The Role Of CXCL5 And Its Receptor CXCR2 In The Pathogenesis Of Necrotizing Enterocolitis

Background:Necrotizing enterocolitis(NEC)is a serious gastrointestinal disease common in neonatal infants,especially in premature and low birth weight infants.Because the pathogenesis of NEC has not been fully elucidated,early diagnosis is very difficult and effective treatment is lacking,which leads to poor prognosis and high mortality of infants with NEC.Clinical studies suggest that premature,formula feeding,intestinal hypoxia-ischemia reperfusion,and abnormal colonization of intestinal flora are the main risk factors for NEC.Exaggerated inflammatory response and intestinal mucosal epithelial barrier damage are key factors in the pathogenesis of NEC.Therefore,how to properly regulate the level of inflammatory response and protect the intestinal epithelial barrier is the key to improve the prognosis of NEC.Early studies have found that the chemokine CXCL5 and its receptor CXCR2 are involved in the regulation of inflammatory diseases,such as inflammatory bowel disease.Since NEC is also a neonatal intestinal inflammatory disease,we focus on how CXCL5 and its receptor CXCR2 regulate the pathogenesis of NEC.Therefore,this study intends to explore the role of CXCL5 and its receptor CXCR2 in the pathogenesis of NEC,in order to provide a new target with potential application value for the prevention,diagnosis and treatment of NEC in clinic.Part Ⅰ:The expression and clinical significance of CXCL5 and its receptor CXCR2 in the development of NECObjective:To investigate the expression and clinical significance of CXCL5 and its receptor CXCR2 in the development of NEC.Methods:Blood samples from infants in NEC and control groups were collected,and plasma samples were separated.The levels of inflammatory cytokines were detected by cytometric bead array(CBA)and enzyme-linked immunosorbent assay(ELISA)methods.The intestinal tissues excised from the infants suffered with NEC were collected and the mRNA expression levels of CXCL5 and its receptor CXCR2 were detected by quantitative real time polymerase chain reaction(qRT-PCR).Results:The baseline data of the infants in control group(n=14)and NEC group(n=13)included in this study were compared.The results showed that there was no statistically significant difference between the two groups.The baseline data of the two groups were comparable.The results of CBA showed that compared with the control group,the plasma levels of IL-6,IL-8 and IL-10 in NEC group were significantly higher(P<0.05),but the plasma levels of IL-1β and TNF-α were not significantly different between the two groups.The results of ELISA showed that compared with the control group,the plasma level of CXCL5 in NEC group was significantly higher,the difference was statistically significant(P<0.05).The results of qRT-PCR showed that compared with control group,the expression of CXCL5 mRNA in the intestinal tissue of NEC group was increased,but the difference was not statistically significant;while the mRNA expression level of CXCR2 in intestinal tissue of NEC group was significantly up-regulated than that of the control group,the difference was statistically significant(P<0.05).Western Blot results showed that the protein level of CXCR2 in NEC intestinal tissue was significantly higher than that in control group(P<0.05).Conclusions:Infants who suffered with NEC were in a state of hyper inflammation,and the release levels of inflammatory cytokines in plasma were significantly increased.With the occurrence of NEC,the expression of CXCL5 and CXCR2 were up-regulated in infants with NEC.The expression of CXCL5 and its receptor CXCR2 were clinically related to the pathogenesis of NEC and may be involved in the regulation of NEC,but the specific role remain to be further studied.Part Ⅱ:Protective effects of regulating CXCR2 expression on experimental NEC animal modelsObjective:To explore the protective effects of regulating CXCR2 on experimental NEC.Methods:Fifty five-day-old Cxcr2 knockout mice and SPF C57BL/6 wild type mice were randomly divided into wild type control group(C-wt group),knockout control group(C-ko group),wild type NEC group(N-wt group)and knockout NEC group(N-ko group).Among them,the control group continued to be breastfed by the mother without any other intervention factors.Experimental NEC were induced by formula feeding,hypoxia and LPS stimulation in the NEC group mice.After 96 hours of induction,the surviving mice were anesthetized and blood and terminal ileum samples were collected for subsequent analysis.Results:After 96 hours of NEC induction,the overall state of mice in each group was compared,suggesting that the mice in C-wt group and C-ko group were in good general condition and increased in weight,while the mice in N-wt group and N-ko group were in worse condition and lost weight than before.The weight loss of mice in N-ko group was lower than that in N-wt group,and the difference was statistically significant(P<0.05).After Cxcr2 gene knockout,the release of CXCL5 in plasma and the expression of CXCR2 receptor in intestinal tissues decreased,which could alleviate the pathological damage of NEC intestinal mucosa,improve intestinal permeability,increase the expression of intestinal barrier protein,and inhibit the release of inflammatory factors IL-6,TNF-a and MCP-1 in plasma.In addition,Western Blot results showed that the expression of p-AKT in intestinal tissue increased significantly in N-wt group,but decreased in N-ko group.Conclusions:Cxcr2 gene knockdown significantly down-regulated the expression of CXCR2 receptor in the intestinal tissue of experimental NEC mice,and inhibited the release of plasma CXCL5,which had protective effects on experimental NEC mice.AKT may be a downstream key signaling molecule associated with this protection,and the specific mechanisms involved in these protective effects still require further investigation.Part Ⅲ:Protective effects of FTY720 targeting CXCL5/CXCR2 axis on experimental NEC animal modelsObjective:To investigate protective effects of FTY720 targeting CXCL5/CXCR2 axis on experimental NEC animal models.Methods:Fifty five-day-old SPF C57BL/6 mice were randomly divided into control group(Ctrl group),NEC group and FTY720 group.Among them,the control group mice continued to be breastfed by the mother without any other intervention factors.In the NEC group,mice were induced by formula feeding,hypoxia and LPS stimulation.In addition to inducing NEC,FTY720(1 μg/g body weight)was given daily as an intervention measure in FTY720 group mice.After 96 hours of induction,the surviving mice were anesthetized and blood and terminal ileum samples were collected for subsequent analysis.Results:After 96 hours of NEC induction,the overall state of mice in each group was compared.The results showed that the weight of control group mice was increased,while the weight of NEC group and FTY720 group mice was decreased.The weight loss of FTY720 group mice was lower than that of NEC group,the difference was statistically significant(P<0.05).The survival of mice after FTY720 treatment was significantly improved compared with NEC group,the difference was statistically significance(P<0.05).Analysis of plasma and intestinal samples of mice in each group showed that compared with NEC group,FTY720 intervention could alleviate intestinal mucosal injury,reduce apoptosis of intestinal epithelial cells,increase the expression of intestinal barrier protein,decrease the release levels of inflammatory factors IL-6,TNF-a,MCP-1 and CXCL5 in plasma,and down-regulate the mRNA expression of inflammatory-related genes(IL-6 and F4/80)in intestinal tissues of experimental NEC mice.FTY720 also significantly decreased the expression of CXCL5/CXCR2 axis in intestinal tissues.Conclusions:FTY720 treatment can effectively reduce the weight loss of experimental NEC mice,improve the survival rate,reduce the degree of intestinal injury,reduce the apoptosis of intestinal mucosal epithelial cells,increase the expression of barrier proteins,inhibit systemic and local inflammatory response levels,and has a certain protective effect on experimental NEC mice.The protective effect of FTY720 on experimental NEC may be related to its targeting down-regulation of CXCL5/CXCR2 axis expression in intestinal tissue.