Targeting HIF1α in intestinal muscularis ameliorates postoperative ileus by reducing nitric oxide generation

Abstract Aim Postoperative ileus (POI) is one of the most common complications after abdominal surgery, especially subsequent to intra-abdominal infection. The underlying mechanism remains unclear. This study is to investigate the role of HIF1α in the progression of POI. Methods Bioinformatics analysis was utilized to analyze the association between HIF1α and POI. A murine model with POI was generated by surgical intestinal manipulation (IM). Gastrointestinal transit (GIT), leukocyte infiltration and inflammatory mediators were measured. Targeting HIF1α with specific inhibitor (2ME2) was performed in both in vivo and in vitro experiment. Human samples were collected for further validation. Results Public RNA sequencing data demonstrated upregulation of HIF1α in murine intestinal muscularis after POI generation. This was confirmed by our murine models as well as human samples, which was accompanied by impaired GIT, higher inflammatory cytokines, inducible nitric oxide synthetase (iNOS) expression and nitric oxide (NO) generation. HIF1α-specific inhibitor 2ME2 reversed inflammatory response in macrophages, suppressed NO production, and eventually ameliorated GI motility. Conclusions HIF1α controls inflammatory response and NO generation in intestinal muscularis, Targeting HIF1α effectively improves GI motility and ameliorates POI development.Fig. 1Gastrointestinal motility injury and inflammatory cell infiltration in the intestinal muscularis after IM-induced POIa Gross pictures of the representational photo in the Sham and IM groups. b FITC-dextran distribution in the intestinal tract of mice 24 h after IM and the genomic center (GC) of FITC-dextran distribution in each experiment group. c Typical HE staining images in each group (×200, ×400). d Typical immunohistochemical staining images with MPO (×200, ×400). Fig. 2 Identification of differentially expressed genes (DEGs) in the GEO dataset GSE134942 a Volcano plot of DEGs. Log2|Foldchange| >1 and P < 0.05 were set as the cutoff values. The red plots represent upregulated DEGs, while the blue plots represent downregulated DEGs. b The top 20 GSEA pathways based on the adjusted P-values. (c–f) Representative results of GSEA analysis of the IM24h group genes. Fig. 3The upregulation of HIF1α was validated in animal models of postoperative ileusa HE staining of the whole layer of the small intestine and the small intestinal muscle layer after the elimination of mucosa. b Representative Western blotting images probed for HIF1α and iNOS extracted from the small intestinal muscularis. c Quantitative RT-PCR analysis of HIF1α, iNOS, IL1β, and COX-2 mRNA levels in murine intestinal muscularis. d The immunofluorescence images of the sham-operated and IM mice intestine. Fig. 4 Validation of HIF1α upregulation in the clinical specimens of patients with intestinal obstruction a Representative Western blotting images probed for HIF1α and iNOS extracted from muscularis in surgically resected intestinal segments. The control group represents the normal intestinal segments of gastric cancer patients that require gastrojejunostomy and small bowel resection. The ileus group represents ischemia intestinal segments from intestinal obstruction patients who underwent bowel anastomosis after the resection of the necrotic bowel. b Quantitative RT-PCR analysis of the HIF1α, iNOS, IL1β, IL6, and COX-2 mRNA levels in human intestinal muscularis. c The immunofluorescence images of the control and ileus clinical intestinal muscularis specimens. Fig. 5Intraperitoneal injection of HIF1α-specific inhibitor 2ME2 relieved gastrointestinal motility injury in vivoa A schematic diagram of the experiment in vivo. b, c FITC-dextran distribution in the intestinal tract of mice 1 day after IM and genomic center (GC) of FITC-dextran distribution in each experiment group. d Representative Western blotting images probed for HIF1α, iNOS, and COX2 extracted from the small intestinal muscularis. e Total NO extracted from the intestinal muscularis homogenate. Fig. 6 2ME2-induced alleviation of the inflammatory cytokines’ expression and NO generation in macrophages a Representative Western blotting images probed for HIF1α, iNOS, and COX2 extracted from 2ME2 treated-RAW264.7 cells subjected to LPS stimulation or hypoxia culture. b Total NO in the cell culture supernatant.