汉防己甲素诱导自噬抑制破骨细胞的分化  

Tetrandrine inhibits osteoclast differentiation by inducing autophagy

作　　者：张博文 李美 张晋宁 杨天翔 程萌旗[3] 陈德胜 Zhang Bowen;Li Mei;Zhang Jinning;Yang Tianxiang;Cheng Mengqi;Chen Desheng(School of Clinical Medicine,Ningxia Medical University,Yinchuan 750004,Ningxia Hui Autonomous Region,China;Key Laboratory of Modernization of Minority Medicine,Ministry of Education,Ningxia Medical University,Yinchuan 750004,Ningxia Hui Autonomous Region,China;Department of Joint Surgery,The Sixth Affiliated People's Hospital of Shanghai Jiao Tong University,Shanghai 200233,China;Department of Orthopedics,People's Hospital of Ningxia Hui Autonomous Region,Yinchuan 750004,Ningxia Hui Autonomous Region,China)

机构地区：[1]宁夏医科大学临床医学院,宁夏回族自治区银川市7500042 [2]宁夏医科大学少数民族医药现代化教育部重点实验室,宁夏回族自治区银川市750004 [3]上海交通大学附属第六人民医院关节外科,上海市200233 [4]宁夏回族自治区人民医院骨科,宁夏回族自治区银川市750004

出　　处：《中国组织工程研究》2023年第28期4473-4479,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金项目(82060408),项目负责人:陈德胜;宁夏回族自治区重点研发计划项目(2021BEG03049),项目负责人:陈德胜。

摘　　要：背景:已有研究表明汉防己甲素可以通过激活AMPK信号通路诱导细胞自噬从而抑制肿瘤细胞增殖,但目前汉防己甲素诱导破骨细胞自噬和分化的相关研究甚少。目的:探究汉防己甲素在不同浓度下对破骨细胞分化和自噬的影响,并分析诱导破骨细胞自噬能否起到抑制其分化的作用。方法:(1)第一部分:使用重组小鼠巨噬细胞集落刺激因子和核因子κB受体活化因子配体两种细胞因子对小鼠RAW264.7巨噬细胞进行诱导,使其向破骨细胞转化。细胞诱导及药物干预分组如下:空白对照组(完全培养基),阳性对照组(含两种细胞因子),低、中、高剂量汉防己甲素干预组(阳性对照组分别添加0.1,0.5,1.0μmol/L汉防己甲素)。抗酒石酸酸性磷酸酶染色法观察成熟破骨细胞数量变化;Western blot法检测组织蛋白酶K(CTSK)、自噬微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)和自噬底物泛素结合蛋白(p62)的蛋白相对表达量;RT-PCR检测CTSK、LC3-Ⅱ和p62的mRNA相对表达量;丹酰尸胺(MDC)染色观察诱导过程中破骨细胞中自噬小体的数量变化。(2)第二部分:选取抑制破骨细分化效果最明显的汉防己甲素浓度(1.0μmol/L)进行回复性验证。各组处理如下:阳性对照组(含两种细胞因子),自噬抑制剂组(巴佛洛霉素处理),药物干预组(汉防己甲素干预),药物干预+自噬抑制剂组。抗酒石酸酸性磷酸酶染色法观察成熟破骨细胞数量变化;Western blot法检测CTSK的蛋白相对表达量。结果与结论:(1)一定浓度汉防己甲素能够对破骨细胞的分化起抑制作用,并且抑制作用随药物浓度升高而提升;(2)汉防己甲素在一定浓度下能够激发破骨细胞分化过程中自噬标志物的表达及减少自噬特异性底物累积,并在破骨细胞分化过程中可刺激自噬小体形成;(3)使用自噬抑制剂后,汉防己甲素抑制破骨细胞分化的作用受到显著影响;(4)综上,一定浓度的汉防己甲素能�BACKGROUND:Previous studies have shown that tetrandrine can inhibit the prolife ration of tumor cells by activating AMPK signaling pathway to induce autophagy.Howeve r,there are few studies on tetrandrine induced autophagy and differentiation of osteoclasts.OBJECTIVE:To explore the effect of tetrandrine at different concentrations on autophagy during osteoclast differentiation,and analyze whether inducing autophagy of osteoclasts can inhibit their differentiation.METHODS:Part one:Recombinant mouse macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand cyto kines were used to induce mouse RAW264.7 macrophages into osteoclasts.Mouse RAW264.7 macrophages were divided into five groups:blank group(complete medium),positive control group(treated by two cytokines),and low-,medium-,and high-concentration drug groups(treated by two cyto kines and added 0.1,0.5,and1.0μmol/L tetrandrine,respectively).Tartrate resistant acid phosphatase staining method was used to detect the number of mature osteoclasts.Western blot assay and RT-PCR were utilized to detect the relative expression levels of proteins of Cathepsin K,autophagy mic rotubule-associated protein 1 light chain 3-Ⅱ,and autophagy substrate ubiquitin-binding protein(p62)at protein and mRNA levels,respectively.Monodansyl cadave rine staining was used to detect the number of autophagosomes in osteoclasts.Part two:The concentration of tetrandrine(1.0 umol/L)with the best inhibito ry effect on osteoclast differentiation was selected for regression verification.Cells were divided into positive control group(treated by two cytokines),autophagy inhibitor group(bafloamycin),drug intervention group(tetrandrine),and drug intervention+authophage inhibitor group.Tartrate resistant acid phosphatase staining was used to detect the number of mature osteoclasts.Western blot assay was utilized to detect the relative protein expression level of Cathepsin K.RESULTS AND CONCLUSION:Tetrandrine at a ce rtain concentration inhibited the differentiation of

关 键 词：汉防己甲素 破骨细胞 自噬 骨溶解 微管相关蛋白1轻链3-Ⅱ CTSK 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R684