The Relationship Between Enteric Virus And Necrotizing Enterocolitis And The Study On The Role Of Murine Norovirus In Neonatal Necrotizing Enterocolitis

PART ?THE RELATIONSHIP BETWEEN ENTERIC VIRUS AND NECROTIZING ENTEROCOLITISObjective: Necrotising Enterocolitis(NEC)is as severe infectious disease in neonates,the pathogenesis is unclear yet,less studies focused on the relationship between virus and NEC and the conclusion is not congruent,we design this study to demonstrate the relationship between virus and NEC.Methods: The neonates who were sent to the Children's Hospital of Chongqing Medical University for hospitalization were enrolled in the study.Fifty-one neonates were enrolled in NEC group according to the NEC diagnostic criteria,and 39 neonates using pair matching way were enrolled as Control group.Stool specimens were collected and analyzed with polymerase chain reaction(PCR)for norovirus(NV),astrovirus(ASV),sapovirus,enterovirus(EV),adenovirus(ADV),EB virus(EBV),cytomegalovirus(CMV).And clinical data of NEC group were analysis according to Virus positive of negative.Results: A total of 51 NEC stool specimens and 39 Control stool specimens were collected.Viral DNA or RNA was detected in 90 stool specimens,representing 35/51(69%)positive of babies in NEC group,and 29/39(74%)in Control group(P > 0.552).NV,ASV,EBV,ADV were detected both in NEC group and Control group,however,EV and HBoV were only detected in NEC group.ASV was the most frequently detected enteric virus,but there was no significance in NEC group(45.1%)and Control group(28.2%),the same as EV(1.9% vs 0%,P>0.05)and EBV(7.8% vs 5.1%,P>0.05).And the prevalence of ADV and HBoV in NEC group were higher than Control group(29.4% vs 7.7%,17.6% vs 0%,P<0.05).However,the prevalence of NV was higher in Control group than NEC group(28.2% vs7.8%,P<0.05).There's no any difference in clinical manifestations such as bloody stool,bowel distention and vomiting between virus positive NEC neonates and virus negative NEC neonates.NEC neonates with ADV and EBV positive presented a higher rate of CRP?8mg/L than ADV and EBV negative NEC neonates(P<0.05).The incidence of CRP?8mg/L in NEC neonates with NV positive were 25%,however,in NEC with NV negative were as high as 46.8%,but without any significance.NEC neonates with ASV positive seemed to have a shorter recovery time than ASV negative NEC neonates,however without any significance[9(6-10)vs 19(6-14.5),P>0.05].Conclusion: The role of different viruses in NEC are not congruent,some of them can be regard as commensal virus in neonate such as astrovirus,and some of them may alleviate the severity of NEC such as norovirus,but some of them maybe the onset of NEC or exacerbate the severity such as ADV and EBV.PART ? THE STUDY ON THE ROLE OF MURINE NOROVIRUS IN NEONATAL NECROTIZING ENTEROCOLITISObjective: murine norovirus(MNV)can play a similar role in intestinal flora to restore the structure of small intestinal villi of sterile mice to normal without excessive inflammatory response,and can promote the expression of IL-10,an anti-inflammatory factor,in mice with intestinal infection caused by Citrobacter,so as to protect the intestinal tract.Combined with our previous findings,we have found that the detection rate and copy number of enteronorovirus in NEC children are lower than those in the control group,The NEC patients with norovirus positive had less inflammation,better prognosis and lower mortality.This part of the study aims to explore whether MNV can protect the intestinal tract of NEC and its related mechanisms by constructing NEC model of MNV infected mice,so as to provide new ideas for clinical treatment.Methods: C57 BL / 6J and Ifnar1-/-newborn mice were randomly divided into three groups: control group,antibiotics(ABX)+ NEC group and ABX + MNV + NEC group.The control group was kept in the same cage with the female rats,without any treatment measures;the ABX + NEC group was given antibiotic mixture solution(including piperacillin sodium,tazobactam sodium,vancomycin,ampicillin sodium and metronidazole)for 10 days to simulate sterility on the first day of life,so as to minimize the impact of intestinal flora on the experiment,and then NEC construction was carried out Model 3 days;ABX + MNV + NEC group was infected with ABX and MNV on the first day of life,NEC model was established for 3 days after 10 days;NEC animal model was established by artificial feeding,anoxia reoxygenation and cold stimulation.At the same time point every day,mice were weighed before and after NEC modeling.Mice were sacrificed for 14 days.Pathological changes of ileocecal intestinal tissue were observed and scored by HE staining.The levels of IL-10,IL-6 and TNF-? in intestinal homogenate were detected by ELISA.The expression levels of MDA-5,IL-6,IL-10,TNF-?,IFN-? and IFN-? genes were detected by q PCR.Results: C57BL/6J mice: Compared with the control group,the weight of ABX+MNV+NEC group and ABX+NEC group decreased obviously and got a intestine damage;Compared with ABX+NEC group,the weight loss of ABX+MNV+NEC group were less [(0.56±0.20)g vs(0.37±0.22)g,P<0.05],and a lower death rate,but without any significance(44% vs 24%,P>0.05).Compared with ABX+NEC group,the ABX+MNV+NEC group exhibited a lower NEC score of histopathologic evaluation(1.720±0.1781 vs.2.640 ±0.1990,P< 0.05),and a lower expression of TNF-?[(46.50±1.35)pg/ml vs(38.14±2.19)pg/ml,P <0.05],but a higher expression of IL-10 level [(114.3±14.75)pg/ml vs(184.8±29.34)pg/ml,P <0.05],the difference of expression of IL-6 was not significant between two groups [(14.83 ±0.85)pg/ml vs(17.75±1.35)pg/ml,P <0.05].Compared with ABX+NEC group,ABX+MNV+NEC group exhibited a higher gene expression of IL-10(0.739±0.1099 vs 1.197 ±0.1279,P <0.05),a lower gene expression of TNF-?(1.453±0.0741 vs 0.790±0.1651,P <0.05),but without any significance of IL-6(1.032±0.2385 vs 0.862±0.0771,P>0.05).Compared with ABX+NEC group,the expression of some key genes of type ? interferon pathway in ABX+MNV+NEC group were increased,which were MDA-5(1.653±0.2347 vs 3.169±0.3658,P<0.05),IFN-?(0.825±0.1552 vs 1.338±0.1570,P <0.05),IFN-?(1.093±0.1366 vs 1.894±0.2338,P <0.05).Ifnar1-/-mice: Compared with the control group,the weight of ABX+MNV+NEC group and ABX+NEC group decreased obviously and got an intestine damage;No statistically significant difference for weight loss between ABX+MNV+NEC group and ABX+NEC group[(0.58±0.03)g vs(0.62±0.05)g,P>0.05],neither did the death rate(28% vs 32%,P>0.05),the NEC score of histopathologic evaluation(2.760 ±0.1939 vs.2.800±0.1732,P>0.05);the expression of IL-10 between ABX+NEC group and ABX+MNV+NEC group[(137.6 ± 26.95)pg/ml vs(118.5 ±14.69)pg/ml,P>0.05] and the expression of TNF-? ?IL-6 [(54.12±9.75)pg/ml vs(52.59±11.04)pg/ml,P>0.05,(20.66±3.40)pg/ml vs(19.43 ± 3.33)pg/ml,P > 0.05,respectively] were not statistically significant.No statistically significant difference for the gene expression of cytokines in ABX +NEC group and ABX+MNV+NEC group [IL-10(1.011±0.0841 vs 0.861±0.0908,P>0.05),TNF-?(1.559±0.1455 vs 1.116±0.1263,P>0.05),IL-6(1.333±0.1454 vs 1.011±0.0772,P>0.05)];neither did the expression of some key genes of type ? interferon pathway between two groups[MDA-5(1.340±0.1820 vs 1.086±0.0803,P>0.05),IFN-?(1.488±0.1488 vs 1.088±0.1515,P>0.05),IFN-?(1.402±0.1351 vs 1.099±0.1316,P>0.05)].With the same intervention of ABX+MNV+NEC,the expression of IL-10 proteins and gene expression of IL-10?MDA-5?IFN-?were higher in C57BL/6J mice than in Ifnar1-/-mice(P<0.05).Conclusion: Murine norovirus can protect the intestine of neonatal necrotizing enterocolitis mice by down regulating TNF-? and up regulating IL-10.