Hydrogen Promotes the M1 Macrophage Conversion During the Polarization of Macrophages in Necrotizing Enterocolitis

Shenghua Yu,ZhiBao Lv,Xueli Wang,Lulu Zheng,Junmei Zhou,Zhimei Gao,Jingyi Shi,Qingfeng Sheng

doi:10.3389/fped.2021.710382

Abstract

Background: Hydrogen is protective against intestinal injury in necrotizing enterocolitis (NEC), mainly through to alleviate inflammation response. The M1 macrophages can promote inflammation. We hypothesized that hydrogen would promote the M1 macrophages conversion during the polarization and reduce the inflammatory factors in NEC.Methods: We used M1 and M2 macrophages induced from RAW264.7 cells and bone marrow-derived macrophages, models of NEC and macrophages derived from spleens, abdominal lymph nodes and lamina propria in model mice. Cytokines, CD16/32 and CD206 were measured by quantitative PCR, flow cytometry. Nuclear factor-κB (NF-κB) p65 were determined by western blot. Histology staining were used to assess the severity of NEC.Results: Macrophages were successfully polarized to M1 or M2 by assessing the expression of inflammatory factors. Pro-inflammatory factors and CD16/32 in M1 macrophages were decreased, and the expression of CD16/32 in lamina propria were inhibited after treatment with hydrogen, but the changes has no effects in other tissues. Hydrogen inhibited the NF-κB p65 in M1 macrophages nucleus and distal ileum of NEC. HE staining showed hydrogen could attenuate the severity of NEC.Conclusion: Hydrogen could attenuate the severity of NEC through promoting M1 macrophages conversion by inhibited the expression of NF-κB p65 in the nucleus.

Highlights

Necrotizing enterocolitis (NEC) is a serious gastrointestinal disease, mainly affecting premature neonates, especially in extremely low-birth-weight infants
RAW264.7 macrophages induced by LPS (20 ng/ml) and IFN-γ (50 ng/ml) at 0, 3, 6, 12, 24, and 48 h were polarized to M1 macrophages, and the axons were small and elongated (Figure 1A)
Raw264.7 macrophages and BMDM were cultured with LPS and IFN-γ, and the messenger RNAs (mRNAs) levels of inducible nitric oxide synthase (Figure 1C), tumor necrosis factor-α (TNF-α) (Figure 1D), and IL-1β (Figure 1E) released by M1 macrophages were significantly upregulated (p < 0.001)

Summary

Introduction

Necrotizing enterocolitis (NEC) is a serious gastrointestinal disease, mainly affecting premature neonates, especially in extremely low-birth-weight infants. Despite over six decades of clinical and basic research, the pathogenesis of NEC remains unclear [1]. Premature infants have immature intestinal and deficient immune function. Some scholars expressed that insufficient immune function is involved in the pathogenesis of NEC, and macrophages play a vital role in the development of NEC as well [4,5,6,7]. Hydrogen is protective against intestinal injury in necrotizing enterocolitis (NEC), mainly through to alleviate inflammation response. We hypothesized that hydrogen would promote the M1 macrophages conversion during the polarization and reduce the inflammatory factors in NEC

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Conclusion