Notoginsenoside R1 Pretreatment Improves The Effect And Mechanism Of Stem Cell Transplantation In The Treatment Of Myocardial Infarction

ObjectiveWith the low efficiency of survival and differentiation after transplantation,stem cell transplantation is still a potential therapy for myocardial infarction.In view of the microenvironment of ischemia and hypoxia after myocardial infarction,improving the survival rate after cell transplantation and promoting stem cell differentiation and maturation are the focus of current research.As two of the most valuable stem cells in clinical application,mesenchymal stem cells（MSCs）have strong exosome function,which can promote tissue repair after transplantation,and has been widely used in clinical research.Induced pluripotent stem cells（i PSs）have the potential of self-renewal and multidirectional differentiation closer to embryonic stem cells,and can effectively induce differentiation into cardiomyocytes.It is called induced pluripotent stem cell derived cardiomyocyte（i PS-CMs）and can avoid ethical problems,so it has a broader clinical application prospect in the future^[1].The aim of this study was to improve the survival and exosome function of MSCs,i PS-CMs by pretreatment with notoginsenoside R1（NGR1）,and to explore the effect and mechanism of MSCs and i PS-CMs transplantation after NGR1 pretreatment in the treatment of MI.It provides theoretical and experimental basis for clinical application of stem cells in the treatment of ischemic heart disease such as MI.MethodsAfter pretreated with NGR1 in vitro,the oxidative stress model of MSCs,was constructed by H₂O₂.The effects of NGR1 on cell proliferation and H₂O₂ induced injury were detected by CCK8 and TUNEL respectively,and the content of ROS in cells was detected by kit.The expression of p-Akt,p-CREB,p-Fox O1 in cells was detected by Western Blot.IGF-1,VEGF,SCF,b FGF content in the cell culture medium,and the expression of mi R-21 in the exosome of the cell culture medium was detected by ELISA.The sequence of the cells in each group was sequenced by transcript group.To investigate the protective effect of NGR1 on MSCs in vitro.IPSs,was cultured in vitro and induced to differentiate into cardiomyocytes.The effect of NGR1 on the proliferation of i PSs-CM was detected by CCK8.The level of p-Akt was detected by Western Blot.The MI model of rats and nude mice was constructed.The cardiac function of each group was detected by echocardiography after transplantation of MSCs or i PS-CMs,pretreated with NGR1,and the myocardial infarction area and collagen fiber deposition in the infarction area were measured by Masson trichromatic staining.Apoptosis was detected by TUNEL assay,the expression of HNA,c Tn T,Cx43,v WF,α-SMA,Lyve-1 in infarction area was detected by immunofluorescence staining,and the expression of IGF-1,VEGF,VEGFC,SCF,b FGF in myocardial tissue was detected by q RT-PCR.To clarify the therapeutic effect and possible mechanism of MSCs or i PS-CMs transplantation after NGR1 pretreatment.ResultsDifferent concentrations of NGR1 could promote the proliferation of MSCs.Under the condition of 300μM H₂O₂ stimulation,0.1μM NGR1 treatment for 24h or 48h could significantly preserve cell activity and protect cells from H₂O₂ damage.1μM NGR1 and10μM NGR1 treatment could also increase the cell activity of MSCs,which is obviously lower than 0.1μM.0.1μM is the best concentration to protect MSCs.After MSCs treatment with NGR1,the levels of p-Akt and p-Fox O1 increased significantly,but the level of p-CREB did not change significantly.After adding PI3K inhibitor LY294002,the levels of p-Akt and p-Fox O1 in each group were similar at each time point.DAPI and TUNEL staining showed that 300μM H₂O₂ could significantly increase the number of apoptotic MSCs and increase the content of ROS in MSCs,while NGR1 could significantly decrease the apoptosis and ROS level of MSCs,while the addition of PI3K inhibitor LY294002 could significantly decrease the apoptosis and ROS level of MSCs.The number of apoptotic MSCs increased significantly and the content of ROS increased significantly.NGR1 can promote the release of IGF-1,VEGF,SCF,b FGF,LY294002 can inhibit the promoting effect of NGR1 on the above cytokines.NGR1 treatment can also increase the level of mi R-21 in MSCs exosome.The results of transcription showed that the signal pathways enriched by NGR1 include pathway related to synthesis,metabolism,apoptosis signal pathway.Such as fatty acid metabolism,T cell receptor signal pathway,Toll-like receptor signal pathway,p53 signal pathway,MAPK signal pathway,VEGF signal pathway and so on.Other pathways are also significantly activated,such as endocytosis,cell cycle.MSCs transplantation could increase the EF and FS values and decrease the LVIDs and LVESV values in MI rats.The EF and FS values were the highest and the LVIDs and LVESV values were the lowest in the MSCs transplantation group pretreated by NGR1.Masson trichromatic staining showed that both MSCs transplantation and NGR1 pretreatment MSCs transplantation could reduce infarction area and collagen deposition.The infarction area and collagen fiber deposition in MSCs transplantation group pretreated with NGR1 were significantly lower than those in PBS group and MSCs transplantation group.The wall thickness of infarction area in PBS group was significantly decreased.Both MSCs transplantation and NGR1 pretreatment MSCs transplantation could increase the wall thickness of infarction area,and the thickness of infarction wall in NGR1 pretreated MSCs transplantation group was significantly higher than that in MSCs transplantation group.The survival of transplanted cells with GFP tracer showed that the number of MSCs in the MSCs transplantation group pretreated by NGR1 was significantly higher than that in the MSCs transplantation group.Tunel staining showed that the number of apoptotic cells in the infarction area was the most in the PBS group,and the number of apoptotic cells in the PBS group was the most.Both MSCs transplantation and NGR1 pretreatment MSCs transplantation could reduce apoptosis.Immunofluorescence staining was used to detect v WF andα-SMA.The results showed that both MSCs transplantation and NGR1-preconditioned MSCs transplantationcould increase the number of microvessels andα-SMA positive vessels in infarction area.Compared with MSCs transplantation group,the number of microvessels andα-SMA positive vessels in infarction area of MSCs transplantation group pretreated with NGR1were significantly increased.The contents of IGF-1,SDF-1 and VEGF in myocardial tissue of each group were detected by Elisa,compared with PBS group,MSCs transplantation could increase the levels of IGF-1,SDF-1 and VEGF,while the level of cytokines in NGR1 pretreated MSCs transplantation group was significantly higher than that in MSCs transplantation group.By inhibiting the signal pathway of GSK3 and Wnt,and controlling cell concentration the differentiation of hi PSs into hi PS-CMs can be effectively induced.Expression of c Tn T in differentiated cells can be detected by immunofluorescence staining.The optimum induction conditions were as follows:1 million cells per 6-well plate hole with6μM CHIR99021.After NGR1 treatment,there was no significant difference in OD value between groups.However,with the increase of drug treatment time,0.1μM,1μM and10μMNGR1 can promote the proliferation of hi PSCMs.After 72 hours of drug treatment,the 0.1μM group promoted the most obvious trend.After hi PS-CMs was treated with NGR1,the level of p-Akt increased significantly.After adding PI3K inhibitor LY294002,the activation of p-Akt was significantly inhibited.At 2 weeks after hi PS-CMs transplantation,the cardiac function index of EF and FS in i PS-CMs group（IC）was significantly improved compared with that in MI group,LVEDV and LVIDd was higher than that in Control group and MI group,and LVESV and LVIDs was lower than that in MI group.The cardiac function index EF and FS of i PS-CMs（NIC）group pretreated by NGR1 was better than that of IC group,and the recovery trend of LVEDV,LVIDd,LVESV,LVIDs index was better than that of IC group.At 4 weeks,the cardiac function index EF and FS of NIC group was significantly improved compared with that of MI group,and was better than that of IC group.Masson trichromatic staining showed that the infarct size and collagen deposition in IC group and NIC group were lower than those in IC group and NIC group.The wall thickness of infarction area in MI group was significantly decreased.Hi PS-CMs transplantation and NGR1 pretreatment i PS-CMs transplantation could increase the wall thickness of infarction area.The thickness of infarction wall in NIC group was significantly higher than that in IC group.The results of TUNEL staining showed that hi PS-CMs transplantation could decrease the number of apoptosis.Compared with hi PS-CMs transplantation,0.1μM NGR1 pretreated hi PS-CMs transplantation could significantly reduce the apoptosis of heart tissue.The results of HNA and c Tn T staining showed that NGR1 pretreatment could promote the survival of transplantation hi PS-CMs,and most of the transplanting i PS-CMs expressed c Tn T.v WF and Lyve-1 staining showed NGR1 pretreatment also increased the vessel density and lymphatic vessel density after i PS-CMs transplantation.The level of SDF-1,VEGFC,IGF-1 in hi PS-CMs transplantation group pretreated with NGR1 was significantly higher than that of hi PS-CMs transplantation group and MI group.ConclusionNGR1 can protect MSCs from apoptosis induced by H₂O₂ and promote the secretion of IGF-1,VEGF,SCF and b FGF.The mechanism may be related to PI3K/Akt/Fox O1signaling pathway.NGR1 pretreatment may enhance the effect of MSCs transplantation on MI by enhancing the survival and paracrine pathway of MSCs.The differentiation of i PS into hi PS-CMs can be effectively induced by the inhibition of GSK3 and Wnt signaling pathways and control of cell concentration.NGR1 has a tendency to promote the proliferation of hi PS-CMs and increase the level of p-Akt.Pretreatment with NGR1can enhance the effect of hi PS-CMs transplantation on MI nude mice,the main mechanism of which may be related to the enhancement of cell survival and paracrine pathway.