Related Experiment Video
Updated: Oct 8, 2025
Depletion and Reconstitution of Macrophages in Mice
Published on: August 1, 2012
NOX4 promotes mucosal barrier injury in inflammatory bowel disease by mediating macrophages M1 polarization through
Chenyang Han1, Yongjia Sheng1, Jin Wang1
1Department of Pharmacy, The Second Affiliated Hospital of Jiaxing University, 314001, China.
Abstract:
NADPH oxidase 4 (NOX4) plays an important role in transporting electrons in the mitochondrial respiratory chain, which is also one major source of ROS. This study investigates the mechanism by which NOX4 promotes the M1 polarization of intestinal macrophages in inflammatory bowel disease (IBD) through ROS. Dextran sulfate sodium (DSS) was used to induce the inflammatory bowel disease (IBD) in wild-type (C57BL/6N, WT) and NOX4 knockout (C57BL/6N-NOX4em1cyagen, KO) mice. Body weights of mice were dynamically monitored and the disease active index (DAI) scores were assessed. H&E staining was performed to examine pathological changes, and immunohistochemical (IHC) staining was conducted to measure the expressions of TJ proteins (ZO-1, Occludin) and CD11c. Tissue ROS labeling was accomplished with ROS probe. More ucosal permeability was assessed by FITC-D. Tissue inflammatory cytokines were detected by enzyme-linked immunosorbent assay (ELISA), while the expressions of TJ proteins (ZO-1, Occludin) were measured through Western Blotting. After NOX4 inhibitor pretreatment of intestinal macrophages in vitro, polarization was induced by lipopolysaccharide (LPS) and IFN-γ, followed by determination of polarization degree. The polarized intestinal macrophages were co-cultured with Caco-2 cells, and their effect on the monolayer cell permeability was evaluated. DSS can induce the intestinal inflammation and mucosal barrier injury in mice. Besides, it can enhance the FITC-D permeability, reduce the TJ protein levels, and promote the CD11c and ROS expressions. In KO mice, intestinal inflammation was alleviated and barrier permeability was reduced. Moreover, the TJ protein levels were higher than those of WT mice, while the CD11c and ROS were down-regulated. In WT mice, the intestinal inflammation and barrier permeability could also be reduced after treatment with NOX4 inhibitor. Overexpression of NOX4 in intestinal macrophages could promote the macrophage M1 polarization while improving the barrier integrity of Caco-2 monolayer cells. NOX4 is capable of promoting M1 polarization of intestinal macrophages through ROS, thereby further aggravating the intestinal inflammation and mucosal barrier injury in IBD. NOX4 has potential as a novel therapeutic target for IBD.
Insights
NADPH oxidase 4 (NOX4) drives M1 macrophage polarization and ROS production, worsening inflammatory bowel disease (IBD) and gut barrier damage. Inhibiting NOX4 shows therapeutic potential for IBD by reducing inflammation and improving barrier function.
Area of Science:
- Immunology and Gastroenterology
- Molecular Biology and Oxidative Stress
Background:
- NADPH oxidase 4 (NOX4) is a significant source of reactive oxygen species (ROS) and implicated in cellular processes.
- Inflammatory bowel disease (IBD) involves complex immune dysregulation and compromised intestinal barrier function.
Purpose of the Study:
- To investigate the role of NOX4 in promoting M1 polarization of intestinal macrophages via ROS in IBD.
- To evaluate NOX4 as a potential therapeutic target for IBD.
Main Methods:
- Induction of IBD in wild-type and NOX4 knockout mice using dextran sulfate sodium (DSS).
- Assessment of disease activity, histopathology, intestinal permeability (FITC-D), tight junction protein expression (ZO-1, Occludin), CD11c, and ROS levels.
- In vitro studies involving NOX4 inhibition in macrophages and assessment of Caco-2 cell monolayer integrity.
Main Results:
- DSS-induced IBD led to increased intestinal inflammation, barrier dysfunction, CD11c, and ROS expression.
- NOX4 knockout mice exhibited reduced inflammation, improved barrier function, and lower CD11c and ROS levels compared to wild-type mice.
- NOX4 inhibition or overexpression modulated macrophage polarization and intestinal barrier integrity in vitro.
Conclusions:
- NOX4 promotes M1 polarization of intestinal macrophages through ROS, exacerbating intestinal inflammation and mucosal barrier injury in IBD.
- Targeting NOX4 represents a promising therapeutic strategy for managing IBD.
