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Pelvic radiation disease (PRD), a frequent side effect in patients with abdominal/pelvic cancers treated with radiotherapy, remains an unmet medical need. Currently available preclinical models have limited applications for the investigation of PRD pathogenesis and possible therapeutic strategies. In order to select the most effective irradiation protocol for PRD induction in mice, we evaluated the efficacy of three different locally and fractionated X-ray exposures. Using the selected protocol (10 Gy/day × 4 days), we assessed PRD through tissue (number and length of colon crypts) and molecular (expression of genes involved in oxidative stress, cell damage, inflammation, and stem cell markers) analyses at short (3 h or 3 days after X-ray) and long (38 days after X-rays) post-irradiation times. The results show that a primary damage response in term of apoptosis, inflammation, and surrogate markers of oxidative stress was found, thus determining a consequent impairment of cell crypts differentiation and proliferation as well as a local inflammation and a bacterial translocation to mesenteric lymph nodes after several weeks post-irradiation. Changes were also found in microbiota composition, particularly in the relative abundance of dominant phyla, related families, and in alpha diversity indices, as an indication of dysbiotic conditions induced by irradiation. Fecal markers of intestinal inflammation, measured during the experimental timeline, identified lactoferrin, along with elastase, as useful non-invasive tools to monitor disease progression. Thus, our preclinical model may be useful to develop new therapeutic strategies for PRD treatment.

Extracellular High-mobility group box 1 (HMGB1) contributes to the pathogenesis of inflammatory disorders, including inflammatory bowel diseases (IBD). Poly (ADP-ribose) polymerase 1 (PARP1) has been recently reported to promote HMGB1 acetylation and its secretion outside cells. In this study, the relationship between HMGB1 and PARP1 in controlling intestinal inflammation was explored. C57BL6/J wild type (WT) and PARP1 -/- mice were treated with DSS to induce acute colitis, or with the DSS and PARP1 inhibitor, PJ34. Human intestinal organoids, which are originated from ulcerative colitis (UC) patients, were exposed to pro-inflammatory cytokines (INFγ + TNFα) to induce intestinal inflammation, or coexposed to cytokines and PJ34. Results show that PARP1 -/- mice develop less severe colitis than WT mice, evidenced by a significant decrease in fecal and serum HMGB1, and, similarly, treating WT mice with PJ34 reduces the secreted HMGB1. The exposure of intestinal organoids to pro-inflammatory cytokines results in PARP1 activation and HMGB1 secretion; nevertheless, the co-exposure to PJ34, significantly reduces the release of HMGB1, improving inflammation and oxidative stress. Finally, HMGB1 release during inflammation is associated with its PARP1-induced PARylation in RAW264.7 cells. These findings offer novel evidence that PARP1 favors HMGB1 secretion in intestinal inflammation and suggest that impairing PARP1 might be a novel approach to manage IBD.

Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory disorders of the gastrointestinal tract. Chronic inflammation is the main factor leading to intestinal fibrosis, resulting in recurrent stenosis, especially in CD patients. Currently, the underlying molecular mechanisms of fibrosis are still unclear. ZNF281 is a zinc-finger transcriptional regulator that has been characterized as an epithelial-to-mesenchymal transition (EMT)-inducing transcription factor, suggesting its involvement in the regulation of pluripotency, stemness, and cancer. The aim of this study is to investigate in vivo and in vitro the role of ZNF281 in intestinal fibrogenesis. Intestinal fibrosis was studied in vivo in C57BL/6J mice with chronic colitis induced by two or three cycles of administration of dextran sulfate sodium (DSS). The contribution of ZNF281 to gut fibrosis was studied in vitro in the human colon fibroblast cell line CCD-18Co, activated by the pro-fibrotic cytokine TGFβ1. ZNF281 was downregulated by siRNA transfection, and RNA-sequencing was performed to identify genes regulated by TGFβ1 in activated colon fibroblasts via ZNF281. Results showed a marked increase of ZNF281 in in vivo murine fibrotic colon as well as in in vitro human colon fibroblasts activated by TGFβ1. Moreover, abrogation of ZNF281 in TGFβ1-treated fibroblasts affected the expression of genes belonging to specific pathways linked to fibroblast activation and differentiation into myofibroblasts. We demonstrated that ZNF281 is a key regulator of colon fibroblast activation and myofibroblast differentiation upon fibrotic stimuli by transcriptionally controlling extracellular matrix (ECM) composition, remodeling, and cell contraction, highlighting a new role in the onset and progression of gut fibrosis.