To investigate whether remimazolam attenuates intestinal ischemia-reperfusion (I/R) injury in mice by regulating ferroptosis through connexin-43 (CX43).

Molecular docking was applied to predict the binding affinity of remimazolam to CX43. A total of 72 SPF-grade adult male C57BL/6J mice (6~8 weeks old, weighing 20~25 g) were subjected. Thirty of them were randomly divided into sham operation group (Sham group), I/R group 1, and I/R+10, 20 and 40 mg/kg remimazolam groups (RM10, RM20 and RM40 groups), with 6 mice in each group. Another 30 mice were randomly assigned into 5 groups (n=6), I/R group 2, erastin group (E group), I/R+40 mg/kg remimazolam group 2 (RM40 group 2), I/R+Fer-1 group (Fer-1 group), and erastin +40 mg/kg remimazolam group (ERM group). The left 12 mice were randomly and equally grouped into I/R+RM+oe-NC group (oe-NC group) and I/R+RM+oe-CX43 group (oe-CX43 group). The Fer-1 group was given an intraperitoneal injection of 5 mg/kg Fer-1 in 1 h prior to reperfusion, the E group was given 10 mg/kg erastin intraperitoneally 1 d before modeling, and all the remimazolam groups, the oe-NC group and the oe-CX43 group were injected intravenously with corresponding doses of remimazolam 30 min pre-modeling, while the oe-NC and oe-CX43 groups were injected with empty vector virus and overexpression of CX43 vector virus, respectively, 48 h before the administration of remimazolam. A mouse intestinal I/R injury model was constructed by clamping the superior mesenteric artery for 45 min and reperfusion for 30 min. The small intestine tissues were harvested and observed for pathological changes, and the intestinal mucosal damage was assessed with Chiu’s score. The contents of Fe²⁺, total iron, malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) were detected by colorimetric assay; the production of reactive oxygen species (ROS) was determined by DHE probe; the expression of ferroptosis-related genes was determined by RT-qPCR; and the expression levels of CX43, GPX4, and SLC7A11 were detected by Western blotting.

Molecular docking indicated that remimazolam had a binding energy of -6.699 kcal/mol with CX43 protein, suggesting good binding affinity between them. Compared with the Sham group, the I/R group 1 showed increases in Chiu’s scores and CX43 expression (P<0.05), along with pathological damage to intestinal tissues, and elevated contents of Fe²⁺, total iron, ROS and MDA (P<0.05), and down-regulated GPX4 and SLC7A11 (P<0.05). Compared with the I/R group 1, Chiu’s score was reduced in the RM40 group, and CX43 was significantly down-regulated (P<0.05), contents of Fe²⁺, total iron, ROS, and MDA were decreased (P<0.05), and expression levels of GPX4 and SLC7A11 were enhanced (P<0.05), and severity of intestinal histological damage was attenuated in both the RM40 and Fer-1 groups. Compared with the E group, the ERM group had the decreases in CX43 expression level (P<0.05), Fe²⁺, total iron, ROS, and MDA contents (P<0.05), and increases in GPX4 and SLC7A11 expression levels (P<0.05), with the improvement in intestinal tissue. Compared with the oe-NC group, overexpression of CX43 resulted in the increased CX43 expression, elevated contents of Fe²⁺, total iron, ROS and MDA (P<0.05) and decreased expression of GPX4 and SLC7A11 (P<0.05), leading to the exacerbated injury in intestinal tissue.

Remimazolam attenuates intestinal I/R injury by inhibiting ferroptosis through down-regulating CX43 expression.