| PART1BackgroundNeck,shoulder,waist and leg pain caused by intervertebral disc degeneration(IVDD)has become a global problem that seriously affects people’s health,and there is still a lack of long-term effective treatments.In recent years,biological treatments dedicated to improving the endogenous repair ability of intervertebral discs have received attention.Nucleus pulposus mesenchymal stem cells(NPMSCs)repair degenerative intervertebral disc tissue through chondrocytic differentiation and paracrine action.SIRTl is a deacetylase,which can prevent cell aging and apoptosis,etc.SIRT1 gene knockout can increase the expression of MCP1 in a variety of diseases including IVDD,thus aggravating the symptoms.Therefore,we suggest that the activation of SIRT1 promotes chondrogenic differentiation of NPMSCs and may reduce the apoptosis of NPMSCs through the MCP1/CCR2axis.Objective:To investigate the effects of SIRT1 on the MCP1/CCR2 signaling axis,chondrogenic differentiation and apoptosis of NPMSCs.MethodsNPMSCs were obtained from IDD patients,then treated with SIRT1 activator,monocyte chemokine protein 1(MCP1)and chemokine receptor 2 inhibitor,and the growth,proliferation,chondrogenic differentiation and apoptosis indexes of NPMSCs were observed Then the IDD model of rats was established and transfected with NPMSCs overexpressing SIRT1.ResultsThe up-regulation of SIRT1 was found to promote the down-regulation of MCP1/CCR2 axis,chondrogenic differentiation and apoptosis in NPMSCs of IDD patients.In addition,MCP1 can reverse chondrogenic differentiation of NPMSCs and induce apoptosis of NPMSCs by overexpression of SIRT1.Increased apoptosis and reduced chondrogenic differentiation of NPMSCs were detected in the rat IDD model,and the expression of SIRT1 was decreased,while the expression of MCP1 and CCR2 was increased.In addition,rats transplanted with NPMSCs overexpressing SIRT1 showed reduced symptoms of IDD.ConclusionsOverexpression of SIRT1 can promote chondrogenic differentiation of NPMSCs and reduce apoptosis,which can alleviate symptoms of IDD in rats by inhibiting the MCP1/CCR2 axis.PART2BackgroundPrevious studies have shown that the activation of SIRT1 promotes chondrogenic differentiation of NPMSCs in IVDD patients by down-regulating the MCP1/CCR2 axis and inhibiting apoptosis.Some studies have shown that MCP1 is highly expressed in degenerative intervertebral discs,but the relevant reports are few.In order to find more targets for the treatment of IVDD,we need to understand the effect and mechanism of MCP-1 on NPMSCs in the process of intervertebral disc degeneration,so we conducted this study.Objective1.Confirmed hyperexpression of MCP1 in degenerative intervertebral discs;2.To study the effects of MCP1 on the proliferation,apoptosis,migration and chondrogenic differentiation of NPMSCs;3.The downstream signaling pathway of MCP1/CCR2 axis was preliminarily discussed.MethodsHuman NPMSCs were successfully isolated and cultured by enzyme digestion,and their morphology,cell phenotype and three-line differentiation were identified.Enzyme-linked immunosorbent assay(ELISA)and fluorescence quantitative PCR were used to detect the changes of MCP-1 transcription and protein expression in response to proinflammatory cytokine stimulation.CCK8 kit is used to test cell proliferation.The mobility of NPMSCs was examined using scratch tests.At the same time,the expression of SOX-9,proteoglycan and Ⅱ collagen was detected.ResultsHuman NPMSCs can significantly increase the expression of MCP-1 at mRNA and protein levels under the stimulation of pro-inflammatory factors.MCP-1 inhibited the proliferation of NPMSCs in a concentration-dependent manner(i.e.,the higher the concentration,the stronger the inhibition).We also found that MCP-1 inhibited chondrogenic differentiation of npMSCs in vitro.This inhibition was demonstrated by the effect of MCP-1 on cartilage pellet size(visual observation)and weight,as well as gene and protein expression changes of proteoglycan and type Ⅱ collagen.In addition,MCP-1 could also promote the expression of CCR2 receptor in NPMSCs(or plasma membrane).CCR2 antagonist RS504393 can significantly reduced the inhibitory effect of MCP-1 on chondrogenic differentiation of nucleus pulposus stem cells.Conclusion MCP1 inhibits chondrogenic differentiation of human NPMSCs through MCP-1/CCR2 axis. |
