Experimental Groups and Treatments

The rats were randomly divided into five groups with six rats in each as follows. 1) The control group (Control), as previous (Yang et al., 2020), the rats were intraperitoneally injected with 2 ml vehicle for 7 days. One hour after the last administration, rats were anesthetized with pentobarbital sodium (50 mg/kg) intraperitoneally, performed thoracotomy, exposed the trachea and then closed the chest immediately. 2) I/R group, the rats were intraperitoneally injected with 2 ml vehicle for 7 days, then operated 1 h after the last administration as previous reported (Yang et al., 2020). Briefly, after the rats were anesthetized with pentobarbital sodium, the trachea was surgically exposed and mechanical ventilation was performed with 40% O₂. Open the chest at the left 3–5 intercostal space, free the left hilar, and arterial clip to block the left hilum for 90 min, then restore blood flow and reperfusion for 4 h. Anesthesia was maintained with inhaled halothane. 3) Kaempferol pre-treatment group (I/R + Kae), as previous report (Yang et al., 2020), the rats were intraperitoneally injected with 50 mg/kg kaempferol for 7 days before I/R operation. Surgery was performed as I/R group immediately 1 h after the last administration. 4) Kaempferol and EX527 combination pre-treatment group (I/R + Kae + EX527), the rats were intraperitoneally injected with EX 527 (5 mg/kg, every 2 days, total 7 days) before I/R operation on the basis of I/R + Kae group as previous report (Yang et al., 2020). 5) Kaempferol and SR-18292 combination pre-treatment group (I/R + Kae + SR-18292), the rats were intraperitoneally injected with 30 mg/kg SR-18292 for consecutive 7 days before I/R operation on the basis of I/R + Kae group. The rats in each group were sacrificed after re-perfusion by removing the ventilator. The left lungs were removed out from the thoracic cavity. One part of left lung was weighed, dried at 60°C for 48 h and weighed again to calculate the wet/dry (W/D) ratio. The remaining left lung was frozen directly at −80°C or fixed with 4% paraformaldehyde.