小檗碱通过JNK信号通路调控大鼠脂肪干细胞成骨向分化

Berberine promotes osteogenic differentiation of rat adipose- derived stem cells through JNK signaling pathway

目的:探讨小檗碱(C_(20)H_(18)NO_(4))对大鼠脂肪干细胞(ADSCs)的成骨分化作用及其相关机制。方法:以5、10、20μmol/L浓度小檗碱培养液处理ADSCs,未处理组为对照组通过MTT法检测细胞增殖活性,应用碱性磷酸酶(ALP)染色及半定量、钙结节染色分析小檗碱对ADSCs成骨分化的影响,采用Western印迹法检测c-Jun氨基末端激酶(JNK)的蛋白磷酸化水平及应用JNK通路抑制剂SP600125前、后成骨相关蛋白Runx2和OCN的表达水平。采用SPSS22.0软件包对数据进行统计学分析。结果:5、10、20μmol/L小檗碱处理大鼠ADSCs13、7天后,细胞增殖活性无显著改变(P>0.05);实验组ALP染色及茜素红染色均较对照组明显深染,实验组ALP蛋白分泌显著上调(P<0.05)。在10μmol/L小檗碱培养液作用下,JNK蛋白磷酸化水平升高,实验组成骨相关蛋白Runx2和OCN表达升高;抑制JNK信号通路后,Runx2和OCN表达下调。结论:小檗碱对ADSCs的增殖无影响,可通过激活JNK信号通路,上调脂肪干细胞的成骨分化效应。

PURPOSE: This study aimed at exploring the effect of berberine(C_(20)H_(18)NO_(4)) on osteogenic differentiation of rat adipose-derived stem cells(ADSCs) and clarifying the related mechanism. METHODS: ADSCs were subjected to 5, 10,20 μmol/L berberine culture solution. The untreated ADSCs were set as the control group. Cell proliferation activity was determined by MTT method. Alkaline phosphatase(ALP) staining, semi-quantitative assay and alizarin red staining(ARS) were applied to analyze the effect of berberine on osteogenic differentiation of ADSCs. The phosphorylation level of c-Jun amino terminal kinase(JNK) protein was tested by Western blot. Runx2, OCN were tested by Western blot before and after application of JNK pathway inhibitor SP600125. SPSS 22.0 software package was used for statistical analysis. RESULTS: There was no significant difference on cell proliferation activity of ADSCs treated with 5, 10 and 20 μmol/L berberine at 1, 3 and 7 day(P>0.05). ALP staining and ARS staining in groups treated by berberine were significantly darker than those of the control group, and ALP protein secretion in the experimental group was significantly up-regulated(P<0.05). The phosphorylation level of JNK was increased after treated with 10 μmol/L berberine culture medium. The expression of osteogenic related proteins Runx2 and OCN was up-regulated in the experimental group. After inhibition of JNK signaling pathway, the ex-pression of Runx2 and OCN was down-regulated. CONCLUSIONS: Berberine has no effect on cell proliferation of ADSCs,and can up-regulate osteogenic differentiation of ADSCs through activation of JNK signaling pathway.