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

doi:10.3969/j.issn.1671-167X.2016.02.003

摘要/Abstract

摘要：

目的：研究关节液及关节软骨中硫化氢（hydrogen sulfide，H2S）及其合成酶的含量和变化，以及H2S对白介素1β（interlukin-1β，IL-1β）诱导的软骨细胞人基质金属蛋白酶-13（matrix metalloproteinase 13，MMP-13）表达的影响。方法：收集在北京大学第一医院行膝关节手术（关节镜手术或膝关节置换术）患者的关节液及废弃的关节软骨，用亚甲基蓝法检测关节液H2S的含量；从废弃的关节软骨中分离出相对正常的软骨细胞，用H2S分子探针方法检测软骨细胞中的内源性H2S，用免疫细胞化学方法检测软骨细胞中H2S合成酶胱硫醚-β-合成酶（cystathionine beta-synthase，CBS）、胱硫醚-γ-裂解酶（cystathionine gamma-lyase，CSE）、巯基丙酮酸硫转移酶（3-mercaptopyruvate sulfurtransferase，MPST）；用Western blot法检测H2S合成酶CBS、CSE、MPST在不同退变程度的软骨组织中的含量。培养相对正常的人软骨细胞至第三代，分为4组：（1）对照组：不加任何药物；（2）IL-β组：单加5 μg/L的IL-1β；（3）IL-1β+H2S组：提前0.5 h加200 μmol/L的NaHS，再加5 μg/L的IL-1β；（4）H2S组：单加200 μmol/L的NaHS。Western blot法检测各组细胞MMP-13蛋白、总核因子κB p65（nuclear factor κB p65，NF-κB p65）及磷酸化NF-κB p65蛋白的含量，Real-time PCR检测各组细胞MMP-13基因的转录情况。结果：退变膝关节的关节液中H2S含量为(14.3±3.3) μmol/L，Outerbridge分级3级软骨组织中CSE表达量显著高于Outerbridge分级1级软骨组织中的表达量（1.67±0.09 vs. 1.26±0.11，P＜0.01），而CBS与MPST的表达量差异无统计学意义（P＞0.05）。IL-1β组MMP-13的表达较正常对照组明显升高，且差异具有统计学意义（1.87±0.67 vs. 0.22±0.10，P＜0.05）, IL-1β+H2S组MMP-13的表达较IL-1β组明显降低，且差异具有统计学意义（0.55±0.11 vs. 1.87±0.67，P＜0.05），H2S组MMP-13的表达较正常对照组差异无统计学意义。 Real-time PCR方法测量了药物干预实验中各组细胞MMP-13基因转录情况，结果显示IL-1β组MMP-13基因转录较正常对照组明显升高，且差异具有统计学意义（31.40±0.31 vs. 1.00±0.00，P＜0.05）, IL-1β+H2S组MMP-13基因转录较IL-1β组明显降低，且差异具有统计学意义（24.41±1.28 vs. 31.40±0.31，P＜0.05）， H2S组MMP-13基因转录较正常对照组无显著性变化。IL-1β组总p65含量较正常对照组明显升高，且差异具有统计学意义（2.13±0.08 vs. 0.73±0.08，P＜0.05）, IL-1β+H2S组总p65含量较IL-1β组明显降低，且差异具有统计学意义（1.24±0.13 vs. 2.13±0.08，P＜0.05）， H2S组总p65含量较正常对照组无显著性变化；IL-1β组磷酸化p65含量较正常对照组明显升高，且差异具有统计学意义（1.30±0.13 vs. 0.19±0.04，P＜0.05）, IL-1β+H2S组磷酸化p65含量较IL-1β组明显降低，且差异具有统计学意义（0.92±0.26 vs. 1.30±0.13，P＜0.05），H2S组磷酸化p65含量较正常对照组无显著性变化。结论： H2S参与了软骨退变，起到部分抑制细胞外基质降解酶MMP-13的作用，从而实现对软骨细胞外基质的保护。

关键词: 硫化氢, 骨关节炎, 基质金属蛋白酶13, 软骨疾病

Abstract:

Objective： To investigate whether endogenous hydrogen sulfide (H2S) was involved in the pathogenesis of osteoarthritis (OA) and its underlying mechanism, to detect H2S and its synthases expression in knee cartilage in patients diagnosed with different severity of OA, and to explore the transcription and expression of gene MMP-13 in chondrocytes treated with IL-1β or H2S. Methods: Synovial fluids of the in-patients with different severity of OA hospitalized in Peking University First Hospital were collected for measurement of H2S 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 H2S molecular probes were used for detection of endogenous H2S generation in the chondrocytes. Immunocytochemistry was used to detect the localization of H2S synthases including cystathionine β-synthase (CBS), cystathionine-γ-lyase (CSE), and mercaptopyruvate 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-1β group, 5 μg/L of IL-1β was 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 H2S 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 H2S in the synovial fluid of degenerative knee was (14.3±3.3) μmol/L. Expressions of endogenous H2S 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+H2S group was significantly decreased than that in the IL-1β group (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-1β group 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+H2S group was significantly decreased than that in the IL-1β group (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-1β group 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-1β group (1.24±0.13 vs. 2.13±0.08, P＜0.05), and that in the H2S group had no significant difference compared with that in the normal control group. The phosphorylated NF-κB p65 in IL-1β group 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-1β group (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: H2S affected the cartilage degeneration by partly inhibiting the degradation of extracellular matrix.

Key words: Hydrogen sulfide, Osteoarthritis, Matrix metalloproteinase 13, Cartilage diseases

中图分类号:

R681.3

潘利平，曹永平，文立成，柴卫兵，杜军保，金红芳，柳佳，杨昕，孟志超，刘恒，崔云鹏，王瑞，吴浩，周星彤，李翔，李卓扬，塔拉提百克. 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): 194-201.

PAN Li-ping, CAO Yong-ping, WEN Li-cheng, CHAI Wei-bing, DU Jun-bao, JIN Hong-fang, LIU Jia, YANG Xin, MENG Zhi-chao, LIU Heng, CUI Yun-peng, WANG Rui, WU Hao, ZHOU Xing-tong, LI Xiang. Hydrogen sulfide in cartilage and its inhibitory effect on matrix metalloproteinase 13 expression in chondrocytes induced by interlukin-1β[J]. Journal of Peking University(Health Sciences), 2016, 48(2): 194-201.

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