大骨节病患者SIRT1异常表达与软骨细胞凋亡的关系研究

The relationship between abnormal expression of SIRT1 and chondrocyte apoptosis in patients with Kashin-Beck disease

目的分析大骨节病(KBD)患者沉默信息调节因子2相关酶类1(SIRT1)的表达及其与软骨细胞凋亡的关系。方法从青海省KBD病区贵德县选取20例KBD患者作为KBD组,同时在病区选取年龄和性别匹配的40例健康受试者作为对照组。采集研究对象的空腹肘静脉血,采用实时荧光定量PCR(real-time PCR)检测外周血SIRT1及硒蛋白基因[谷胱甘肽过氧化酶(GPX)2、GPX3、硫氧还蛋白还原酶(TXNRD)1、TXNRD3、碘甲腺原氨酸脱碘酶Ⅰ(DIO1)、硒磷酸合成酶2(SPS2)]mRNA水平;并采用曲线拟合法进行KBD患者外周血SIRT1与硒蛋白基因表达关联分析。同时,体外培养人正常软骨细胞,分为对照组(未经任何处理)、单纯白藜芦醇(RES)组(验证RES对SIRT1的激活作用)、叔丁基过氧化氢(tBHP)损伤组(软骨细胞氧化损伤模型)和RES保护组(RES预保护后进行tBHP损伤),采用real-time PCR检测各组细胞SIRT1,硒蛋白基因及凋亡相关基因[B淋巴细胞瘤-2基因(BCL2)、BCL2相关X蛋白(BAX)、核转录因子κB(NF-κB)p65、肿瘤抑制基因P53]mRNA水平。结果人群研究中,KBD组外周血SIRT1 mRNA水平(1.12±0.38)低于对照组(1.87±0.97),差异有统计学意义(t=3.31,P=0.002)。经曲线拟合分析,KBD组外周血GPX3、TXNRD1和TXNRD3 mRNA水平随SIRT1 mRNA水平的升高均有所升高(R^(2)=0.48、0.66、0.95,均P<0.001);DIO1 mRNA水平随SIRT1 mRNA水平的升高呈现先下降后上升的趋势(R^(2)=0.51,P=0.024);GPX2、SPS2 mRNA水平随SIRT1 mRNA水平的升高均未见显著变化趋势(R^(2)=0.16、0.12,P=0.064、0.114)。细胞实验中,与对照组(1.00±0.10)比较,单纯RES组SIRT1 mRNA水平(1.79±0.07)较高(P<0.05)。与tBHP损伤组比较,RES保护组硒蛋白基因GPX3、TXNRD1、TXNRD3、DIO1 mRNA水平均较高(均P<0.05);凋亡相关基因BAX、P53 mRNA水平及BAX/BCL2比值均较低,BCL2、NF-κB p65 mRNA水平均较高(均P<0.05)。结论KBD患者SIRT1低表达;RES激活SIRT1后可能通过上调硒蛋白基因表达来增强软骨细胞抗氧化能力,从而抑制软骨细胞凋亡。

Objective To analyze the expression of silent information regulator 2-related enzyme 1(SIRT1)and its relationship with chondrocyte apoptosis in patients with Kashin-Beck disease(KBD).Methods Twenty patients with KBD were selected as the KBD group from Guide County,Qinghai Province,and 40 healthy subjects matched by age and sex were selected as the control group.Fasting elbow venous blood of the study subjects was collected,and peripheral blood mRNA levels of SIRT1 and selenoprotein genes[glutathione peroxidase(GPX)2,GPX3,thioredoxin reductase(TXNRD)1,TXNRD3,iodothyronine deiodinaseⅠ(DIO1),and selenophosphate synthetase 2(SPS2)]were detected by real-time PCR.The correlation between SIRT1 expression and selenoprotein genes in peripheral blood of KBD patients was analyzed by curve fitting method.Meanwhile,normal human chondrocytes cultured in vitro were divided into control group(without any treatment),resveratrol(RES)group(to verify the activation effect of RES on SIRT1),tert-butyl hydroperoxide(tBHP)injury group(oxidative injury model of chondrocyte),and RES protection group(tBHP injury after RES pre protection).The mRNA levels of SIRT1,selenoprotein genes,and apoptosis-related genes[B lymphoblastoma-2 gene(BCL2),BCL2-associated X protein(BAX),nuclear transcription factorκB(NF-κB)p65,and tumor suppressor gene P53]in each group of cells were detected by real-time PCR.Results In the population study,the peripheral blood SIRT1 mRNA level in the KBD group(1.12±0.38)was lower than that of control group(1.87±0.97),and the difference was statistically significant(t=3.31,P=0.002).According to curve fitting analysis,the mRNA levels of GPX3,TXNRD1,and TXNRD3 in peripheral blood of KBD group increased with the increase of SIRT1 mRNA level(R^(2)=0.48,0.66,0.95,P<0.001).The level of DIO1 mRNA showed a trend of decreased first and then increased with the increase of SIRT1 mRNA level(R^(2)=0.51,P=0.024).The mRNA levels of GPX2 and SPS2 showed no significant change trend with the increase of SIRT1 mRNA level(R^(2)=0.16,0.12,P=0.064,0.114).In cell studies,compared with the control group(1.00±0.10),the SIRT1 mRNA level in the RES group(1.79±0.07)was higher(P<0.05).Compared with tBHP injury group,the RES protection group had higher mRNA levels of selenoprotein genes GPX3,TXNRD1,TXNRD3,and DIO1(P<0.05);the mRNA levels of apoptosis-related genes BAX,P53 and the ratio of BAX/BCL2 were lower,while the mRNA levels of BCL2 and NF-κB p65 were higher(P<0.05).Conclusions KBD patients have low expression of SIRT1.And RES activation of SIRT1 may enhance the antioxidant capacity of chondrocyte by up-regulating the expression of selenoprotein genes,thus inhibiting chondrocyte apoptosis.