紫檀芪干预人软骨细胞的氧化应激性凋亡  

Effect of pterostilbene on oxidative stress induced apoptosis in chondrocytes

作　　者：林义才[1] 吴正远 罗颖丽[3] 姚军[1,4] Lin Yicai;Wu Zhengyuan;Luo Yingli;Yao Jun(Department of Osteoarticular Surgery,The First Affiliated Hospital of Guangxi Medical University,Nanning 530021,Guangxi Zhuang Autonomous Region,China;Department of Orthopaedics and Hand Surgery,The First Affiliated Hospital of Guangxi Medical University,Nanning 530021,Guangxi Zhuang Autonomous Region,China;Department of Anesthesiology,The First Affiliated Hospital of Guangxi Medical University,Nanning 530021,Guangxi Zhuang Autonomous Region,China;Guangxi Collaborative Innovation Center For Biomedicine,The First Affiliated Hospital of Guangxi Medical University,Nanning 530021,Guangxi Zhuang Autonomous Region,China)

机构地区：[1]广西医科大学第一附属医院骨关节外科,广西壮族自治区南宁市530021 [2]广西医科大学第一附属医院创伤骨科手外科,广西壮族自治区南宁市530021 [3]广西医科大学第一附属医院麻醉科,广西壮族自治区南宁市530021 [4]广西医科大学第一附属医院协同创新中心,广西壮族自治区南宁市530021

出　　处：《中国组织工程研究》2020年第32期5092-5096,共5页Chinese Journal of Tissue Engineering Research

基　　金：广西壮族自治区卫生和计划委员会自筹经费科研项目(Z20180944),项目负责人:林义才;广西自然科学基金(2018GXNSFAA050082)。

摘　　要：背景:已有研究表明多种抗氧化剂可抑制炎性因子生成而表现出抗关节炎作用,紫檀芪是一种强大的天然抗氧化剂,但其在软骨细胞抗氧化应激作用中的研究目前尚无相关报道。目的:探讨紫檀芪对人软骨细胞氧化应激性凋亡的影响。方法:体外培养正常人关节软骨细胞,使用含不同质量浓度紫檀芪(7.8-32000μg/L)的培养基继续培养24 h,确定紫檀芪的最佳浓度。实验分为对照组,紫檀芪组(含有125μg/L紫檀芪的培养基),H2O2组(0.2 mmol/L的H2O2),H2O2+紫檀芪组(含有125μg/L紫檀芪的培养基预处理2 h后,更换含H2O2与125μg/L紫檀芪的培养基继续培养),分别处理24 h。通过MTT实验观察细胞增殖率,应用苏木精-伊红染色观察细胞形态及数量、FDA/PI染色检测细胞活性、番红O染色观察蛋白聚糖水平的变化,以及通过RT-PCR检测软骨细胞分化标志基因聚集蛋白聚糖、Ⅱ型胶原酶1表达变化。研究方案取得广西医科大学第一附属医院伦理委员会批准,批号201805008。结果与结论:①MTT实验显示,紫檀芪在15.6-250μg/L可显著促软骨细胞生长,尤其是在为125μg/L时促进作用最明显;②苏木精-伊红染色和FDA/PI染色结果显示,在H2O2组中软骨细胞的数量减少,在H2O2+紫檀芪组中细胞数量较单独H2O2刺激组显著增加,细胞活性提高;③番红O染色显示紫檀芪促进软骨细胞分泌蛋白聚糖,抑制H2O2对软骨细胞的不利作用;④RT-PCR结果表明紫檀芪可促进氧化应激损伤的软骨细胞高表达聚集蛋白聚糖、Ⅱ型胶原酶1基因,改善软骨细胞分化功能;⑤结果说明,紫檀芪能够促进软骨细胞增殖,抑制氧化应激引起的人关节软骨细胞凋亡。BACKGROUND:A variety of antioxidants exhibit anti-arthritis effects by inhibiting the production of inflammatory factors.Pterostilbene is a powerful natural antioxidant;however,there is no report on its effect against oxidative stress in chondrocytes.OBJECTIVE:To investigate the effect of pterostilbene on oxidative stress induced apoptosis in human chondrocytes.METHODS:Normal human articular chondrocytes were cultured in medium containing different concentrations of pterostilbene(7.8-32000μg/L)for 24 continuous hours to determine the optimal concentration of pterostilbene.Normal human articular chondrocytes cultured in vitro were randomized into control group,pterostilbene group(treatment with 125μg/L pterostilbene),hydrogen peroxide(H2O2)group(treatment with 0.2 mmol/L H2O2),and H2O2 plus pterostilbene group(pretreatment with 125μg/L pterostilbene followed by continuous treatment in the medium containing 125μg/L pterostilbene and 0.2 mmol/L H2O2).After treating for 24 hours,the cell proliferation rate was detected by MTT experiment,the cell morphology and number by hematoxylin-eosin staining,the cell activity was measured by FDA/PI staining,and the changes of proteoglycan content were observed by saffron O staining.The expression of chondrogenesis marker genes aggrecan and type II collagenase 1 was detected by RT-PCR.An approval for the study protocol was validated by the Ethic Committee of the First Affiliated Hospital of Guangxi Medical University with an approval No.201805008.RESULTS AND CONCLUSION:The results of MTT assay showed that pterostilbene could significantly promote chondrocyte growth at 15.6-250μg/L,especially at 125μg/L.The results of hematoxylin-eosin staining and FDA/PI staining further showed that pterostilbene could inhibit H2O2-induced chondrocyte apoptosis,promote chondrocyte proliferation,and increase cell viability.The results of saffron O staining showed that pterostilbene promoted the secretion of proteoglycan by chondrocytes and inhibited the adverse effects of H2O2 on chondrocyte

关 键 词：软骨 关节软骨 紫檀芪 软骨细胞 凋亡 氧化应激 实验 

分 类 号：R446[医药卫生—诊断学] R496[医药卫生—临床医学]