The Effects Of TNF-α On The Biological Functions Of Rat Nucleus Pulposus Mesenchymal Stem Cells

Background Degenerative disc disease(DDD)is one of the common chronic diseases,intervertebral disc degeneration(IVDD)is the basic pathogenesis of these diseases.However,the exact mechanism of disc degeneration is still unclear,which hindered the development of advanced bio-therapy methods.The latest theory claimed that numerous factors have involved in the progression of IVDD,both eventurally resulting in the chaos of IVD structure and malfunction of its endogenous stem cell,thus the balance of IVD homeostasis could not be further maintained.Nucleus pulposus mesenchymal stem cells(NPMSCs)were found in the nucleus pulposus tissue as precursors of nucleus pulposus cells,and were considered as the most valuable stem cell resource for the treatment of IVDD.According to previous studies,we have found that the bio-functions of NPMSCs was inhibited during the intervertebral disc degeneration process,which viewed as an important trigger for DDD.Inflammation is one of the key factors of DDD.It plays an important role during the development of disc degeneration.TNF-a(Tumor Necrosis Factor-alpha)is the most abundant inflammatory factor in the degenerative intervertebral disc,which exert negatively affect to inner cells by induce their senescence and apoptosis.However,what effect did TNF-a have on the biological behavior of NPMSCs has not been systematically investigated.Accordingly,the aim of this study was to investigate the regulatory effects of TNF-a at high and low concentrations on the biological behavior of healthy rat NPMSCs,including apoptosis proliferation,migration,and nucleus pulposus differentiation.Objectives The aim of this study was to in vitro investigate the effect of different concentrations of TNF-a on the biological functions of NPMSCs,including apoptosis,proliferation,migration and nucleus pulposus differentiation.Methods NPMSCs were collected from the tail discs of ten male Sprague-Dawley rats(3 months old)Limiting Dilution Method was used for isolating NPMSCs,and the acquired cells were characterized by a multi-lineage differentiation assay and flow cytometry.Then,NPMSCs were treated with different concentration of TNF-a(0-200 ng/mL).Then we used Annexin V/PI flow cytometry analysis to detected the apoptosis of NPMSCs.CCK-8,EdU assay,cell cycle test were used to examine the proliferation capacity of NPMSCs.Migration ability of NPMSCs was detected by wound healing assay and transwell migration assay.Pellets method was used to induce nucleus pulposus differentiation of NPMSCs,and immunohistochemical staining,RT-PCR and western-blotting were used to examine the NP phenotypic genes and proteins.The cells were further treated with the inhibitor Bay 11-7082 to determine the role of the nuclear factor-kappa B(NF-κB)pathway in the mechanism underlying the differentiation process.Results Nucleus pulposus mesenchymal stem cells were successfully isolated and identified,treatment with a high concentration of TNF-a(50-200 ng/mL)induced the apoptosis of NPMSCs,The CCK-8 assay showed TNF-a at low concentrations(0.1 ng/ml,1ng/ml,10ng/ml)could significantly promoted the proliferation of NPMSCs at day 5 to day 10.Consistently,the cell cycle test also indicated that with increase dose of TNF-a,the percent of G1 phase cells decreased and S/G2 phase cells increased,the rate of EdU positive cells also increased,these results illustrated that these concentration promoted the proliferation rate of NPMSCs and had a concentration effect.The results of wound healing assay showed exposure to TNF-a enhanced NPMSCs migration,leading to a significant decrease in the scratch gap area compared to untreated cells(P<0.05),and transwell assay revealed the parallel effect of TNF-a.These results suggested that low concentration of TNF-a could promoted the migration of NPMSCs.RT-PCR and Western Blotting showed the synthesis of collagen Ⅱ,aggrecan,Krt-19,HIF-la,Shh and Sox-9 were lower than control group after treated with TNF-a,which means the differentiation ability of NPMSCs been inhibited.Moreover,we observed that the NF-κB signaling pathway is activated during the TNF-a-regulated differentiation of NPMSCs,and the NF-κB signal inhibitor Bay 11-7082 could reverse the adverse effect of TNF-a on the differentiation of NPMSCs.Conclusions The effect of TNF-a on NPMSCs is quite complex.In conclusion,we discovered that high concentrations of TNF-a(50ng/ml-200ng/ml)could induce the apoptosis of NPMSCs,while low concentrations(0.1ng/ml-10ng/ml)promoted the proliferation and migration but inhibited the differentiation of NPMSCs.Our findings disclosed the dynamic biological reactivity of NPMSCs during IVD degeneration process,thus provide new sight toward the prevention and treatment of IVD degeneration.