ERK1/2信号蛋白和蛋白降解酶在骨关节炎患者软骨/软骨下骨中的表达及其意义

Expression and significance of ERK1/2 signal protein and protein-degrading enzyme in cartilage and subchondral bone of osteoarthritis patients

目的探讨细胞外信号调节激酶1/2(ERK1/2)信号蛋白与骨关节炎(OA)致病相关蛋白降解酶在软骨/软骨下骨中的表达及其意义。方法收集2017年12月-2019年12月石河子大学医学院第一附属医院关节外科收治的50例OA患者的膝关节胫骨平台样本以及创伤科因外伤截肢的10例患者的健康膝关节胫骨平台样本(作为对照)。采用HE染色观察软骨退变情况,利用OARSI-Modified Manking评分评估软骨破坏程度,并根据评分将OA患者分成轻度组(n=31)与重度组(n=19)。采用免疫组化染色和Western blotting检测ERK1/2、pERK1/2、MMP-3、MMP-9及ADAMT5在软骨/软骨下骨中的表达,实时定量PCR(RT-qPCR)检测软骨/软骨下骨中ERK1/2、MMP-3、MMP-9及ADAMT5 mRNA的表达,micro-CT测定软骨下骨微结构的改变情况。结果HE染色显示,轻度组软骨变薄,软骨细胞排列紊乱,软骨下骨厚度增加;重度组软骨几乎完全消失,软骨下骨明显暴露并变形,钙化软骨增厚。OARSI评分结果显示,与对照组相比,轻度组、重度组OARSI评分明显升高[(8.14±0.56)分、(23.33±0.17)分vs.(3.53±0.11)分,P<0.05],且重度组高于轻度组(P<0.05)。免疫组化染色、Western blotting及RT-qPCR检测结果显示,与对照组相比,轻度组和重度组软骨/软骨下骨中pERK1/2、MMP-3、MMP-9及ADAMT5蛋白表达水平增高(P<0.05),MMP-3、MMP-9及ADAMT5 mRNA相对表达水平明显增高(P<0.05),但各组ERK1/2 mRNA和蛋白相对表达水平无明显差异(P>0.05)。Micro-CT扫描结果显示,轻度组和重度组软骨下骨的微结构严重紊乱并伴有细胞增殖;与对照组比较,轻度组和重度组软骨/软骨下骨中骨体积分数(BV/TV)、骨小梁厚度(Tb.Th)、骨小梁数量(Tb.N)、骨密度(BMD)明显增高,骨小梁间距(Tb.Sp)明显缩小(P<0.05)。结论OA患者软骨/软骨下骨中pERK1/2、MMP-3、MMP-9及ADAMT5均呈现高表达且软骨下骨微结构发生改变,pERK1/2表达增加可能是蛋白降解酶活性增强及软骨下骨微结构改变的原因之一。

Objective To study the expression and significance of extracellular signaling protein-regulated kinase 1/2(ERK1/2)signal protein and protein-degrading enzyme in cartilage and subchondral bone of osteoarthritis(OA)patients.Methods From December 2017 to December 2019,50 knee tibial plateau samples of OA patients in the Joint Surgery Department and 10 healthy knee tibial plateau samples of patients with traumatic amputation(as control)in the Trauma Department were collected from the First Affiliated Hospital of Medical College of Shihezi University.The cartilage degeneration was observed by HE staining,and the degeneration of articular cartilage was detected by OARSI Modified Manking score.OA patients were divided into mild group and severe group according to the score.The expressions of ERK1/2,pERK1/2,MMP-3,MMP-9 and ADAMT5 protein in cartilage and subchondral bone were detected by immunohistochemical staining and Western blotting.The expressions of ERK1/2,MMP-3,MMP-9 and ADAMT5 mRNA in cartilage and subchondral bone were detected by real-time PCR(RT-qPCR),and the changes of subchondral bone microstructure were measured by micro-CT.Results HE staining showed that the cartilage became thinner,the chondrocytes arranged disorderly,and the thickness of subchondral bone increased in the mild group;the cartilage almost completely disappeared,the subchondral bone obviously exposed and deformed,and the calcified cartilage thickened in the severe group.The results of OARSI score showed that compared with control group,the OARSI scores were significantly higher[(8.14±0.56)points and(23.33±0.17)points vs.(3.53±0.11)points,P<0.05]in the mild group and severe group,and the score of the severe group was significantly higher than that of the mild group(P<0.05).Immunohistochemical staining and Western blotting results showed that compared with control group,the protein expression levels of pERK1/2,MMP-3,MMP-9 and ADAMT5 in cartilage and subchondral bone in the mild group and severe group increased(P<0.05),the mRNA expression levels of MMP-3,MMP-9 and ADAMT5 in cartilage and subchondral bone in mild group and severe group obviously increased(P<0.05),but there was no significant difference in the expression level of ERK1/2 mRNA and protein between the groups(P>0.05).MicroCT scanning showed that the microstructure of subchondral bone in the mild and severe groups was seriously disordered with cell proliferation.Compared with control group,BV/TV,Tb.Th,Tb.N and BMD in cartilage and subchondral bone in mild group and severe group significantly increased,and Tb.Sp significantly decreased(P<0.05).Conclusions pERK1/2,MMP-3,MMP-9 and ADAMT5 are highly expressed in OA cartilage and subchondral bone,and the microstructure of subchondral bone is changed.The increased expression of pERK1/2 may be one of the reasons for the increased activity of cartilage-degrading enzymes and the changes of subchondral bone microstructure.