Adult male C57/BL mice (7–9 weeks old; weight, 27.1±2.3 g) were purchased from Changsheng Biotechnology Co., Ltd. (Benxi, Liaoning, China). All the mice had free access to food and water; they were kept in a 12-h alternating light and dark facility at 25°C ± 1°C (humidity 50%–70%). The tests involving animals were performed following the guidelines of the Animal Ethics Committee of Cangzhou Central Hospital.
Grouping and intestinal ischemia-reperfusion (IR) induced ALI
In the first stage, animals were randomly grouped into five groups: sham (n =24); intestinal ischemia-reperfusion (IR) + vehicle (n =24); IR + C-176 (350 nmol, 4.7 mg/kg) (n =18); IR + C-176 (550 nmol, 7.3 mg/kg) (n =24) and IR + C-176 (750 nmol, 10 mg/kg) (n =18). In the second stage, animals were randomly divided into two groups: IR + C176 + compound C (n =12), and IR + C176 + vehicle (n =12).
Mice were anesthetized with pentobarbital (10 mg/kg) via intraperitoneal injection. An oral endotracheal tube for the mouse was established then connected with a ventilator (Model: volume-controlled; tidal volume: 8 mL/kg; frequency: 120 beats/min). Throughout the experiment, the animals were kept on a warm blanket to maintain the body temperature within 37–38℃. Based on previous studies , the IR model was established as follows: (1) a lower midline laparotomy was performed; (2) the superior mesenteric artery was identified and occluded below the celiac trunk with an arterial microclamp, and intestinal ischemia was confirmed by the paleness of the jejunum and ileum; (3) the clamp was removed after 60 min; (4) 0.5 mL of sterile saline at 37℃ was injected into the peritoneal cavity; (5) the incision was sutured and blocked using ropivacaine. At the first stage, a selective STING inhibitor, three dosages of C-176 (4.7 mg/kg, 7.3 mg/kg, and 10 mg/kg) (HY-112906, MedChemExpress, NJ, USA) were administered 30 minutes before surgical exposure via intraperitoneal injection. In the second stage, a selective antagonist of AMPK, compound C (25 mg/kg) (HY-13418A, MedChemExpress), was administered via subcutaneous injection 30 min after initiation of IR. Both C-176 and compound C were dissolved with 10% DMSO (HY-Y0320, MedChemExpress) and 90% corn oil (HY-Y1888, MedChemExpress). Notably, the sham animals underwent a similar procedure without clamps.
Assessment of lung injury
Three hours after IR, mice (n =6) were euthanized through cervical dislocation under 8% sevoflurane. The surface of the left lungs was wiped after rinsing with saline. The weight was recorded as wet weight (W). After drying at 70℃ for 24 h, the dry weight (D) was recorded. Total lung water content (TLW) was calculated using this equation: TLW =(W-D)/D × 100%.
Mice (n =6) were anesthetized under 3–4% sevoflurane and injected with 0.5 mL of normal saline via the trachea. Then, the liquid of bronchoalveolar lavage was gently aspirated and bronchoalveolar lavage was performed twice. After centrifugation with 4000×g at 4 ℃ for 10 min, the supernatant from the lavage solution was obtained as bronchoalveolar lavage fluid (BALF). The content of BALF protein was measured using the BCA assay based on the manufacturers’ instructions (P0012, Beyotime, Shanghai, China).
Hematoxylin and eosin (H&E) staining and Masson staining
Three hours after IR, mice (n =6) were anesthetized under 3-4% sevoflurane and perfused with cold saline via ventriculus sinister–aorta. Mice were perfused with 10% paraformaldehyde after the clear saline was released from the right auricle. After fixing with 10% paraformaldehyde for 48 h, left lungs were cut into 5-μm paraffin coronal sections for hematoxylin and eosin (H&E) and Masson staining as previously published [18, 19]. The slides were observed under a light microscope (BX51; Olympus, Tokyo, Japan). Three fields (magnification, ×200) in one slice (3 slices in one group) were randomly selected. Lung injury was analyzed by an experienced investigator blinded to the group, and recorded as normal (0), mild (1), moderate (2), or severe (3), based on histological parameters, including alveolar edema, diffuse alveolar hemorrhage, and congestion, intra-alveolar infiltration of inflammatory cells. Additionally, the percentage of Masson-stained collagen was measured using Image J (1.37v, Wayne Rasband, available through the National Institutes of Health).
Slices (thickness: 5-μm) mentioned above were used for immunofluorescence staining. After boiling with sodium citrate at 100℃ for 20 min, the cooled sections were incubated with 1% Triton X-100 for 20 min and blocked with the QuickBlockTM Blocking Buffer (P0260, Beyotime) at 25℃ for 1 h. After washing with PBS three times, sections were overnight incubated with rabbit anti-Gasdermin-D (GSDMD) (K009328P, Solaibio, Beijing, China) at 4 °C. After rising with PBS, the slices were incubated with goat anti-rabbit secondary antibodies (P0208, Beyotime) at room temperature for 1 h, then coated with anti-fluorescence quenching sealing solution with DAPI (P0131, Beyotime) for 5 min and sealed. Terminal deoxynucleotide transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) (C1062, Beyotime) assay was performed as per the manufacturer’s protocol to detect pulmonary apoptosis. Six fields with a magnification of ×200 in 3 slices were randomly selected from each group. Under a fluorescence microscope (BX53, Olympus, Tokyo, Japan), the percentage of GSDMD-positive cells and TUNEL-positive cells were calculated using the Image J (1.37v, Wayne Rasband, available through the National Institutes of Health).
After three hours of IR, mice (n =6) were anesthetized under 3–4% sevoflurane and perfused with cold saline via ventriculus sinister–aorta until clear fluid flowed out from the right atrial appendage. Lung tissues were quickly kept on ice. The lung tissues were removed, homogenized on ice; the total protein was extracted and quantified through the BCA assay. Each sample containing 40 μg of mixed with loading buffer was boiled at 100℃ for 15 minutes. The sample was separated with 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated with QuickBlockTM blocking buffer (P0235, Beyotime) at 25℃ for 10 min, then rinsed with Western wash buffer (P0023C, Beyotime) for 5 min to 3 times. Anti-rabbit p-AMPK (dilution: 1:500, ab133448, Abcam, Cambridge, UK), anti-rabbit AMPK (dilution: 1:1000, ab32047, Abcam, Cambridge, UK), anti-rabbit cleaved-caspase-1 (dilution: 1:500, ab179515, Abcam, Cambridge, UK), anti-rabbit cleaved-caspase-3 (dilution: 1:500, ab32351, Abcam, Cambridge, UK) and GADPH (dilution 1:1000, K106389P, Solaibio) were used for overnight incubation of PVDF membranes at 4 ℃. After rising with Western washing buffer 3 times, the membranes were incubated with goat anti-rabbit secondary antibody (dilution 1:1000, A0562, Beyotime) at 25 ℃ for 1 h. After washing 3 times with Western washing buffer, protein bands were detected using the BeyoECL Moon (P0018, Beyotime). The ratio between the gray value of the target protein to GAPDH bands (internal reference) was calculated using Image J.
All results were expressed as the Means ± standard deviation (SD). Statistical analysis was performed using student’s t-test or one-way analysis of variance (ANOVA) for multiple comparisons. Differenceswith P values of less than 0.05 (P<0.05) were considered statistically significant.