GADD45β在骨关节炎中的作用及其对MKK7/JNK途径的影响  

Role of GADD45βin osteoarthritis and its effect on MKK7/JNK pathway

作　　者：王远瑞 孙强 陈永锋 漆伟 康飞科 WANG Yuanrui;SUN Qiang;CHEN Yongfeng;QI Wei;KANG Feike(Department of Orthopaedics,The First Affiliated Hospital of Air Force Medical University,Xi'an 710032,China)

机构地区：[1]空军军医大学第一附属医院骨科,陕西西安710032

出　　处：《西部医学》2024年第8期1097-1106,共10页Medical Journal of West China

基　　金：陕西省重点研发计划项目(2022-SF-229)。

摘　　要：目的探讨生长抑制和DNA损伤诱导基因45β(GADD45β)在骨关节炎(OA)发生发展中的作用及机制。方法将大鼠软骨细胞分为对照组、IL-1β组、NC-OE+IL-1β组、GADD45βOE+IL-1β组、NC-sh+IL-1β组和GADD45βsh+IL-1β组。除对照组之外,其他组细胞均采用10 ng/mL的IL-1β处理24 h。采用MTT法和TUNEL法检测软骨细胞增殖和凋亡。将SD大鼠分为假手术组、OA组、NC-OE+OA组和GADD45βOE+OA组,每组12只。假手术组大鼠只进行假手术操作,其他组大鼠均为前交叉韧带切断加内侧半月板部分切除法诱导的OA模型大鼠。假手术组和OA组大鼠关节腔内注射磷酸盐缓冲液,NC-OE+OA组和GADD45βOE+OA组大鼠关节腔内分别注射NC-OE和GADD45βOE慢病毒,每周注射1次,共4周。采用番红O-固绿染色观察大鼠软骨降解并进行OARSI评分,采用TUNEL染色检测大鼠软骨细胞凋亡。采用RT-qPCR检测GADD45β、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、基质金属蛋白酶(MMP)3、MMP13、IL-1β、IL-6、TNF-α的mRNA水平。采用Western blot检测GADD45β、丝裂原活化蛋白激酶激酶7(MKK7)、p-MKK-7、c-Jun氨基末端激酶(JNK)1/2/3和p-JNK1/2/3的蛋白水平。结果与对照组比较,IL-1β组软骨细胞的GADD45βmRNA和蛋白水平降低,相对细胞活力和Bcl-2 mRNA水平均降低,TUNEL阳性率以及Bax、MMP3、MMP13、IL-1β、IL-6和TNF-α的mRNA水平均升高,MKK-7和JNK1/2/3的相对磷酸化水平升高(P<0.05)。与IL-1β组和NC-OE+IL-1β组比较,GADD45βOE+IL-1β组软骨细胞的GADD45βmRNA和蛋白水平升高,相对细胞活力和Bcl-2 mRNA水平均升高,TUNEL阳性率以及Bax、MMP3和MMP13、IL-1β、IL-6和TNF-α的mRNA水平均降低,MKK-7和JNK1/2/3的相对磷酸化水平降低(P<0.05)。与IL-1β组和NC-sh+IL-1β组比较,GADD45βsh+IL-1β组软骨细胞的GADD45βmRNA和蛋白水平降低,相对细胞活力和Bcl-2 mRNA水平均降低,TUNEL阳性率以及Bax、MMP3、MMP13、IL-1β、IL-6和TNF-α的mRNA水平均升高,MKObjective To investigate the role of growth arrest DNA damage-inducible gene 45beta(GADD45β)in the development and mechanism of osteoarthritis(OA).Methods Rat chondrocytes were divided into control group,interleukin-1β(IL-1β)group,NC-OE+IL-1βgroup,GADD45βOE+IL-1βgroup,NC-sh+IL-1βgroup and GADD45βsh+IL-1βgroup.Except the control group,cells in other groups were treated with 10 ng/mL IL-1βfor 24 h.Chondrocyte proliferation and apoptosis were detected by MTT and TUNEL methods.Rats were divided into Sham group,OA group,NC-OE+OA group and GADD45βOE+OA group(n=12).Rats in sham group undergoing sham operation,and rats in other group were OA model rats induced by anterior cruciate ligament amputation and medial meniscectomy.Phosphate buffer was injected into the joint cavity of Sham group and OA group.NC-OE and GADD45βOE+OA lentivirus were injected into the joint cavity of NC-OE and GADD45βOE+OA group,respectively.The injection was given once a week for 4 weeks.The cartilage degradation was observed by safranin O-fast green staining and OARSI score was performed.Chondrocyte apoptosis was detected by TUNEL staining.mRNA levels of GADD45β,B-cell lymphoma/leukemia-2(Bcl-2),Bcl-2 associated X protein(Bax),matrix metalloproteinase(MMP)3,MMP13,IL-1β,IL-6,and tumor necrosis factor-α(TNF-α)were detected by RT-qPCR.Protein levels of GADD45β,mitogen-activated protein kinase kinase 7(MKK7),p-MKK-7,c-Jun amino-terminal kinase(JNK)1/2/3 and p-JNK1/2/3 were detected by Western blot.Results Compared with control group,the mRNA and protein levels of GADD45βdecreased in IL-1βgroup(P<0.05),the relative cell viability and Bcl-2 mRNA levels decreased(P<0.05),TUNEL positive rate and the mRNA levels of Bax,MMP3,MMP13,IL-1β,IL-6 and TNF-αincreased(P<0.05),and the relative phosphorylation levels of MKK-7 and JNK1/2/3 increased(P<0.05).Compared with IL-1βgroup and NC-OE+IL-1βgroup,the mRNA and protein levels of GADD45βincreased in GADD45βOE+IL-1βgroup(P<0.05),the relative cell viability and Bcl-2 mRNA levels increased(

关 键 词：骨关节炎 生长抑制和DNA损伤诱导基因45β C-JUN氨基末端激酶 软骨 炎症 细胞凋亡 

分 类 号：R684.3[医药卫生—骨科学]