感觉神经元外泌体引起髓核干细胞衰老促进椎间盘退变发生的作用机制

Sensory neuronal exosomes induce the senescence of nucleus pulposus stem cells and promote the occurrence of intervertebral disc degeneration

目的探讨感觉神经元外泌体(sensory neuron-derived exosomes,nExo)介导椎间盘退变的作用及分子机制。方法构建大鼠椎间盘退变(intervertebral disc degeneration,IDD)模型,将nExo注入椎间盘,4周后应用组织学染色评估椎间盘退变程度及免疫荧光染色评估组织中髓核干细胞的衰老表型。应用nExo或外源性硫氧还蛋白1(thioredoxin 1,TXN)处理髓核干细胞24 h,利用免疫印迹、流式细胞周期分析、衰老相关β-半乳糖苷酶染色评估细胞的衰老表型。转录组学分析筛选并验证介导nExo调控细胞衰老的关键分子。向大鼠椎间盘退变模型中注入nExo和TXN,4周后评估椎间盘退变程度。结果nExo提高了大鼠椎间盘组织退变等级,IDD组髓核组织中TEK+p16+及TEK+p21+双阳细胞比例分别为36.32%±4.04%和33.69%±4.56%,IDD+nExo组提升至56.41%±5.26%和50.14%±8.49%(t=7.420,P<0.001;t=4.184,P<0.002);nExo促进髓核干细胞的p16及p21蛋白表达,并提高了衰老相关β-半乳糖苷酶染色阳性细胞率,自7.32%±1.73%提高至58.22%±11.38%(t=7.658,P=0.002);nExo下调了G2/M期细胞百分比,从18.10%±1.32%下调至1.60%±0.67%(t=19.290,P<0.001)。转录组学分析显示空白对照组和nExo组差异基因与细胞衰老密切相关,与数据库中的衰老基因集交集筛选出关键基因,即TXN。nExo下调髓核干细胞中的TXN,而补充外源性TXN下调了衰老相关蛋白的表达,降低衰老相关β-半乳糖苷酶染色阳性细胞率从58.84%±3.99%降低至21.68%±8.16%(t=7.048,P=0.021);将G2/M期细胞百分比从1.21%±0.34%上调至15.26%±2.60%(t=9.259,P=0.001)。TXN降低了椎间盘组织退变等级,nExo组退变组织学评分为(14.33±0.82)分,nExo+TXN组为(8.17±1.17)分,差异有统计学意义(t=10.590,P<0.001);增加细胞外基质的含量,nExo组为(10.94±4.35)μg/mg,nExo+TXN组提高至(50.55±12.16)μg/mg,差异有统计学意义(t=7.512,P<0.001);减少组织中TEK+p16+及TEK+p21+双阳细胞比例,nExo组分别为54.92%±4.21%和60.31%±9.02%,nExo+TXN组下降至27.93%±3.26%和33.75%±8.07%,差异有统计学意义(t=12.430,P<0.001;t=5.375,P<0.001)。结论nExo通过下调髓核干细胞中的TXN,促进细胞衰老和椎间盘退变。

Objective To investigate the role and molecular mechanism of sensory neuron-derived exosomes(nExo)in mediating intervertebral disc degeneration(IDD).Methods A rat IDD model was constructed,with nExo injected into the intervertebral disc.After 4 weeks,the degenerative grades of operated discs were evaluated using histological staining,while the senescent phenotype of nucleus pulposus stem cells(NPSC)in the tissue was evaluated using immunofluorescence staining.For in vitro experiments,24 hours after the treatment of nExo to NPSC,immunoblotting,flow cytometry,or senescence-associatedβ-galactosidase staining was applied to evaluate the senescent phenotype of NPSC.Transcriptomics analysis was applied to identify the key molecules that mediate nExo-induced cells senescence.After 4 weeks of injecting nExo and TXN into the rat tail disc degeneration model.Results nExo increased the degenerative grades of IDD and increased the proportion of TEK+p16+and TEK+p21+cells(from 36.32%±4.04%,33.69%±4.56%in IDD group to 56.41%±5.26%,50.14%±8.49%in IDD+nExo group,respectively;t=7.420,P<0.001;t=4.184,P<0.0019,respectively)in the disc tissue.Besides,nExo promoted the expression of p16 and p21 in NPSC and increased the percentage of cells with positive senescence-associatedβ-galactosidase staining(from 7.32%±1.73%to 58.22%±11.38%,t=7.658,P=0.002),while the percentage of G2/M cells was downregulated(from 18.10%±1.32%to 1.60%±0.67%,t=19.290,P<0.001).Transcriptomic analysis showed that the differential genes of CTRL vs.nExo were closely related to cell senescence,and TXN was screened by intersecting the differential gene set with the cellular senescence gene sets from the published database.Furthermore,we verified that nExo decreased the content of TXN in NPSC,while exogenous TXN downregulated the expression of p16 and p21 in NPSC,reduced the positive cell rate of senescence-associatedβ-galactosidase staining(from 58.84%±3.99%to 21.68%±8.16%,t=7.048,P=0.021),increased the percentage of G2/M cells(from 1.21%±0.34%to 15.26%±2.60%,t=9.259,P=0.001).TXN significantly reduced the grade of disc tissue degeneration(histological score:14.33±0.82 in the nExo group;8.17±1.17 in the nExo+TXN group,t=10.590,P<0.001),significantly increased the content of extracellular matrix(from 10.94±4.35μg/mg to 50.55±12.16μg/mg,t=7.512,P<0.001),and reduced the proportion of TEK+p16+and TEK+p21+double-positive cells(from 54.92%±4.21%and 60.31%±9.02%to 27.93%±3.26%and 33.75%±8.07%,respectively;t=12.430,P<0.001;t=5.375,P<0.001,respectively).Conclusion nExo promotes cell senescence and IDD by downregulating TXN in NPSC.