Naringin alleviates intestinal mucosal injury in mice with intra-abdominal infection by inhibiting non-canonical pyroptosis through P2X7 receptor

To investigate the therapeutic efficacy and underlying mechanism of naringin (Ng) for intestinal mucosal injury in mice with intra-abdominal infection (IAI).

A mouse IAI model was established by intraperitoneal injection (i.p.) of lipopolysaccharide (LPS). Sixty-four healthy male mice were randomly assigned to 4 groups (n=16): Control group (0.2 mL PBS), LPS group (LPS 10 mg/kg), Ng group (50 mg/kg Ng+10 mg/kg LPS), and BzATP group (5 mg/kg BzATP +50 mg/kg Ng+10 mg/kg LPS). Ng and BzATP were administered i.p. 1 h before modeling, while the Control group received an equivalent volume of PBS. At 24 h post-modeling, 6 mice from each group were randomly selected and sacrificed for ileum collection, and the histopathological changes in the ileal mucosa and Chiu's score were assessed using HE staining. Immunofluorescence assay was employed to investigate the expression and distribution of intestinal mucosal P2X7, pyroptosis effector protein GSDMD, and macrophage marker CD68. Western blotting was conducted to detect the expression of P2X7 and pyroptosis-related proteins NLRP3, Caspase11, GSDMD, and GSDMD-N protein in ileum tissue. ELISA was utilized to measure the contents of inflammatory factors IL-1β, IL-18, and IL-10 in the ileum. The mouse sepsis score (MSS) was recorded within 7 d.

In comparison to the Control group, the LPS group exhibited significantly higher MSS and Chiu's scores (P < 0.05), along with inflammation-associated damage to the ileal mucosa; Immunofluorescence assay displayed consistent and intensified distribution of intestinal mucosal P2X7, GSDMD, and CD68 (P < 0.05); Levels of pro-inflammatory factors IL-1β and IL-18 were increased, while that of anti-inflammatory factor IL-10 was decreased (P < 0.05); Additionally, the protein expression of P2X7, NLRP3, Caspase11, GSDMD, and GSDMD-N in ileum tissue was increased (P < 0.05). In contrast to the LPS group, the Ng group displayed a decrease in MSS; HE staining and Chiu's scores indicated a reduction in inflammatory damage to the intestinal mucosa; Moreover, fluorescence signals for P2X7, GSDMD, and CD68 were decreased (P < 0.05). Levels of IL-1β and IL-18 were decreased (P < 0.05), while that of IL-10 was elevated (P < 0.05). The expression of P2X7, NLRP3, Caspase11, GSDMD, and GSDMD-N protein was decreased (P < 0.05). The results of the BzATP group were in contrast to those of the Ng group.

Elevoted expression of P2X7 receptor and Caspase11-mediated non-canonical pyroptosis aggravates the intestinal mucosal injury IAI mice. Ng may ameliorate the injury in IAI mice by suppressing P2X7 receptor and modulating non-canonical pyroptosis.