间歇性高糖环境通过激活过氧化物酶增殖物激活受体γ下调成骨细胞Wnt信号通路

Intermittent high glucose environment downregulate the Wnt signaling pathway of osteoblasts by activating peroxisome proliferator activated receptor γ

目的研究Wnt信号转导通路在间歇性高糖环境所致成骨细胞损伤中的作用机制。方法 (1)将对数生长期的第3代成骨细胞随机分为3组:5.5mmol·L^(-1)正常糖浓度组,25 mmol·L^(-1)持续性高糖组,25~5 mmol·L^(-1)QD间歇性高糖组。半定量RT-PCR法检测细胞中过氧化物酶增殖物激活受体γ(PPARγ)和骨形成蛋白(BMP-2)的mRNA表达情况;以免疫印迹法检测细胞中PPARγ和BMP-2蛋白表达情况。(2)正常糖浓度成骨细胞作为空白对照组、间歇性高糖成骨细胞作为模型组。在此基础上,分别给予阴性药物——Wnt蛋白阻滞剂Dickkopf 1(DKK-1)蛋白、阳性药物——Wnt蛋白激动剂氯化锂(Li Cl),分为DKK-1对照组、DKK-1模型组、Li Cl对照组、Li Cl模型组,干预3周后,评价PPARγ、BMP-2及Wnt相关蛋白β-catenin及Cyclin D1蛋白表达情况。结果 (1)干预3周后,间歇性高糖组与高糖组、正常糖浓度组的BMP-2mRNA灰度值分别是0.49±0.03,0.83±0.04,0.96±0.02;这3组的PPARγmRNA灰度值分别是0.94±0.02,0.86±0.05,0.77±0.04,组间比较差异均有统计学意义(均P<0.05),存在时间依赖性。(2)模型组与空白对照组比较,PPARγ表达升高,而β-catenin、Cyclin D1及BMP-2蛋白表达降低;DKK-1模型组与DKK-1对照组相比,PPARγ表达升高,而β-catenin、Cyclin D1及BMP-2蛋白表达明显降低;氯化锂组与DKK-1组相比,PPARγ表达降低、而β-catenin、Cyclin D1及BMP-2蛋白表达都明显升高,上述指标组间两两比较,差异均有统计学差异(均P<0.05)。结论间歇性高糖环境所致成骨细胞损伤可能与PPARγ激活后Wnt信号转导通路被抑制有关。

Objective To explore the role of the Wnt signaling pathway in the molecular mechanism of osteoblasts damage mediated by intermittent high glucose（ Glu）. Methods（ 1） The third generation osteoblasts during the logarithmic growth period were divided into 3 groups： normal glucose group（ 5. 5 mmol · L^（-1） Glu）,persistent high glucose（ 25 mmol·L^（-1） Glu） and intermittent high glucose group（ 5. 5-25 mmol·L^（-1） Glu）. After three weeks of drug intervention,peroxisome proliferator-activated receptor gamma（ PPARγ） and bone morphogenetic protein（ BMP） 2 mRNA expressions of osteoblasts were detected by semi-quantitative RT-PCR and Western blot.（ 2） The normal glucose was served as blank control group,and intermittent high glucose group asmodel group. The cultured osteoblasts were cultured and divided into following groups（ DKK-1 control group,DKK-1 model group,Li Cl control group,Li Cl model group） in which added Wnt signaling pathway activator lithium chloride（ Li Cl） or blocker Dickkopf 1 protein（ DKK-1）. After three weeks of drug intervention,the expressions of peroxisome proliferator-activated receptor（ PPARγ）,BMP-2 and Wnt-related proteins such as beta-catenin and Cyclin D1 were detected by Western blot. Results（ 1） After 3 weeks of intervention,the gray value of BMP-2 mRNA in intermittent high glucose,the high glucose group,and normal glucose group were 0. 49 ± 0. 03,0. 83 ± 0. 04,0. 96 ± 0. 02,the gray value of PPARγ in the three groups were 0. 94 ± 0. 02,0. 86 ± 0. 05,0. 77 ± 0. 04 respectively,the differences between groups had statistical significance（ all P 0. 05）. It is in time-dependent manner.（ 2） Comparison between model group with the blank control group found that with the intervention time increased,the expression of the PPARγincreased and β-catenin,Cyclin D1,BMP-2 decreased with statistical significance（ all P 0. 05）. Comparison between DKK-1 group and Li Cl group,the expression of the factors show the opposite trend. Conclusion The damage of intermittent high glucose to osteoblasts may be related to the inhibition of Wnt signal transduction pathway activated by PPARγ.