大麻二酚对衰老小鼠骨髓间充质干细胞成骨分化影响的体外细胞实验

李琳; 朱彪; 田萧羽; 党瑞杰; 赵立升; 杨烁; 孙磊; 温宁

doi:10.3969/j.issn.2095-5227.2022.01.015

大麻二酚对衰老小鼠骨髓间充质干细胞成骨分化影响的体外细胞实验

Effect of cannabidiol on osteogenic differentiation of senescent mice bone marrow mesenchymal stem cells in vitro

摘要

摘要:
背景衰老状态的骨髓间充质干细胞(bone marrow mesenchymal stem cells，BMSCs)成骨分化能力降低，影响正常骨代谢。细胞自噬在衰老过程及成骨代谢中发挥重要作用，大麻二酚(cannabidiol，CBD)能促进自噬，但CBD能否通过调节自噬进而促进衰老BMSCs成骨分化仍不清楚。
目的探究CBD对衰老小鼠BMSCs成骨分化的影响以及细胞自噬在其中的作用。
方法复苏并培养小鼠BMSCs，使用D-半乳糖(D-gal)诱导体外细胞衰老模型，将细胞随机分为对照组(Vehicle)、衰老模型组(D-gal)和CBD干预组(D-gal+CBD)。qRT-PCR检测衰老相关基因P16和P21的表达，CCK-8法测定不同条件下BMSCs的增殖活性。加入BMSCs自噬抑制组(D-gal+CBD+氯喹)，对四组细胞进行成骨诱导后，Western blot检测自噬相关蛋白P62及LC3B的表达情况，qRT-PCR评估成骨相关基因的表达改变，碱性磷酸酶(alkaline phosphatase，ALP)检测试剂盒测定ALP活性。
结果经D-gal处理后的BMSCs高表达衰老基因P16及P21，细胞增殖能力降低，P62蛋白表达升高，LC3B-Ⅱ蛋白表达降低，ALP、Runt相关转录因子2和骨钙素的mRNA表达水平及ALP活性下降(P＜0.05)；而CBD干预显著下调衰老BMSCs中P16及P21的表达，增强细胞增殖活性(P＜0.05)。同时，CBD降低P62水平，显著提升LC3B-Ⅱ水平，促进成骨相关基因的转录表达并提高ALP活性(P＜0.05)。而当使用氯喹抑制细胞自噬后，CBD促进衰老BMSCs成骨分化的作用减弱(P＜0.05)。
结论 CBD通过激活自噬促进衰老小鼠BMSCs成骨分化。

Abstract:
Background Bone marrow mesenchymal stem cells (BMSCs) in senescent state have a reduced capacity for osteogenic differentiation, which hazards to normal bone metabolism. Autophagy plays an important role in cellular senescence process and osteogenic metabolism. Cannabidiol (CBD) activates autophagy, but whether CBD can promote osteogenic differentiation of senescent BMSCs via regulating autophagy remains unclear.
Objective To investigate the effect of CBD on osteogenic differentiation of senescent mice BMSCs and the role of cellular autophagy.
Methods Mice BMSCs were resuscitated and cultured. D-galactose (D-gal) was used to induce an in vitro senescence model. Cells were randomly divided into control (Vehicle) group, senescence model (D-gal) group and CBD intervention (D-gal+CBD) group. qRT-PCR was performed to detect the mRNA expression levels of senescence-related genes, and CCK-8 assay was used to detect BMSCs proliferation activity. BMSCs in autophagy inhibition group (D-gal+CBD+Chloroquine) were added, and after osteogenesis induction of the four cells groups, the expression levels of autophagy-related proteins, P62 and LC3B, were detected by Western blot. qRT-PCR was performed to assess the mRNA expression levels of osteogenesis-related genes, and ALP activity was determined by alkaline phosphatase (ALP) assay kit.
Results BMSCs treated with D-gal overexpressed senescence-related genes P16 and P21, and exhibited decreased cell proliferation activity. The protein expression level of P62 increased while the protein expression level of LC3B-Ⅱ reduced, and the mRNA expression levels of ALP, Runx2, OCN and ALP activity were also decreased (P＜0.05); In contrast, CBD intervention down-regulated the mRNA expression levels of P16 and P21 in senescent BMSCs. CBD significantly decreased P62 and increased the LC3B-Ⅱ protein levels, promoted the mRNA expression levels of ALP, Runx2 and OCN, and significantly enhanced ALP activity (P＜0.05). In addition, the activation of autophagy and osteogenic differentiation of senescent BMSCs promoted by CBD were weakened by the autophagy inhibitor chloroquine (P＜0.05).
Conclusion CBD promotes osteogenic differentiation of senescent mice BMSCs via activating autophagy.

HTML全文

参考文献(29)

施引文献

资源附件(0)