| Background Intestinal ischemia-reperfusion(I/R)injury is a common clinical event which caused by different pathophysiological factors,such as acute mesenteric ischemia,intestinal obstruction,bowel transplantation surgery and so on.Due to the occult incidence of intestinal ischemia-reperfusion injury and the lack of effective treatment,the mortality is very high.Exploring the strategies to reduce intestinal I/R injury is of great significance to improve organ recovery and patient survival.Bone marrow mesenchymal stem cells(BMSCs)have a positive effect on a variety of injuries,including intestinal I/R injury.It is found that this effect is mainly related to the BMSCs derived exosomes(BMSC-exo),however,the role and mechanism of BMSC-exo in intestinal I/R injury need to be further explored.Exosomes can carry mi RNAs and regulate a variety of biological processes through targeted genes.Mir-144-3p was found to be highly expressed in BMSC-exo,and has a closely relationship with the physiological process of hypoxia.Mir-144-3p can target and regulate PTEN expression,and PTEN mediated Akt/Nrf2 antioxidant stress pathway plays an important role in improving renal I/R injury.However,whether BMSC-exo can regulate PTEN/Akt/ Nrf2 signaling pathway through mi R-144-3p and to reduce intestinal I/R injury remains to be further explored.Therefore,this study intends to make an in-depth study on the effect of BMSC-exo on intestinal I/R injury,and explore its possible mechanism based on mi R-144-3p and PTEN/Akt/Nrf2 signal pathway.Methods1.Taken bone marrow from mouse femur and tibia,BMSCs were cultured by adherent cell culture.Observed the morphology of BMSCs under microscope,the expressions of CD29,CD34,CD45 and CD105 in BMSCs were detected by flow cytometry,identify the osteogenic,adipogenic and chondrogenic differentiation ability of BMSCs.Exosomes were extracted from the culture supernatant of BMSCs of 3-6 generations by ultracentrifugation,and identified exosome by transmission electron microscope,nanoparticle tracking analyzer and Western blot.2.PKH-67 labeled exosomes were co-cultured with DAPI labeled intestinal epithelial cell-6(IEC-6)for 6 h,12 h and 24 h,observing the uptake of exosomes in ice-6cells.The intestinal epithelial cell-6 cultured in cell incubator for 12 hours of hypoxia and 6 hours of reoxygenation to establish oxygen-glucose deprivation/reoxygenation(OGD/R)model.After corresponding treatments,CCK-8 method was used to detect the cell viability of each group,flow cytometry was used to detect the apoptosis rate,the expression levels of SOD,MDA and ROS were detected by ELISA,western blot used to detect the expression levels of PTEN,Akt,p-Akt and Nrf2 in IEC-6 cells.Superior mesenteric artery of mice ischemia for 45 min and reperfusion for 2 h to establish intestinal I/R model.Mice were randomly divided into 5 groups: control group,I/R group,I/R + MSC group,I/R + MSC-exo group,I/R + MSC-GW4869(GW4689 is an inhibitor of exosome biogenesis and release,except the control group,intestinal I/R models were prepared in other groups).HE staining was used to observe the pathological injury of intestinal tissue of mice in each group,Chiu's score was used to evaluate the degree of injury,TUNEL was used to detect the apoptosis of intestinal tissue,the expression levels of SOD,MDA,ROS,DAO and TNF-? were detected by ELISA,the expressions of PTEN,Akt and p-Akt in intestinal tissues were detected by Western blot,the expression of Nrf2 in intestinal tissues was measured by immunofluorescence staining.3.RT-PCR was used to detect the expression level of mi R-144-3p in control group,OGD/R group and BMSC exo group.Dual-luciferase reporter verified the targeted regulatory relationship between mi R-144-3p and PTEN.mi R-144-3p mimic/NC mimic and mi R-144-3p inhibitor/NC inhibitor were transfected into BMSCs by gene transfection technology,and the corresponding exosomes were extracted.IEC-6 cells were randomly divided into 7 groups: control group,OGD/R group,OGD/R+WT-Exo group,OGD/R+NC mimic-Exo group,OGD/R+mi R-144 mimic-Exo group,OGD/R+NC inhibitor-Exo group and OGD/R+mi R-144 inhibitor-Exo group(OGD / R cell models were established in all groups except the control group).Mice were randomly divided into 7 groups: control group,I/R group,I/R+WT-Exo group,I/R+NC mimic-Exo group,I/R+mi R-144 mimic-Exo group,I/R+NC inhibitor-Exo group,I/R+mi R-144 inhibitorExo group(except the control group,intestinal I/R models were prepared in other groups).After corresponding treatment,the cell viability,intestinal histopathological injury,intestinal cell apoptosis,the expression of SOD,MDA and ROS,as well as the expression of PTEN,Akt,p-Akt and Nrf2 in each group were detected(the relevant detection methods are described above).Results1.BMSCs are spindle,fibrous and irregular in shape.Alizarin red S staining,oil red O staining and Allicin blue staining confirmed that BMSCs had the ability of osteogenesis,adipogenesis and chondrogenic differentiation.The expression of CD29 and CD105 was positive,while the expression of CD34 and CD45 was negative in BMSCs.BMSC-exo is round or oval,the average particle size was 79.70 nm,the expression of surface markers TSG101,CD63 and CD9 were positive.2.Exosomes entered IEC-6 cells at 6 h,12 h and 24 h.Compared with the OGD/R group,BMSC-exo significantly increased cell viability and the expression of SOD,pAkt and Nrf2 in cells(P < 0.05),reduce cell apoptosis and the expression of MDA,ROS and PTEN in cells(P < 0.05).Compared with the I/R group,BMSC-exo significantly reduced intestinal histopathological injury,decrease intestinal tissue apoptosis,down regulate the expression of MDA,ROS,DAO,TNF-? and PTEN(P < 0.05),up regulate the expression of SOD,p-Akt and Nrf2(P < 0.05).The application of GW4869 reversed the effect of BMSCs on the changes of these indexes(P < 0.05).3.Compared with the control group,the expression of mi R-144-3p in OGD/R group were significantly decreased(P < 0.001).mi R-144-3p targets and negatively regulates PTEN expression.Compared with the OGD/R or I/R group,WT-Exo,NC mimicExo,mi R-144 mimic-Exo,NC inhibitor-Exo and mi R-144 inhibitor-Exo all significantly increased cell viability and the expression of SOD,p-Akt and Nrf2 in cells and intestinal tissues(P < 0.05),reduce the pathological injury and apoptosis of intestinal tissue,and the expression of MDA,ROS and PTEN in cells and intestinal tissue(P <0.05).Compared with NC mimic-Exo,mi R-144 mimic-Exo has a stronger regulatory effect(P < 0.05).The application of mi R-144 inhibitor partially reversed the regulatory effect of NC inhibitor(P < 0.05).Conclusion1.In this study,BMSCs were successfully isolated and cultured from mouse bone marrow.2.BMSC-exo were successfully extracted.3.BMSCs alleviate intestinal I/R injury through their derived exosomes.4.The effect of BMS-exo on reducing intestinal I/R injury is related to mi R-144-3p,BMSC-exo regulates oxidative stress mediated by PTEN/Akt/Nrf2 signaling pathway through mi R-144-3p,so as to improve intestinal I/R injury. |
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