山竹醇通过NF-κB信号通路抑制IL-1β诱导的髓核细胞炎症和细胞外基质降解

Garcinol inhibiting the inflammation and extracellular matrix degradation in IL-1βinduced nucleus pulposus cells through NF-κB signaling pathway

目的探讨山竹醇(Gar)对白介素-1β(IL-1β)诱导的髓核细胞炎症和细胞外基质降解的影响。方法提取大鼠髓核细胞,用含不同浓度山竹醇联合或者不联合IL-1β的新鲜培养基进行培养,根据细胞处理方式分为Control组、IL-1β组、IL-1β+Gar 2.5μM组、IL-1β+Gar 5μM组。采用MTT法检测大鼠髓核细胞活力,采用Western blot、定量RT-PCR和免疫荧光染色检测大鼠髓核细胞的基质代谢、炎症因子水平及相关信号通路蛋白。结果山竹醇在0~10μM剂量下对大鼠髓核细胞无细胞毒性;IL-1β可诱导髓核细胞活力降低(P<0.05),剂量为2.5~10μM的山竹醇能够改善IL-1β刺激下的髓核细胞活力(P<0.05)。IL-1β刺激后髓核细胞中MMP-3、MMP-9、MMP-13、ADAMTS-4和ADAMTS-5 mRNA表达水平显著提高(P<0.05),而山竹醇能抑制IL-1β刺激后的髓核细胞中MMP-3、MMP-9、MMP-13、ADAMTS-4和ADAMTS-5 mRNA表达水平(P<0.05)。IL-1β下调了髓核细胞中ColⅡ和Aggrecan mRNA表达水平(P<0.05),而山竹醇则显著改善了IL-1β刺激下髓核细胞中ColⅡ和Aggrecan mRNA表达水平(P<0.05)。IL-1β可显著提高髓核细胞中TNF-α、IL-6、IL-8、COX-2和iNOS mRNA表达水平(P<0.05),而山竹醇可抑制IL-1β诱导的TNF-α、IL-6、IL-8、COX-2和iNOS mRNA表达水平(P<0.05)。Western blot结果显示,IL-1β激活了髓核细胞中的NF-κB信号通路,而山竹醇逆转了IL-1β对NF-κB p65的活化作用(P<0.05),同时逆转了IL-1β引起的IκBα蛋白水平降低(P<0.05)。免疫荧光染色结果显示,IL-1β刺激下髓核细胞核中发生p65核转移,山竹醇显著抑制了p65向核的转移。结论山竹醇可通过抑制NF-κB信号通路,减轻IL-1β诱导的大鼠髓核细胞炎症反应,降低分解代谢水平,具有治疗椎间盘退行性变(IDD)的潜能。

Objective To investigate the effect of garcinol(Gar)on the inflammation and extracellular matrix degradation in nucleus pulposus cells induced by Interleukin-1β(IL-1β).Methods The rat nucleus pulposus cells were extracted and cultured with fresh media containing different concentrations of garcinol with or without IL-1β,and the nucleus pulposus cells were divided into the Control group,the IL-1βgroup,the IL-1β+Gar 2.5μM group and the IL-1β+Gar 5μM group according to cell processing methods.Cell viability of rat nucleus pulposus cells was detected by MTT method,and the matrix metabolism,levels of inflammatory factors and proteins of the related signaling pathway were detected by Western blot,quantitative RT-PCR and immunofluorescence staining.Results Garcinol had no cytotoxicity to rat nucleus pulposus cells at the concentration of 0μM to 10μM;IL-1βcould induce a decrease in the viability of nucleus pulposus cells(P<0.05),and garcinol at the concentration of 2.5μM to 10μM could improve the viability of nucleus pulposus cells simulated by IL-1β(P<0.05).The mRNA expression levels of MMP-3,MMP-9,MMP-13,ADAMTS-4,and ADAMTS-5 in nucleus pulposus cells were significantly increased after IL-1βstimulation(P<0.05),while garcinol could inhibit the mRNA expression levels of MMP-3,MMP-9,MMP-13,ADAMTS-4,and ADAMTS-5 after IL-1βstimulation(P<0.05).IL-1βdown-regulated the mRNA expression levels of ColⅡand Aggrecan in nucleus pulposus cells(P<0.05),while garcinol significantly improved the mRNA expression levels of ColⅡand Aggrecan in nucleus pulposus cells stimulated by IL-1β(P<0.05).IL-1βcould significantly increase the mRNA expression levels of TNF-α,IL-6,IL-8,COX-2 and iNOS in nucleus pulposus cells(P<0.05),while garcinol could inhibit the mRNA expression levels of TNF-α,IL-6,IL-8,COX-2 and iNOS stimulated by IL-1β(P<0.05).Western blot results showed that IL-1βactivated the NF-κB signaling pathway in nucleus pulposus cells,while garcinol reversed the activation function of IL-1βon NF-κB p65(P<0.05),and reversed the decrease of the level of IκBαprotein stimulated by IL-1βat the same time(P<0.05).Immunofluorescence staining results showed that IL-1βstimulated p65 nuclear transfer occurred in the nucleus pulposus cells,but garcinol significantly inhibited the transfer of p65 to the nucleus.Conclusion Garcinol can reduce the inflammatory response and the catabolism level in rat nucleus pulposus cells induced by IL-1βthrough NF-κB signaling pathway,which has the potential to treat intervertebral disc degeneration(IDD).