ROS介导棕榈酸诱导的肾小球足细胞骨架及裂孔隔膜损伤

Role of ROS in palmitic acid induced actin cytoskeleton and slit diaphragm injury in podocytes

摘要:

目的：探讨棕榈酸（palmitic acid，PA）对足细胞肌动蛋白纤维骨架及裂孔隔膜蛋白损伤的影响。方法：应用PA刺激体外培养的条件永生性小鼠肾小球足细胞株，分为对照组（1% BSA）和PA（150 ?滋mol/L）组。采用Alexa 549-phalloidin染色观察足细胞肌动蛋白纤维骨架的变化；免疫荧光染色法观察检测足细胞裂孔隔膜蛋白Nephrin和Podocin的表达；油红“O”、透射电镜以及BODIPY染色检测PA刺激下足细胞内脂质积聚情况。应用不同浓度PA刺激足细胞，分为对照组（1% BSA）、50 ?滋mol/L PA组、150 ?滋mol/L PA组、300 ?滋mol/L PA组。采用Western blot检测足细胞裂孔隔膜蛋白Nephrin和Podocin的表达。进一步应用氧自由基清除剂N-乙酰半胱氨酸（N-acetyl cysteine，NAC）预处理足细胞，分为对照组（1% BSA）、PA（150 ?滋mol/L）组、NAC组、PA（150 ?滋mol/L）+ NAC组。采用DCFH-DA染色检测细胞内活性氧（reactive oxygen species，ROS）产生；Alexa 549-phalloidin染色观察足细胞肌动蛋白纤维骨架的变化；Western blot检测足细胞裂孔隔膜蛋白Nephrin和Podocin的表达。结果：Western blot结果示300 ?滋mol/L PA组足细胞内Nephrin和Podocin 的表达（0.360±0.030，0.900±0.040）分别与对照（Ctr）组（1.000±0.030，1.000±0.030）和50 ?滋mol/L PA组（0.912±0.020，0.900±0.040）相比都明显减少（P=0.000，P=0.000；P=0.000，P=0.003）。免疫荧光显示与Ctr组相比，PA组足细胞Nephrin（t=6.014，P=0.010）和Podocin（t=6.171，P=0.010）蛋白的表达减少。使用NAC预处理足细胞清除ROS，与PA组（1.21±0.04，1.30±0.03）相比，PA+NAC组Nephrin（2.87±0.05）和Podocin（2.69±0.06）的表达减少（P=0.000，P=0.000）。结论：ROS介导了棕榈酸诱导的足细胞骨架及裂孔隔膜损伤。

Abstract:

Objective：To investigate the effect of palmitic acid（PA） on actin cytoskeleton and slit diaphragm injury in podocytes. Methods：Conditioned immortalized mouse glomerular podocyte strains stimulated with PA in vitro were divided into control group （1% BSA） and PA（150 ?滋mol/L） group. Alexa 549-phalloidin staining was used to observe the change of the actin cytoskeleton of the podocyte. Immunofluorescence staining was used to detect the expression of Nephrin and Podocin. The oil red “O”，transmission electron microscope and BODIPY staining were used to detect intracellular lipid accumulation with PA stimulation. The podocytes were stimulated with different concentrations of PA and divided into control group（1% BSA），50 ?滋mol/L PA group，150 ?滋mol/L PA group，and 300 ?滋mol/L PA group. The expression of Nephrin and Podocin was measured by Western blot. The podocytes were further treated with oxygen free radical scavenger N-acetylcysteine（NAC） and divided into control group（1% BSA），PA（150 ?滋mol/L） group，NAC group，PA（150 ?滋mol/L） + NAC group. DCFH-DA staining was used to detect the generation of reactive oxygen species（ROS） in the cells. Alexa 549-phalloidin staining was used to observe the changes of the actin cytoskeleton of the podocyte. Western blot was used to detect the expression of Nephrin and Podocin. Results：Western blot results showed that the expression of Nephrin and Podocin in the podocyte of the 300 ?滋mol/L PA group（0.360±0.030，0.900±0.040），was significantly decreased（P=0.000，P=0.000；P=0.000，P=0.003） compared with those of control group（1.000±0.030，1.000±0.030） and the 50 ?滋mol/L PA group（0.912±0.020，0.900±0.040）. Immunofluores－cence revealed a decrease in the expression of Nephrin（t=6.014，P=0.010） and Podocin（t=6.171，P=0.010） protein in the podocyte of the PA group compared with those of control group. With the pretreatment of podocytes with NAC to clear ROS，expression of Nephrin（2.87±0.05） and Podocin（2.69±0.06） decreased in PA+NAC group（P=0.000，P=0.000），when compared with that of PA group（1.21±0.04，1.30±0.03）. Conclusion：ROS mediated palmitic acid induces cytoskeletal structure injury in podocytes.