CHI3L1拮抗IL-1β诱导髓核细胞炎症反应的机制研究

CHI3L1 protects against IL-1β-induced inflammatory response of nucleus pulposus cells in intervertebral disc degeneration:A mechanistic study

目的:初步探究炎症相关分泌蛋白CHI3L1在椎间盘退变(IDD)中的作用及机制。方法:通过收集腰椎间盘突出症患者手术中的椎间盘样本作为退变组,同时收集腰椎外伤患者手术中的椎间盘样本作为对照组(根据Pfirrmann分级进行收集),通过免疫组化检测两组样本髓核细胞中CHI3L1的表达水平。在体外通过炎症因子IL-1β构建退变模型,利用质粒转染过表达或者siRNA干预CHI3L1的表达水平并检测髓核细胞基质代谢相关基因的表达水平变化。通过不同浓度的CHI3L1重组蛋白干预髓核细胞,并检测其在IL-1β诱导下对髓核退变的影响。利用Transwell实验验证CHI3L1对髓核细胞基质代谢的实际作用。结果:通过免疫组化检测发现CHI3L1主要表达于椎间盘的髓核区域,且正常髓核细胞均有表达,而退变髓核细胞表达显著上调。体外细胞实验显示,过表达CHI3L1后髓核细胞基质合成相关基因ACAN、COL2、CHSY的表达均显著高于对照组(均P<0.05),而炎症相关基因MMP1、MMP3、MMP13、ADAMTS4以及ADAMTS5的表达均显著低于对照组(均P<0.05)。干扰CHI3L1表达后结果出现明显反向变化(均P<0.05)。通过添加不同量CHI3L1的重组蛋白,炎症刺激下髓核细胞基质合成相关基因的表达随着剂量增加而逐渐上升,但其存在最大效应浓度。同时通过Transwell小室侵袭实验发现,过表达CHI3L1显著抑制了IL-1β致髓核细胞分泌降解细胞外基质因子的能力,使得穿膜细胞显著减少(P<0.05),而干扰CHI3L1后则出现相反结果(P<0.05)。结论:炎症相关基因CHI3L1具有髓核特异性,是在炎症刺激下上调的能够保护髓核细胞抵抗IL-1β的诱导退变作用的分泌性分子,具有保护椎间盘髓核细胞退变的作用。

Objective:To preliminarily investigate the role and action mechanism of inflammation-related secreted protein CHI3L1 in intervertebral disc degeneration(IDD).Methods:Based on Pfirrmann grading,intervertebral disc samples collected from patients with lumbar disc herniation were assigned to the degeneration group,and those collected from patients with lumbar trauma were assigned to the control group.The CHI3L1 expression level in nucleus pulposus cells of tissue samples in the two groups was examined by immunohistochemistry.In vitro,a degeneration model was established by IL-1βinduction.In this model,the CHI3L1 level was interfered by knockdown or overexpression via transfection of plasmid or siRNA,and then the expression levels of nucleus pulposus cell matrix metabolism-related genes were measured.The effects of IL-1βinduction on nucleus pulposus degeneration were further examined after the nucleus pulposus cells were treated with different concentrations of recombinant CHI3L1 protein.Finally,the actual effect of CHI3L1 on the matrix metabolism of nucleus pulposus cells was verified by Transwell assay.Results:Immunohistochemistry showed that CHI3L1 was chiefly expressed in the nucleus pulposus area of the intervertebral discs,and was expressed both in normal and degenerative nucleus pulposus cells except for a significantly up-regulation in the latter.In vitro cell experiments showed that after overexpression of CHI3L1,the expression of nucleus pulposus cell matrix synthesis-related genes ACAN,COL2,and CHSY were significantly higher than those in the control group(all P<0.05),while the inflammation-related genes MMP1,MMP3,MMP13,ADAMTS4 and ADAMTS5 were significantly lower than those in the control group(all P<0.05).After interference of the CHI3L1 expression,these observations changed in a significant reverse direction(all P<0.05).By adding varying amounts of recombinant CHI3L1protein,the expression of nucleus pulposus matrix synthesis-related genes under inflammatory stimulation gradually increased along with dose,but was subjected to a maximum effect concentration.Meanwhile,Transwell chamber invasion assay showed that overexpression of CHI3L1 significantly inhibited the ability of IL-1β-induced nucleus pulposus cells to secrete and degrade extracellular matrix factors,resulting in a significant decrease in migratory cells(P<0.05);however,the opposite was observed after interference of CHI3L1(P<0.05).Conclusion:The inflammation-related gene CHI3L1 is a nucleus pulposus-specific secretory molecule that can be up-regulated under inflammatory stimulation to protect the nucleus pulposus cells against degeneration induced by IL-1β,and thereby protect the nucleus pulposus cells of the intervertebral disc from degeneration.