黄芪多糖抑制氧化应激致大鼠软骨细胞损伤及凋亡的作用机制分析  

Action mechanism of Astragalus polysaccharide on inhibiting chondrocyte injury and apoptosis in rats caused by oxidative stress

作　　者：肖剑伟 周唯践[2] 蔡旭 郭粉莲 陈新鹏 洪易炜 曾苗雨 叶志中 XIAO Jian-wei;ZHOU Wei-jian;CAI Xu(Department of Rheumatology,Rheumatology Specialized Hospital in Futian District,Shenzhen Guangdong 518000,China;Department of Rheumatology,Yunnan Hospital of Traditional Chinese Medicine,Kunming Yunnan 650000,China)

机构地区：[1]深圳市福田区风湿病专科医院风湿科,广东深圳518000 [2]云南省中医医院风湿科,云南昆明650000

出　　处：《临床和实验医学杂志》2020年第10期1040-1044,共5页Journal of Clinical and Experimental Medicine

基　　金：国家自然科学基金(编号:81102266);广东省医学科学技术研究基金(编号:A2018089);深圳市医疗卫生三名工程(编号:SZSM201602087);2018福田区卫生公益性科研项目(编号:FTWS2018066)。

摘　　要：目的探究黄芪多糖(alp)抑制氧化应激致大鼠软骨细胞损伤及凋亡的作用机制。方法选取SPF级雌性3周龄大鼠,分为活性氧自由基培养组(ROS组)、黄芪多糖组(ROS+alp组),另设空白对照组。ROS组取其软骨细胞,加入活性氧自由基培养;ROS+alp组在ROS组的基础上加入浓度为30 mg/L的黄芪多糖处理;空白对照组不作处理。使用CCK-8法测定不同浓度(0 mg/L、20 mg/L、50 mg/L、100 mg/L、200 mg/L)黄芪多糖对大鼠细胞生存率的影响;采用蛋白质印记法(Western Blot)检测大鼠软骨细胞中Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2基因(Bcl-2)、基质金属酶蛋白3(MMP3)、MMP13、CLO2蛋白表达情况;采用Hoechst 33342染色法测定软骨细胞凋亡率。结果alp 0 mg/L时,细胞大量凋亡,生存率不到40%,随着alp的浓度的增加,大鼠软骨细胞存活率显著升高;与空白对照组相比,ROS组大鼠细胞Bcl-2、CLO2蛋白表达显著下降(P<0.01),MMP3、MMP13、Bax蛋白表达显著上升(P<0.01);与ROS组大鼠细胞相比,ROS+alp组大鼠细胞CLO2、Bcl-2蛋白表达显著上升(P<0.01),MMP3、MMP13、Bax蛋白表达显著下降(P<0.01)。Hoechst 33342染色结果显示,空白对照组软骨细胞核染色分布均匀,呈弥散均匀的低密度荧光,ROS组大鼠细胞荧光分布较为杂乱且荧光密度较高,ROS+alp组大鼠细胞分布也较为均匀,荧光密度也较低。结论在一定范围内黄芪多糖呈剂量依赖性显著降低大鼠软骨细胞内ROS水平,上调抑制凋亡因子,下调凋亡因子,减少大鼠软骨细胞的凋亡,达到缓解软骨细胞损伤的效果。Objective To explore action mechanism of Astragalus polysaccharide on inhibiting chondrocyte injury and apoptosis in rats caused by oxidative stress.Methods SPF level female 3-week-old rats were enrolled in this study and were divided into reactive oxygen species(ROS)culture group,Astragalus polysaccharide group and blank control group.In ROS culture group,chondrocytes were collected to add into ROS for culture.Astragalus polysaccharide group was treated with 30 mg/L astragalus polysaccharide on the basis of ROS culture group.The effects of Astragalus polysaccharide on cell survival rate was determined by CCK-8 method.The expressions of Bax,Bcl-2,MMP3,MMP13 and CLO2 protein in chondrocytes of rats were detected by Western Blot.Chondrocyte apoptosis rate was determined by Hoechst 33342 staining.Results At alp 0 mg/L,the cells undergo massive apoptosis and the survival rate is less than 40%.As the concentration of alp increases,the survival rate of rat chondrocytes increases significantly.Compared with blank group,expressions of Bcl-2 and CLO2 protein were significantly decreased after given ROS(P<0.01),while expressions of MMP3,MMP13 and Bax protein were significantly increased(P<0.01).Compared with those in rat cells given ROS,expressions of CLO2 and Bcl-2 protein were increased significantly after given ROS and Astragalus polysaccharide(P<0.01),while expressions of MMP3,MMP13 and Bax protein were significantly decreased(P<0.01).Hoechst 33342 staining results showed that the blank control group had evenly distributed chondrocyte nuclear staining with uniformly distributed low-density fluorescence.The ROS group rat cells had a more turbulent fluorescence distribution and higher fluorescence density,and the ROS+alp group rat cells had a more uniform distribution Fluorescence density was also low.Conclusion Astragalus polysaccharide can significantly reduce ROS level in rat chondrocytes,up-regulate antiapoptotic factors,down-regulate apoptotic factors,reduce apoptosis of rat chondrocytes to alleviate chondrocyte dam

关 键 词：大鼠 黄芪多糖 软骨细胞 氧化应激 细胞凋亡 

分 类 号：R285.5[医药卫生—中药学]