Kartogenin Enhances The Antioxidant Functions Of Nucleus Pulposus Mesenchymal Stem Cells And Promotes Differentiation To Nucleus Pulposus-like Cells

Background: Neck,shoulder,waist and leg pain caused by intervertebral disc degeneration(IDD)is a common disease in orthopedic clinics,which causes great burden to patients and society.At present,both conservative treatment and surgical treatment have limitations,and biological treatment methods have been continuously explored and gradually applied in clinical practice.The decrease in the number of nucleus pulposus cells(NPCs)in the intervertebral disc is considered to be the main reason for IDD.The discovery of nucleus pulposus mesenchymal stem cells(NPMSCs)is considered to have great research potential.Kartogenin(KGN)is a newly discovered small molecule compound that can induce mesenchymal stem cells(MSCs)to differentiate into cartilage,and can enhance the antioxidant stress capacity of nucleus pulposus cells(NPCs).Based on this,it is of great significance to explore the role of KGN in NPMSCs.Objective: To explore the toxic effect of KGN on NPMSCs and its antioxidant stress effect;RT-PCR and Western Blot were used to verify the ability of KGN to induce the differentiation of NPMSCs into NPCs.Construct a new hydrogel with good biosafety,and meet the ability of delivering sustained-release KGN.Methods: Firstly 20 SD male rats(3 months old,200-300g)were killed,and the NP tissue of the rats was extracted to culture NPMSCs,which were identified as MSCs by three-line differentiation and flow cytometry.Then,CCK-8 method was used to detect the cytotoxicity of different concentrations of KGN on NPMSCs,and the next experimental concentration of KGN was set at 10 μM.The cell viability of NPMSCs under serum deprivation and oxidative stress was measured by CCK-8 method.NPMSCs were cultured in groups,blank control group and KGN culture group,respectively.The phenotypic changes of NPMSCs after culture in the two groups were detected by RT-PCR and Western blot.Thirdly,Construct polypeptide hydrogel loaded with KGN,study the properties of the hydrogel through scanning electron microscopy and rheological experiments,and study the toxicity of the hydrogel to NPMSCs through CCK-8 method.Results: The cultured adherent cells can be effectively induced into osteogenic differentiation,chondrogenic differentiation and adipogenic differentiation through threeline differentiation,and have the potential of three-line differentiation.At the same time,CD29,CD90 and CD44 are highly expressed,and CD34,CD45 and CD11 b are low expressed,so they can be identified as NPMSCs.Conclusion can be drawn through CCK-8 test and flow cytometry,KGN at 10 μM concentration had no obvious cytotoxicity to NPMSCs(p>0.05).KGN can effectively delay the decrease of NPMSCs activity induced by serum deprivation and hydrogen peroxide(p<0.05).KGN can effectively induce the overexpression of aggregate,collagen II,GLUT1,KRT19 and HIF-1α in NPMSCs(p<0.05),indicating that NPMSCs are induced to differentiate into nucleus pulposus like cells.The results of naked eye observation,scanning electron microscope and rheological experiment show that the polypeptide solution can load KGN to form hydrogel by selfassembly.The CCK-8 experiment shows that the nano polypeptide hydrogel loaded with KGN has no obvious cytotoxicity to NPMSCs(p>0.05).Conclusions: NPMSCs were successfully isolated,cultured and identified from rat caudal vertebrae in this experiment.10 μM KGN has no obvious toxic effect on MPMSCs,indicating that KGN is a safe potential drug for use in intervertebral discs.KGN can effectively delay the decline of NPMSCs activity caused by oxidative stress and serum deprivation.The intervertebral disc is in an acidic and nutritional state,indicating that KGN can be used as a potential drug and can effectively improve the potential of use in the intervertebral disc.KGN can effectively induce NPMSCs to differentiate into NPCs,indicating that KGN has the ability to supplement the lost NPCs by inducing the differentiation of NPMSCs in the intervertebral disc,and has the potential to treat IDD.In this study,we successfully constructed a kind of hydrogel that can load KGN nano polypeptide,and this hydrogel has no cytotoxicity to NPMSCs,providing theoretical support for later use in intradiscal administration.