北京大学学报（医学版）2016，Vol.48Issue(2)：194-202,9.DOI:10.3969/j.issn.1671-167X.2016.02.003
软骨中硫化氢含量及其对白介素1β诱导的软骨细胞基质金属蛋白酶13表达的抑制作用
Hydrogen sulfide in cartilage and its inhibitory effect on matrix metalloproteinase 13 expression in chondrocytes induced by interlukin-1β
潘利平 1刘恒 1崔云鹏 1王瑞 1吴浩 1周星彤 1李翔 1李卓扬 1塔拉提百克•买买提居马 1曹永平 1立成 1柴卫兵 1杜军保 2金红芳 2柳佳 2杨昕 1孟志超1
作者信息
- 1. 北京大学第一医院骨科，北京 100034
- 2. 北京大学第一医院儿科，北京 100034
- 折叠
摘要
Abstract
Objective:To investigate whether endogenous hydrogen sulfide (H2 S)was involved in the pathogenesis of osteoarthritis (OA)and its underlying mechanism,to detect H2 S and its synthases ex-pression in knee cartilage in patients diagnosed with different severity of OA,and to explore the transcrip-tion and expression of gene MMP-13 in chondrocytes treated with IL-1βor H2S.Methods:Synovial fluids of the in-patients with different severity of OA hospitalized in Peking University First Hospital were collected for measurement of H2 S content using methylene blue assay.Articular cartilages of the patients who underwent knee arthroplasty were collected for the cell culture of relatively normal chondrocytes.The chondrocytes were cultured to the P3 generation and H2 S molecular probes were used for detection of endogenous H2 S generation in the chondrocytes.Immunocytochemistry was used to detect the localization of H2 S synthases including cystathionine β-synthase (CBS),cystathionine-γ-lyase (CSE),and mercap-topyruvate sulfurtransferase (MPST)in OA chondrocytes.Western blot was used to quantify the protein expressions of CSE,MPST,and CBS in cartilage tissues of the patients who were diagnosed with OA and underwent knee arthroplasty.The relatively normal human chondrocytes were cultured to passage 3 and then divided into 4 groups for different treatments:(1 )the normal control group,no reagent was added;(2)the IL-1βgroup,5 μg/L of IL-1βwas added;(3)the IL-1β+H2S group,200 μmol/L of NaHS was added 30 min before adding 5 μg/L of IL-1β;(4)the H2 S group,200 μmol/L of NaHS was added. The transcription and expression of gene MMP-13 in chondrocytes of each group were determined with Real-time PCR and Western blot,respectively.And the total NF-κB p65 and phosphorylated NF-κB p65 in chondrocytes were detected with Western blot.Results:The content of H2 S in the synovial fluid of degenerative knee was (14.3 ±3.3)μmol/L.Expressions of endogenous H2 S and its synthases including CBS,CSE and MPST were present in the cytoplasm of chondrocytes.CSE protein expression in Grade 3 (defined by outerbridge grading)cartilage tissues was significantly increased as compared with that of Grade 1 cartilage tissues (1.67 ±0.09 vs.1.26 ±0.11,P<0.05).However,no significant difference of CBS or MPST expression among the different groups was observed.The expression of MMP-13 protein in the IL-1βgroup was significantly higher than that in the normal chondrocytes (1 .87 ±0.67 vs.0.22 ± 0.10,P<0.05 ),and that in the IL-1β+H2 S group was significantly decreased than that in the IL-1βgroup (0.55 ±0.11 vs.1.87 ±0.67,P<0.05),and that in the H2S group had no significant difference compared with that in the normal control group.The transcription of MMP-13 protein in the IL-1βgroup was significantly higher than that in the normal chondrocytes (31.40 ±0.31 vs.1.00 ±0.00,P<0.05), and that in the IL-1β+H2 S group was significantly decreased than that in the IL-1βgroup (24.41 ± 1.28 vs.31.40 ±0.31,P<0.05),and that in the H2S group had no significant difference compared with that in the normal control group.The total NF-κB p65 in the IL-1βgroup was significantly higher than that in the normal chondrocytes (2.13 ±0.08 vs.0.73 ±0.08,P<0.05),and that in the IL-1β+H2S group was significantly decreased than that in the IL-1βgroup (1 .24 ±0.13 vs.2.13 ±0.08,P<0.05 ),and that in the H2 S group had no significant difference compared with that in the normal control group.The phosphorylated NF-κB p65 in IL-1βgroup was significantly higher than that in the normal chondrocytes (1.30 ±0.13 vs.0.19 ±0.04,P<0.05),and that in IL-1β+H2S group was significantly decreased than that in the IL-1βgroup (0.92 ±0.26 vs.1.30 ±0.13,P<0.05),and that in the H2S group had no significant difference compared with that in the normal control group.Conclusion:H2 S affected the cartilage degeneration by partly inhibiting the degradation of extracellular matrix.关键词
硫化氢/骨关节炎/基质金属蛋白酶13/软骨疾病Key words
Hydrogen sulfide/Osteoarthritis/Matrix metalloproteinase 13/Cartilage diseases分类
医药卫生引用本文复制引用
潘利平,刘恒,崔云鹏,王瑞,吴浩,周星彤,李翔,李卓扬,塔拉提百克•买买提居马,曹永平,立成,柴卫兵,杜军保,金红芳,柳佳,杨昕,孟志超..软骨中硫化氢含量及其对白介素1β诱导的软骨细胞基质金属蛋白酶13表达的抑制作用[J].北京大学学报（医学版）,2016,48(2):194-202,9.
