重组脂连蛋白通过非AMPK依赖的蛋白激酶C途径抑制小鼠星形胶质细胞炎症相关蛋白表达

Recombinant adiponectin inhibits the expression of inflammation-associated proteins from mouse astrocytes via AMPK-independent PKC pathway

目的探讨重组脂连蛋白(APN)是否通过蛋白激酶C(PKC)及AMP活化蛋白激酶(AMPK)途径在小鼠星形胶质细胞氧糖剥夺/复氧(OGD/R)模型中发挥抗炎作用。方法MA1800小鼠星形胶质细胞分别采用氧糖剥夺(OGD)2、4、6、8、12、24 h及(20、50、100、200、400、600、800、1000、1200、1600、2000)μmol/L氯化钴(CoCl2)进行OGD/R处理,采用Western blot法检测PKC、AMPK的磷酸化水平,确定后续实验中OGD/R处理条件。将细胞分为正常对照组、OGD/R处理组、单纯APN处理组、OGD/R联合APN处理组、AMPK抑制剂联合OGD/R和APN处理组。采用Western blot法检测PKCα、磷酸化的广谱PKC[p-PKC(pan)]、AMPKα、磷酸化的AMPKα(p-AMPKα)以及含pyrin结构域NOD样受体家族3(NLRP3)炎性体和细胞培养液上清液中肿瘤坏死因子α(TNF-α)水平。结果在OGD处理6 h后复氧以及OGD处理中CoCl2为400μmol/L时,PKC磷酸化水平达到峰值。与对照组相比,OGD/R组PKCα、p-AMPKα水平,NLRP3炎性体及培养细胞上清中TNF-α水平显著增加;与模型组相比,APN处理组和AMPK抑制剂联合APN处理组的p-PKC(pan)水平、NLRP3水平及培养基中的TNF-α水平降低,但对APN处理组AMPKα的磷酸化无显著影响。结论重组APN可能通过非AMPK依赖的PKC途径抑制小鼠星形胶质细胞炎症相关蛋白表达。

Objective To investigate whether recombinant adiponectin peptide(APN)plays an anti-inflammatory role in the oxy-glucose deprivation/reoxygenation(OGD/R)model of astrocytes via the protein kinase C(PKC)and AMP dependent protein kinase(AMPK)pathway.Methods The phosphorylation levels of PKC and AMPK in astrocytic cell line MA1800 was assessed by Western blotting under the condition of OGD/R with the time gradient(2,4,6,8,12,24 hours)or the condition of cobalt chloride(CoCl2)treatment with the concentration gradient(20,50,100,200,400,600,800,1000,1200,1600,2000μmol/L)to determine the optimal condition of OGD/R in the following study.The cells were grouped into a normal control group,OGD/R treatment group,APN only group,OGD/R combined with APN treatment group,and AMPK inhibition combined with OGD/R and APN treatment group.Western blotting was used to detect the levels of PKCα,phospho-PKC(pan),AMPKα,phospho-AMPKα,NLRP3 inflammasome in the cells,and tumor necrosis factorα(TNF-α)in the conditioned medium of cell culture.Results After OGD/R or 400μmol/L CoCl2 treatment for 6 hours,the phosphorylation level of p-PKC reached a peak.Compared with the control group,the levels of PKCα,p-AMPKα,AMPK,NLRP3 and TNF-αin the supernatant increased significantly in the experimental groups.Compared with the ODG/R group,the phosphorylation level of PKC,the expression of TNF-αand NLRP3 significantly decreased in the APN treated groups,while the phosphorylation of AMPK remained unchanged.Conclusion APN treatment can inhibit the expression of inflammatory factors in astrocyte model of OGD/R via AMPK-independent PKC signaling pathway.