| Objective: Ulcerative colitis(UC)is a chronic inflammatory bowel disease caused by heredity,immune and environmental factors,which can lead to intestinal inflammatory changes and immune disorders.Studies have shown that Hypoxic(HP)preconditioning enhances the therapeutic effect of bone marrow mesenchymal stem cells(BMSCs)and their derived Exosomes(Exos)in various diseases.At the same time,compound sophorae decoction has been proven to be effective in the treatment of UC,but the specific mechanism of action is unclear.The aim of this study was to investigate the effects of hypoxia on the activity,migration ability and apoptosis of BMSCs and the expression of miRNA in Exos.Subsequently,the mechanism of mitophagy in UC intestinal epithelial cells promoted by HP-Exos and the intervention effect of compound sophorae decoction was further investigated.Methods: The BMSCs and Exos were identified by flow cytometry,transmission electron microscopy(TEM),NTA and Western Blot(WB).BMSCs and Exos were extracted after lentivirus transfection and hypoxia pretreatment.The effects of hypoxia on BMSCs were studied by CCK8,wound healing experiment and flow cytometry.The effects of hypoxia on Exos gene expression were further studied by miRNA sequencing.HE/PAS staining,WB,q RT-PCR,immunofluorescence staining,flow cytometry and other methods were used to study the effect of HP-Exos on improving intestinal mucosal injury in UC rats.Subsequently,the target and pathway of the treatment of UC by compound sophorae decoction were screened by network pharmacology,and the metabolites of compound sophorae decoction were analyzed by UPLC-MRM.Finally,special staining of mitochondrial membrane potential,TEM,flow cytometry,ELISA,WB and other techniques were used to investigate the mechanism of compound sophorae decoction combined with HP-Exos in the treatment of DSS-induced UC rats.Results: Hypoxia pretreatment for 24 h significantly enhanced the activity and migration ability of BMSCs and inhibited their apoptosis,but the hypoxia effect was reversed after HIF-1α knockdown.The results showed that BMSCs cultured with high purity.BMSC-Exos and HP-Exos were identical in morphology and surface labeling,but hypoxia could change the miRNAs and proteins carried by Exos.Among them,miR-24-3p,miR-199a-5p and miR-140-3p are known to have significant differences.Compared with Exos,HP-Exos could more effectively relieve the pathological injury and barrier destruction of UC colon tissue,inhibiting ROS accumulation,cell apoptosis and DNA damage in intestinal epithelial cells,regulating T cell differentiation and balancing Th1/Th17/Treg cells.HP-Exos can promote mitophagy of LPS-induced HT-29 cells and DSS-induced intestinal epithelial cells of UC rats,inhibit oxidative stress,apoptosis and inflammatory factor expression,but HIF-1α silence significantly reversed the regulatory effect of HP-Exos.Network pharmacological studies have found that compound sophorae decoction can target HIF-1α and may regulate mitophagy through HIF-1α-BNIP3.This study verified the hypothesis through vivo and vitro experiments that compound sophorae decoction combined with HP-Exos can enhance its regulatory effect on mitophagy and better alleviate intestinal mucosal damage of UC.Conclusions: Hypoxia enhances the therapeutic effect of BMSCs and Exos.HP-Exos can regulate mitophagy,ROS accumulation,apoptosis and T-cell immune balance of UC intestinal epithelial cells through HIF-1α-BNIP3 pathway,and compound sophorae decoction combined with HP-Exos has a better effect in the treatment of UC.The above research results provide the basis and ideas for finding more effective treatment of UC.The miRNA modified by HP-Exos has not been studied in detail in this study.The study of miRNA can provide a more comprehensive understanding of the therapeutic effect of HP-Exos,which is worthy of further exploration. |
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