川芎嗪对椎间盘退变髓核细胞的作用机制

The Effect of Ligustrazine on Degeneration of Nucleus Pulposus Cells in Intervertebral Disc and Its Possible Mechanisms

目的探讨川芎嗪对椎间盘退变髓核细胞的作用机制。方法选择成年雄性SD大鼠10只,提取并培养椎间盘髓核细胞,随机分为对照组、诱导组、川芎嗪低浓度组、川芎嗪中浓度组、川芎嗪高浓度组,分别加入0. 9%氯化钠注射液、10μg/L的白细胞介素(interleukin,IL)-1β、10μg/L的IL-1β+10μmol/L的川芎嗪、10μg/L的IL-1β+20μmol/L的川芎嗪、10μg/L的IL-1β+50μmol/L的川芎嗪。采用CCK-8检测髓核细胞增殖倍数,流式细胞学检测髓核细胞凋亡率,免疫印迹检测凋亡相关蛋白(Bcl-2、Bax、Caspase-3)、髓核细胞外基质蛋白(AGG、COLⅡ和SOX-9)和p65蛋白相对表达水平。结果与对照组比较,诱导组细胞增殖倍数、Bax、AGG、COLⅡ和SOX-9蛋白表达水平减少,细胞凋亡率、Bcl-2和Caspase-3蛋白表达水平及磷酸化p65(p-p65)、p-p65/p65水平明显升高,差异有统计学意义(P <0. 05);与诱导组比较,川芎嗪低、中、高浓度组细胞增殖倍数、Bax、AGG、COLⅡ和SOX-9蛋白水平逐渐增高,细胞凋亡率、Bcl-2和Caspase-3蛋白表达水平及p-p65、p-p65/p65水平逐渐降低,差异有统计学意义(P <0. 05)。结论川芎嗪可通过抑制IL-1β诱导的髓核细胞凋亡和p-p65缓解椎间盘退变。

Objective This study aims to explore the mechanism of ligustrazine(LCT)in the degeneration of nucleus pulposus cells(NPCs)in intervertebral disc.Methods NPCs in intervertebral disc were isolated from scoliosis disc of10adult male SD rats.After the separation,the cells were randomly divided into five groups:the control group(NPCs,0.9%sodium chloride),the induction group(10μg/L interleukin-1β,IL-1β),and low-,medium-and high-concentration ligustrazine group(10μg/L IL-1β+10,20,50μmol/L LCT).The proliferation folds of NPCs was detected by CCK-8,and the apoptosis rate of NPCs was detected by flow cytometry.The relative expression levels of apoptosis-related protein(Bcl-2,Bax and Caspase-3),extracellular matrix protein(AGG,COLⅡand SOX-9)and p-p65protein were measured by western blot.Results Compared with control group,the cell proliferation folds,the protein expression level of Bax,AGG,COLⅡand SOX-9were decreased in cells in induction group,whereas apoptosis rate,protein expression levels of Bcl-2and Caspase3,levels of phospho-p65(p-p65),and p-p65/p65ratio were increased significantly,and the difference was statistically significant(P<0.05).Compared with induction group,the cell proliferation fold,the protein levels of Bax,AGG,COLⅡand SOX-9,in LCT low-,medium-and high-concentration groups were incrementally increased.However,apoptosis rate,the protein expression levels of Bcl-2and Caspase-3and p-p65,p-p65/p65ratio were gradually decreased,and the difference was statistically significant(P<0.05).Conclusion Ligustrazine inhibits the IL-1β-induced apoptosis and p-p65to alleviate intervertebral disc degeneration.