Mechanisms Of Functional Self-Assembled Polypeptide Hydrogel RADKPS Inhibiting Nucleus Pulposus Mesenchymal Stem Cells Degeneration

Part 1BackgroundIntervertebral disc degeneration(IVDD)is one of the main causes of low back pain in clinic.For the pain caused by IVDD,the conservative treatment based on medicine and the comprehensive treatment based on surgery are mainly used in clinic,but neither can completely solve the impact of low back pain.In recent years,the research on biological treatment of intervertebral disc degeneration has gradually increased,and some achievements have been made.Especially in recent years,mesenchymal stem cells have been used for biological treatment of intervertebral discs.Our team conducted a series of experiments on the biological scaffold structure and related bioremediation of the functionalized self-assembled peptide gel scaffold RADKPS in the early stage.However,the specific repair mechanism of RADKPS on intervertebral disc degeneration has not been fully clarified.This study will explore the relationship between RADKPS hydrogel and the scorch of nucleus pulposus mesenchymal stem cells,and clarify the possible molecular mechanism.Objective:1.Establishing a model of lipopolysaccharide(LPS)induced apoptosis of nucleus pulposus mesenchymal stem cells(NPMSCs2.To explore the expression of ERK1/2 and RhoA signaling pathway in NPMSCs;3.To explore the effects of ERK1/2 and RhoA signal pathway inhibitors PD98059 and CCG-1423 on NPMSCs after RADKPS;Methods:Human NPMSCs were isolated by differential adherent method,stem cell phenotypes were identified by flow cytometry,and three-line differentiation ability was determined.The gelling properties of RADA 16-1 and RADKPS were observed.The effect of RADKPS on the proliferation of NPMSCs was detected by CCK-8 method.The expression of ERK1/2 and p-ERKI/2 proteins in human normal and degenerative intervertebral disc tissues was analyzed by immunohistochemistry.Real-time quantitative PCR and WB test were used to evaluate the changes of NPMSCs matrix after LPS treatment,the expression of pyrolysin,and the impact on potential signal pathways ERK1/2 and RhoA.Then evaluate the effect of RADKPS on the expression of pyrolytic protein and signal pathway protein in NPMSCs,and evaluate the effect on signal pathway protein and RADKPS after adding signal pathway inhibitors PD98059 and CCG-1423.Results:NPMSCs isolated and passed to P3 generation showed spindle-like long spindle shape under microscope.Real-time quantitative PCR experiment confirmed that LPS promoted the scorch related gene IL-1β And down-regulated the expression of extracellular matrix(ECM)related gene sox-9(p<0.001).Immunohistochemical analysis showed that the expression of ERX1/2 and P-ERKl/2 in degenerative intervertebral disc tissue decreased.We evaluated the effect of RADKPS on the proliferation of NPMSCs from the perspective of two-dimensional and threedimensional culture.We found that RADKPS can promote the proliferation of NPMSCs in two-dimensional and three-dimensional culture.WB experiment showed that RADKPS inhibited the expression of pyrolytic related protein,promoted the expression of p-ERK1/2(p<0.001)and RhoA(p<0.01),and promoted the expression of type II collagen(p<0.01)and sox-9(p<0.01).PD98059 and CCG-1423 inhibited its expression.Conclusion:This study shows that RADKPS hydrogel effectively protects NPMSCs from scorch death through ERK1/2 and RhoA signaling pathways.The results also showed that RADKPS promoted the proliferation of NPMSCs in a time-dependent manner,and could protect NPMSCs from LPS mediated scorch in a three-dimensional cultured hydrogel model.Therefore,RADKPS hydrogel can be used as a potential treatment for intervertebral disc degeneration.Part 2Background:Previous experiments have confirmed that RADKPS hydrogel reduced the damage of NPMSCs by inhibiting scorch death,which is to achieve the purpose of treating intervertebral disc degeneration by inhibiting inflammatory changes induced by programmed death.From the perspective of cell’s own potential,its self-repairing ability has not been improved.Autophagy is a potential self-protection mechanism of cells,and moderate autophagy of cells is conducive to the recovery and growth of cells.This chapter discusses the relationship between RADKPS hydrogel and NPMSCs autophagy,and explores its possible molecular mechanismObjective:To explore the relationship between RADKPS and autophagy of NPMSCs,to explore the long-term release rhythm of RADKPS,and explore the possible signal pathway and molecular mechanism between RADKPS and autophagyMethods:Human primary NPMSCs were isolated and the morphology and growth status of the passage cells were observed.The differentiation ability of stem cells was identified,and the hydrogel dissolution and release experiments verified the release rhythm and slow release effect of RADA 16-1 and RADKPS.The cell proliferation experiment explored the effects of serum starvation,AKT signal inhibitor LY294002 and RADKPS on the proliferation of NPMSCs.Real-time quantitative PCR was used to detect the effect of RADKPS on the expression of autophagy-related gene p62.WB test to evaluate serum hunger and IL-1β and the effect of 3%DMSO on autophagy of NPMSCs.The effect of RADKPS on autophagy of NPMSCs and AKT signaling pathway.Results:NPMSCs cultured to P3 had good ability of adipogenesis,osteogenesis and chondrogenic differentiation.After 1,3 and 7 days of dissolution and release experiments,the average release rates of RADA16-1 were 0.3450,0.5197 and 0.6972,and the average release rates of RADKPS were 0.1163,0.3902 and 0.5001.Through statistical test,the slow-release efficiency of RADKPS can have statistical significance compared with RADA 16-1 at three time points of 1 day,3 days and 7 days.This shows that RADKPS has a higher storage efficiency for drugs.CCK-8 experiment showed that RADKPS hydrogel could promote the proliferation of NPMSCs and alleviate the damage of serum starvation on NPMSCs.Real-time quantitative PCR showed that RADKPS inhibited the expression of p62 mRNA.WB experiment confirmed that it is consistent with 10ng/ml IL-1β Compared with 3%DMSO,1%FBS,0%FBS and 10 p mol/L Rapamycin can induce autophagy of NPMSCs.However,1%FBS also upregulated the expression of apoptotic protein Bax,autophagy activator Rapamycin reduced the expression of Bax,and autophagy inhibitor 3-MA reversed this change and increased the expression of Bax.Like Rapamycin,RADKPS hydrogel can promote the expression of autophagy related protein Beclin-1 and p-AKT protein.AKT signal pathway inhibits the expression of LY294002 and p-AKT,and can inhibit the expression of Beclin-1.This suggests that AKT signaling pathway may participate in the autophagy process of NPMSCsConclusion:RADKPS hydrogel has a good function of sustained release drug,significantly increasing the half-life of BMP-7 core fragment(KPSS),and increasing the drug action time.RADKPS hydrogel can alleviate the damage of NPMSCs caused by serum starvation.RADKPS hydrogel can promote autophagy of NPMSCs,which may be related to the activation of AKT signaling pathway.