摘要
目的评估大骨节病(Kashin-Beck disease,KBD)患者氧化应激水平及氧化损伤对沉默信息调节因子2相关酶类1(silent information regulator 2 homolog 1,SIRT1)表达的影响,探讨氧化应激与SIRT1在KBD软骨损伤中的作用。方法2017年5月,从青海省贵德县选取20例KBD患者作为KBD组,同时选取40例健康受试者作为对照组,采集5 ml肘静脉血,离心后留存上层血浆,酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)法检测谷胱甘肽过氧化物酶(glutathione peroxidase,GPX)活性和活性氧(reactive oxygen species,ROS)水平;并采用实时荧光定量PCR(RT-qPCR)法检测SIRT1 mRNA水平。选择150μmol/L叔丁基过氧化氢(tert-butyl hydroperoxide,tBHP)损伤软骨细胞;并用不同浓度亚硒酸钠(Na_(2)SeO_(3))干预软骨细胞后检测细胞活力,选择适宜浓度Na_(2)SeO_(3)进行预保护。提取软骨细胞总RNA和DNA,采用RT-qPCR法检测SIRT1、DNA甲基转移酶1(DNA methyltransferase 1,DNMT1)mRNA水平以及SIRT1启动子区DNA甲基化水平;同时,采用Hoechst 33342染色方法检测软骨细胞凋亡情况。结果KBD组患者血浆GPX活性[(35.48±8.82)U/g·Hb]低于对照组[(40.43±6.68)U/g·Hb,t=-2.43,P=0.018],ROS水平[(577.10±96.92)U/ml]高于对照组[(526.44±62.63)U/ml,t=2.13,P=0.043]。GPX活性与SIRT1 mRNA水平呈正相关(r_(s)=0.44,P=0.005),ROS水平与SIRT1 mRNA水平呈负相关(r_(s)=-0.39,P=0.006)。150μmol/L tBHP(tBHP损伤组)作用48 h,软骨细胞存活率下降至(55.27±2.96)%;0.10μg/ml Na_(2)SeO_(3)预保护(硒保护组)后软骨细胞存活率明显高于tBHP损伤组(P<0.05)。与对照组比较,tBHP损伤组软骨细胞SIRT1 mRNA水平较低,SIRT1启动子区DNA甲基化水平、DNMT1 mRNA水平、细胞凋亡率均较高(均P<0.05);与tBHP损伤组比较,硒保护组软骨细胞SIRT1 mRNA水平较高,SIRT1启动子区DNA甲基化水平、DNMT1 mRNA水平、细胞凋亡率均较低(均P<0.05)。细胞凋亡率与SIRT1 mRNA水平呈负相关(r_(s)=-0.78,P=0.004),与SIRT1启动子区DNA甲基化水平呈正相关(r_(s)=0.76,P=0.006)。结论KBD患者氧化应激水平增加,可能与SIRT1低表达相关。氧化损伤可能通过催化SIRT1启动子区DNA甲基化,下调SIRT1表达,促进软骨细胞凋亡。
Objective To investigate the role of oxidative stress and silent information regulator 2 homolog 1(SIRT1)in cartilage injury in Kashin-Beck disease(KBD)by evaluating the level of oxidative stress and the effect of oxidative injury on SIRT1 expression in patients with KBD.Methods In May 2017,Twenty patients with KBD were selected from Guide County of Qinghai Province as the KBD group,and 40 healthy subjects were selected as the control group,5 ml elbow venous blood was collected,centrifuged,and the upper plasma was retained.The glutathione peroxidase(GPX)activity and reactive oxygen species(ROS)level were determined by enzyme linked immunosorbent assay(ELISA),and SIRT1 mRNA level was determined by real-time fluorescence quantitative PCR(RT-qPCR).Meanwhile,150μmol/L tert-butyl hydroperoxide(tBHP)was selected to damage chondrocytes;and different concentrations of sodium selenite(Na_(2)SeO_(3))were used to intervene in chondrocytes to detect cell viability,and appropriate concentration of Na_(2)SeO_(3) was selected for pre protection.Total RNA and DNA of chondrocytes were extracted.The mRNA levels of SIRT1,DNA methyltransferase 1(DNMT1),and the DNA methylation level in the SIRT1 promoter region were determined by RT-qPCR.At the same time,Hoechst 33342 staining was used to detect chondrocyte apoptosis.Results The plasma GPX activity[(35.48±8.82)U/g·Hb]in KBD group was lower than that in control group[(40.43±6.68)U/g·Hb,t=-2.43,P=0.018],and the ROS level[(577.10±96.92)U/ml]was higher than that in control group[(526.44±62.63)U/ml,t=2.13,P=0.043].GPX activity was positively correlated with SIRT1 mRNA level(r_(s)=0.44,P=0.005),while ROS level was negatively correlated with SIRT1 mRNA level(r_(s)=-0.39,P=0.006).After 48 hours of treatment with 150μmol/L tBHP(tBHP injury group),the survival rate of chondrocytes decreased to(55.27±2.96)%;and the survival rate of chondrocytes pre-protected with 0.10μg/ml Na_(2)SeO_(3)(selenium protection group)was significantly higher than that of tBHP injury group(P<0.05).Compared with control group,the SIRT1 mRNA level of chondrocytes in tBHP injury group was significantly decreased;while the DNA methylation level in the SIRT1 promoter region,DNMT1 mRNA level and cell apoptosis rate were significantly increased(P<0.05).Compared with tBHP injury group,the selenium protection group had higher levels of SIRT1 mRNA in chondrocytes,lower levels of DNA methylation in the SIRT1 promoter region,DNMT1 mRNA,and cell apoptosis rate(P<0.05).The apoptosis rate was negatively correlated with SIRT1 mRNA level(r_(s)=-0.78,P=0.004),and positively correlated with the DNA methylation level in the SIRT1 promoter region(r_(s)=0.76,P=0.006).Conclusions KBD patients have increased levels of oxidative stress,which may be associated with low expression of SIRT1.Oxidative injury may down-regulate SIRT1 expression and promote chondrocytes apoptosis by catalyzing DNA methylation in the SIRT1 promoter region.
作者
杨晓莉
李强
张迪
徐翠香
靳占奎
熊咏民
Yang Xiaoli;Li Qiang;Zhang Di;Xu Cuixiang;Jin Zhankui;Xiong Yongmin(Shaanxi Provincial Key Laboratory of Infection and Immune Diseases,Shaanxi Provincial People's Hospital,Xi'an 710068,China;School of Public Health,Xi'an Jiaotong University Health Science Center,Key Laboratory of Trace Elements and Endemic Diseases,National Health Commission of the People's Republic of China,Xi'an 710061,China;Department of Orthopedics,Shaanxi Provincial People's Hospital,Xi'an 710068,China)
出处
《中华地方病学杂志》
北大核心
2024年第10期783-789,共7页
Chinese Journal of Endemiology
基金
国家自然科学基金(82073494)
陕西省自然科学基金(2023-JC-YB-790)
陕西省人民医院人才支持项目(2022JY-53)
陕西省人民医院科技发展孵化基金(2023YJY-20)
