The Effect Of Rh-EPO In AMPK-mTOR-ULK1_ser757pathway In Necrotizing Enterocolitis

Background:Neonatal necrotizing enterocolitis（NEC）mainly occurs in the ileum and colon of very low birth weight infants;excessive activation of autophagy may cause damage to intestinal cells.Increased phosphorylation of adenosine monophosphate activated protein kinase（AMPK）can promote autophagy.AMPK-mTOR-ULK1ser757 plays an important role as an important autophagy pathway in vivo.In addition to the treatment of premature anemia,recombinant human erythropoietin also plays an important role in intestinal autophagy.Objective:In this study,the intestinal tissue of NEC children,the neonatal rat model of NEC and IEC-6 cells of intestinal epithelial cells of rats were used as subjects.A variety of experimental techniques were used to study the role of autophagy and autophagy pathway AMPK-mTOR-ULK1ser757 in NEC from different levels of the whole,cells and molecules.To verify whether rh-EPO could reduce the severity of NEC by reducing the autophagy of NEC,and whether rh-EPO could inhibit the autophagy pathway of AMPK-mTOR-ULK1ser757 by reducing the phosphorylation level of AMPK,so as to play a protective role in the intestine.Method:Part I---Expression of autophagy-related proteins in neonatal necrotizing small intestine The intestinal tube tissue species of 10 cases of very low birth weight infants diagnosed with NEC parallel surgery in the Neonatology Department of our hospital from March 2020 to December 2021 were divided into NEC group and control group according to the severity of intraoperative necrosis and postoperative pathological grading,and the intestinal tube specimens were stained by HE staining,the expression of autophagy-related proteins beclin1 and p62 was detected by immunohistochemistry,and the protein expression of AMPK-mTOR-ULK1ser757 pathway was compared immunoblot in the two groups.Part II---The establishment of NEC animal model Newborn Sprague-Dawley（SD）rats were selected and neonatal necrotizing enterocolitis（NEC）modeling was established by combining hypoxia（8% oxygen and92% nitrogen mixture,low oxygen for 60 seconds）,cold（4 °C,10 minutes）,and different concentrations of lipopolysaccharide（LPS）gassing at 24 hours,48 hours and72 hours after birth.The execution was carried out at 96 hours after birth,and the ileal part of the upper part of ileocecal junction was retrieved.The best mold making method was selected by comparing the general situation,weight change and survival rate,and the pathological morphology of the intestinal tract and the Nadler grade（Nadler ≥level 2 considering the success of NEC molding）of different groups.Part III—The effect of autophagy on NEC and the role of erythropoietin in autophagy The newborn rats were divided into control group,NEC group,NEC + EPO group,NEC + rapamycin group,NEC + rapamycin + EPO group according to different treatment methods.At 24 hours,48 hours,and 72 hours after birth,different treatment methods were carried out according to the group,and the blood was taken for centrifugation in the heart at 96 hours,the serum was isolated for backup,and the upper part of the ileocecal junction was retrieved after death.Serum was used in ELISA to detect inflammatory factors IL-6,IL-1β,TNF-α.Autophagosomes were observed by electron microscopy,pathology and Nadler score were observed by HE staining,Expression of autophagy-related proteins beclin1,p62 and apoptosis-related proteins beclin1,p62 and apoptosis-related proteins casspase-3 and bcl-2 were detected by immunohistochemistry,and autophagy-related proteins beclin1,p62 and apoptosis-related proteins casspase-3 and bcl-2 were detected by western blotting.Protein expression of the autophagy pathway AMPK-mTOR-ULK1ser757 in the NEC group and the NEC + EPO group was compared using western blots.Part IV---The role of rh-EPO in the AMPK-mTOR-Ulk1ser757 pathway in IEC-6intestinal epithelial cells IEC-6 cells were divided into control group,LPS group,LPS + EPO group,LPS +AICAR group and LPS + AICAR + EPO group according to different treatment methods.Apoptosis was observed by flow cytometry,and the expression of autophagy proteins beclin1,p62 and apoptotic-related proteins in different groups was observed by immunofluorescence,RT-q PCR,and western blotting;the effect of EPO on the phosphorylation level of AMPK was observed by western blotting,and whether the autophagy pathway of AMPK-mTOR-ULK1ser757 could be inhibited by inhibiting the autophagy pathway of AMPK-mTOR-ULK1ser757.Result:Part I---Expression of autophagy-related proteins in neonatal necrotizing small intestine The intestinal tissue Nadler grade in the NEC group was higher than that in the control group,and the expression of the autophagy-related protein beclin1 was increased,and the expression of p62 protein was decreased,and the differences were significant.The expression of p-AMPK in the NEC group was higher than that in the control group,and the expression of p-mTOR and p-ULK1ser757 was lower than that in the control group,and the differences were significant.Part II---Establishment of an animal model of neonatal necrotizing enterocolitis The method of hypoxia and cold combined with 20mg/kg LPS intragastric administration was an effective method for establishing NEC model which success rate was over 80%.Part III---The effect of autophagy on NEC and the role of rh-EPO in NEC autophagy Compared with the control group,beclin1 expression was increased,p62 expression was reduced,autophagosomes were increased by electron microscopy,Nadler scores were increased,caspase-3 expression was increased,bcl-2 expression was decreased,inflammatory factor TNF-α,IL-6,IL-1β expression were increased in NEC group;compared with NEC group,beclin1 expression was increased,p62 expression was decreased,autophagosomes were increased under electron microscopy,and Nadler scores were increased,caspase-3 expression was increased,bcl-2 expression was decreased,inflammatory factors TNF-α,IL-6,and IL-1β were increased in NEC +rapamycin group;compared with NEC + rapamycin group,beclin1 expression was decreased,p62 expression was increased,autophagosomes were decreased under electron microscopy,Nadler scores were decreased,caspase-3 expression was decreased,bcl-2 expression was increased,inflammatory factor TNF-α,IL-6,IL-1β were decreased in NEC+ rapamycin + EPO group;compared with NEC group,the expression of p-AMPK was decreased,p-mTOR and p-ULK1ser757 were increased in NEC+EPO group.Part IV---Mechanism of action of rh-EPO on AMPK-mTOR-Ulk1 pathway in IEC-6intestinal epithelial cells Compared with the control group,beclin1 expression was increased,p62 expression was decreased,caspase-3 expression was increased,and bcl-2 expression was decreased in LPS group;compared with the LPS group,beclin1 expression was decreased,p62 expression was increased,caspase-3 expression was decreased,and bcl-2 expression was increased in LPS + EPO group;compared with the LPS group, p-AMPK expression was increased,p-mTOR and p-ULK1ser757 expression were decreased,beclin1 expression was increased,p62 expression was decreased,caspase-3expression was increased,bcl-2 expression was decreased in LPS + AICAR group;compared with LPS + AICAR group,p-AMPK expression was decreased,p-mTOR and p-ULK1ser757 expression were increased,beclin1 expression was decreased,p62 expression was increased,caspase-3 expression was decreased,Bcl-2 expression was increased in LPS + AICAR + EPO group.Conclusion:Intestinal tissue autophagy in the NEC group was enhanced,and AMPK-mTOR-ULK1ser757 may be involved in autophagy of the intestine.The method of cold hypoxia combined with LPS（20mg/kg）gastric filling is a mold making method for establishing NEC models,and the success rate of mold making exceeds 80%.Autophagy activation may lead to intestinal apoptosis,increased inflammatory factors,rh-EPO may reduce autophagy by participating in the AMPK-mTOR-ULK1ser757 pathway,thereby reducing the severity of NEC.Autophagy may be involved in the apoptosis of IEC-6 cells,rh-EPO may reduce the apoptosis of IEC-6 cells by inhibiting autophagy,the mechanism of which may be by inhibiting the AMPK phosphorylation level of the AMPK-mTOR-ULK1ser757 pathway,thus playing a protective role for intestinal epithelial cells.