SB 216763

Primary human fibroblasts were obtained after circumcision of foreskins and approval of the protocol by the ethics committee of TMMU. Isolation protocols have been previously described.^{50 (link)} In brief, foreskin tissues were cut into 1–2 cm² pieces after subcutaneous tissue removal and digested for 1 h at 37 °C in a digestion medium containing 1 mg/ml dispase (Roche, Basel, Switzerland). The epidermis was then stripped and digested for another 1 h at 37 °C in the digestion medium consisting of DMEM with 0.25% collagenase I (Worthington Biochemical, Beijing, China). The digested cells were then passed through a 75-μm cell strainer, centrifuged, and re-suspended in Iscove's modified Dulbecco's Medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (all products obtained from Beyotime). Cells were grown to >90% confluence, trypsinized (trypsin-EDTA 0.5% w/v, Hyclone), and re-plated for experiments.
Mouse granulation tissue cells were isolated as previously described.^{34 (link)} Briefly, granulation tissues were harvested 10 days after wounding and cut into 1 cm² pieces and digested for 1 h at 37 °C in a digestion medium containing 0.25% collagenase I (Worthington Biochemical). The following protocols and culture conditions were the same as those used for human fibroblast isolation and culture. Tumor cell lines (A549 and MG63) were purchased from ATCC (CCL-185 and CRL-1427) and cultured under the same conditions as those for human fibroblasts. LY294002 (50 μM; Sigma, St. Louis, MO, USA) and SB216763 (10 μM; Sigma) were used to inhibit PI3K and GSK-3β, respectively.

After a midline laparotomy, mice were injected with heparin (100 μg/kg) and an atraumatic clip was used to interrupt arterial and portal venous blood supply to the cephalad liver lobes. After 90 minutes of ischemia, the clip was removed to initiate hepatic reperfusion. Sham controls underwent the same procedure but without vascular occlusion. Mice were sacrificed after 6 hours to 7 days of reperfusion, and liver and serum samples were collected. Serum ALT levels were measured with an autoanalyzer by ANTECH Diagnostics. Portions of liver specimens were fixed in 10% buffered formalin and embedded in paraffin. Liver sections (4 μm) were stained with H&E. The severity of liver IRI was graded blindly using the Suzuki criteria on a scale from 0 to 4. No necrosis, congestion, or centrilobular ballooning is given a score of 0, whereas severe congestion and >60% lobular necrosis is given a score of 4. Anti–IL-10 Abs (0.5mg/mouse; clone JES5-2A5, Bio-Express) were administered i.p. 1 hour prior to inducing the liver ischemia. Gsk3 inhibitor SB216763 (25 μg/g; Sigma) was administered i.p. at 24 hours, 3 days, and 5 days after reperfusion.

The animal exposure to PM_2.5 was conducted using a modified whole-body animal exposure model, as detailed in our previous publication [15 (link)]. The temperature, pressure, and flow rate within the chamber were maintained at optimal levels. To monitor the concentrations of PM, oxygen (O₂), and carbon monoxide (CO), as well as the temperature and humidity inside the chamber, a PRANA air-sourced CAIR air quality monitor was employed. The experimental animals used in the present study were divided into two major groups: Group A (composed of 42 rats), exposed to PM_2.5 for 21 days; and Group B (composed of 42 rats), exposed to PM_2.5 for 1 day. Group A and B were further subdivided into 7 groups composed of 6 animals per group.
Group A: (21-day exposure study): 1: Normal; 2: IR control; 3: PM_2.5 exposure control (PM_C): Exposure to PM_2.5 at a concentration of 250 µg/m³ for 3 h daily for 21 days; 4: PM_2.5 exposure followed by IR induction (PM_IR): Exposure to PM_2.5 followed by IR induction; 5: GSK3β_IR: 0.7 mg/kg of GSK3β inhibitor: SB216763 was administered intraperitoneally followed by IR induction; 6: PM+ GSK3β_C: Exposure to PM_2.5 at a concentration of 250 µg/m³ for 3 h daily for 21 days followed by 0.7 mg/kg of SB216763 administration and normal perfusion for 120 min; 7: PM+ GSK3β_IR: Exposure to PM_2.5 at a concentration of 250 µg/m³ for 3 h daily for 21 days followed by SB216763 administration and IR induction.
Group B: (1-day exposure study): Normal; 2: IR control; 3: PM_2.5 exposure control (PM 1day_C): Exposure to PM_2.5 at a concentration of 250 µg/m³ for 1 h; 4: PM_2.5 exposure followed by IR induction (PM 1day_IR): Exposure to PM_2.5 followed by IR induction; 5: GSK3β_IR: 0.7 mg/kg of GSK3β inhibitor: SB216763 was administered intraperitoneally followed by IR induction; 6: PM 1day+ GSK3β_C: Exposure to PM_2.5 at a concentration of 250 µg/m³ for 1 h followed by 0.7 mg/kg of SB216763 administration and normal perfusion for 120 min; 7: PM 1 day + GSK3β_IR: Exposure to PM_2.5 at a concentration of 250 µg/m³ for 1 h followed by SB216763 administration and IR induction.