Different Concentrations Of Sa12b Improve The Biological Activity Of Human Degenerated Nucleus Pulposus Mesenchymal Stem Cells In Acid Environment By Inhibiting ASICs

Background:Low back pain（LBP）is one of the most common public health problems and the leading cause of disability worldwide.The study found that intervertebral disc degeneration（IVDD）was one of the important reasons.With the progress of IVDD,many changes would occur in the microenvironment of the intervertebral disc（IVD）,such as hypoxia,low p H,hypertonicity and low nutrition,etc.Among them,low p H could decrease the biological activity of nucleus pulposus mesenchymal stem cells（NP-MSCs）,which could differentiate into nucleus pulposus cells in IVDD.Acid-sensitive ion channels（ASICs）,an important member of the epithelial sodium channel/degenerin family（DEG/ENa C）,are vital receptor for changes in the vivo acid environment.The acid environment in IVDD could regulate the biological activity of NP-MSCs by mediating ASICs.Recent studies have found that Sa12 b,a wasp peptide that could non-specifically inhibit ASICs,reversibly inhibits the current peak of ASICs in the rat dorsal root ganglion（DRG）in a concentration-dependent manner.However,whether it affects the biological activity of NP-MSCs by mediating ASICs in the acid environment of IVDD,thereby delaying IVDD and improving LBP is still unclear.Objective:1.To observe the effect of different concentrations of Sa12 b on the biological activity of human degenerated NP-MSCs in an acidic microenvironment;2.To observe the expression of ASICs after different concentrations of Sa12 b affected the biological activity of human degenerated NP-MSCs in an acidic microenvironment,and to explore the relevant mechanisms.Methods:1.Isolate NP-MSCs from the nucleus pulposus tissue of patients with lumbar disc herniation during nucleus pulposus enucleation,and conduct cell identification and purification through cell morphology observation,tri-lineage differentiation and flow cytometry phenotype;2.The biological activity such as cell proliferation and apoptosis of human degraded NP-MSCs under acidic microenvironment treated with different concentrations of Sa12 b were detected by cytotoxicity assay,apoptosis assay and Annexin V assay;3.Quantitative Real-time PCR（q PCR）and Western blotting（WB）were used to detect the extracellular matrix-related（ECM）genes（Collagen Ⅱ,Aggrecan,SOX-9）expression of human degenerated NP-MSCs after affected by different concentrations of Sa12 b in the acidic microenvironment;4.q PCR was used to detect the gene expression of ASICs subunits（ASIC1,ASIC2,ASIC3,ASIC4）and stem cell related genes（Oct4,Nanog,Jag1,Notch1）of the human degenerated NP-MSCs affected by different concentrations of Sa12 b in the acidic microenvironment.5.Flow cytometry and laser confocal microscopy were used to detect [Ca2+]I in human degenerated NP-MSCs affected separately by Sa12 b,Amilorade and the simultaneous addition of Sa12 b and amiloride in an acidic microenvironment.Results:1.The cells isolated and cultured from nucleus pulposus tissue grow in spindle shape and adhere to the wall,and could be expanded in vitro.The results of flow cytometry show that the cells high expression of CD73（98.1%）,CD90（97.5%）,CD105（98.3%）and low expression of HLA-DR（3.01%）,CD34（1.73%）,CD45（1.29%）,and can differentiated into osteogenic,adipogenic and chondrogenic differentiation.Therefore,the isolated and cultured cells were identified as NP-MSCs;2.Compared with the normal culture environment（p H=7.4）,the proliferation of human degenerated NP-MSCs cultured in the acidic environment（p H=6.2）was significantly inhibited（p < 0.001）.However,After different concentrations of Sa12b（0,2,4,6,8 μ g/μ l）,the proliferation inhibition of human degenerative NP-MSCs was significantly improved（p < 0.05）,the apoptosis rate gradually decreased（p < 0.001）,and the rate of apoptosis was gradually decreased（p < 0.001）when changed to the acidic environment（p H=6.2）.And the best state was reached when the concentration was 8μg/μ l;3.With the increase of Sa12 b concentration,the extracellular matrix-related genes（Collagen Ⅱ,Aggrecan,SOX-9）in human degenerated NP-MSCs in acidic microenvironment gradually increased（p<0.01）,and Sa12 b was likely to promote the remodeling of ECM for IVDD;4.With the increase of Sa12 b concentration,the subunits of ASICs（ASIC1,ASIC2,ASIC3,ASIC4）in human degenerated NPMSCs in acidic microenvironment（p H=6.2）gradually decreased（p < 0.05）.And compared with the control group,the expression of stem cell-related genes（Oct4,Nanog,Jag1,Notch1）was significantly increased（p < 0.05）5.Compared with the control group,the [Ca2+] i in the treatment group containing both Sa12 b and amiloride was significantly reduced（p<0.0001）.Sa12 b or amiloride treatment group followed,and the control group was the highest.Conclusion:1.Different concentrations of Sa12 b could improve the biological activity of human degenerated NP-MSCs in an acidic microenvironment,and the optimum concentration was 8 μ g/μ l;2.Different concentrations of Sa12 b reduce [Ca2+] i by inhibiting AISCs,and it is most likely to improve the biological activity of NP-MSCs through Notch signaling pathway in the acidic environment of IVDDThis study provided a new idea for the biological treatment of IVDD.Sa12 b has a great potential in delaying IVDD and treating low back pain.