硫酸氨基葡萄糖对白细胞介素1β诱导人骨关节炎软骨细胞合成前列腺素E2的影响

Effects of glucosamine sulfate on IL-1β induced production of prostaglandin E2 by hunmn osteoarthritic chondrocytes

目的 探讨硫酸氨基葡萄糖（glueosamine sulfate，GS）对白细胞介素1β（IL-1β）诱导体外培养的人骨关节炎软骨细胞（human osteoarthritic chondroeyte，HOC）合成前列腺素E2（prostaglandin E2，PGE2）的影响及其作用机制。方法 取10例骨关节炎患者行全膝关节置换术的股骨髁和胫骨平台软骨标本，酶消化法获取软骨细胞进行体外培养。在原代或第二代的HOC培养液中加入IL-1β（5×10^-3μg／L）和不同浓度的GS（0．2、2和20mmol／L）作用24h（对照组不加IL-1β和GS），首先应用ELISA法检测GS对IL-1β诱导HOc合成PGE2的影响，然后采用RT—PCR法和Western蛋白印迹法分别检测其对IL-1β诱导HOC表达COX-2mRNA和蛋白的影响。结果 对照组HOC培养液中PGE2浓度为（109．46±17．48）pg／ml，IL-1β刺激后软骨细胞合成PGE2为（3607．22±239．34）pg／ml，其差异有统计学意义（P〈0．01）。0．2、2和20mmol／L GS以浓度依赖的方式抑制IL-1β诱导HOC合成PGE2（P〈0．05），其浓度分别为（2594．23±185．18）、（1057．53±126．81）和（565．43±52．79）pg／ml；单独使用20mmol／L GS时软骨细胞合成PGE2为（169．94±30．03）pg／ml，与对照差异无统计学意义（P〉0．05）。IL-1β刺激后，HOC表达COX-2mRNA和蛋白上调（P〈0．01），GS能以浓度依赖的方式抑制IL-1β诱导HOC表达COX-2mRNA和蛋白上调（P〈0．01）。结论 GS抑制HOC在IL-1β诱导下分泌炎性介质PGE2，其机制是下调调控它们表达的COX-2 mRNA和蛋白的表达；这可能是GS既能缓解症状和又能保护软骨的机制之一。

Objective To study the effects of glucosamine sulfate on IL-1β induced activation of human osteoarthritic chondrocytes in vitro and to elucidate the possible mechanism of action of the drug. Methods Chondrocytes were harvested from 10 osteoarthritis patients undergoing TKR operation, and cultured in monolayer cell. Human recombinant IL-1β（5 × 10^-3 μg/L）and GS in different concentrations （0. 2 mmol/L,2 mmol/L,20 mmol/L）were administered into culture medium of the first or second passage cells. PGE2 was detected by ELISA , COX-2 mRNA expression and protein secretion were measured by RT-PCR and Western-blot respectively. Results The concentration of PGF2 in control was（109. 46±17.48） pg/ml. After stimulation of HOC with IL-1β for 24 hours, PGE2 level raised to（3607. 225±239. 34）pg/ml （P〈0.01）. After pretreatment with 0.2 mmol/L,2 mmol/L and 20 mmol/L GS, PGE2 concentration increased to（2594.23 ± 185.18） pg/ml, （1057.53 ± 126. 81） pg/ml, （565.43 ± 52. 79） pg/ml respectively, showing a dose-dependent inhibiting effect（P〉0. 05）. However, GS alone could .not modulate the production of PGE2 （P〉0.05）. Stimulation of HOC with IL-1β also resulted in rise of COX-2 mRNA and protein（P〈0.01） while pretreatment with GS inhibited IL-1β induced COX-2 mRNA expression（P〈0. 01）and COX-2 protein secretion（P〈 0. 01 ）. Conclusions GS affords protection against IL-1β induced inflammatory mediators PGE2 production in HOC by regulating the expression of their respective enzymes. Our result reveals the possible mechanism of GS as a symptom- and structure-modifying drug in the treatment of OA.