| ObjectiveThrough the establishment of murine NEC model,the changes of intestinal permeability,macrophage polarization and related inflammatory factors during the development of NEC were dynamically observed,and the possible mechanism of macrophage polarization was explored,so as to provide a certain theoretical basis for the clinical treatment and nursing of NEC.MethodsC57BL/6 mice were used to build murine NEC model.Premature mice were randomly divided into control group(n=50)and NEC group(n=50).Mice in the control group were breastfed by mothers,and mice in the NEC group were exposed to hypoxia,hypothermia,hypertonic feeding and lipopolysaccharide treatment.Ten were killed in each group at each time point.Intestinal tissues from the lower duodenum to the colon were collected at 0h,24h,48h,72h and 96h.1.HE staining was used to observe the morphology of intestinal tissue at 0h,24h,48h,72h and 96h.2.Intestinal permeability of mice at 0h,24h,48h,72h and 96h was detected by FD70gavage feeding.3.TUNEL staining was used to detect the apoptosis of intestinal epithelial cells at96h.4.Flow cytometry was used to detect the proportion of M1 and M2 macrophages in the intestine of mice at 0h,24h,48h,72h and 96h.5.ELISA was used to detect the concentrations of cytokines IL-1β,IL-6,IL-12,IFN-γand HMGB-1 at 0h,48h and 96h.6.The protein expression levels of JAK2,STAT3,p-JAK2 and p-STAT3 in the polarization signal transduction pathway of macrophages were detected by Westernblot at 96h.Results1.Results of murine NEC modelMice in the control group were in good growth and development;Mice in the NEC group showed different degrees of feeding difficulties,growth retardation,thin body,poor mental state,abdominal distension,abnormal defecation and other conditions.2.The structure and function of intestinal tract of mice(1)HE staining showed there was no intestinal damage in the control group during the modeling process.The intestinal tissue damage of mice in the NEC group was gradually aggravated.(2)The intestinal permeability of mice in the NEC group was gradually enhanced during the modeling process.Compared with the control group at the same time point,the plasma optical density of mice in the NEC group was significantly increased at 48h,72h and 96h(p<0.05).(3)TUNEL showed increased intestinal epithelial cell apoptosis at 96h in the NEC group(1.9%±1.1%vs.7.6%±2.6%,p<0.01).3.Macrophage changes(1)The percentage of intestinal M1 macrophages in the NEC group increased gradually and reached the peak value at 48h,then decreased,but still at a high level.Compared with the control group at the same time point,the percentage of intestinal M1 macrophages in the NEC group was significantly increased at 48h,72h and 96h(p<0.01).(2)The percentage of intestinal M2 macrophages in the NEC group gradually decreased and reached the peak value at 48h,then increased,but still at a low level.Compared with the control group at the same time point,the percentage of intestinal M2 macrophages in NEC group was significantly increased at 48h and 72h(p<0.01).(3)The percentage of intestinal CD86~+CD206~+macrophages in the NEC group increased gradually.Compared with the control group at the same time point,the percentage of CD86~+CD206~+macrophages in intestinal tract of mice in NEC group was significantly increased at 48h,72h and 96h(p<0.01).(4)The ratio of M1/M2 macrophages in intestinal tissues of mice in NEC group increased gradually and reached the peak value at 48h,then decreased,but still at a high level.Compared with the control group at the same time point,the ratio of intestinal M1/M2 macrophages in NEC group was significantly increased at 24h,48h,72h and 96h(p<0.01).4.Cytokine expressionCompared with the control group at the same time point,the concentrations of inflammatory cytokines IFN-γ,IL-1β,IL-6,IL-12 and HMGB-1 in intestinal tissue of mice in the NEC group were all significantly increased at 48h and 96h(p<0.01).5.Activation of signaling pathwayThe expression of p-JAK2(0.35±0.13 vs.1.00±0.25)and p-STAT3(0.36±0.12 vs.0.84±0.14)protein in the NEC group were significantly increased 96h compared with the control group(p<0.05).Conclusions1.The impaired intestinal structure and function are the morphological basis and initial factors of NEC.2.During the development of NEC,the ratio of M1 and M2 macrophages is out of balance,which is manifested as a significant increase of pro-inflammatory M1macrophages accompanied by the release of a large number of inflammatory cytokines.3.Macrophage polarization in NEC may be regulated by the JAK2/STAT3 signaling pathway.4.The results of this study have certain enlightening significance for improving the nursing quality of premature infants and preventing the occurrence of NEC. |
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