外泌体miR-885-5p靶向Hmgb1调控急性胰腺炎细胞凋亡的实验研究OA

Objective:To investigate the role and mechanism of exosomal miR-885-5p targeting High mobility group protein b1(Hmgb1)on acute pancreatitis cell apoptosis.Methods:The untreated rabbit pancreatic immortalized follicular cells were used as the Control group,and rabbit pancreatic follicular immortalized cells were treated with sainfoin at a concentration of 1×10-7 mol/L combined with 10 μg/mL lipopolysaccharide for 24 h to establish an in vitro model of acute pancreatitis(AP),and the su-pernatants of the cells in the Control group and the model group were separated from the exosomes,and the morphology of exo-somes was observed by transmission electron microscopy.Exosomes were isolated from the supernatants of Control and model group cells,the morphology of exosomes was observed by transmission electron microscopy,the particle size analysis of exo-somes was performed by nanoparticle tracking analyzer,the exosomal protein markers were detected by Western blot,The expres-sion of miR-885-5p in exosomes was detected by RT-qPCR.Construct miR-885-5p overexpression lentiviral and empty viral vec-tors,miR-885-5p low-expression lentiviral and empty viral vectors were transfected with model cells to establish miR-885-5p mim-ics group and mimics NC group,miR-885-5p inhibitor group and inhibitor NC group.RT-qPCR detected the mRNA expression of miR-885-5p,Hmgb1,Caspase-3,Bax and Bcl-2.Western blot detected the protein expression of Hmgb1,Caspase-3,Bax,and Bcl-2 in the cells of each group.Flow cytometry detected the apoptosis rate of cells in each group.Hoechst staining was used to ob-serve the apoptosis of cells.Dual luciferase reporter assay verified the targeting relationship between miR-885-5p and Hmgb1.Re-sults:The diameters of cellular exosomes in both Control and severe AP groups were about 50-200 nm,with tea-to-shaped outer vesicles and double-membrane structure.Western blot results showed that CD63,HSP70,and TSG101 were expressed;and the expression of exosomal miR-885-5p was up-regulated in the severe AP group(P<0.01).Compared to the Control group,miR-885-5p expression was up-regulated in the severe AP group(P<0.01),mRNA and protein expression of Hmgb1,Cas-pase-3,and Bax was up-regulated(P<0.01),mRNA and protein expression of Bcl-2 was decreased(P<0.01),and apopto-sis rate was increased(P<0.01).Compared with the mimics NC group,miR-885-5p miR-885-5p expression was up-regulated in the miR-885-5p mimics group(P<0.01),the mRNA and protein expression of Hmgb1 and Caspase-3(P<0.01),the mRNA expression of Bax(P<0.01)as well as the protein expression of Bax(P<0.05).The mRNA and protein expression of Bcl-2 was decreased(P<0.05),and the apoptosis rate was increased(P<0.01).Compared with the inhibitor NC group,miR-885-5p expression was down-regulated in the miR-885-5p inhibitor group(P<0.05),mRNA and protein expression of Hmgb1 and Bax(P<0.01),mRNA expression of Caspase-3(P<0.05)and protein expression(P<0.01),elevated mRNA and protein expres-sion of Bcl-2(P<0.01),and decreased apoptosis(P<0.01).Dual luciferase gene detection reporter assay showed Hmbg1 as the target gene of miR-885-5p.Conclusion:Exosomal miR-885-5p was highly expressed in severe acute pancreatitis model cells.The up-regulation of miR-885-5p expression promoted apoptosis,while inhibition of miR-885-5p expression reversed this effect.The mechanism may be related to targeting HMGB1,which promotes the expression of Bax and Caspase-3,and inhibits the expression of Bcl-2.