| Background:Pulmonary arterial hypertension（PAH）is a group of serious progressive pulmonary remodeling diseases,which often leads to increased pulmonary vascular resistance and right ventricular failure,and eventually leads to the death of patients.In 2021,the New England Journal of Medicine estimated that 1%of the world’s total population and as many as 10%of the elderly over 65 suffered from pulmonary hypertension.The patient has severe late symptoms,poor quality of life and high mortality.In recent years,targeted drugs have developed rapidly,but the mortality rate of PAH patients has not decreased significantly,and the five-year mortality rate remains at around 50%.Although some PAH patients can achieve the purpose of relieving symptoms or even curing diseases through operations such as pulmonary endarterectomy,balloon pulmonary angioplasty,Potts shunt,pulmonary artery denervation,balloon atrial septostomy,lung transplantation and combined heart-lung transplantation,the number of them is limited due to various factors such as the difficulty of surgery and the small number of transplant donors.Mesenchymal stem cells（MSCs）are a kind of pluripotent stem cells derived from mesoderm,and have become the most widely studied stem cells in the field of stem cell transplantation because of their wide clinical application prospects and little moral and ethical controversy.After MSCs is transplanted into the body and returned to the injured lung,it can repair the damaged pulmonary vascular endothelial cells through directional differentiation,and can also secrete cytokines such as VEGF,chemokines,growth factors and extracellular vesicles through paracrine to improve pulmonary vascular remodeling,thus treating PAH.At present,the overall results of various studies on the treatment of PAH by MSCs are very promising.However,there are two important problems in the treatment process.One problem is how to deliver the transplanted cells to the injured site.This process is called homing.MSCs has the characteristic of specific homing to the injured tissue,but the amount of homing in the target site is very small.The homing rate of MSCs directly determines its therapeutic effect.SDF-1/CXCR4 axis is the key signal to mediate MSCs homing.The injured tissue cells release SDF-1 chemokine as ligand,and CXCR4 as receptor exists on the surface of MSCs,which can migrate to the injured tissue cells along the concentration gradient of SDF-1.Therefore,it is very important to increase the content of CXCR4 protein on the surface of MSCs to increase homing.Secondly,after MSCs is transplanted into the body,it faces a severe oxidative stress environment,and there are many mechanisms of pulmonary hypertension that can produce severe oxidative stress.Excessive or persistent stimulation of oxidative stress can reduce the function of MSCs and increase the apoptosis of MSCs.The fewer MSCs survive,the worse the curative effect.Anti-apoptosis protein Bcl-2 can inhibit cell apoptosis.At present,a large number of experiments use Bcl-2 to reflect the degree of apoptosis,and increasing Bcl-2 can reduce apoptosis.Therefore,it is a hot issue to try to use different methods to treat MSCs,reduce the apoptosis of MSCs,increase the homing and paracrine function of MSCs,and finally improve the curative effect of MSCs transplantation by activating different pathways.HIF pathway is the star pathway of the 2019 Nobel Prize in Physiology or Medicine.The most important HIF family is HIF-1α.After the activation of HIF pathway,HIF-1α and HIF-1β form HIF-1 dimer,which acts as a transcription factor and is closely related to apoptosis,cell migration and secretion of various factors.YC-1 can completely inhibit the expression of HIF-1α at the post-transcriptional level,and is a specific blocker of HIF-1α.Prostaglandin E1（PGE1）is a naturally occurring prostaglandin.In this study,PGE1 was used to treat MSCs,and the effects of PGE1 on apoptosis,homing and paracrine of MSCs under oxidative stress environment and the role of HIF signaling pathway were explored.To verify whether PGE1 pretreated MSCs and transplanted into PAH rats can improve the therapeutic effect of PAH.Part oneIn vitro study on PGE1 reducing apoptosis and increasing homing of MSCs by regulating HIF-1α.Objective:To investigate the effects of PGE1 on apoptosis,homing and paracrine of MSCs under oxidative stress and the role of HIF signaling pathway in it.Methods:1.MSCs was extracted from rat lower limb bone and identified by flow cytometry.2.MSCs was treated with different concentrations of PGE1（5ng/mL,10ng/mL,20ng/mL）for 24 hours,and the levels of CXCR4,Bcl-2 and VEGF were detected by Western blot（WB）and ELISA,so as to explore the most suitable experimental concentration in the future.3.Choose 10 ng/mL as the concentration of PGE1 in the follow-up experiment,and treat it with 100μM YC-1 for 12 hours before treating it with PGE1 to explore the relationship between PGE1 and HIF pathway.In order to study the survival and migration of MSCs under oxidative stress,the medium was changed after pretreatment with PGE1 and YC-1 for 24 hours,and then treated with 200μM H2O2for 12 hours to simulate the oxidative stress environment.4.The experiment was divided into four groups:control group,group H2O2,group H2O2+PGE1,and group H2O2+PGE1+YC-1.4.Through WB experiment,the contents of HIF-1α,CXCR4 and Bcl-2 protein in each group were detected.5.The degree of apoptosis in each group was evaluated by Annexin V-FITC/PI double staining flow cytometry.6.Through Transwell migration test,the migration and homing of each group were studied.7.The expression of CXCR4 on MSCs surface was analyzed by flow cytometry.8.The concentration of VEGF in each group was measured by ELISA.Results:1.MSCs was successfully extracted and cultured in vitro,and the cell population used in the experiment was confirmed by flow cytometry,and the cell purity was high,which could meet the requirements of subsequent experiments.2.With the increase of PGE1 concentration,the levels of Bcl-2,CXCR4 and VEGF secretion in MSCs gradually increased.When 20ng/mL is used,the effect is not obviously increased compared with 1Ong/mL,so the concentration of PGE1 is 1Ong/mL in subsequent experiments.3.In the control group,the amount of apoptosis of MSCs was very small,and WB suggested that the level of Bcl-2 protein was high and that of HIF-1α protein was low.Apoptosis in H2O2group increased obviously,and WB indicated that Bcl-2 decreased obviously and HIF-1α protein changed little.Compared with H2O2+PGE1 group,the apoptosis of H2O2group decreased,and WB indicated that the levels of Bcl-2 and HIF-1α protein increased.Compared with H2O2+PGE1+YC-1 group,the apoptosis of H2O2+PGE1 group increased,and WB indicated that the levels of Bcl-2 and HIF-1α protein decreased.H2O2can induce the apoptosis of MSCs,PGE1 can reduce the apoptosis of MSCs,and YC-1 can reverse the protective effect of PGE1.4.Transwell migration test showed that the migration of MSCs induced by SDF-1α in control group and H2O2group was less,and the migration of MSCs in H2O2+PGE1 group was significantly higher than that in H2O2group.The migration of MSCs in H2O2+PGE1+YC-1 group was less than that in H2O2+PGE1 group.5.WB experiment and flow cytometry showed that the content of CXCR4 in MSCs cell and cell surface in H2O2+PGE1 group was higher than that in H2O2group.Compared with H2O2+PGE1+YC-1 group,the content of CXCR4 in MSCs cells and on the cell surface decreased.6.ELISA experiment showed that the VEGF secreted by MSCs in H2O2+PGE1 group was significantly higher than that in H2O2group.The VEGF secreted by MSCs in H2O2+PGE1+YC-1 group was lower than that in H2O2+PGE1 group.Conclusion:PGE1 pretreatment of MSCs can increase the protein content of Bcl-2 and reduce the apoptosis of MSCs under oxidative stress by activating HIF-1α pathway.Increase the protein content of CXCR4 and increase the migration of MSCs to the injured site;It can also increase the secretion of VEGF by MSCs by activating HIF-1α pathway.Part twoIn vivo study of PGE11 improving the therapeutic effect of MSCs on pulmonary arterial hypertension by regulating HIF-1α,Objective:to verify whether PGE1 pretreated MSCs and transplanted into PAH rats can improve the therapeutic effect of PAH.Methods:1.In order to trace cells in vivo,MSCs was stained with CM-DIL before transplantation.2.The experimental rats were randomly divided into five groups:（ⅰ）the control group（n=6）was injected with normal saline intraperitoneally,and PBS was injected into the tail vein three days later;（Ⅱ）MCT group（n=6）,MCT was injected intraperitoneally,and PBS was injected into the tail vein 3 days later;（Ⅲ）MCT+Non-PCMSC group（n=12）,MCT was injected intraperitoneally,and untreated MSC was injected through tail vein three days later;（Ⅳ）MCT+PGE1-PCMSC group（n=12）,MCT was injected intraperitoneally,and PGE1-treated MSC was injected into the tail vein three days later;（Ⅴ）MCT+PGE1+YC-1-PCMSC group（n=12）,MCT was injected intraperitoneally,and MSC treated with PGE1+YC-1 was injected into the tail vein three days later.3.One week after MSC transplantation,the proportion of MSC homing to rat lung tissue was observed by fluorescence microscope to detect the homing ability of MSC.4.One week after 4.MSC transplantation,the levels of MDA and VEGF in rat blood were measured by ELISA.5.Four weeks after the injection of MCT4,the systolic pressure,diastolic pressure and mean pressure of pulmonary artery were measured.Measure and calculate the ratio of right ventricle and left ventricle+interventricular septum weight[RV/（LV+S）].Lung tissu e was stained with HE,and the wall thickness index（%WT）and relative wall area index（%WA）of pulmonary arterioles were measured and calculated.Results:1.Compared with the control group,the serum MDA level of rats in MCT group increased significantly,while that in MCT+Non-PCMSC group decreased slightly;MCT+PGEI-PCMSC group decreased significantly;MCT+PGE1+YC-1-PCMSC group increased.2.Compared with the control group,the serum VEGF level in MCT group was slightly lower.MCT+Non-PCMSC increased slightly;MCT+PGE1-PCMSC increased significantly;MCT+PGE1+YC-1-PCMSC group decreased slightly.3.In MCT+Non-PCMSC group,there were very few homing MSCs;In MCT+PGE1-PCMSC group,the number of homing MSC increased significantly.However,the number of homing MSC in MCT+PGE1+YC-1-PCMSC group was significantly lower than that in MCT+PGE1-PCMSC group.4.Compared with the control group,the mean pulmonary artery pressure and systolic blood pressure of rats in MCT group increased significantly;After injection of untreated MSC,it decreased slightly;After injection of MSC treated with PGE1,it decreased significantly;After injection of PGE1+YC-1 treated MSC,it was significantly higher than that of MCT+PGE1-PCMSC group.5.The right ventricular hypertrophy index of rats in MCT group was significantly higher than that in control group;In MCT+Non-PCMSC group,it decreased slightly;MCT+PGE1-PCMSC group decreased significantly;The MCT+PGE1+YC-1-PCMSC group was significantly higher than that of MCT+PGE1-PCMSC group.6.The vascular wall thickness index and relative wall area index of rats in MCT group were significantly higher than those in control group.In MCT+Non-PCMSC group,it decreased slightly;MCT+PGE1-PCMSC group decreased significantly;The MCT+PGE1+YC-1-PCMSC group was significantly higher than that of MCT+PGE1-PCMSC group.Conclusion:Oxidative stress in rats with pulmonary hypertension is very strong.PGE1 can activate HIF pathway after preconditioning MSC.MSC transplantation can increase the homing of MSC,significantly reduce the average pulmonary artery pressure,systolic blood pressure,vascular wall thickness index and relative wall area index of rats,which is better than transplantation of untreated MSC.YC-1 can block this effect by inhibiting HIF pathway in MSC.Innovation and significanceThis study confirmed that PGE1 pretreatment of MSCs can reduce the apoptosis of MSC under oxidative stress,increase the migration of MSCs to the injured site and increase the secretion of VEGF by activating HIF-1α pathway.PGE1 pretreated MSCs and transplanted into PAH rats can obviously improve the therapeutic effect of PAH.This topic provides a theoretical basis and new ideas for improving the effect of MSCs transplantation. |
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