MMP13和LRP1在大骨节病患者关节软骨细胞自噬功能异常中的作用及机制研究

The role and mechanism of MMP13 and LRP1 in abnormal autophagy function of articular chondrocytes in patients with Kashin-Beck disease

目的探讨基质金属蛋白酶13(MMP13)和低密度脂蛋白受体相关蛋白1(LRP1)对大骨节病患者关节软骨细胞自噬功能的影响。方法自陕西省地方病防治研究所获取4例大骨节病患者和4例对照受试者的关节软骨样本,采用免疫组织化学(IHC)法检测软骨组织中MMP13和LRP1的表达水平;体外提取并培养软骨细胞,分别采用实时荧光定量PCR(qRT-PCR)和蛋白免疫印迹(WB)法检测软骨细胞LRP1以及自噬相关基因[自噬基因Beclin1(BECN1)、微管相关蛋白1轻链3(LC3)],软骨损伤相关基因[MMP13、半胱氨酸蛋白酶3(CASP3)],软骨细胞分化相关基因[Ⅱ型胶原α1链(COL2A1)、Y染色体性别决定区-盒转录因子9(SOX9)]的mRNA和蛋白表达水平。另提取3例大骨节病患者膝关节样本的软骨细胞,采用RNA干扰(RNAi)技术进行MMP13基因沉默实验,并采用qRT-PCR和WB法检测上述基因mRNA和蛋白表达水平。此外,采用LRP1的拮抗剂受体相关蛋白(RAP)对人正常软骨细胞(C28/I2细胞)中LRP1进行封闭,并采用qRT-PCR和WB法检测软骨细胞LRP1、自噬、分化和软骨损伤相关基因的mRNA和蛋白表达水平。结果IHC结果显示,大骨节病患者软骨组织表、中、深层的MMP13表达水平(1.67±0.21、0.59±0.15、0.51±0.12)均显著高于对照受试者(0.25±0.03、0.26±0.04、0.06±0.01),差异均有统计学意义(t=-11.38,P<0.001;t=-3.82、-6.26,P=0.019、0.003);LRP1表达水平(0.10±0.02、0.03±0.01、0.17±0.03)均显著低于对照受试者(1.63±0.40、0.44±0.12、0.34±0.08),差异均有统计学意义(t=6.61、5.61、3.64,P=0.003、0.005、0.022)。大骨节病患者软骨细胞中MMP13、CASP3、SOX9的mRNA和蛋白表达水平均显著高于对照受试者,差异均有统计学意义(均P<0.05);LRP1、LC3、COL2A1的mRNA表达水平均显著低于对照受试者,差异均有统计学意义(均P<0.05)。在大骨节病患者软骨细胞中沉默MMP13基因后,LRP1、BECN1、LC3、CASP3、COL2A1和SOX9的mRNA和蛋白表达水平均无显著变化(均P>0.05)。采用RAP封闭LRP1后,软骨细胞LRP1、BECN1、LC3、MMP13、COL2A1和SOX9的蛋白表达水平均明显低于对照组(均P<0.05)。结论MMP13与大骨节病患者关节软骨细胞的自噬异常无直接联系;封闭LRP1后软骨细胞自噬相关基因BECN1、LC3的表达降低。

Objective To investigate the impact of matrix metalloproteinase 13(MMP13)and low-density lipoprotein receptor-related protein 1(LRP1)on autophagy of articular chondrocytes in patients with Kashin-Beck disease(KBD).Methods Human articular cartilage samples obtained from 4 KBD patients and 4 control subjects were collected from Shaanxi Institute for Endemic Disease Prevention and Control,and the expression levels of MMP13 and LRP1 in cartilage tissue were determined using immunohistochemistry(IHC).Chondrocytes were extracted and cultured in vitro,the mRNA and protein expression levels of LRP1 and the autophagy related genes[Beclin 1(BECN1),microtubule associated protein 1 light chain 3(LC3)],cartilage injury related genes[MMP13,caspase-3(CASP3)],chondrocyte differentiation related genes[collagen typeⅡalpha 1 chain(COL2A1),and SRY-box transcription factor 9(SOX9)]were detected by real-time fluorescence quantitative PCR(qRT-PCR)and Western blot(WB),respectively.Chondrocytes from 3 KBD patients were extracted,and MMP13 gene silencing experiment was performed by RNA interference(RNAi)technology,the mRNA and protein expression levels of the above genes were detected by qRT-PCR and WB,respectively.In addition,the antagonist receptor associated protein(RAP)of LRP1 was used to block the LRP1 of human normal chondrocytes(C28/I2 cells),and qRT-PCR and WB were used to detect the mRNA and protein expression levels of LRP1,chondrocyte autophagy,differentiation and cartilage injury related genes,respectively.Results The IHC results showed that the expression levels of MMP13(1.67±0.21,0.59±0.15,0.51±0.12)in the surface,middle,and deep layers of cartilage tissue of KBD patients were significantly higher than those of control subjects(0.25±0.03,0.26±0.04,0.06±0.01),and the differences were statistically significant(t=-11.38,P<0.001;t=-3.82,-6.26,P=0.019,0.003).The expression levels of LRP1(0.10±0.02,0.03±0.01,0.17±0.03)were significantly lower than those of control subjects(1.63±0.40,0.44±0.12,0.34±0.08),and the differences were statistically significant(t=6.61,5.61,3.64,P=0.003,0.005,0.022).The mRNA and protein expression levels of MMP13,CASP3,SOX9 in chondrocytes of KBD patients were significantly higher than those of control subjects,and the differences were statistically significant(P<0.05).The mRNA expression levels of LRP1,LC3,COL2A1 were significantly lower than those of control subjects,and the differences were statistically significant(P<0.05).After silencing the MMP13 gene in chondrocytes of KBD patients,there were no significant differences in the mRNA and protein expression levels of LRP1,BECN1,LC3,CASP3,COL2A1,and SOX9(P>0.05).After blocking LRP1 with RAP,the protein expression levels of LRP1,BECN1,LC3,MMP13,COL2A1 and SOX9 in chondrocytes were significantly lower than those in control group(P<0.05).Conclusions There is no direct correlation between MMP13 and abnormal autophagy of articular chondrocytes in KBD patients.After blocking LRP1,the expression of the autophagy related genes BECN1 and LC3 in chondrocytes is decreased.