小檗碱在高糖及糖基化终末产物诱导足细胞损伤中的保护作用

Protective effect of berberine in podocyte injury induced by high glucose and advanced glycosylation end products

目的探讨小檗碱对糖基化终末产物(AGE)和高糖诱导下足细胞损伤及其骨架蛋白的影响及机制。方法以条件永生性人足细胞作为研究对象,于含10%胎牛血清及100 U/Lγ-干扰素的RPMI 1640培养液中进行体外培养,细胞增殖并诱导分化后进行分组处理。分别用高糖(30 mmo/L)、AGE(100μg/ml)、小檗碱(10μmo/L)处理48 h后,激光共聚焦检测技术观察纤维状肌动蛋白(F-actin),球状肌动蛋白(G-actin)变化;原位细胞免疫组化检测cspase-3,nephrin表达。采用SPSS13.0统计软件包进行统计学分析。结果共聚焦显微镜下观察显示,高糖及AGE作用下,足细胞F-actin出现重排,G-actin易位,小檗碱干预后有所恢复。免疫组化结果显示,对照组及高糖组几乎未见capase-3阳性表达,但高糖+AGE组,capase-3呈阳性表达(F=99.339,P<0.001);高糖+AGE组nephrin表达显著降低(F=165.84,P<0.001),与对照组及高糖组比较,差异均有统计学意义。小檗碱作用后,高糖+AGE组capase-3的水平下降(F=6.927,P=0.048),nephrin表达水平升高(F=165.84,P=0.025),差异均有统计学意义。结论在持续的高糖和AGE作用下,可引起足细胞骨架蛋白F-actin、G-actin重构及分布异常,并诱导足细胞凋亡,小檗碱能改善高糖和AGE引起的足细胞骨架蛋白损伤,并抑制足细胞的凋亡,其机制可能与nephrin的参与有关。

Objective To investigate the protective effect of berberine in podocyte injury and cytoskeleton change induced by high glucose and advanced glycosylation end-products （AGE）.Methods The conditionally immortalized human glomerular podocytes were used for study,and cultured in RPMI 1640 medium containing 10% fetal bovine serum and 100 U/L gamma-interferon. After proliferation and differentiation induction,the podocytes were treated for 48 hours in groups,including high glucose （30 mM） group,AGE （100μg/mL）group,and berberine （10μM）group,respectively.The changes of F-actin and G-actin in podocytes were observed with laser confocal microscope;and the expressions of capase-3 and nephrin were detected with in-situ immunocytochemistry.For statistical analysis,the SPSS 13.0 statistical software package was used.Results The laser confocal microscope showed that F-actin reorganized,and G-actin dislocated in the high glucose plus AGE group,which recovered partly after berberine treatment. Immunohistochemical results showed that the control group and the high glucose group hardly displayed any positive expression of capase-3.But the high glucosre plus AGE group displayed positive expression of capase-3 （F=99.339,P〈0.001）,and significantly decreased expression of nephrin （F=165.84,P〈0.001）,the differences of which were statistically significant compared with those of the control group and the high glucose group.After treatment with berberine,the high glucose plus AGE group showed lower expression of capase-3 （F=6.927,P=0.048）,but higher expression of nephrin （F=165.84,P=0.025）,the differences of which were statistically significant.Conclusion High glucose and AGE induced F-actin reoganization and G-actin dislocation,and stimulated apoptosis in podocytes,which were inhibited＆amp;nbsp;by berberine,whose mechanism may involve the participation of nephrin.