Naringin Protect Nucleus Pulposus-derived Mesenchymal Stem Cell From Apoptosis And Alleviate Intervertebral Disc Degeneration Via Activating Of PI3K/Akt Signaling Pathway

Background and Objective: In 2018,the Lancet reported that the low back pain was the leading cause of disability in most countries over the last thirty years,and its incidence is on the rise.It stressed that low back pain and neck pain should be a priority for future research on prevention and therapy.The majority of low back pain symptoms are caused by intervertebral disc degeneration(IDD),but the pathological mechanism has not yet been clarified.Recent studies have shown that the apoptosis of nucleus pulsus-derived mesenchymal stem cell(NPMSC)is an important cause of IDD.We explored the biological effect of naringin on the apoptosis and differentiation of human NPMSC(hNPMSC)and its possible mechanism.It will provide a theoretical basis for drug therapy to delay IDD.Methods: Samples of nucleus pulposus from patients with lumbar IDD were collected during operation,and intramedullary ligaments and fibrous ring tissues were separated and removed under microscope to obtain the nucleus pulposus tissues.The cells were isolated and cultured aseptically in vitro.The cells were identified by cell morphology observation,cell surface antigen phenotype detection,and the ability to induce osteogenesis,lipogenesis and chondrogenic differentiation.Naringin was used at different concentrations(0 μg/m L,0.8μg/m L,4 μg/m L,20 μg/m L,100 μg/m L and 200 μg/m L)to interfere with the P3 generation hNPMSC.The effect of different concentrations of naringin on cell viability was evaluated by CCK-8 assay and the optimal concentration was selected.The P3 hNPMSC were divided into control group(normal medium),naringin group(medium containing 20 μg/m L naringin)and LY294002 group(medium containing 20 μg/m L naringin and PI3K/Akt signaling inhibitor LY294002),and the hNPMSCs in each group were incubated for 6 days separately.Apoptosis was analyzed using an Annexin V-FITC/PI Apoptosis Detection Kit according to the instructions by the flow cytometry instrument.According to the manufacturer’s instructions,cells were stained with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL),and then photograph was observed under a fluorescence microscope.Total proteins were extracted and the protein concentration was then measured.The pro-apoptotic proteins Caspase-3 and Bax and anti-apoptotic proteins Bcl-2,and PI3K/Akt signaling pathway related protein p-Akt,Akt and p53 were detected by western blot.The expressions of collagen type II and aggrecan were detected by RTPCR.Results: The primary cells isolated from the nucleus pulposus of human degenerative intervertebral disc grew adherent to the wall,showing irregular polygon at first,disk-like cell nucleus,clearly demarcated,obvious nucleoli,and weak cytoplasm.After passing,it was mainly spindle-shaped.Flow cytometry showed that the cell surface is highly expressed with the stem-cell related positive surface antigen molecules CD73,CD90 and CD105 and lower expressed of CD45 and CD34 which were usually negative in MSC.For adipogenic differentiation,the cells showed dot red lipid droplets when dyed with oil red "O".For osteogenic differentiation,the cells formed highly visible calcium deposits.For chondrogenic differentiation,the cells displayed accumulated of sulfated proteoglycans after stained with toluidine blue staining.These characteristics were consistent with the criteria for mesenchymal stem cell identification of international society of cell therapeutics.CCK-8 results showed that the activity of hNPMSC increased after treatment with 0.8 μg/m L-200 μg/m L naringin,and the effect was strongest at 20 μg/m L.Compared with the control group,the apoptosis rate,TUNEL staining positive cell rate,protein expressions of p53,caspase-3 and Bax were significantly decreased in the naringin group,while the expressions of p-akt and anti-apoptotic protein bcl-2 were significantly increased.However,this protect effect can be reversed with LY294002 intervention.RT-PCR results showed that m RNA expressions of collagen type II and aggrecan in the hNPMSC of the naringin group were significantly increased compared with those of the control group.The m RNA expressions of collagen type II and aggrecan were significantly lower in LY294002 group than those of the naringin group.Conclusion: Naringin can inhibit the apoptosis of hNPMSC derived from the degenerative disc and promote the differentiation of hNPMSC into nucleus pulposus by activating PI3K/Akt signaling pathway.