Neutrophilic Inflammation and Reactive Oxygen Species Production Promotes Necrotizing Enterocolitis induced Lung Injury in Mice

Abstract

Abstract This study was designed to investigate the role and mechanism of neutrophils in lung injury in mice with NEC. Combinational treatment with formula milk, hypoxia, and LPS was performed to establish NEC in 5-day-old mice. The pathological changes in intestinal and lung tissues were examined by HE staining. The terminal ileum was taken out for RNA-seq. Ly6G, NE, MPO staining, and flow cytometry were used for qualitative and quantitative analysis of neutrophils in lung tissues respectively. The mRNA relative expression levels of inflammatory factors in intestinal and lung tissues were detected by RT-qPCR; the ROS release level in neutrophils stimulated by fMLP was detected by an enzyme-labeled instrument. The role of ROS in NEC-induced lung injury was further validated by the NAC, a ROS-specific inhibitor. The expression levels of genes in the Keap1—Nrf2 pathway were determined by RT-qPCR. Results showed that compared with the Ctrl group, the intestinal and lung tissues of the mice in the NEC group were significantly damaged. The mRNA relative expression levels of inflammatory factors and anti-oxidant genes were increased, while the expression levels of anti-ROS genes were downregulated. Mouse intestinal RNA-seq results suggested a strong enrichment in the neutrophil degranulation pathway. The levels of NE, MPO, and ROS released by neutrophils were significantly higher than those in the Ctrl group. And the ROS released by neutrophils is involved in NEC-induced lung injury by regulating the Keap1-Nrf2 pathway as the inhibition of the release of ROS can alleviate the injury of intestinal and lung tissues. Thus our study demonstrates that NAC has therapeutic potential for NEC-induced lung injury.