补骨脂素对大鼠腰椎间盘软骨细胞炎性退变的影响

Effect of Psoralens on Cartilage Degeneration in Lumbar Intervertebral Disc of Rats

目的探讨补骨脂素延缓椎间盘退变的内在机制。方法体外分离1月龄雌性SD大鼠腰椎间盘软骨板,采用组织块法培养,通过HE染色、甲苯胺蓝染色和免疫荧光进行细胞辨别、鉴定。通过细胞增殖实验和RT-PCR法进行补骨脂素最佳浓度筛选。取第3代生长状况良好的椎间盘软骨细胞,以每孔1×105的浓度将细胞置于6孔板内,分为空白对照组、白细胞介素-1β(IL-1β)诱导组(10ng/ml)、补骨脂素组(IL-1β10ng/ml+补骨脂素最佳浓度),每组3孔,检测各组Ⅱ型胶原基因(Col2a1)、聚集蛋白聚糖(Aggrecan)、血小板反应蛋白解整合素金属肽酶5(ADAMTS-5)、IL-1β和环氧化酶-2(COX-2)mRNA的表达。结果补骨脂素浓度在100、150、200、400μM时,细胞活性较浓度为0时明显减弱(P<0.01)。补骨脂素在12.5、25μM时较浓度为0时能上调Col2a1 mRNA表达(P<0.05或P<0.01)。补骨脂素组Aggrecan mRNA高于空白对照组(P<0.01);与IL-1β诱导组比较,补骨脂素组Col2a1 mRNA升高,ADAMTS-5 mRNA降低(P<0.01)。结论补骨脂素可以一定程度缓解IL-1β诱导的椎间盘软骨细胞的退变进程,并对IL-1β炎性信号通路的相关因子产生影响。

Objective To observe the effect of Psoralens on cartilage degeneration in lumbar intervertebral disc of rats induced by interleukin-1β（IL-1β） and research its internal mechanism of delaying disc degeneration Methods The cartilages of lumbar intervertebral disc in 1-month-old female SD rats were isolated in vitro. The cells were cultured by tissue explant method and the cells were identified by HE staining, toluidine blue staining and immu- nofluorescence. The optimal concentration of Psoralens was screened by cell proliferation assay and RT-PCR method. The 3rd generation cartilage cells were selected and placed in a 6-well plate at a concentration of 1 × 10^5/well. The cells were divided into the blank control group, IL-1 [5-induced group （10 ng/ml IL-1 β） and Psoralens group （10 ng/ ml IL-1 β, added with the optimal concentration of Psoralens）, with 3 wells in each group. The expressions of type II collagen gene （Co12al）, Aggrecan, A Disintegrin and Metalloproteinase with Thrombospondin Motifs 5 （ADAMTS- 5）, IL-1 β and cyclooxygenase-2 （COX-2） mRNA were detected. Results Comparing with concentration of zero, the cell activity of Psoralens was significantly weakened at concentrations of 100, 150, 200 and 400 μM （ P 〈0.01 ）. Comparing with concentration of zero, the expression of Col2al mRNA was significantly increased at concentrations of 12. 5 and 25 μM （P 〈0. 05 or P 〈0. 01）. The expression higher than that in the blank control group （ P 〈 0. 01 ）. of Aggrecan mRNA in the Psoralens group was significantly Comparing with the IL-l-induced group, the expression of Col2al mRNA was significantly increased but the expression of ADAMTS-5 mRNA was significantly decreased in the Psoralens group （P 〈0. 01） Conclusion Psoralens can alleviate IL-1 β-induced cartilage degeneration of inter- vertebral disc and affect the related factors of IL-1 β signaling pathway