紫草素干预破骨细胞生成及破骨相关基因的表达  

Shikonin intervenes with osteoclast formation and osteoclast-related gene expression

作　　者：薄雨佳 林静[1,2] 王莉平 赵今 Bo Yujia;Lin Jing;Wang Liping;Zhao Jin(Department of Dentistry and Endodontics,First Affiliated Hospital(Affiliated Stomatological Hospital)of Xinjiang Medical University,Urumqi 830054,Xinjiang Uygur Autonomous Region,China;Institute of Stomatology of Xinjiang Uygur Autonomous Region,Urumqi 830054,Xinjiang Uygur Autonomous Region,China)

机构地区：[1]新疆医科大学第一附属医院(附属口腔医院)牙体牙髓科,新疆维吾尔自治区乌鲁木齐市830054 [2]新疆维吾尔自治区口腔医学研究所,新疆维吾尔自治区乌鲁木齐市830054

出　　处：《中国组织工程研究》2022年第32期5126-5131,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家重点实验室专项资金(SKLOD2021OF04),项目负责人:赵今。

摘　　要：背景:破骨细胞的活化在牙周炎的发生发展中起重要作用,紫草素因其毒副作用较小并具有抗炎等优点,可将其应用于牙周炎基础治疗后的辅助药物治疗。目的:探讨紫草素对核因子κB受体活化因子配体(RANKL)诱导的破骨细胞生成及破骨相关基因表达的影响。方法:采用CCK-8法检测不同浓度紫草素(0,0.062 5,0.125,0.25,0.5,1,2μmol/L)对RAW264.7细胞的毒性,筛选出安全浓度;用不同质量浓度的核因子κB受体活化因子配体(0,30,50,100μg/L)诱导RAW264.7细胞向破骨细胞分化,经抗酒石酸酸性磷酸酶染色观察破骨细胞数目,筛选出最佳核因子κB受体活化因子配体质量浓度;给予核因子κB受体活化因子配体和不同浓度的紫草素干预后,通过抗酒石酸酸性磷酸酶染色和抗酒石酸酸性磷酸酶活性检测其对破骨生成的影响;通过实时定量PCR技术检测破骨细胞标志性基因基质金属蛋白酶9、组织蛋白酶K、核因子κB受体活化因子、活化T细胞核因子和原癌基因c-Fos的m RNA表达。结果与结论:①高于0.5μmol/L的紫草素显著抑制RAW264.7细胞的生长(P <0.05);② 50μg/L的核因子κB受体活化因子配体为最佳诱导浓度;③紫草素呈浓度依赖性的抑制体外破骨细胞生成(P <0.05);④紫草素呈浓度依赖性的抑制基质金属蛋白酶9、组织蛋白酶K、核因子κB受体活化因子、活化T细胞核因子和原癌基因c-Fos等参与破骨细胞分化的标志性基因表达(P <0.05);⑤结果表明,紫草素能够在体外呈浓度依赖性的通过抑制破骨相关基因的表达,进而抑制核因子κB受体活化因子配体诱导的破骨细胞的生成。BACKGROUND:The activation of osteoclast plays an important role in the occurrence and development of periodontitis.In recent years,shikonin has been used as an adjuvant drug after periodontal non-surgical treatment because of its small side effects and anti-inflammatory properties.OBJECTIVE:To discuss the effects of shikonin on osteoclast fo rmation induced by receptor activator for nuclear factor-KB ligand(RANKL) and osteoclast-related gene expression.METHODS:The toxicity of different concentrations of shikonin(0,0.062 5,0.125,0.25,0.5,1,and 2 μmol/L) to RAW264.7 cells was detected by cell counting kit-8 assay,and the optimal concentration was determined.RAW264.7 cells were induced into osteoclasts by different concentrations of RANKL(0,30,50,100 μg/L).The number of osteoclasts was revealed by tartrate resistant acid phosphatase staining,and the optimal concentration of RANKL was determined.After treatment with different concentrations of shikonin,the effects of RANKL on osteoclast formation were detected by tartrate resistant acid phosphatase staining and to rtrate resistant acid phosphatase activity.The mRNA expressions of osteoclast marker genes,including matrix metalloproteinase 9,cathepsin K,receptor activator for nuclear facto r-KB,nuclear factor of activated T cells and proto-oncogene c-Fos,were detected by real-time quantitative PCR.RESULTS AND CONCLUSION:Shikonin at a concentration of higher than 0.5 μmol/L significantly inhibited the growth of RAW264.7 cells(P <0.05).The optimal induction concentration of RANKL was 50 μg/L.Shikonin inhibited osteoclast formation in a concentration-dependent manner(P<0.05).Shiko nin also inhibited the expression of matrix metalloproteinase 9,cathepsin K,receptor activator for nuclear factor-KB,nuclear factor of activated T cells,and proto-oncogene c-Fos in a concentration-dependent manner(P<0.05).To conclude,shikonin can inhibit RANKL-induced osteoclast formation by inhibiting the expression of osteoclast-related genes in a concentration-dependent manner in vitro.

关 键 词：紫草素 破骨细胞生成 牙周炎 RAW264.7 核因子ΚB受体活化因子配体 诱导 活化T细胞核因子 原癌基因C-FOS 

分 类 号：R459.9[医药卫生—治疗学] R781.4[医药卫生—临床医学]