摘要
目的:探讨骨化三醇[1,25(OH)_(2)D_(3)]对创伤性骨关节炎(PTOA)大鼠膝关节软骨的保护作用及其分子机制。方法:构建PTOA大鼠模型。将20只SD大鼠随机分为对照组、假手术组、PTOA组和1,25(OH)_(2)D_(3)组,每组5只。采用免疫组织化学(IHC)染色法检测膝关节软骨IL-10受体(IL-10R)和基质金属蛋白酶-13(MMP-13)的表达,番红O染色法测定大鼠胫骨关节头软骨的损伤程度。分离大鼠骨髓干细胞(BMSCs),将细胞分为未分化组、分化组、1,25(OH)_(2)D_(3)干预组和1,25(OH)_(2)D_(3)+IL-10R抗体组。采用荧光定量PCR(RT-q PCR)法检测细胞中MMP-13和胶原蛋白10a1(Col10a1)m RNA表达,western blotting法检测磷酸化(p)-JAK、JAK、转化生长因子-β1(TGF-β1)、SOX9、Smad2和Runx2蛋白表达,TUNEL染色法检测细胞凋亡率。结果:IHC染色显示,与对照组相比,PTOA组IL-10R表达水平降低,MMP-13表达水平升高(均P<0.05);与PTOA组相比,1,25(OH)_(2)D_(3)组IL-10R表达水平升高,MMP-13表达水平降低(均P<0.05)。番红O染色结果显示,PTOA组软骨层厚度低于对照组(P<0.05),1,25(OH)_(2)D_(3)组软骨层厚度高于PTOA组(均P<0.05)。与分化组相比,1,25(OH)_(2)D_(3)干预组细胞MMP-13m RNA相对表达水平降低,Col10a1 m RNA相对表达水平及p-JAK、TGF-β1、SOX9、Smad2和Runx2蛋白表达水平升高(均P<0.05),TUNEL阳性细胞率降低(P<0.05)。与1,25(OH)_(2)D_(3)干预组比较,1,25(OH)_(2)D_(3)+IL-10R抗体组细胞部分逆转了上述指标(P<0.05)。结论:1,25(OH)_(2)D_(3)可能通过上调膝关节软骨IL-10R表达,抑制软骨细胞凋亡,从而发挥保护PTOA大鼠膝关节软骨的作用。
Objective:To investigate the protective effect and molecular mechanism of calcitriol[1,25(OH)_(2)D_(3)]on knee articular cartilage in rats with post-traumatic osteoarthritis(PTOA).Methods:The PTOA rat model was established.Twenty SD rats were randomly divided into control group,sham operation group,PTOA group and 1,25(OH)_(2)D_(3) group,with 5 rats in each group.Immunohistochemistry(IHC)was used to detect the expression of IL-10 receptor(IL-10R)and matrix metalloproteinase-13(MMP-13)in knee articular cartilage.Safranin O staining method was used to determine the degree of knee articular cartilage injury.Rat bone marrow stem cells(BMSCs)were isolated and divided into undifferentiation group,differentiation group,1,25(OH)_(2)D_(3) intervention group and 1,25(OH)_(2)D_(3)+IL-10R antibody group.The expression of MMP-13 and collagen 10a1(Col10a1)mRNA in cells were detected by real-time fluorescence quantitative PCR(RT-qPCR).Western blotting was used to detect the expression of phospho(p)-JAK,JAK,transforming growth factor-β1(TGF-β1),SOX9,Smad2 and Runx2 and the apoptosis rate was detected by TUNEL assay.Results:IHC staining results showed that compared with the control group,the expression level of IL-10R decreased and the expression level of MMP-13 increased in the PTOA group(both P<0.05).Compared with the PTOA group,the expression level of IL-10R increased and the expression level MMP-13 decreased in the 1,25(OH)_(2)D_(3) group(both P<0.05).Safranin O staining results showed that the thickness of cartilage layer in the PTOA group was lower than that in the control group (P<0.05), and the thickness of cartilage layer in the 1,25(OH)_(2)D_(3) group was higher than that in the PTOAgroup (both P<0.05). Compared with the differentiation group, the relative expression level of MMP-13 mRNAin the 1,25(OH)_(2)D_(3) intervention group decreased, and the relative expression level of Col10a1 mRNA and the proteinexpression levels of p-JAK, TGF-β1, SOX9, Smad2 and Runx2 increased (all P<0.05). The rate of TUNELpositive cells decreased (P<0.05). Compared with the 1,25(OH)_(2)D_(3) intervention group, 1,25(OH)_(2)D_(3)+IL-10R antibodygroup cells partially reversed the above indicators (P<0.05). Conclusion: The 1,25(OH)_(2)D_(3) may play aprotective role in knee articular cartilage of rats with PTOA by up-regulating the expression of IL-10R in knee articularcartilage and inhibiting chondrocyte apoptosis.
作者
吕青
翟生
黄涛
Lyu Qing;Zhai Sheng;Huang Tao(Orthopedic Center of the Fifth Affiliated Hospital of Xinjiang Medical University,Urumqi 830011,China)
出处
《广西医科大学学报》
CAS
2023年第10期1692-1698,共7页
Journal of Guangxi Medical University
基金
新疆维吾尔自治区自然科学基金资助项目(No.2021D01C427)。
