| BackgroundThe intervertebral disc(IVD),an important tissue of the human body,is composed of cartilage endplates,nucleus pulposus and fibrous rings.Different degrees of degeneration will occur in the IVD with aging,which will lead to a series of clinical diseases,such as spinal stenosis,disc herniation and spinal segment instability.These put a great burden on patients and society.A variety of factors contribute to the degeneration of the IVD,including genetic factors,the age,nutritional condution and biomechanical factors.Since the IVD is a kind of connective tissue without blood vessles in adults,its nutritional source is mainly dependent on the infiltration from the cartilage end plate and the fibrous ring.Studies have shown that nutritional deficiency can induce the apoptosis of cartilage endplate cells,which will further leads to cartilage endplate degeneration,and ultimately the degeneration of the entire disc.Meanwhile,in the rabbit nucleus pulposus cells,nutrient deprivation can induce apoptosis by up-regulating the expression of BNIP3(Bcl-2/adenovirus E1 B 19-kDa-interacting protein 3).However,the exact molecular mechanisms of cartilage end plate degeneration caused by nutritional deficiency are not clear yet.Therefore,the investigation of the uderlying mechanism of nutrient deprivation caused cartilage endplate degeneration has important scientific significance and social value for the prevention and treatment of intervertebral disc degeneration.ObjectiveThe nutrient deprivation model of cells in vitro was established by simulating vertebral blood supply and peripheral microcirculation in vertebral body.The purpose of this study is to investigate the effects of nutrient deprivation on cartilage endplate-derived stem cells(CESCs)and the possible role of BNIP3 signaling pathway in the process.Thus,the mechanisms of the degeneration of the disc can be further clarified.MethodsThe degenerated intervertebral disc cartilage end plate specimens were obtained from eight cases in the clinic.The cartilage cells were isolated from the end plates,after which the CESCs were screened by fibronectin(FN).Then,cells were further cultured and replicated after identifing the surface antigen markers of the stem cells.Cells from the third-generation were selected as control or experimental group for culturing 24 h and 48 h in the experiment,respectively.The control group was placed in the normal culture condition(DMEMF12,5 mM glucose,21% O2 and 10% serum),while the experimental group was incubated in nutrient deprivation condition(DMEMF12,sugar free,1% O2 and serum free).After the treatment,the apoptotic rate was detected by flow cytometry,BNIP3 gene and protein expression levels were detected by real-time fluorescence quantitative PCR and Western Blot respectively,and the binding of BNIP3 protein to CESCs mitochondria was detected by immunofluorescence staining.In addition,changes of mitochondrial transmembrane potential(MMP)was also detected by JC-1 staining and caspase-3 activity was detected by the activity assay kit.Finally,BNIP3 siRNA was used to interfere with the expression of BNIP3 gene in CESCs,while the negative control group of interference was treated with scramble siRNA.After 24 hof interference process,the four groups of cells were cultured under normal conditions or nutrient deprivation conditions for 48 h.And then the apoptosis rate and the expression of BNIP3 were detected.Results1.Cell identification results: CESCs obtained from 8 clinical specimens were identified by stem cell markers,the CD90,CD105,CD73 and CD44 were positive,and Neg CKTL(including HLA-DR,CD19,CD34,CD11 b and CD45)were negative,indicating that CESCs had the characteristic of stem cell.2.The result of cell apoptosis and BNIP3 expression: the apoptosis rate of CESCs in experimental group(24 h: 24.47% ± 0.97%,48 h: 28.98% ± 0.47%)was significantly higher than that in control group(17.26% ± 1.84%,p<0.05).The expression of BNIP3 gene and protein in experimental group was significantly higher than that in control group(p<0.05).The results showed that nutrient deprivation could up-regulate the expression of BNIP3 and increase of apoptosis rate of CESCs.3.After interfering with the expression of BNIP3 gene,the apoptosis rate and BNIP3 expression were detected again.The apoptosis rate(23.72% ±1.21%)of CESCs which was interfered BNIP3 expression was significantly lower than that of the control group(34.05% ±1.43%,p<0.05).The expression of BNIP3 protein was significantly down-regulated in the intefered group compared with the control siRNA group.The results suggest that interfering with the BNIP3 gene can partially inhibit apoptosis caused by nutrient deprivation.4.Immunofluorescence results: nutritional deprivation could up-regulate the expression of BNIP3 protein in CESCs and promote the binding of BNIP3 protein to mitochondria to reduce MMP.5.Caspase-3 activity: there was no significant difference in caspase-3 activity between groups(p>0.05).The results suggest that nutrition deficiency promotes apoptosis of CESCs in a caspase-independent manner.ConclusionNutrition deficiency may promote CESC apoptosis partially through upregulating BNIP3 and the increased bingding of BNIP3 to mitochondira casing decreased MMP,which might lead to activation of the BNIP3-related pathway and apoptosis of CESCs in a caspase-independent manner.[1]... |
PDF Full Text Request