摘要
目的探讨瘦素(leptin)经JAK2/STAT3通路对核心结合因子β亚基(core-binding factorβ-subunit,CBFβ)的调控作用及其促进软骨细胞凋亡的分子机制。方法选取5例因膝关节骨关节炎行全膝关节置换的患者(骨关节炎组)及5例外伤截肢患者(截肢组),术中取膝关节软骨。采用Western-blot技术检测leptin、CBFβ、基质金属蛋白酶1(matrix metalloproteinases,MMP1)和MMP13的蛋白表达。通过流式细胞术筛选确定leptin的最佳作用时间和浓度。将软骨细胞按处理方式分为:对照组(未接受leptin处理的软骨细胞)、leptin组(接受50 ng/ml leptin处理)、阴性leptin组(转染无意义序列作为对照)以及leptin+shCBFβ组(通过转染shCBFβ来抑制CBFβ的表达),检测四组细胞凋亡及MMP1、MMP13的表达水平。将软骨细胞按处理方式分为:对照组(未处理)、leptin组(予50 ng/ml leptin刺激48 h)、AG490组(JAK2/STAT3抑制剂AG490处理)及leptin+AG490组(AG490预处理2 h,50 ng/ml leptin刺激48 h),检测四组CBFβ、MMP1、MMP13的蛋白表达及细胞凋亡率。结果截肢组软骨leptin相对表达量为0.66±0.06、CBFβ为0.69±0.06、MMP1为0.74±0.05、MMP13为0.41±0.03,低于骨关节炎组的1.04±0.10、1.06±0.09、0.95±0.04、0.99±0.09,差异均有统计学意义(P<0.05)。Leptin的最佳作用时间和浓度分别为48 h和50 ng/ml。对照组、leptin组、阴性leptin组及leptin+shCBFβ组MMP1和MMP13表达水平的差异均有统计学意义(P<0.05);其中leptin组较对照组高、leptin+shCBFβ组较leptin组低(P<0.05);四组软骨细胞凋亡率分别为4.55%±1.30%、22.52%±2.03%、22.03%±2.01%、5.15%±0.915%,差异有统计学意义(F=114.066,P<0.001),其中leptin组较对照组高、leptin+shCBFβ组较leptin组低(P<0.05)。对照组、leptin组、AG490组及leptin+AG490组CBFβ、MMP1和MMP13表达水平的差异有统计学意义(P<0.05),其中leptin组较对照组高、leptin+AG490组较leptin组低(P<0.05)。四组软骨细胞凋亡率分别为5.19%±0.94%、31.52%±2.63%、5.51%±1.41%、10.47%±0.85%,差异有统计学意义(F=117.104,P<0.001),其中leptin组较对照组高、leptin+AG490组较leptin组低(P<0.05)。结论Leptin通过JAK2/STAT3通路促进CBFβ表达,从而导致关节软骨细胞的凋亡和细胞外基质的降解。
Objective To investigate the regulatory effect of leptin via the JAK2/STAT3 pathway on the core-binding factor β-subunit(CBFβ)and its molecular mechanism in promoting chondrocyte apoptosis.Methods A total of five patients undergoing total knee arthroplasty due to knee osteoarthritis(OA group)and five patients undergoing amputation due to trauma(amputation group)were enrolled,and knee cartilage samples were obtained intraoperatively.Western blotting was used to detect the protein expression levels of leptin,CBFβ,matrix metalloproteinase-1(MMP1),and MMP13.Flow cytometry was performed to determine the optimal treatment duration and concentration of leptin.Chondrocytes were divided into the following groups based on treatment conditions:control group(untreated chondrocytes),leptin group(chondrocytes treated with 50 ng/ml leptin),negative leptin group(chondrocytes transfected with a non-targeting sequence as a control),and leptin+shCBFβgroup(chondrocytes transfected with shCBFβto inhibit CBFβexpression).Apoptosis and the expression levels of MMP1 and MMP13 were analyzed in the four groups.Additionally,chondrocytes were categorized into the following groups for further analysis:control group(untreated cells),leptin group(cells stimulated with 50 ng/ml leptin for 48 h),AG490 group(cells treated with the JAK2/STAT3 inhibitor AG490),and leptin+AG490 group(cells pretreated with AG490 for 2 h followed by 50 ng/ml leptin stimulation for 48 h).The protein expression levels of CBFβ,MMP1,and MMP13,as well as the apoptosis rate,were examined in the four groups.Results The relative expression levels of leptin,CBFβ,MMP1,and MMP13 in the amputation group were 0.66±0.06,0.69±0.06,0.74±0.05,and 0.41±0.03,respectively,which were significantly lower than those in the OA group(1.04±0.10,1.06±0.09,0.95±0.04,and 0.99±0.09,respectively)(P<0.05).The optimal treatment duration and concentration of leptin were determined to be 48 h and 50 ng/ml,respectively.The expression levels of MMP1 and MMP13 significantly differed among the control,leptin,negative leptin,and leptin+shCBFβgroups(P<0.05).Specifically,the leptin group showed higher expression levels compared to the control group,while the leptin+shCBFβgroup exhibited lower expression levels than the leptin group(P<0.05).The apoptosis rates of chondrocytes in the four groups were 4.55%±1.30%,22.52%±2.03%,22.03%±2.01%,and 5.15%±0.91%,respectively,with significant differences(F=114.066,P<0.001).The apoptosis rate in the leptin group was significantly higher than that in the control group,while the leptin+shCBFβgroup exhibited a significantly lower apoptosis rate than the leptin group(P<0.05).Similarly,significant differences were observed in the expression levels of CBFβ,MMP1,and MMP13 among the control,leptin,AG490,and leptin+AG490 groups(P<0.05).The expression levels in the leptin group were higher than those in the control group,while the leptin+AG490 group exhibited lower expression levels compared to the leptin group(P<0.05).The apoptosis rates of chondrocytes in the control,leptin,AG490,and leptin+AG490 groups were 5.19±0.94%,31.52±2.63%,5.51±1.41%,and 10.47±0.85%,respectively,with significant differences(F=117.104,P<0.001).The apoptosis rate in the leptin group was significantly higher than that in the control group,while the leptin+AG490 group exhibited a significantly lower apoptosis rate than the leptin group(P<0.05).Conclusion Leptin promotes CBFβexpression via the JAK2/STAT3 pathway,leading to chondrocyte apoptosis and extracellular matrix degradation.
作者
杨嘉飞
周竹君
李光第
黄远
张沕
董亮宏
Yang Jiafei;Zhou Zhujun;Li Guangdi;Huang Yuan;Zhang Mi;Dong Lianghong(Department of Pediatric Surgery,Affiliated Hospital of Guizhou Medical University,Guiyang 550001,China;Department of Respiratory Medicine,Xiuwen People's Hospital,Guiyang 550001,China;Department of Orthopaedics,Affiliated Hospital of Guizhou Medical University,Guiyang 550001,China;Department of Orthopaedics,Tongren People's Hospital,Tongren 554300,China;Department of Orthopaedics,Qiandongnan People's Hospital,Kaili 556000,China;Department of Orthopedics,Qingzhen People's Hospital,Guiyang 550001,China)
出处
《中华骨科杂志》
北大核心
2025年第7期436-445,共10页
Chinese Journal of Orthopaedics
基金
国家自然科学基金地区项目(82160432)
贵州省卫健委科学技术基金(gzwkj2024-036)
贵州省科技厅基础研究(自然科学)项目[黔科合基础ZK(2022)一般427]。
关键词
骨关节炎
膝
瘦素
核心结合因子β亚基
信号传导
Osteoarthritis,knee
Leptin
Core binding factor beta subunit
Signal transduction
