NF-κB抑制剂减低LPS致兔腰椎间盘髓核细胞锌指蛋白A20表达升高

NF-κB inhibitor reduces the expression of zinc finger protein A20 in rabbit lumbar intervertebral disc nucleus pulposus cells induced by lipopolysaccharide

目的观察脂多糖(LPS)刺激退变兔椎间盘髓核细胞前后,锌指蛋白A20(A20)、NF-κB及相关炎性因子的表达及其相互关系。方法获取正常和退变兔腰椎间盘髓核细胞,分为正常组、退变组、LPS刺激组和NF-κB抑制剂组,HE染色观察椎间盘髓核及纤维环的形态,免疫组化检测椎间盘A20、NF-κB p65蛋白及Ⅱ型胶原(COL-Ⅱ)的表达。Real-time PCR分别检测各组A20、IL-1β、TNF-α、NF-κB和COL-ⅡmRNA的表达。Western blot观察A20、p65和COL-Ⅱ的表达。ELISA检测细胞培养上清液中IL-1β、TNF-α的表达。结果退变组髓核细胞数量明显减少,髓核细胞聚集成团,COL-Ⅱ表达下降,A20和p65蛋白表达升高;与正常组相比,退变组A20、TNF-α、IL-1β和p65表达在mRNA及蛋白水平均明显升高,COL-Ⅱ表达降低(P<0.05);LPS刺激组中A20、TNF-α、IL-1β和p65表达较退变组更显著,COL-Ⅱ降低明显(P<0.05);NF-κB抑制剂组较LPS刺激组A20、TNF-α、IL-1β和p65表达回降,COL-Ⅱ表达回升(P<0.05)。结论髓核细胞炎性反应与兔椎间盘退变的发生和发展密切相关,其中A20可能发挥了重要作用。

Objective To observe the expression of zinc finger protein A20 （A20） , NF-κB and related inflammatory factors before and after lipopolysaeeharide （ LPS ） stimulates degeneration of rabbit intervertebral disc nucleus pulposus ceils. Methods The normal and degenerative nucleus pulposus cells were isolated and cultured, then divided into normal group, degenerative group, LPS stimulation group and NF-κB inhibition group. HE staining observe the morphological changes of nucleus pulposus and annulus fibrosus, immunohistochemistry was used to detect the expression of A20, NF-κB/p65 and COL- Ⅱ. Real-time PCR was employed to analyze the expression of A20, IL-1β ,TNF-α,NF-κB and COL-Ⅱ, Western blot was used to observe the A20 protein, p65 and C OL-Ⅱexpression in the four groups, and TNF-α, IL- 1β in cell supernatant was determined by ELISA. Results The number of nucleus pulposus cells significantly decreased, aggregation occured in the degenerative group. COL-Ⅱ was obvious lower and A20, p65 significantly higher than that in normal group by immunohistochemical staining. Compared with the normal group, A20, TNF-α, IL-1β,1365 expression was significantly increased and COL-Ⅱ decreased in the mRNA and protein levels in degenerative group. Above indexes changed more significant in LPS stimulation group than in degenerative group. The expression of A20, TNF-α, IL-1β, p65 in the NF-κB inhibitor group was lower than that in the LPS group, and the expression of type Ⅱ collagen increased （P 〈 0. 05 ）. Conclusions Intervertebral disc inflammatory response is closely related to the development of intervertebral disc degeneration, A20 may play an important role.