健骨方对破骨细胞形成和成骨细胞增殖分化的影响  

Effect of Jiangu Decoction on osteoclast formation and osteoblast proliferation and differentiation

作　　者：许华珍 黄丹娥 郑柳怡 姚楠 蔡大可 甘海宁 黄雪君 胡子旋 赵自明 陈玉兴 XU Huazhen;HUANG Dane;ZHENG Liuyi;YAO Nan;CAI Dake;GAN Haining;HUANG Xuejun;HU Zixuan;ZHAO Ziming;CHEN Yuxing(The Fifth Clinical College of Guangzhou University of Chinese Medicine,Guangzhou 510405;Guangdong Provincial Second Hospital of Traditional Chinese Medicine(Guangdong Provincial Engineering Technology Research Institute of Traditional Chinese Medicine),Guangzhou 510095;Guangdong Provincial Key Laboratory of Research and Development in Traditional Chinese Medicine,Guangzhou 510095,China)

机构地区：[1]广州中医药大学第五临床医学院,广东广州510405 [2]广东省第二中医院(广东省中医药工程技术研究院),广东广州510095 [3]广东省中医药研究开发重点实验室,广东广州510095

出　　处：《中国骨质疏松杂志》2022年第12期1728-1734,共7页Chinese Journal of Osteoporosis

基　　金：国家自然科学基金青年科学基金项目(82104473);广东省省基础与应用基础研究基金青年基金(2019A1515110484);广东省中医药局科研项目(20201017)。

摘　　要：目的探讨健骨方水提物对破骨细胞分化及成骨细胞增殖分化的影响。方法制备健骨方水提物,通过MTT法测定药物对骨髓单核巨噬细胞(BMMs)细胞的毒性,采用核因子κB受体活化因子配体(RANKL)诱导BMMs分化形成破骨细胞,加入不同浓度药物进行干预,采用抗酒石酸酸性磷酸酶(TRACP)染色法测定破骨细胞分化抑制作用,采用Western Blot法测定RANKL诱导的NF-κB破骨细胞分化信号通路,运用RT-qPCR法测定信号通路下游破骨细胞分化关键基因NFATc1、C-FOS等的mRNA表达水平。以MC3T3-E1细胞作为前体成骨细胞,加入不同浓度药物进行干预,通过CCK8法测定细胞增殖能力、PNPP法检测碱性磷酸酶(ALP)活性、茜素红S染色法测定细胞矿化能力。结果MTT法结果显示,健骨方细胞有毒性浓度大于500μg/mL(P<0.05),破骨细胞分化抑制IC 50为1.25μg/mL。机制研究显示健骨方显著下调了RANKL-NF-κB信号通路中的p-P65、P53的蛋白表达(P<0.05),显著抑制了通路下游C-FOS、NFATc1等的mRNA表达水平(P<0.01,P<0.05)。此外,成骨细胞活性检测显示,健骨方能明显促进MC3T3-E1细胞增殖、提高ALP活性及增加成骨细胞钙化的能力。结论健骨方具有抑制破骨细胞分化和促进成骨前体细胞增殖、分化、矿化的药效作用。其作用与抑制破骨细胞分化RANKL-NF-κB信号通路及其下游C-FOS、NFATc1等基因,上调成骨细胞分化促进因子CAL1A2、SPARC和FOSL1基因的表达有关。Objective Investigation of the effect of aqueous extract of Jiangu Decoction on osteoclast differentiation and osteoblast proliferation and differentiation.Methods Preparation of JGF aqueous extract.MTT method was applied to study the toxicity of Jiangu Decoction on bone marrow mononuclear macrophages(BMMs)cells.The differentiation of BMMs into osteoclasts was induced by using nuclear factor-κB receptor activating factor ligand(RANKL),and different concentrations of the drug were added.Anti-tartrate acid phosphatase(TRACP)staining was used to determine the inhibition of osteoclastogenesis of Jiangu Decoction.The signaling pathway of NF-κB osteoclast differentiation induced by RANKL was determined by Western blot,and the RT-qPCR method was used to determine the mRNA expression levels of NFATc1,C-FOS,etc.,which are key genes of osteoclastogenesis downstream of the signaling pathway.MC3T3-E1 cells were used as precursor osteoblasts,and different concentrations of drugs were added for intervention.The proliferation ability of cells was measured by the CCK8 method.Alkaline phosphatase(ALP)activity was detected by the PNPP method,and the mineralization ability of cells was measured by alizarin red S staining.Results MTT assay showed that the cytotoxic concentration of Jiangu Decoction was greater than(P<0.05)500μg/mL,and the IC 50 of osteoclastogenesis inhibition was 1.25μg/mL.Mechanistic studies showed that Jiangu Decoction significantly downregulated the protein expression of p-P65 and P53 in the RANKL-NF-κB signaling pathway(P<0.05)significantly inhibited the downstream of the pathway C-FOS,NFATC,etc.mRNA expression levels downstream of the pathway(P<0.01,P<0.05).In addition,the osteoblast activity assay showed that Jiangu Decoction could significantly promote the proliferation ability of MC3T3-E1 cells,improve ALP activity and increase the calcification ability of osteoblasts.Conclusion JGF has the pharmacological effect of inhibiting osteoclast differentiation and promoting osteogenic precursor cell prolifera

关 键 词：健骨方 破骨细胞 成骨细胞 骨质疏松症 中医药 

分 类 号：R285.5[医药卫生—中药学]