沙苑子苷A对关节软骨细胞凋亡的影响  

Effect of Complanatoside A on the apoptosis of articular chondrocytes

作　　者：尹路 蒋川锋 陈俊杰 易明 王子赫 石厚银 汪国友 沈骅睿 Yin Lu;Jiang Chuanfeng;Chen Junjie;Yi Ming;Wang Zihe;Shi Houyin;Wang Guoyou;Shen Huarui(Southwest Medical University,Luzhou 646000,Sichuan Province,China;The Affiliated Traditional Chinese Medicine Hospital,Southwest Medical University,Luzhou 646000,Sichuan Province,China)

机构地区：[1]西南医科大学,四川省泸州市646000 [2]西南医科大学附属中医医院,四川省泸州市646000

出　　处：《中国组织工程研究》2025年第8期1541-1547,共7页Chinese Journal of Tissue Engineering Research

基　　金：泸州市人民政府-西南医科大学科技战略合作项目(2021LZXNYD-J31),项目负责人:沈骅睿;四川省科技计划专项(2022YFS0609),项目负责人:石厚银;四川省中医药管理局项目(2023MS446),项目负责人:沈骅睿;2023年度西南医科大学校级科研项目(2023ZYYJ05),项目负责人:沈骅睿。

摘　　要：背景:软骨细胞凋亡是骨关节炎发生发展的重要因素,沙苑子苷A具有类黄酮作用,可抑制多种细胞凋亡,但其对软骨细胞凋亡的影响及作用机制尚不明确。目的:基于Wnt/β-catenin信号通路探讨沙苑子苷A与软骨细胞凋亡的内在关联及作用机制。方法:①收集膝关节置换术中截取的股骨及胫骨部分软骨组织,体外分离培养及鉴定软骨细胞;②通过CCK-8检测沙苑子苷A在0-160μmol/L范围内是对软骨细胞的最佳干预浓度;③将软骨细胞分为空白组、硝普钠(1.5 mmol/L)诱导组、硝普钠(1.5 mmol/L)+沙苑子苷A(5μmol/L)组。采用CCK-8、流式细胞技术检测各组细胞活力、凋亡率;免疫荧光染色检测各组细胞中Ⅱ型胶原蛋白、SOX9表达;Western Blot检测细胞凋亡相关蛋白及Wnt/β-catenin通路蛋白的表达。结果与结论:①体外提取的细胞经培养、染色鉴定为软骨细胞;②沙苑子苷A在0-80μmol/L浓度范围内对软骨细胞无明显细胞毒性,在2.5-10μmol/L浓度范围内可明显提高软骨细胞活力,且当浓度为5μmol/L时作用较为显著;③硝普钠诱导组软骨细胞凋亡率高于空白组,硝普钠+沙苑子苷A组的细胞凋亡率低于硝普钠诱导组;④硝普钠诱导组软骨细胞Ⅱ型胶原蛋白、SOX9荧光强度弱于空白组,硝普钠+沙苑苷A组Ⅱ型胶原蛋白、SOX9的荧光强度高于硝普钠诱导组;⑤硝普钠诱导组Bax、Caspase-3、基质金属蛋白酶13、Wnt3a、Wnt5a和β-catenin的蛋白表达量高于空白组,Bcl-2蛋白表达低于空白组;硝普钠+沙苑子苷A组除Bcl-2的蛋白表达高于硝普钠诱导组外,上述其他蛋白表达均低于硝普钠诱导组。结果表明沙苑子苷A对软骨细胞凋亡具有一定的抑制作用,可调节凋亡相关蛋白、促进软骨细胞调节因子表达,推测可能通过抑制Wnt/β-catenin信号通路发挥作用。BACKGROUND:Chondrocyte apoptosis is an important factor in the development of osteoarthritis,and Complanatoside A has a flavonoid effect,which can inhibit apoptosis of various cells,but its effect on chondrocyte apoptosis and the mechanism of action are not clear.OBJECTIVE:To investigate the intrinsic association and mechanism of Complanatoside A in chondrocyte apoptosis based on the Wnt/β-catenin signaling pathway.METHODS:(1)The cartilage tissues of the femur and tibia transected during knee arthroplasty were collected,and chondrocytes were isolated,cultured in vitro,and identified.(2)Cell counting kit-8 was used to detect the optimal intervention concentration of Complanatoside A in the concentration range of 0-160μmol/L.(3)Chondrocytes were divided into blank group,sodium nitroprusside(1.5 mmol/L)-induced group,and sodium nitroprusside(1.5 mmol/L)+Complanatoside A(5μmol/L)group.The viability and apoptosis rate of the cells in each group were detected by cell counting kit-8 and flow cytometry.The expression of type II collagen and SOX9 was detected by immunofluorescence staining.The expression of apoptosis-related proteins and Wnt/β-catenin pathway proteins was detected by western blot assay.RESULTS AND CONCLUSION:The cells extracted in vitro were cultured and stained,and were clearly identified as chondrocytes.Complanatoside A had no obvious cytotoxicity to chondrocytes in the concentration range of 0-80μmol/L,and significantly improved the chondrocyte viability in the concentration range of 2.5-10μmol/L,especially when the concentration was 5μmol/L.The apoptotic rate of chondrocytes was higher in the sodium nitroprusside-induced group than the blank control group,while the apoptotic rate was lower in the sodium nitroprusside+Complanatoside A group than the sodium nitroprusside-induced group.The fluorescence intensity of type II collagen and SOX9 in chondrocytes was weaker in the sodium nitroprusside-induced group than the blank control group,while the fluorescence intensity of type II collagen and SOX9

关 键 词：骨关节炎 软骨细胞 沙苑子苷A 细胞凋亡 WNT/Β-CATENIN信号通路 

分 类 号：R496[医药卫生—康复医学] R318[医药卫生—临床医学] R681