0.36） %in LPS group, （1.78 ±0.33） %in LPS ＋ DEX group. Mean fluorescence intensity （MFI） of F-actin was 925 ±136 in control group, 924 ±148 in DEX group, 217 ±44.4 in LPS group, 634 ±71.9 in LPS＋ DEX group. The protein expressions of TRPC6, nephrin and desmin were 0.33 ＋ 0.09, 1.14 ±0.15 and 0.32 ±0.05 respectively in the control group, 0.36±0.08, 1.10 ±0.15 and 0.38±0.04 respectively in DEX group, 0.81 ＋ 0.13, 0.39 ± 0. 11 and 1.01 ±0.15 respectively in LPS group, 0.61 ±0.12, 0.68 ±0.10 and 0.84 ±0.14 respectively in LPS ＋ DEX group. Corn pared with the control group and DEX group, Ca^2＋ concentration and podocyte apoptosis percentage were increased, F-actin was reduced, the expressions of TRPC6 and desmin were upregulated, the expression of nephrin was downregulated in LPS group.Compared with LPS group, Ca^2＋ concentration and podoeyte apoptosis percentage were decreased, F-aetin was elevated, the expressions of TRPC6 and desmin were downregulated, the expression of nephrin was upregulated in LPS＋ DEX group.Conclusions DE？（ reverses LPSyinduced overexpressing of TRPC6 and the high levels of intraeellular Ca^2＋ concentration in podocytes, and has a protective effect on podocytes.