Fibulin-3调控组织金属蛋白酶抑制因子-3影响椎间盘髓核细胞衰老的机制研究

Fibulin-3 Regulates Tissue Inhibitor of Metalloproteinases 3 to Inhibit Senescence in Intervertebral Disc Nucleus Pulposus Cells

目的探讨fibulin-3通过调控组织金属蛋白酶抑制因子-3(tissue inhibitor of metalloproteinase 3,TIMP-3)表达而影响椎间盘髓核细胞衰老的分子机制。方法(1)收集37例椎间盘术后患者的髓核组织及影像学资料,按照Pfirrmann分级评估椎间盘的退变程度,衰老相关β-半乳糖苷酶(senescence-associatedβ-galactosidase,SA-β-gal)染色法确定髓核细胞的衰老程度,Western blot和ELISA检测fibulin-3蛋白水平,探讨fibulin-3与椎间盘退变和髓核细胞衰老之间的关系;(2)体外培养人椎间盘髓核细胞(nucleus pulposus cells,NPCs),观察连续传代过程中NPCs的增殖(CCK8法)和衰老(SA-β-gal染色法)情况,检测fibulin-3的表达水平以及促炎症因子和基质蛋白酶的表达情况。通过添加外源性fibulin-3验证其对NPCs增殖和衰老的影响;(3)通过基因过表达技术验证fibulin-3对NPCs凋亡和增殖的作用,结合凋亡抑制剂进行双向验证;(4)利用生物信息学分析fibulin-3与TIMP家族的关系,通过过表达fibulin-3并沉默TIMP-3基因的实验,验证其在NPCs衰老中的作用。结果(1)在37例患者的椎间盘退变样本中,按Pfirrmann分级,退变等级越高,fibulin-3的表达越低。Spearman相关性分析显示椎间盘分级与NPCs的衰老等级负相关(r=-0.87,P<0.001),与fibulin-3的表达量负相关(r=-0.79,P<0.001);(2)随着NPCs传代次数增加,fibulin-3的表达逐渐降低,细胞增殖能力减弱,促炎症因子和基质蛋白酶的表达增高。添加外源性fibulin-3后,细胞形态和生长状态得到保持,细胞衰老显著抑制,促炎症因子和基质蛋白酶的表达明显降低;(3)基因过表达实验显示,fibulin-3减少了NPCs的凋亡,促进了细胞增殖,从而抑制了NPCs的衰老;(4)生物信息学分析显示fibulin-3与TIMP家族的TIMP-3存在显著关联。进一步实验验证发现,过表达fibulin-3能够增强TIMP-3的表达,而沉默TIMP-3基因后,fibulin-3对NPCs衰老的抑制作用显著减弱。这表明fibulin-3通过调控TIMP-3来抑制基质金属蛋白酶活性,影响细胞外基质的合成和降解,最终抑制髓核细胞的衰老。结论本研究表明,fibulin-3通过调控TIMP-3在椎间盘髓核细胞衰老过程中发挥重要作用。具体机制包括:fibulin-3上调TIMP-3的表达,抑制基质金属蛋白酶活性,减少细胞外基质的降解,从而促进细胞外基质的合成;此外,fibulin-3通过减少细胞凋亡和促进细胞增殖,进一步抑制髓核细胞的衰老。因此,fibulin-3和TIMP-3在维持椎间盘健康和延缓退变过程中具有潜在的治疗意义。

Objective To investigate the effect of fibulin-3 on the senescence of intervertebral disc nucleus pulposus cells(NPCs)through the regulation of tissue inhibitor of metalloproteinases 3(TIMP-3)expression and to elucidate the molecular mechanisms involved.Methods 1).The nucleus pulposus tissues and imaging data of 37 patients who had undergone intervertebral disc surgery were collected.The degree of degeneration of the intervertebral discs were classified according to the Pfirrmann grading system.The senescence degree of NPCs was determined using senescence-associatedβ-galactosidase(SA-β-gal)staining.Fibulin-3 expression levels were determined using Western blot and ELISA.The relationship between fibulin-3 and disc degeneration and NPCs senescence was investigated.2).Human intervertebral disc NPCs were cultured in vitro.The proliferation and senescence of NPC across continuous passage were observed via CCK-8 assay and SA-β-gal staining,respectively.Fibulin-3 expression levels and the expression of inflammatory cytokines and matrix metalloproteinases were assessed.Exogenous fibulin-3 was added to verify its effect on the proliferation and senescence of NPCs.3).The effect of fibulin-3 on the apoptosis and proliferation of NPCs was verified through gene overexpression,which was used in combination with an apoptosis inhibitor for bidirectional verification.4).Bioinformatics analysis was performed to explore the relationship between fibulin-3 and the TIMP family.Experiments overexpressing fibulin-3 and silencing the TIMP-3 gene were performed to verify their role in NPCs senescence.Results 1).The intervertebral disc degeneration samples from 37 patients were classified according to the Pfirrmann grading system.The higher the degeneration grade,the lower fibulin-3 expression.Spearman correlation analysis showed that the disc grade was negatively correlated with the NPC senescence grade(r=-0.87,P<0.001)and fibulin-3 expression(r=-0.79,P<0.001).2).As the passage number of NPCs increased,fibulin-3 expression gradually decreased,cell proliferation ability weakened,and the expression of inflammatory cytokines and matrix metalloproteinases increased.After exogenous fibulin-3 was added,cell morphology and growth status were maintained,cell senescence was significantly inhibited,and the expression of inflammatory cytokines and matrix metalloproteinases was markedly reduced.3).Gene overexpression experiments showed that fibulin-3 reduced NPC apoptosis and promoted cell proliferation,thereby inhibiting NPC senescence.4).Bioinformatics analysis revealed a significant association between fibulin-3 and TIMP-3 of the TIMP family.Further experiments confirmed that overexpressing fibulin-3 enhanced TIMP-3 expression,while silencing the TIMP-3 gene significantly weakened the inhibitory effect of fibulin-3 on NPCs senescence.This indicates that,through regulating TIMP-3,fibulin-3 inhibits the activity of matrix metalloproteinases,affects the synthesis and degradation of the extracellular matrix,and ultimately inhibits NPCs senescence.Conclusion This study demonstrates that fibulin-3 plays a crucial role in inhibiting the senescence of intervertebral disc NPCs by regulating TIMP-3.The specific mechanisms involved are as follows,fibulin-3 upregulates TIMP-3 expression,inhibits matrix metalloproteinase activity,and reduces extracellular matrix degradation,thereby promoting extracellular matrix synthesis.Additionally,fibulin-3 inhibits NPCs senescence by reducing apoptosis and promoting cell proliferation.Therefore,fibulin-3 and TIMP-3 have potential therapeutic significance in maintaining intervertebral disc health and delaying degeneration.