摘要
目的:探讨骨痹通消颗粒对糖皮质激素致骨髓间充质干细胞损伤的干预作用。方法:体外培养人骨髓间充质干细胞,显微镜下观察细胞形态,对照组完全培养基培养,模型组使用梯度浓度地塞米松(10-5 mol/L、10-6 mol/L、10-7 mol/L)溶液诱导损伤,实验组在模型组基础上加入不同浓度骨痹通消颗粒含药血清干预培养。油红O染色检测脂滴,茜素红染色检测成骨钙结节,CCK-8法检测细胞活力,Western blotting检测ALP、OCN、p38和MEK蛋白表达水平,RT-qPCR分析各组ALP mRNA、OCN mRNA、p38 mRNA和MEK mRNA的表达情况。结果:镜下观察及成骨成脂分化鉴定为人骨髓间充质干细胞,地塞米松对人骨髓间充质干细胞的活力有影响,其中10-5 mol/L对细胞活力影响最大(P<0.01),不同浓度含药血清可干预这一损伤,并且促进细胞增殖,其中体积分数为5%时作用最为明显(P<0.01)。茜素红染色后镜下观察含药血清体积分数为5%的染色区域钙结节数量最为密集;与模型组比较,5%含药血清组ALP、OCN、p38和MEK蛋白的表达水平升高(P<0.01)。同时ALP mRNA、OCN mRNA、p38 mRNA和MEK mRNA的表达水平升高(P<0.01)。结论:骨痹通消颗粒含药血清能促进人骨髓间充质干细胞的增殖及矿化,可有效减少Dex对细胞的损伤,并上调成骨相关因子ALP和OCN的表达,其作用机制可能与激活MAPK通路有关。
Objective:To investigate the mechanism of action of Gubi Tongxiao granules in interfering with glucocorticoid-induced bone marrow mesenchymal stem cell injury.Methods:Human bone marrow mesenchymal stem cells were cultured in vitro,and the cell morphology was observed under the microscope.The control group was cultured in complete medium,while the model group was induced to be damaged by using graded concentrations of dexamethasone solution(10-5 mol/L,10-6 mol/L,10-7 mol/L).The experimental group was intervened in the culture based on the model group with the addition of different concentrations of Gubi Tongxiao granules-containing serum.Oil red O staining was used to detect lipid droplets,and Alizarin red staining was used to detect osteogenic calcium nodules.CCK-8 method was used to detect cell viability.Western blotting was used to detect the expression levels of ALP,OCN,p38,and MEK proteins,and RT qPCR was used to analyze the expression of ALP mRNA,OCN mRNA,p38 mRNA,and MEK mRNA in each group.Results:The cells were identified as human bone marrow mesenchymal stem cells under microscopic observation and osteogenic and adipogenic induction.Dexamethasone showed an impact on the viability of human bone marrow mesenchymal stem cells,with 10-5 mol/L having the greatest effect on cell viability(P<0.01).After the intervention of different volume fractions of the serum containing Gubi Tongxiao granules,the decrease of cell viability caused by GCs was inhibited,and the effect was most obvious at a volume fraction of 5%(P<0.01).After the microscopic observation of alizarin red staining,the number of calcium nodules was most dense in the stained area when the volume fraction of the serum containing the drug was 5%.Compared with the model group,the expression levels of ALP,OCN,p38 and MEK proteins were increased in the 5%drug-containing serum group(P<0.01),and the expression levels ALP mRNA,OCN mRNA,p38 mRNA and MEK mRNA were also increased(P<0.01).Conclusion:Gubi Tongxiao granules-containing serum can promote the proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells,effectively reduce the damage of dexamethasone to the cells,and up-regulate the expression of osteogenesis-related factors,ALP and OCN,whose mechanism of action may be related to the activation of the MAPK pathway.
作者
陈家康
周正新
朱磊
王壮壮
朱彩玉
CHEN Jiakang;ZHOU Zhenxin;ZHU Lei;WANG Zhuangzhuang;ZHU Caiyu(The First Clinical Medical College of Anhui University of Chinese Medicine,Hefei Anhui 230022,China;The First Affiliated Hospital of Anhui University of Chinese Medicine,Heifei Anhui 230011,China)
出处
《中医药导报》
2024年第11期48-52,81,共6页
Guiding Journal of Traditional Chinese Medicine and Pharmacy
基金
安徽省自然科学基金面上项目(2008085MH281)
安徽省教育厅高等学校自然科学研究项目(2023AH040109)。
关键词
股骨头坏死
人骨髓间充质干细胞
地塞米松
糖皮质激素
骨痹通消颗粒
femoralhead necrosis
human bone marrow mesenchymal stem cells
dexamethasone
glucocorticoid
Gubi Tongxiao granules
