姜黄素对脂多糖诱导炎症致软骨细胞凋亡及增殖能力的影响

Effects of curcumin on apoptosis and proliferation of inflammatory chondrocytes induced by lipopolysaccharide

背景:在骨关节炎的相关研究中,姜黄素被证实具有抗炎抗氧化及抗凋亡的作用,但相关机制的研究还不够深入。目的:探讨姜黄素对脂多糖诱导的软骨细胞凋亡、增殖的影响,以及潜在的作用机制,为治疗骨关节炎疾病提供新思路。方法:取4周龄SD大鼠,进行软骨细胞的体外分离培养,甲苯胺蓝染色进行细胞鉴定。采用脂多糖诱导法建立体外软骨细胞炎症模型。将软骨细胞分为5组:对照组(普通培养基);模型组(含5μg/L脂多糖培养基);低剂量姜黄素组(含5μg/L脂多糖+5μmol/L姜黄素培养基);中剂量姜黄素组(含5μg/L脂多糖+10μmol/L姜黄素培养基);高剂量姜黄素组(含5μg/L脂多糖+20μmol/L姜黄素培养基)。培养24h后,ELISA法检测各组细胞上清液中白细胞介素1β和转化生长因子α的水平;流式细胞仪检测细胞凋亡状况;MTT法进行细胞增殖检测;Western blot法检测自噬相关蛋白及ERK1/2的表达变化。结果与结论:(1)经脂多糖诱导处理的软骨细胞上清液中白细胞介素1β和转化生长因子α的水平均显著高于对照组(P <0.01),各姜黄素组细胞上清液中白细胞介素1β和转化生长因子α的水平较模型组显著降低(P <0.05,P <0.01),且呈剂量依赖性;(2)经24 h培养后,模型组细胞凋亡率显著高于对照组(P <0.01),与模型组相比,各姜黄素处理的细胞凋亡率有所降低,且20μmol/L姜黄素处理的细胞凋亡率降低最为显著(P<0.01);(3)MTT法检测结果显示,与对照组相比,模型组细胞增殖能力显著降低(P<0.01);与模型组相比,经不同浓度的姜黄素处理细胞后,细胞的增殖能力得以恢复,且20μmol/L姜黄素处理的细胞恢复效果最好;(4)Western blot法检测结果显示,与模型组相比,各姜黄素组细胞自噬能力明显增强,自噬标志物LC3-II及相关蛋白Beclin-1表达量均显著升高(P <0.05,P <0.01),且p-ERK1/2蛋白表达量显著升高(P <0.05);(5)结果提示,20μmol/L姜黄素能显著缓解脂多糖诱导的软骨细胞炎症反应,抑制软骨细胞凋亡,并对脂多糖诱导的炎症软骨细胞增殖抑制具有一定的拮抗作用,这可能与姜黄素通过ERK1/2途径激活的自噬分子调控紧密相关。

BACKGROUND:Curcumin has been shown to play antioxidative,anti-inflammatory and anti-apoptotic roles in osteoarthritis,but the underlying mechanisms remain unclear.OBJECTIVE:To investigate the effects of curcumin on apoptosis and proliferation of chondrocytes induced by lipopolysaccharide,and to explore the potential mechanisms,so as to provide new ideas for the treatment of osteoarthritis.METHODS:Chondrocytes were isolated from the 4-week-old Sprague-Dawley rats,cultured,and identified by toluidine blue staining.The chondrocyte inflammation model in vitro was established by lipopolysaccharide induction.The cells were divided into five groups:control group(common medium);model group(medium with 5μg/L lipopolysaccharide);low-dose group(medium with 5μg/L lipopolysaccharide+5μmol/L curcumin);medium-dose group(medium with 5μg/L lipopolysaccharide+10μmol/L curcumin);high-dose group(medium with 5μg/L lipopolysaccharide+20μmol/L curcumin).After 24 hours,the expression levels of interleukin 1βand transforming growth factorαin the cellular supernatant were detected by ELISA method.The apoptotic cells were detected by flow cytometry.Cell proliferation was tested by MTT assay.The expression levels of autophagy-related proteins and extracellular regulated protein kinases 1/2 were determined by western blot assay.RESULTS AND CONCLUSION:The levels of interleukin 1βand transforming growth factorαin the supernatant of chondrocytes induced by lipopolysaccharide were significantly higher than those in the control group(P<0.01).The levels of interleukin 1βand transforming growth factorαin the supernatant treated with curcumin were lower than those in the model group,which was on a dose-dependent manner(P<0.05,P<0.01).After 24 hours of treatment,apoptotic rate of cells in the model group was significantly higher than that in the control group(P<0.01).Compared with the model group,the cell apoptotic rate in each curcumin group was decreased,especially the high-dose group(P<0.01).MTT assay showed that the cell proliferation ability in the model group was significantly lower than that in the control group(P<0.01).Compared with the model group,the cell proliferation ability in each curcumin group was restored,and the effect was the best in the high-dose group.The results of western blot showed that the autophagic ability and the levels of autophagy marker(LC3-II)and autophagy related protein(Beclin-1)were significantly increased in each curcumin group compared with the model group(P<0.05,P<0.01),and the expression level of p-ERK1/2 protein was significantly increased(P<0.05).Our findings suggest that 20μmol/L curcumin cannot only inhibit apoptosis and relieve inflammatory of chondrocytes induced by lipopolysaccharide,but also has a certain antagonistic effect on lipopolysaccharideinduced inhibition of inflammatory chondrocyte proliferation,which may be via regulating the ERK1/2 pathway-activated autophagy.