Study Of The Mechanisms Of Hydrogen On Macrophage Polarization And The Effects In Necrotizing Enterocolitis Mice

Necrotizing enterocolitis is the most common gastrointestinal critical disease that affected most infants born prematurely,and especially with very low birth weight.Due to lacking of the early diagnostic makers,and changing quickly of NEC progression,the strategy of intervention needed by NICU physicians and gernal surgeons.However,the effective treatment is still on the road.With the development of perinatal medicine and the skills of pediatrics nearly 20 to 30 years,the survival rate of preterm and very low birth weight is increasing,as the result of the high rate of the incidence of NEC.Although the risk factors including prematurity,hypoxic-ischemia injury,abnormal bacterial colonisation and formula feeding,the exactly etiology and the pathogenesis of NEC is not yet clear.To investigate the the possible molecular mechanism of hydrogen on macrophage polarization and the roles of hydrogen in mice model of necrotizing enterocolitis,then we proceed to the following research:Part 1:The study of molecular mechanism of macrophage polarization regulated by hydrogen Objective: To study the possible molecular mechanism of hydrogen on macrophage polarization,macrophage was polarized to the type of M1 and M2.Methods: In vitro exprements RAW264.7 and BMDM macrophages were polarized to the type of M1 and M2 with LPS,IFN-γ and IL-4.Macrophages viability was detected by CCK-8,and the markers of the different types of macrophage were detected by flow cytometry,and inflammatory cytokines were detected by RT-PCR,and the nuclear transcription factors-κB was detected by western bolt,and the migration capacity was detected by wound scratch assay.Results: Macrophages were polarized to M1 by LPS(20 ng/ml)and IFN-γ(50 ng/ml),the axons were small and elongated;and polarized to M2 by IL-4(20 ng/ml),the axons were large.No significant of hydrogen on the viability of macrophages was observed over the priods of hydrogen incubation.The levels of mRNA including iNOS,TNF-α and IL-1β in macrophages were significantly up-regulated,and the levels of mRNA including Arg-1,IL-10 and YM-1 in M2 macrophages were significantly up-regulated(P <0.05).After treatment with hydrogen,the markers of macrophsges CD16/32 was decreased and the expression of NF-κB(P65)decreased significantly.The migration ability of M1 macrophages was inhibited.However,there was no significant effect of hydrogen on the polarization and function in M2 macrophages(P> 0.05).Conclusion: Hydrogen decrease the levels of mRNA of inflammatory factors in M1 macrophages and decrease its migration ability by reducing the translocation of nuclear factor NF-κB(P65).Part 2:The effects of hydrogen on necrotizing enterocolitis in newborn mice Objective: To observe the effect of hydrogen on the macrophage polarization in the mice model of necrotizing enterocolitis.Methods: 8-10 days old C57BL6/J mice were used to imitate the mice model of NEC by exposure to asphysia and cold stress with formula feeding.Histological changes of the diatant ileum and the general apperarance of mice were observed.The types of different macrophage and the inflammatory cytokines in spleens,lymphnodes and lamina propria was analyzed by flow cytometry.The expression of NF-κB was detected by western blot.Results: Hydrogen attenuated the severity of mice affected NEC,and reduced the morbidity and mortality of NEC,and decreased the levels of mRNA of inflammation in intestinal wall and changed the types of macrophages by reducing the translocation of nuclear factor NF-κB(P65).Conclusion: Hydrogen reduce the severity of NEC mice,and regulated the types of macrophage in mice and reduce the expression of inflammatory factors by reducing the translocation of nuclear factor NF-κB(P65).