虾青素调控tRF-ValAAC对骨关节炎软骨细胞生物学功能的影响及其机制  

Effect and mechanism of astaxanthin in regulating tRF-VaLAAC on the biological functions of Chondrocytes in osteoarthritis

作　　者：朱春晖 刘刚 陈伟 翟晨骏 ZHU Chunhui;LIU Gang;CHEN Wei;ZHAI Chenjun(Trauma Center,Changzhou Second People's Hospital Affiliated to Nanjing Medical University,Changzhou 213003,Jiangsu,China;Department of Orthopaedics,Yixing People's Hospital,Wuxi 214200,Jiangsu,China)

机构地区：[1]南京医科大学附属常州第二人民医院创伤中心,江苏常州213003 [2]宜兴市人民医院骨科,江苏无锡214200

出　　处：《中华骨与关节外科杂志》2024年第10期921-930,共10页Chinese Journal of Bone and Joint Surgery

基　　金：国家自然科学基金(82201716);常州市卫生健康人才国外研修资助项目(GW2023021);常州市第九批科技计划项目(CJ20220072);2022年第三十九批人才专项资金(2022级卫生健康青苗人才资助)(CZQM2022009);常州市卫生健康委员会科技项目(QN202118)。

摘　　要：目的:探讨虾青素调控tRF-ValAAC对骨关节炎(OA)软骨细胞生物学功能的影响及机制。方法:选取常州第二人民医院2021年12月至2023年9月行全膝关节置换术的OA患者及膝关节损伤且无OA患者各10例,胰酶消化法提取上述患者骨关节软骨细胞,分别作为OA组和无OA组;另取OA患者骨关节软骨细胞用20μmol/L虾青素处理,作为OA+AST组。OA组软骨细胞分别转染tRF-ValAAC模拟物(tRF-ValAAC-mimic)、tRF-ValAAC抑制剂(tRF-ValAAC-inhibitor),并在用20μmol/L虾青素处理过的OA软骨细胞中转染tRF-ValAAC-inhibitor。处理结束后,采用CCK-8法检测细胞增殖活性;流式细胞术检测细胞凋亡;酶联免疫吸附试验(ELISA)法检测白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)和肿瘤坏死因子α(TNF-α)水平;实时荧光定量PCR(qRT-PCR)检测细胞中tRF-ValAAC基因的表达;Western blot实验检测氧化应激相关酶[超氧化物歧化酶1(SOD1)、谷胱甘肽过氧化物酶4(GPX4)]及细胞外基质(ECM)相关蛋白[Ⅱ型胶原蛋白(COL2)、聚集蛋白聚糖(ACAN)和基质金属蛋白酶13(MMP-13)]水平。结果:与无OA组比较,OA组软骨细胞内tRF-ValAAC的基因表达显著降低(P<0.05),而应用虾青素干预可上调其表达(P<0.05)。与对照组比较,OA组软骨细胞增殖率降低、细胞凋亡率升高(P均<0.05);与OA组比较,OA+AST组软骨细胞增殖率升高、凋亡率降低(P均<0.05);转染tRF-ValAAC-mimic后,OA软骨细胞增殖率升高、凋亡率降低(P均<0.05);在用虾青素处理过的OA软骨细胞中转染tRF-ValAAC-inhibitor后,细胞增殖率升高、凋亡率降低(P均<0.05)。与对照组比较,OA组IL-6、IL-1β和TNF-α水平升高;与OA组比较,OA+AST组IL-6、IL-1β、TNF-α水平降低(P均<0.05);转染tRF-ValAAC-mimic后,OA软骨细胞IL-6、IL-1β和TNF-α水平降低(P<均0.05);在用虾青素处理过的OA软骨细胞中转染tRF-ValAAC-inhibitor后,IL-6、IL-1β和TNF-α水平升高(P均<0.05)。与对照组比较,OA组MMP-13蛋白水平升高Objective:To explore the effect and mechanism of astaxanthin in regulating tRF-ValAAC on the biological functions of chondrocytes in osteoarthritis(OA).Methods:Chondrocytes were isolated from the articular cartilage of 10 OA patients who underwent total knee arthroplasty and 10 control patients with knee trauma but without OA at the Second People's Hospital of Changzhou from December 2021 to September 2023.The cells were extracted using trypsinization and designated as the OA group and the control group,respectively.Chondrocytes from OA patients were treated with 20μmol/L astaxanthin and designated as the OA+AST group.In the OA group,chondrocytes were transfected with tRF-ValAAC mimics or inhibitors,while in the OA+AST group,chondrocytes were transfected with tRF-ValAAC-inhibitors.After treatment,cell proliferation was assessed using the CCK-8 assay,and apoptosis was measured by flow cytometry.Levels of interleukin-6(IL-6),interleukin-1β(IL-1β)and tumor necrosis factorα(TNF-α)were measured using enzyme-linked immunosorbent assay(ELISA).The expression of tRF-ValAAC was detected by quantitative PCR(qPCR),and Western blot was performed to evaluate oxidative stress-related enzymes[superoxide dismutase 1(SOD1),glutathione peroxidase 4(GPX4)]and extracellular matrix(ECM)related proteins[typeⅡcollagen(COL2),aggrecan(ACAN),and matrix metalloproteinase-13(MMP-13)].Results:Compared with the control group,tRF-ValAAC expression in OA chondrocytes was significantly decreased(P<0.05),while astaxanthin treatment significantly upregulated its expression(P<0.05),and OA chondrocytes showed decreased proliferation and increased apoptosis(all P<0.05).Compared with the OA group,the OA+AST group presented increased proliferation and decreased apoptosis(all P<0.05).Transfection with tRF-ValAAC mimics in OA chondrocytes increased proliferation and decreased apoptosis(all P<0.05).Transfection with tRF-ValAAC inhibitor in astaxanthin-treated OA chondrocytes increased proliferation and reduced apoptosis(all P<0.05).IL-6,IL-1βand TN

关 键 词：虾青素 tRF-ValAAC 软骨细胞 细胞损伤 氧化应激 

分 类 号：R684.3[医药卫生—骨科学]