I型组蛋白去乙酰化酶抑制剂在阻断软骨退化中的作用

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摘要目的：探讨I型组蛋白去乙酰化酶（HDAC）抑制剂阻断软骨退化的作用及其可能机制。方法：通过内侧半月板不稳术（DMM）构建SD大鼠关节炎（OA）模型，选取广谱性HDAC抑制剂曲古抑菌素A（TSA）进行体内干预，采用关节软骨番红O染色及关节OA评分法观察TSA对软骨退化的影响。体外培养软骨肉瘤细胞SW-1353，选取不同浓度的TSA及选择性HDAC抑制剂丙戊酸（VPA）和MS-275进行干预，采用Western blot检测核心组蛋白（H3、H4）和α-微管蛋白去乙酰化水平，验证各HDAC抑制剂的活性。体外培养人源性关节软骨细胞（HACs）及SW-1353细胞，选取以上HDAC抑制剂并结合基质金属蛋白酶（MMP）诱导剂白细胞介素-1α（IL-1α）干预HACs；采用siRNA特异性干扰I型HDAC各亚型的表达，结合IL-1α干预SW-1353；采用RT-PCR检测2种细胞中MMP1及MMP13的表达，分析各亚型HDAC抑制对MMP表达的影响。利用外植体软骨退化（PNC外植体）试验，选取以上HDAC抑制剂及制瘤素M进行干预，检测糖胺聚糖和胶原蛋白，体外观察各HDAC抑制剂对软骨退化的影响。结果：TSA明显减轻OA模型SD大鼠胫骨内侧平台及股骨内侧髁的软骨退化程度（P=0.015）。TSA、VPA及MS-275可显著提高核心组蛋白（H3、H4）和α-微管蛋白乙酰化水平（P＜0.05），并呈浓度依赖性，即TSA、VPA及MS-275干预浓度越高，组蛋白乙酰化水平越高，HDAC抑制剂的活性越高。TSA、VPA及MS-275可显著抑制HACs细胞中IL-1α诱导的MMP1及MMP13的表达（P＜0.001），并亦呈浓度依赖性。I型HDAC（HDAC-1、HDAC-2、HDAC-3及HDAC-8）亚型被特异性干扰后，SW-1353细胞中IL-1α诱导的MMP13的表达明显减少（P＜0.001）。体外实验证实TSA、VPA及MS-275可显著抑制软骨中制瘤素M诱导的糖胺聚糖和胶原蛋白水平（P＜0.05），并呈浓度依赖性。结论：I型HDACs被抑制剂抑制或特异性消耗均能抑制软骨细胞MMP1及MMP13的表达，抑制PNC外植体糖胺聚糖和胶原蛋白水平，其可能是通过抑制MMP、糖胺聚糖和胶原蛋白的表达而发挥对软骨退化的保护作用。

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关键词 ： I型组蛋白去乙酰化酶抑制剂, 关节炎, 软骨退化, 曲古抑菌素A

Abstract：Objective: To investigate the protective effect and possible mechanism of I type HDAC inhibitor in cartilage degradation. Methods: An unstable dementabilization of the medial meniscus (DMM) model was used to assess the inhibition of HDAC by trichostatin A (TSA) in vivo. Cytokines, TSA, MS-275, valproic acid and siRNA were applied to human articular chondrocytes (HACs) and sw-1353 hondrosarcocma cells. And RT-PCR was used to confirm how they influence the expression of metalloproteinase. The activity of HDAC inhibitor was tested by western blot. The absorption of cartilage in vitro was examined by a pig’s nasal cartilage explant test. Results: Systemic administration of TSA in the developmental process (DMM) can protect cartilage. TSA, valproic acid, and MS-275 in human articular chondrocytes (HACs) inhibited the expression of MMP1 and MMP13 which was induced by cytokine, and after knocking out each type I HDAC, MMP13 expression which was induced by interleukin-1 decreased. Conclusion: MS-275 inhibition to type I HDAC (HDAC-1, HDAC-2, HDAC-3) and HDACs specificity consumption can inhibit the expression of metalloproteinase induced by cytokines in chondrocytes and PNC explants, thereby inhibiting cartilage absorption. These observations indicate that specific type I HDACs inhibitors can block cartilage degradation to some extent.

Key words： deacetylase inhibitor osteoarthritis cartilage degeneration trichostatin A

收稿日期: 2018-07-30

基金资助:江西省科技支撑计划项目（20172BBG70118）。

通讯作者: 顾岳全，副主任医师，Email：117820182@qq.com。

作者简介: 顾霄鹏（1987-），男，浙江舟山人，主治医师，硕士。

链接本文:

https://xb.wmu.edu.cn/CN/ 或 https://xb.wmu.edu.cn/CN/Y2018/V48/I12/881

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