| Objective: Sepsis is a life-threatening organ dysfunction caused by the imbalance of body response caused by infection,with high morbidity and mortality.It is also one of the prime causes of pediatric mortality and the occupation of medical resources worldwide.Septic shock is the subtype of sepsis,and sepsis-induced myocardial depression is one of the complications of septic shock and the main cause of death.The pathogenesis of myocardial depression is varied and complex,among which the expression level of some specific miRNA in plasma is closely related with the severity of sepsis.Exosomes carrying miRNAs play important roles in intercellular communication and information transfer.Among them,exosomes from placenta-derived mesenchymal stem cells(PDMSC)have attracted increasing attentions due to their unique advantages in carrying specific miRNAs involved in the repair of damaged organs.The role of miR-126a-3p in the repair of myocardial ischemic injury has been confirmed by a number of studies,but its role in sepsis-induced myocardial depression and the related molecular mechanisms have not been reported.Based on current research background,this study intended to transfect PDMSC with agomiR-126a-3p and agomiRNC,and extracted their exosomes.The adolescent rat and H9c2 model of septic myocardial depression were generated by LPS treatment.Exosomes from placental mesenchymal stem cell(Exo-agomiR-126a-3p)loaded with miR-126a-3p were administered to investigate the effect and mechanism of sepsis-induced myocardial depression induced in vivo and in vitro.Methods: Part Ⅰ: The effects of Exo-agomiR-126a-3p on myocardial apoptosis and pyroptosis induced by sepsis(1)We performed primary culture of PDMSC and identified CD29、CD73、CD90、CD105 、 CD34 、 CD45 and HLA-DR expression by flow cytometry.PDMSC was transfected with agomiR-126a-3p and agomiR-NC for 48 h.We Extracted the exosomes from the supernatant.The expressions of CD9,CD63,CD81,TSG101 and Calnexin in PDMSC-Exos were detected by Western blot.The morphology of PDMSC-Exos was observed by transmission electron microscopy.The range of particle size of PDMSCExos was determined by Nanoparticle-Tracking Analysis(NTA).The expression level of miR-126a-3p in exosomes was detected by Real-time PCR.(2)The model of sepsis-induced myocardial depression was generated by intraperitoneal injection of LPS.The Wistar rats were divided into 4 groups randomly as following: Control group,LPS group,LPS+Exo-agomiR-NC group,and LPS+ExoagomiR-126a-3p group.At 24 h before modeling,rats in the interventional group were given caudal intravenous injection of Exo-agomiR-126a-3p and Exo-agomiR-NC,respectively.After 6h of LPS administration,we detected the cardiac function.We used Real-time PCR to detect the expression level of miR-126a-3p.ELISA was used to detect the levels of IL-1β and IL-18.Serum LDH,CK-MB and c Tn I levels were detected by related kits.The pathological changes of myocardial tissue in each group were compared by HE staining.Myocardial cell apoptosis was detected by TUNEL staining.The expression of apoptosis-related proteins,as well as NLRP3,ASC,GSDMD-N and cleaved caspase-1 in myocardial tissue were detected by immunofluorescence and Western blot.(3)H9c2 rat cardiomyocytes were co-treated with Exo-agromiR-126a-3p and ExoagromiR-NC for 48 h,and miR-126a-3p levels in H9c2 cardiomyocytes were detected by Real time PCR.After intervention with exosome blockers,the expression of miR-126a-3p in H9c2 myocardial cells was detected.We treated H9c2 cells with LPS and ExoagromiR-126a-3p,as well as LPS and Exo-agromiR-NC,respectively.Flow cytometry was used to detect the apoptosis of H9c2 cardiomyocytes,and Western blot was used to detect the expression of apoptosis related genes and the expression of NLRP3,ASC,GSDMD-N and cleared caspase-1.Additionally,ELISA detected the levels of IL-1β and IL-18 in cell supernatant.Part Ⅱ: Study on the mechanism of sepsis-induced myocardial depression by placental mesenchymal stem cell exosomes loaded with miR-126a-3pThe expression levels of Akt,p-Akt,NF-κB(cytoplasm),NF-κB(nucleus),p-IκBαand IκBα in myocardial tissue of rats and H9c2 rat cardiomyocytes of each group were detected by Western blot.To investigate the regulatory mechanism by which placental mesenchymal stem cell exosomes loaded with miR-126a-3p affect myocardial depression induced by sepsis at the animal level and the cellular level.We performed the Western blot to detect the expression levels of Akt,p-Akt,NF-κB(cytoplasm),NF-κB(nucleus),p-IκBα and IκBα in myocardial tissue of each group in vivo.Also,H9c2 cells were treated with LPS and Exo-agromiR-126a-3p,as well as LPS and Exo-agromiR-NC,respectively and the aforementioned indicators were detected by Western blot.Part Ⅲ: Verification of the regulatory relationship between miR-126a-3p and target gene,and the functional research of target gene.(1)Bioinformatics software was used to screen the target genes of miR-126a-3p.Target binding prediction of miR-126a-3p and target genes.Binding of miR-126a-3p to target genes verified by dual luciferase assay.H9c2 cells were treated with Exo-agomiR-126a-3p and Exo-agomiR-NC for 48 h,respectively.The expression levels of target genes in H9c2 cells were detected by Real-time PCR and Western Blot(2)H9c2 cells were transfected with target gene overexpression and empty vector.The transfected H9c2 cells were treated with LPS and Exo-agomiR-126a-3p,and apoptosis was detected by flow cytometry.The expression levels of NLRP3,ASC,GSDMD-N,cleaved caspase-1 were detected by Western blot.ELISA was used to detect the levels of IL-1β and IL-18 in cell supernatant.Results: Part Ⅰ: Exosomes from Placental mesenchymal stem cell loaded with miR-126a-3p can inhibit myocardial apoptosis and pyroptosis which induced by LPS and improve myocardial depression induced by sepsis(1)The identify of PDMSC displayed,as CD29,CD73,CD90 and CD105 were positively expressed;CD34,CD45 and HLA-DR were negatively expressed.Under transmission electron microscope,exosomes showed cup-bracket lipid bilemolecular membrane structure.NAT showed that the ranges of exosome particle size was from 90 nm to 200 nm.The results from Western blot showed that CD9,CD63,CD81,TSG101 and Calnexin were not expressed in the supernatant of PDMSC cells.Moreover,CD9,CD63,CD81 and TSG101 were highly expressed in Exo-agomiR-NC group and ExoagomiR-126a-3p group,while Calnexin was not expressed.Mi R-126a-3p was expressed in both Exo-agomiR-NC group and Exo-agomiR-126a-3p group,and the expression was significantly up-regulated in Exo-agomiR-126a-3p group(P<0.01).(2)Cardiac function test showed that the LVEDP was significantly increased,the+dp/dtmax and the-dp/dtmax were significantly decreased(P<0.01).Exo-agomiR-126a-3p intervention mitigated the above changes.Mi R-126a-3p showed low expression in myocardial tissue of LPS group,and the expression of miR-126a-3p was significantly up-regulated in the interventional group with Exo-agomiR-126a-3p(P<0.01).The detection of LDH,CK-MB and c Tn I in serum dispalyed that the levels of all in LPS group were significantly up-regulated compared with control group(P<0.01).All these indicators were downregulated after intervened by Exo-agomiR-126a-3p(P<0.05).HE staining results showed that the myocardial cells in the control group were well arranged,but the myocardial cells in LPS group were edema,inflammatory cells infiltrated,and the intercellular space widened.After intervened by Exo-agomiR-126a-3p,the pathological changes were significantly reduced.TUNEL results showed that compared with the control group,the apoptotic rate of myocardial cells in LPS group was significantly increased(P<0.01).Compared with LPS group,the apoptosis rate in LPS+Exo-agomiR-126a-3p group was significantly decreased(P<0.05).The results from immunofluorescence and Western blot showed that the expressions of Bax and cleaved caspase-3 were significantly up-regulated,and the expression of Bcl-2 was significantly down-regulated in LPS group(P<0.01).After intervened by Exo-agomiR-126a-3p,the expression of Bax and cleaved caspase-3 were significantly down-regulated,and the expression of Bcl-2 was significantly up-regulated(P<0.01).The results from immunofluorescence and Western blot also showed that the expression of NLRP3,ASC,GSDMD-N,and cleaved caspase-1 in LPS group were significantly up-regulated(P<0.01).After intervened by Exo-agomiR-126a-3p,the above proteins were significantly decreased(P<0.01);ELISA results showed that compared with the control group,the levels of IL-1β and IL-18 were significantly up-regulated in LPS group and significantly down-regulated after intervened by Exo-agomiR-126a-3p.(3)The results of Real-time PCR displayed that compared with control group and Exo-agomiR-NC group,the level of miR-126a-3p in Exo-agomiR-126a-3p group was up-regulated after LPS and Exo-agomiR-126a-3p treatment in vitro,as well as LPS and Exo-agomiR-NC treatment,respectivel(P<0.01).The expression of miR-126a-3p in H9c2 cells co-cultured with PDMSC transfected with agomiR-126a-3p was significantly up-regulated(P<0.01).After GW4869 was applied to block exosome secretion,the expression of miR-126a-3p was significantly down-regulated(P<0.01),suggesting that exosomes could transport miR-126a-3p to H9c2 cells.Flow cytometry showed that apoptosis rate was significantly increased in LPS group compared with control group.The results from Western blot analysis of apoptosis-related proteins showed that LPS induced high expression of pro-apoptosis-related proteins,while low expression of anti-apoptosis-related protein.Exo-agomiR-126a-3p can reverse the changes of the above apoptotic proteins.Compared with control group,NLRP3,ASC,GSDMD-N and cleaved caspase-1 were higher in LPS group than in control group(P<0.01).ELISA results showed that the expressions of IL-1β and IL-18 were significantly increased in LPS group compared with control group,which were significantly decreased compared with LPS group after intervened by Exo-agomiR-126a-3p.Part Ⅱ: Placental mesenchymal stem cell exosomes loaded with miR-126a-3p regulate sepsis-induced myocardial depression via the AKT/NF-κB signaling pathway.Compared with the control group,the expressions of P-Akt and NF-κB p65 in cytoplasm about LPS group were down-regulated(P<0.01),and the expressions of NF-κB and p-IκBα in nucleus were up-regulated(P<0.01).Compared with LPS group,the expressions of P-Akt and NF-κB p65 in cytoplasmic cells about LPS+Exo-agomiR-126a-3p group were up-regulated(P<0.01),while the expressions of NF-κB and p-IκBα in nucleus were down-regulated(P<0.01).Part Ⅲ : The target gene for miR-126a-3p was screened from the database as PIK3R2,and regulatory relationship was verified as well as the function of the target gene was studied(1)The database screened the target gene of miR-126a-3p including PIK3R2 and predicted the target binding site of between miR-126a-3p and PIK3R2.Luciferase assay verified the targeted binding of miR-126a-3p to PIK3R2.The results from Western blot analysis showed that compared with PDMSC-agomiR-NC+H9c2 group,the levels of PIK3R2 in PDMSC-agomiR-126a-3p+H9c2 group were significantly down-regulated after intervened by GW4869(P<0.01).Compared with PDMSC-agomiR-NC+H9c2group,the levels of PIK3R2 in PDMSC-agomiR-126a-3p+H9c2+GW4869 group was significantly up-regulated(P<0.01).(2)Flow cytometry showed that compared with vector group,the apoptosis rate of PIK3R2 overexpression group was increased(P<0.05).The results from Western blot analysis showed that compared with vector group,the expressions of NLRP3,ASC,GSDMD-N,cleaved caspase-1 in PIK3R2 overexpression group were significantly upregulated(P<0.01).Compared with the vector group,the levels of IL-1β and IL-18 in PIK3R2 overexpression group were significantly up-regulated by ELISA.Conclusion: 1.Exosomes from placenta-derived mesenchymal stromal cells loaded with miR-126a-3p could inhibit the occurrence of myocardial apoptosis and pyroptosis,and alleviate myocardial deression induced by sepsis in vivo and in vitro.2.Exosomes from placenta-derived mesenchymal stromal cells loaded with miR-126a-3p can activate Akt and inhibit NF-κB pathway by targeting PIK3R2,thereby inhibiting myocardial apoptosis and pyroptosis induced by sepsis in vivo and in vitro,respectively.3.Exosomes from placenta-derived mesenchymal stromal cells contain miR-126a-3p and can transport miR-126a-3p to act on myocardial cells. |
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