外源性Klotho对糖尿病肾病小鼠成骨细胞甲状旁腺激素刺激的分化活性调控作用

Regulatory effect of Klotho on changes of osteoblast differentiation activity induced by PTH(1-34)in diabetic nephropathy mice

目的观察外源性Klotho对糖尿病肾病小鼠成骨细胞甲状旁腺激素(PTH)刺激的分化活性的调控作用。方法18周龄KK-Ay糖尿病肾病模型小鼠及C57BL/6J对照小鼠各12只,处死后取股骨,采用骨片贴壁分离筛选法获取成骨细胞,分为A、B组,KK-Ay小鼠成骨细胞为A组,C57BL/6J小鼠成骨细胞为B组。A组成骨细胞分别加入不同浓度的PTH(1-34)、Klotho,分别记为A1、A2、A3、A4、A5组,其中A1组加入0μg/mL的PTH(1-34),A2组加入0.05μg/mL的PTH(1-34),A3组加入0.1μg/mL的PTH(1-34),A4组加入0.2μg/mL的PTH(1-34),A5组加入0.1μg/mL的PTH(1-34)和100 ng/mL的Klotho。B组成骨细胞参照A组各亚组给予相同处理,分别记为B1组、B2组、B3组、B4组、B5组。采用ELISA法检测各组细胞中环磷酸腺苷(cAMP),采用蛋白印迹法检测各组细胞中磷酸化蛋白激酶A(p-PKA)、磷酸化细胞外调节蛋白激酶(p-ERK)1/2,作为成骨细胞对PTH刺激反应性的指标。结果A1组、A2组、A3组、A4组细胞中cAMP浓度、p-PKA相对表达量、p-ERK1/2相对表达量组间相比,P均>0.05。B1组、B2组、B3组、B4组细胞中cAMP浓度、p-PKA相对表达量、p-ERK1/2相对表达量组间相比,P均<0.05。A5组细胞中cAMP浓度、p-PKA相对表达量、p-ERK1/2相对表达量与B5组相比,P均>0.05。结论正常小鼠成骨细胞对PTH刺激反应性呈剂量依赖性上升,而糖尿病肾病小鼠成骨细胞对PTH刺激无反应;加入外源性Klotho可促进糖尿病肾病小鼠成骨细胞对PTH的刺激反应。

Objective To investigate the regulatory effect of exogenous Klotho on the activity of osteoblasts in diabetic nephropathy mice induced by recombinant parathyroid hormone(PTH)(1-34).Methods Twelve 18-week-old KK-Ay diabetic nephropathy model mice and 12 C57BL/6J mice were sacrificed to collect their femora,and osteoblasts were cultured and identified by the method of adherent separation and screening of bone fragments.Osteoblasts were then divided into two groups:the osteoblasts from KK-Ay mice were taken as the group A and osteoblasts from C57BL/6J mice as group B.The osteoblasts in the group A were treated with different concentrations of PTH(1-34)and Klotho,which were marked as groups A1,A2,A3,A4,andA5.Osteoblasts in the group A1 were added with 0μg/mL PTH(1-34),group A2 with 0.05μg/mL PTH(1-34),group A3 with 0.1μg/mL PTH(1-34),group A4 with 0.2μg/mL PTH(1-34),and group A5 with 0.1μg/mL PTH(1-34)and 100 ng/mL Klotho.The osteoblasts in the group B were divided into groups B1,B2,B3,B4,and B5,which were treated with the same methods of subgroups of group A.Intracellular cyclic adenosine monophosphate(cAMP)levels were detected by ELISA,and the levels of phosphorylated PKA(p-PKA)and phosphorylated extracellular signal-regulated kinase(p-ERK)1/2 in osteoblasts were detected by Western blotting,which were taken as the indicators of osteoblasts’response to PTH stimulation.Results There were no significant differences in the levels of intracellular cAMP,p-PKA or p-ERK1/2 between groups A1,A2,A3,and A4(all P>0.05).Significant differences were found in the levels of intracellular cAMP,p-PKA and p-ERK1/2 between groups B1,B2,B3,and B4(all P<0.05).There were no significant differences in the levels of intracellular cAMP,p-PKA,or p-ERK1/2 between group A5 and group B5(all P>0.05).Conclusions The osteoblasts of normal mice show a dose-dependent increase in the response to PTH stimulation,while the osteoblasts of diabetic nephropathy mice do not respond to PTH stimulation.Adding Klotho can improve the osteoblasts’response to PTH stimulation in diabetic nephropathy mice.