滑膜细胞炎性因子水平及miR-145/MKK4分子轴的关联性

Correlation between inflammatory factors in synovial cells and microRNA-145/mitogen-activated protein kinase 4 molecular axis

背景:炎症因子被证明在软骨细胞和双指关节细胞中表达异常,靶向炎症途径可能是治疗骨关节炎的有效策略,但目前鲜有关于滑膜细胞炎症反应的研究报道。目的:探讨~(99)Tc-MDP通过miR-145/MKK4分子轴对滑膜细胞产生炎性因子的影响。方法:①骨关节炎成纤维样滑膜细胞(HFLS-RA细胞)经过不同质量浓度~(99)Tc-MDP药物处理48 h,qRT-PCR实验检测HFLS-RA细胞中miR-145以及炎性因子caspase 1、白细胞介素1β、白细胞介素18和肿瘤坏死因子α的表达水平,CCK-8实验检测HFLS-RA细胞存活率;②确定~(99)Tc-MDP干预最佳质量浓度后,将HFLS-RA细胞分为4组:NC组(空白对照组,不经任何处理)、~(99)Tc-MDP组(13μg/L~(99)Tc-MDP处理HFLS-RA细胞48 h)、~(99)TcMDP+miR-145 inhibitor组(13μg/L~(99)Tc-MDP处理HFLS-RA细胞48 h同时在细胞中转染miR-145 inhibitor)、~(99)Tc-MDP+miR-145 inhibitor+sh-MKK4组(13μg/L~(99)Tc-MDP处理HFLS-RA细胞48 h同时在细胞中转染miR-145 inhibitor+sh-MKK4),qRT-PCR实验检测HFLS-RA细胞中miR-145以及炎性因子caspase 1、白细胞介素1β、白细胞介素18和肿瘤坏死因子α的表达水平,CCK-8实验检测HFLS-RA细胞存活率。结果与结论:①~(99)Tc-MDP可显著抑制HFLS-RA细胞炎性因子caspase 1、白细胞介素1β、白细胞介素18和肿瘤坏死因子α表达和细胞存活率,且半抑制浓度IC_(50)=13μg/L;②~(99)Tc-MDP可显著上调HFLS-RA细胞中miR-145的表达,下调MKK4表达(P<0.01);③miR-145靶向MKK4,且过表达miR-145可显著抑制MKK4的表达;④与~(99)Tc-MDP组相比,~(99)Tc-MDP+miR-145 inhibitor组可显著抑制HFLS-RA细胞中miR-145的表达(P <0.05),促进细胞中炎性因子的表达(P<0.05)和细胞增殖(P<0.05),相比较于~(99)Tc-MDP+miR-145 inhibitor+sh-MKK4组,~(99)Tc-MDP+miR-145 inhibitor组可明显促进HFLS-RA细胞增殖(P<0.05)和炎性因子的表达(P<0.05);⑤结果表明,~(99)Tc-MDP通过miR-145靶向下调MKK4抑制HFLS-RA细胞产生炎性因子,缓解骨关节炎发生。

BACKGROUND:Inflammatory cytokines have been shown to be abnormally expressed in chondrocytes and double-knuckle cells.Targeting inflammato ry pathways may be an effective strategy for the treatment of osteoarthritis,but few repo rts focus on inflammato ry responses in synovial cells.OBJECTIVE:To investigate the effect of 99Tc-MDP on synovial cells-produced inflammato ry facto rs through microRNA-145(miR-145)/mitogen-activated protein kinase 4(MKK4)molecular axis.METHODS:Osteoarthritis fibro blast-like synovial cells(HFLS-RA cells)were treated with 99Tc-MDP of different mass concentrations for 48 hours.The expression levels of miR-145 and inflammato ry facto rs,caspase 1,inte rle u kin(IL)-1,IL-18 and tumor nec rosis factor-α(TNF-α)in HFLS-RA cells were measured by qRT-PCR,and the survival rate of HFLS-RA cells was measured by cell counting kit-8.After determining the optimal mass concentration for 99Tc-MDP intervention,HFLSRA cells were divided into four gro u ps:blank control group(without any treatment),99Tc-MDP group(13μg/L 99Tc-MDP treated HFLS-RA cells for 48 hours),99Tc-M DP+miR-145 inhibitor group(13μg/L 99Tc-M DP treated HFLS-RA cells followed by tra nsfection with miR-145 inhibitor),99Tc-M DP+miR-145 inhibito r+shMKK4 group(13μg/L 99Tc-MDP treated HFLS-RA cells followed by transfection with miR-145 inhibitor+sh-MKK4).The expression levels of miR-145 and the inflammatory factors,caspase 1,IL-1,IL-18 and TNF-α,in HFLS-RA cells were measured by qRT-PCR,and the survival rate of HFLS-RA cell was measured by cell counting kit-8.RESULTS AND CONCLUSION:99Tc-MDP significantly inhibited the expression of caspase 1,IL-1,IL-18 and TNF-αas well as the cell survival rate of hFLSRA cells.The 50%inhibiting concentration of 99Tc-MDP was 13μg/L.99Tc-MDP significantly upregulated the expression of miR-145,and downregulated the expression of MKK4 expression in HFLS-RA cells(P<0.01).MiR-145 to rgeted MKK4,and ove rexpression of miR-145 could significantly inhibit the expression of M KK4.Compared with the 99Tc-MDP group,the 99Tc-M DP+miR-145 inhibitor group significantly inhibited the expression of miR-145 in HFLS-RA cells(P<0.05)and promoted the expression of inflammato ry facto rs in the cells(P<0.05)and cell prolife ration(P<0.05).Compared with the 99Tc-MDP+miR-145 inhibito r+sh-MKK4 group,the 99Tc-MDP+miR-145 inhibitor group could significantly promote HFLS-RA cell proliferation(P<0.05)and increase the expression of inflammatory factors in the cells(P<0.05).To conclude,99Tc-MDP relieves osteoarthritis by inhibiting HFLS-RA cell-produced inflammatory factors via the downregulation of MKK4 by miR-145.