CP-25调控GRK2/p38MAPK信号在血管紧张素Ⅱ诱导小鼠系膜细胞增殖中的作用

The role of CP-25 on the proliferation induced by angiotensinⅡin mouse mesangial cells through regulating GRK2/p38MAPK signals

目的研究芍药苷-6′-O-苯磺酸酯(CP-25)在血管紧张素Ⅱ(AngⅡ)诱导的肾小球系膜细胞(MCs)增殖中的作用及相关机制。方法体外培养SV40 MES 13系膜细胞,以AngⅡ诱导MCs增殖,高内涵成像显微镜检测不同浓度CP-25(10、100、1000 nmol/L)对AngⅡ诱导的MCs增殖的影响;Western blot检测MCs中G蛋白偶联受体激酶-2(GRK2)和磷酸化p38(p-p38)蛋白表达水平;激光共聚焦显微镜检测GRK2和p-p38蛋白的荧光信号并分析二者共定位率;成像流式细胞仪检测MCs中GRK2入胞质的细胞比例。结果与对照组比较,AngⅡ可诱导MCs的增殖,增加GRK2和p-p38蛋白表达,上调GRK2和p-p38MAPK共定位率,提高GRK2入胞比例,差异有统计学意义(P<0.01)。CP-25(10、100、1000 nmol/L)可不同程度地抑制AngⅡ诱导的增殖,抑制GRK2、p-p38蛋白表达及GRK2和p-p38的共定位水平,降低GRK2入胞比例。结论CP-25可抑制AngⅡ诱导的MCs增殖,其机制与调节GRK2/p38信号有关。

Objective To study the effects and mechanisms of paeoniflorin-6′-O-benzenesulfonate(CP-25)on the proliferation of mesangial cells(MCs)induced by angiotensin(Ang)Ⅱ.Methods The mesangial cell line SV40 MES 13 was cultured in vitro and stimulated with AngⅡ(50 nmol/L)for 2 days.CP-25(10,100,1000 nmol/L)was added at same time.After co-cultured 48 h,the proliferation of MCs was measured by high-content imaging microscope.The protein expression levels of GRK2 and phosphorylated p38(p-p38)in MCs were detected by Western blot.The fluorescence signaling of GRK2 and p-p38 protein was measured with laser confocal microscope and its colocalization rates were analyzed.The proportion of GRK2 into the cytoplasm was detected with cytometer imaging flow.Results Compared to the control group,the results showed that AngⅡcould significantly promote the MCs proliferation,increase the protein expression levels of GRK2 and p-p38,up-regulate the colocalization rates of GRK2 and p-p38,and increase the proportion of GRK2 into the cytoplasm.CP-25(10,100,1000 nmol/L)could inhibit the proliferation induced by AngⅡto varying degrees,reduce the protein expression levels of GRK2,p-p38 and the co-localization rates of GRK2 and p-p38,and down-regulate the ratios of GRK2 into cytoplasm.Conclusion CP-25 can inhibit MCs′proliferation stimulated with AngⅡ,and its mechanism may be related to GRK2/p38MAPK signaling pathway.