Effects Of Macrophages On The Phenotype Change Of ICCs In Neonatal Necrotizing Enterocolitis

PART ONEMacrophage activation n TNF-alpha expression and phenotype change of ICCs in neonatal necrotizing enterocolitisObjective:To study the role of macrophages and TNF-a and ICCs in neonatal necrotizing enterocolitis.Methods:A comparative study of normal small intestine tissue and NEC small intestine tissue were conducted by means of HE staining, immunofluorescence, Immunohistochemistry, western blot and RT-PCR.Results:HE staining-Villi were arranged regularly, and there was no edema or hyperemia in normal small intestine. In contrast, villi necrosis, hyperemia and a lot of imflammatory cells were found in NEC small intestine. Immunofluorescence-There was a few CD68 positive macrophages in normal small intestine. In contrast, there was a large number of M1 macrophages, but no M2 macrophages, in NEC small intestine. Immunohistochemistry and western blot-The TNF-a protein expression level of NEC group was significantly higher than that of control group. TNF-a was mainly distributed in the mucosa and the region between the circular and longitudinal muscle. In the normal small intestine, ICCs were found in the circular muscle, the longitudinal muscle and the region between them. In contrast, ICCs disappeared in the NEC group. RT-PCR-The mRNA expression level of CD68, iNOS and TNF-a of NEC group were significantly higher than that of control group, which were consistent with the results of protein expression. On the contrary, The mRNA expression level of c-kit of NEC group was significantly lower than that of control group.Conclusion:A large quantity of M1 macrophages infiltrated in NEC small intestine. At the same time, the expression level of TNF-a increased obviously. In the progress of NEC, the change of ICCs phenotype may be related with disorder of intestine movement.PART TWOEffects of macrophages on the phenotype change of ICCs in the model of NECObjective:To study the activation of macrophages, the expression of TNF-α, and the phenotypic change of ICCs in the animal model; and to explore the effect of macrophages on the expression of TNF-α, and the phenotype of ICCs.Methods:Neonatal mice were randomly divided into 4 groups:control group, NEC group, NEC + NS group, and NEC + CL group. Control group:no hypoxia, injection or cold stimulation was conducted. NEC group:the NEC model was constructed by the methods of hypoxia and cold stimulation. NEC + NS group:60μl normal saline were injected into the abdomen of the mice before the progress of hypoxia and cold stimulation. NEC + CL group: 60μl liposomal clodronate were injected into the abdomen of the mice before the progress of hypoxia and cold stimulation. The small intestine specimens were collected at the time of the second day (early stage), the fourth day (progressive stage), the eighth day (recovery stage) and the fifteenth day (intestinal recovery). Pathological evaluation after HE staining was performed to measure the intestinal injury and the incidence of NEC in each group. Activation of macrophages, expression of TNF-α, and phenotypic change of ICCs were detected by methods of immunofluorescence, immunohistochemistry, western blot and RT-PCR.Results:Clodronate liposome can significantly inhibit the expression of M1 macrophages at the early and recovery stage. But this kind of inhibition became weakened at the progressive stage. At the early stage, the lower expression of M1 macrophages could relieve intestine injury, decrease the incidence of NEC and the expression of TNF-α obviously. At the recovery stage, The lower expression of M1 macrophages could also decrease the expression of TNF-α and promote the recovery of intestine and ICCs.Conclusion:M1 macrophages play an important role in the intestine inflammatory progress of NEC. Inhibition of M1 macrophages in the progress of NEC can not only decrease the expression of TNF-α and relieve the injury of intestine and ICCs, but also promote the recovery of intestine and ICCs at the recovery stage.PART THREEEffects of TNF-a on the phenotype change of ICCsObjective:To study the mechanism of TNF-a inhibiting c-kit expression, which caused the phenotype change of ICCs.Methods:B6 mice were randomly divided into 4 groups within 24 hours after birth, which include control group, blank group, TNF-a group and miRNA-222 antagomir group. In the control group, the intestine specimens were cultured without TNF-a. In the blank group, the intestine specimens passed the cultivation step. In the TNF-a group, the intestine specimens were cultured with TNF-a of different concentration. In the miRNA-222 antagomir group, miRNA-222 antagomir were injected into the abdomen of mice 24 hours before the step of cultivation with different concentration of TNF-α. The expression of miRNA-222 and c-kit were measured by the means of RT-PCR and western blot 48 hours later. Dual luciferase assay was conducted to identify whether c-kit is a target gene for miRNA-222.Results:At the concentration of 100-500ng/ml, TNF-a could increase the expression of miRNA-222, and inhibit the expression of c-kit mRNA and protein. At the concentration of 100-200ng/ml, the inhibitory effect of TNF-a can be reversed by miRNA-222 antagomir. This kind of reversion disappeared when the concentration of TNF-a is above 200ng/ml. Dual luciferase assay confirmed that c-kit is the target gene of miR-222. MiR-222 can inhibit the expression of c-kit by binding with c-kit mRNA.Conclusion:TNF-a can inhibit the expression of c-kit by up regulating miRNA-222, which can be reversed by miRNA-222 antagomir within a certain range.