| Objective:By establishing multivariate necrotizing enterocolitis(NEC) model in newborn rats(formula fed + hypoxia + cold stress + LPS fed) and being fed fish oil via orogastric gavage and intraperitoneal injection of salubrinal(which is a selective inhibitor of eukaryotic initiation factor-2a dephosphorylation) for intervention, at the same time, to study the dynamic expression of the related molecules PERK, p-PERK, e IF2а, p-e IF2а,CHOP in the PERK/e IF2а/CHOP signaling pathway of endoplasmic reticulum stress(ERS), to further explore the role of ERS signaling pathway in the pathogenesis of NEC and reveal the possible protective molecular mechanism of fish oil in NEC model, and in order to provide new ideas and experimental basis for clinical prevention and treatment on NEC.Materials and Methods:240 neonate Sprague-Dawley(SD) rats aged 1 day, body mass 5~10g, regardless of male or female, breastfeeding to 7 days of age,body mass 13~18g, were randomly divided into six groups: NEC group(NEC, n=40), fish oil prevention group(FO, n=40), salubrinal prevention group(SAL, n=40), salubrinal and fish oil combined intervention group(SF,n=40), control group(CON1,n=40), DMSO group(CON2,n=40).NEC group:From postnatal 8 day, every day neonatal rats fed with formula,experienced hypoxia and cold stress, and LPS fed via orogastric gavage to induce NEC model,the process lasted for 3days,at the same time fed normal saline(NS)(0.6ml /100 g.d) via orogastric gavage and intraperitoneal injection of NS(1 ml / 100 g.d) once a day. FO group: SD neonatal rats on the 8th day for three consecutive days to establish NEC model(as stated above), while daily fed with fish oil(the concentration of fish oil was 35%, dose of fish oil was 0.6ml/100 g.d) via orogastric gavage and intraperitoneal injection of the same amount of NS once.SAL group: SD neonate rats on the 8th day for three consecutive days to establish NEC model(as stated above), while daily fed with the same amount of NS via orogastric gavage and intraperitoneal injection of Salubrinal(1mg/kg) once. SF group: SD neonate rats on the 8th day for three consecutive days to establish NEC model(as stated above), while daily fed with fish oil(the concentration of fish oil was 35%, dose of fish oil was 0.6ml/100 g.d) via orogastric gavage and intraperitoneal injection of Salubrinal(1mg/kg) once. CON1 group: 7-day-old newborn SD rats continued to stay with their mothers and breast fed, and from the postnatal 8th day fed with the same amount of NS via orogastric gavage and intraperitoneal injection of the same amount of NS once for three days.CON2 group: 7-day-old newborn SD rats continued to stay with their mothers and breast fed, and from the postnatal 8th day fed the same amount of NS via orogastric gavage and intraperitoneal injection of 1% DMSO(1ml / 100 g.d)once for three days.Observed rats’ general situation at 0h, 12 h, 24 h, 48 h and 72 h of the modeling process and incidence of NEC at 72 h in six groups. Eight rats selected from each group randomly were decapitated respectively at 0h, 12 h, 24 h, 48 h and 72 h in six groups, obtained duodenum inferior to rectum superior intestinal tissue, observed rats’ general situation, the changes of intestinal pathology by HE staining and the intestinal tissue injury scores to assess the degree of intestinal injury. Expression of PERK, p-PERK,e IF2а,p-e IF2а,CHOP proteins were detected by Western-blot technique and the expression levels of CHOP m RNA gene by reverse transcription-polymerase chain reaction(RT-PCR).Results:1. The performance and general condition of the neonatal rats in six groupsIn CON1 and CON2 groups, the neonatal rats had normal activities.In NEC group,the neonatal rats had decreased activities, decreased responsiveness, tachypnea, slow bodyweight gain, reduced subcutaneous fat, loose skin,abdominal distension, Yellow-green viscous watery faeces, frequent defecation after stimulus, which was worsened by sustained stimulation.In SAL, SF and FO groups,the neonatal rats had fairly normal activities, slightly increased frequency of defecation and fairly normal appearance of faeces when compared with NEC group.2. The changes of the intestinal gross morphology, ileal pathomorphology and tissue injury pathological scores in six groupsThe gut of CON1 and CON2 groups were pale yellow, elastic, complete and smooth,without blackening and dilatation. The gut of NEC group was dark purple or black,and had gross signs of NEC such as lumen expansion, wall thinning and local thrombosis, aminority of which had pneumatosis. The gut of SF and FO groups had better gross morphology compared with SAL group.When observed under microscope,the intestinal of CON1 and CON2 groups had normal structure. The intestinal of group NEC was mild mucosal and / or swelling of the lamina propria separated at 12 h, and gradually progressed to partial villous necrosis, moderate to severe mucosal layer and intrinsic layer was separated, inflammatory cell infiltration and other changes at 72 h.The intestinal of SAL,SF and FO groups were mild swelling of the mucosal and lamina propria or separated at24 h, and gradually progressed to Local intestinal villi detachment, mild mucosal layer and intrinsic layer was separated, inflammatory cell infiltration and other changes at 72 h.The pathological score of NEC group was significantly higher than CON1 and CON2 groups at12 h,24h,48 h and 72h(P <0.05).The pathological scores of SAL, SF and FO groups were significantly higher than CON1 and CON2 group at 24 h, 48 h and 72h(P <0.05).The pathological scores of SAL,SF and FO groups were significantly lower than NEC group at12 h,24h,48 h and 72h(P <0.05).3.The incidence of NEC at 72 h in six groupsThe CON1 and CON2 groups had no NEC. The incidence of NEC in NEC group was87.5%. The incidence of NEC in SAL group was 50%. The incidence of NEC in SF and FO groups was 37.5%. The incidence of NEC, SAL, SF and FO groups was significantly higher than CON1 and CON2 groups(P <0.01).The incidence of SAL, SF and FO groups was significantly lower than NEC group(P <0.01).4. The dynamic expression of PERK, p-PERK, e IF2а, p-e IF2а, CHOP protein of intestinal tissues in six groups4.1 The dynamic expression of PERK, p-PERK protein of intestinal tissues in six groups.There were no significant differences in PERK protein expression among six groups at12 h, 24 h, 48 h and 72h(P>0.05). There were no significant differences in PERK protein expression at 12 h, 24 h, 48 h and 72 h of each group(P>0.05). There were no significant differences in p-PERK protein expression at 12 h, 24 h, 48 h and 72 h of CON1 and CON2groups(P>0.05).The p-PERK protein expression in NEC and SAL groups was gradually increased from 12 h, and reach peak at72 h. The p-PERK protein expressionsin SF and FO groups was gradually increased from 12 hand reach the peak at 24 h after a downward trend.The p-PERK protein expression of NEC, SAL, SF and FO groups was significantlyhigher than control groups at 12 h, 24 h, 48 h and 72h(P<0.05). There were no significant differences in p-PERK protein expression between NEC group and SAL groups at 12 h,24h, 48 h and72h(P> 0.05). The p-PERK protein expression of SF and FO groups was significantly lower than NEC group at 12 h, 24 h, 48 h and 72h(P <0.05). The p-PERK protein expression of SF and FO groups was significantly lower than SAL group at 12 h,24h, 48 h and 72h(P <0.05). There were no significant differences in p-PERK protein expression between SF group and FO group at 12 h, 24 h, 48 h and 72h(P> 0.05).4.2 The dynamic expression of e IF2а, p-e IF2а protein of intestinal tissues in six groupsThere were no significant differences in e IF2а protein expression among six groups at12 h, 24 h, 48 h and 72h(P>0.05). There were no significant differences in e IF2а protein expression at 12 h, 24 h, 48 h and 72 h of each group(P>0.05).There were no significant differences in p-e IF2а protein expression at 12 h, 24 h, 48 h and 72 h of CON1 and CON2groups(P>0.05).The p-e IF2а protein expression in NEC, SAL,SF and FO groups was gradually increased from 12 h, and reach peak at 72 h.The p-e IF2а protein expression of NEC, SAL, SF and FO groups was significantly higher than control groups at 12 h, 24 h,48h and 72h(P<0.05).There were no significant differences in p-e IF2а protein expression between SAL group and NEC group at 0h and 12h(P>0.05).The p-e IF2а protein expression in SAL group was significantly higher than NEC group at 24 h, 48 h and72h(P<0.05).The p-e IF2а protein expression in SF and FO groups was significantly lower than NEC group at 12 h, 24 h, 48 h and 72h(P<0.05).The p-e IF2а protein expression in SF and FO groups was significantly lower than SAL group at 12 h, 24 h, 48 h and72h(P<0.05).There were no significant differences in p-e IF2а protein expression between SF group and FO group at 12 h, 24 h, 48 h and 72h(P> 0.05).4.3 The dynamic expression of CHOP protein of intestinal tissues in six groupsThere were no significant differences in CHOP protein expression at 12 h, 24 h, 48 h and72h of CON1 and CON2 groups(P>0.05).The CHOP protein expression in NEC group was gradually increased from 12 h, and reach peak at72 h.The CHOP protein expression in SAL group was gradually increased from 12 h, and reach peak at 72 h.The expression of CHOP protein in SF and FO groups gradually increased from 48 h, and reach peak at 72 h. The CHOP protein expression of NEC group was significantly higher than control groups at12 h, 24 h, 48 h and 72h(P<0.05).The CHOP protein expression of SAL group was significantly higher than control groups at 24 h, 48 h and 72h(P<0.05).The CHOP proteinexpression of SF and FO groups was significantly higher than control groups at 24 h, 48 h and 72h(P<0.05).The CHOP protein expression of SAL, SF and FO groups was significantly lower than NEC group at 12 h, 24 h, 48 h and 72h(P<0.05).The CHOP protein expression of SF and FO groups was significantly lower than SAL group at 24 h, 48 h and72h(P<0.05).There were no significant differences in CHOP protein expression between SF group and FO group at 12 h, 24 h, 48 h and 72h(P>0.05).5. The dynamic expression of CHOP m RNA of intestinal tissues in six groupThere were no significant differences in CHOPm RNA expression at 12 h, 24 h, 48 h and72h of CON1 and CON2 groups(P>0.05).The CHOP m RNA expression in NEC group was gradually increased from 12 h, and reach peak at72 h. The CHOP m RNA expression in SAL group was gradually increased from 12 h, and reach peak at 72 h.The CHOP m RNA expression in SF and FO groups was gradually increased from24 h, and reach peak at72 h.The CHOPm RNA expression of NEC and SAL groups was significantly higher than control groups at12 h, 24 h, 48 h and 72h(P<0.05).The CHOPm RNA expression of SF and FO groups was significantly higher than control groups at 24 h, 48 h and 72h(P<0.05).The CHOPm RNA expression of SAL, SF and FO groups was significantly lower than NEC group at 12 h, 24 h, 48 h and 72h(P<0.05).The CHOPm RNA expression of SF group was significantly lower than SAL group at 24 h, 48 h and 72h(P<0.05).The CHOPm RNA expression of FO group was significantly lower than SAL group at 24 h and72h(P<0.05).There were no significant differences in CHOPm RNA expression between SF group and FO group at 12 h, 24 h, 48 h and 72h(P> 0.05).Conclusions:1.Formula-fed, hypoxia, cold stress, and LPS-fed via orogastric gavage induce NEC model.2. During the process of NEC model, the expression of the related molecules p-PERK,p-e IF2а, CHOP in the PERK/e IF2а/CHOP signaling pathway of ERS were increased, with prolonged modeling stimulation showed a progressive increase. The p-e IF2а protein expressions in SAL group was increased, but the expression of CHOP protein and m RNA in SAL group was reduced, with the intestinal injury reduced. It suggests that PERK /e IF2а / CHOP signal pathway may participate in NEC, and salubrinal probably by inhibiting p-e IF2а dephosphorylation, and inhibiting CHOP gene and protein expression to work.3. In NEC rat model, fish oil may reduce the protein expression of PERK, p-PERK,e IF2а, p-e IF2а, CHOP and m RNA expression of CHOP, further reduce the damage of intestinal. It suggests that the protection effect of fish oil may be related to inhibition of PERK / e IF2а / CHOP signal pathway. |
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