To investigate the role of quercetin (QUE) in ferroptosis during intestinal ischemia-reperfusion (IR) injury and elucidate its underlying mechanisms.

① Potential target genes of QUE were predicted using the TCMSP, PharmMapper, and SwissTargetPredictive databases. Target genes associated with intestinal IR injury and ferroptosis were collected from GeneCards, PharmGKB, and OMIM databases. After overlapping genes were identified and analyzed, protein-protein interaction (PPI) networks were constructed using the STRING database and then visualized with Cytoscape 3.10.0. Molecular docking was performed to validate the binding conformations between QUE and key targets. ② In vivo experiments were conducted to verify QUE’s protective effects against intestinal IR injury. Thirty-six SPF-grade male C57BL/6J mice (6~8 weeks old, body weight: 22±2 g) were randomly divided into Sham, Sham+QUE, IR, IR+QUE, IR+QUE+erastin (IR+QUE+Era), and IR+QUE+ kevetrin hydrochloride (IR+QUE+KH) groups, with 6 mice in each group. Mouse model of intestinal IR injury was induced by 45 min ischemia of the superior mesenteric artery followed by 60 min reperfusion. HE staining was used to observe histopathological changes in the intestinal tissues. ELISA was employed to the serum or intestinal contents of diamine oxidase (DAO), pro-inflammatory cytokines (TNF-α, IL-6, IL-1β), and ferroptosis markers [glutathione (GSH) and Fe²⁺] Western blotting was utilized to detect the protein expression of glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long-chain family member 4 (ACSL4), and tumor protein 53 (p53).

① Network pharmacology identified 460 QUE targets, 1552 intestinal IR injury targets, and 1967 ferroptosis-related targets, and 92 overlapping genes were identified as potential therapeutic targets. Molecular docking revealed a strong binding affinity between QUE and p53 (binding energy:-6.8 kcal/mol). ② In vivo experiments demonstrated that the IR+QUE group exhibited reduced intestinal damage and lower Chiu’s score (P<0.05), decreased serum DAO content but elevated intestinal DAO content (P<0.05), decreased levels of TNF-α, IL-6, and IL-1β in the serum and intestinal tissues (P<0.05), reduced Fe²⁺ accumulation, and increased GSH content (P<0.05), and up-regulated GPX4 (P<0.05) and down-regulated ACSL4 and p53 expression (P<0.05) at protein level when compared with the IR group. While, the administration of ferroptosis agonist Era, or p53 agonist KH resulted in diminished therapeutic effects of QUE (P<0.05) when compared with the IR+QUE group.

QUE alleviates intestinal IR injury by inhibiting ferroptosis, which may be associated with its down-regulating p53 expression.

Intestinal ischemia-reperfusion (IR) injury is a common and critical pathological process in clinical practice. Ferroptosis, a form of cell death characterized by iron-dependent accumulation of uncontrolled lipid peroxidation, has been confirmed to be involved in intestinal IR injury. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key molecule in regulating ferroptosis, and its activation can inhibit this form of cell death, thereby alleviating tissue injury. Sulforaphane (SFN), a classical Nrf2 activator, exerts antioxidant protective effects. This study aims to investigate whether SFN alleviates intestinal IR injury by activating the Nrf2 pathway to inhibit ferroptosis.

① SFN-related target genes, ferroptosis-related genes, and intestinal IR injury-related genes were retrieved and integrated from online databases, including TCMSP, GeneCards and OMIM, and the intersection genes were identified and presented in a Venn diagram. Then the intersection genes were imported into STRING to construct a protein-protein interaction (PPI) network, and topological analysis was performed using Cytoscape (v3.9.1) to screen core targets. Molecular docking was employed to predict the binding affinity between SFN and the key target Nrf2. ② Twenty-four SPF-grade male C57BL/6 mice (6 to 8 weeks old, weighing 22±2g) were randomly divided into 4 groups (n=6): Sham group, IR group, IR+SFN group, and IR+SFN+ML385 (a specific Nrf2 inhibitor) group. Except for the Sham group, the other groups were inflicted with clamping the superior mesenteric artery for 45 min followed by reperfusion for 30 min to establish an IR injury model. The IR+SFN and IR+SFN+ML385 groups were given an intraperitoneal injection of SFN (5 mg/kg) 1 h before clamping, while the IR+SFN+ML385 group additionally received intraperitoneal injection of ML385 (30 mg/kg) 2 h before clamping. After 30 min of reperfusion, small intestinal tissues were harvested. Histopathological changes were observed by light microscopy after HE staining, and the severity of intestinal injury was evaluated using Chiu’s score. Levels of malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) were measured using biochemical reagent kits. Western blotting was performed to detect the protein expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), glutathione peroxidase 4 (GPX4), Nrf2, and heme oxygenase-1 (HO-1).

① Network pharmacology analysis identified 162 SFN target genes, 2772 ferroptosis-related genes, and 2100 intestinal IR injury-related genes, with 43 intersection genes. PPI network topological analysis revealed 36 core genes, including Nrf2. Molecular docking showed that SFN could form a binding conformation with Nrf2, with a binding energy of -3.3 kcal/mol. ② In vivo results demonstrated that compared with the Sham group, the IR group exhibited significantly aggravated intestinal mucosal injury, as evidenced by an increased Chiu’s score (P<0.05), elevated MDA level, and decreased SOD and GSH activities (P<0.05). Meanwhile, the expression of GPX4 was downregulated while that of ACSL4 was upregulated (P<0.05), suggesting the occurrence of ferroptosis in intestinal IR. SFN pretreatment ameliorated the above pathological and biochemical changes, upregulated Nrf2 and HO-1 expression (P<0.05), restored GPX4 expression and downregulated ACSL4 expression (P<0.05). The protective effects of SFN and its regulatory effects on the Nrf2/HO-1 axis and ferroptosis-related proteins were partially reversed by the addition of ML385 (P<0.05).

SFN can alleviate intestinal IR injury, and its effect may be related to enhancing the antioxidant response associated with the Nrf2/HO-1 signaling axis and ameliorating ferroptosis-related molecular changes.

To investigate the anesthetic effects and adverse effects of cypropofol and propofol combined with alfentanil, respectively, for gastroenteroscopy.

A total of 162 patients who underwent elective gastroenteroscopy at the Gastrointestinal Endoscopy Center of the First Hospital of Lanzhou University from January to February 2024 were enrolled, including 86 males and 76 females, at an age of 18~65 years old, with a BMI value of 18~30 kg/m², and ASA grade ≤Ⅱ. They were randomly divided into propofol group (Group P) and cypropofol group (Group C), with 81 cases in each group. All patients were sedated with 0.7 μg/kg alfentanil, and in 30 s later, 2 mg/kg propofol and 0.4 mg/kg cypropofol was intravenously dripped into Group P and Group C, respectively. When the modified alertness/sedation score (MOAA/S) ≤1, a gastroscope was started to insert. The related indicators, including total procedure time, successful cases of sedation, induction time and awakening time, heart rate, blood pressure, and pulse oximetry saturation were recorded, occurrence of adverse reactions such as hypotension, respiratory depression, injection pain, intraoperative body movement, nausea and vomiting were observed, and the satisfaction of endoscopists and of patients to anesthesia were recorded and compared between the 2 groups.

There were no statistical differences in the success rate of sedation, induction time and awakening time between the 2 groups. The patients of the Group C had more stable intraoperative vital signs, statistically lower incidences of injection pain, respiratory depression and hypotension (P < 0.05), and increased satisfaction for anesthesia (P < 0.05) when compared with those in Group P. No obvious difference were observed in the satisfaction of endoscopist to anesthesia between the 2 groups.

In combination with small-dose alfentanil, 0.4 mg/kg cypropofol shows similar sedation effect as 2 mg/kg propofol in gastroenteroscopy, with comparable induction and awakening time. Cypropofol has more advantages in stable intraoperative vital signs, less adverse effects such as low blood pressure, respiratory depression and injection pain, higher the patient satisfaction, which is worthy of clinical promotion.

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.