资木瓜中莽草酸通过减少RBL-2H3细胞脱颗粒抑制大鼠软骨细胞分化  

Shikimic acid extracted from Chaenomeles speciosa reduces chondrocyte differentiation by inhibiting RBL-2H3 cell degranulation

作　　者：陈桂婷 郑倩倩[1] 余婕 闫梦真 周婷婷 曹后平 李世刚[1] CHEN Guiting;ZHENG Qianqian;YU Jie;YAN Mengzhen;ZHOU Tingting;CAO Houping;LI Shigang(Three-Level Laboratory for Traditional Chinese Pharmacologic(Tumor)Research,Medical College,China Three Gorges University,Yichang 443002,China)

机构地区：[1]三峡大学医学院中药药理(肿瘤)科研三级实验室,湖北宜昌443002

出　　处：《细胞与分子免疫学杂志》2020年第10期890-896,共7页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81274166,81673665);湖北省卫生健康委员会联合基金重点项目(WJ2019H531)。

摘　　要：目的研究资木瓜中总提物莽草酸(SA)通过抑制RBL-2H3细胞脱颗粒从而减少软骨细胞向肥厚软骨细胞的分化作用。方法采用甲苯胺蓝染色法,类胰蛋白酶免疫组织化学染色法进行软骨细胞的鉴定;软骨细胞培养组分为正常软骨细胞对照组,化合物48/80(C48/80)活化RBL-2H3细胞培养上清液处理组,(3、10、30)μg/mL SA分别作用后的RBL-2H3细胞培养上清液处理组。噻唑蓝(MTT)法检测SA和RBL-2H3细胞上清液对软骨细胞的毒性作用;Western blot法检测软骨细胞2型胶原蛋白(Col2)和Col10的蛋白表达;免疫组织化学染色法检测软骨细胞Col10蛋白水平;ELISA检测软骨细胞培养上清液基质金属蛋白酶13(MMP13)、可溶性核因子κB受体激活蛋白配体(sRANKL)及骨保护素(OPG)含量,二甲基亚甲蓝(DMB)比色法检测糖胺多糖(GAG)的含量。结果(0~30)μg/mL SA对软骨细胞生长无显著影响,与C48/80活化RBI-2H3细胞培养上清液处理组比较,随着SA剂量的增加,软骨细胞中Col2、GAG蛋白表达增加,Col10、MMP13的表达明显降低、sRANKL/OPG的比值降低。结论SA通过抑制RBL-2H3细胞脱颗粒减少软骨细胞向肥厚软骨细胞的分化。Objective To investigate the reducing effects of shikimic acid from the total extract of Chaenomeles speciose on the differentiation of chondrocytes into hypertrophic chondrocytes by inhibiting RBL-2H3 cell degranulation.Methods The chondrocytes were identified by toluidine blue staining and tryptase immunohistochemical staining.The chondrocytes were divided into normal chondrocytes control group,C48/80 activated RBL-2H3 cell culture supernatant treatment group,3,10 and 30μg/mL SA activated RBL-2H3 cell culture supernatant treatment groups.The toxicity of SA and RBL-2H3 cell supernatant were detected by MTT assay.Western blotting was used to detect the expression of collagen typeⅡ(Col2)and collagen typeⅩ(Col10)in chondrocytes.The levels of matrix metalloproteinase 13(MMP13),soluble nuclear factor B receptor activated protein ligand(sRANKL)and bone protective factor(OPG)were determined by ELISA,and glycosaminoglycan polysaccharide(GAG)were tested by dimethylmethylene blue(DMB)colorimetry.Results(0~30)μg/mL SA had no significant effects on the growth of chondrocytes.Compared with the C48/80 activated RBI-2H3 cell supernatant treatment group,the expression of Col2 and GAG proteins increased significantly,while the expression of Col10 and MMP13 and the ratio of sRANKL/OPG decreased significantly in the SA treatment groups in a dose-dependent manner.Conclusion SA can effectively reduce the differentiation of chondrocytes into hypertrophic chondrocytes by inhibiting RBL-2H3 cell degranulation.

关 键 词：资木瓜 莽草酸(SA) RBL-2H3细胞 软骨细胞 分化 

分 类 号：R392.12[医药卫生—免疫学] R285.5[医药卫生—基础医学]