Tnf α

Ferric sulfate and ferrous sulfate heptahydrate were purchased from Energy Chemical Technology Co., Ltd. (Shanghai, China). Ferric citrate was purchased from Sigma‐Aldrich (St. Louis, MO). Fetal bovine serum (FBS) was purchased from Invitrogen (Carlsbad, CA). Dulbecco's Modified Eagle's Medium (DMEM) was obtained from Sigma (Yuzhong District, Chongqing, China). Antibodies against p21 and C646, a p300/CREB‐binding protein (CBP) inhibitor, were purchased from Abcam Trading Co. Ltd. (Shanghai, China). N‐acetyl‐l‐cysteine (NAC), dihydroethidium (DHE), and a ROS Detection Kit were purchased from Beyotime Company (Jiangsu, China). Prussian Blue staining solution was acquired from Leagene Biological Technology Co. Ltd. (Beijing, China). Antibodies against p53, acetylated p53, CD86, and CD206 were from Cell Signaling Technology (Danvers, MA), and IL‐1, IL‐10, TGF‐β, and TNF‐α were purchased from GeneTex (San Antonio, TX). p21, p300, CBP, p65, phosphorylated p65, and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) were purchased from Abcam (Cambridge, MA). FITC‐CD86 and APC‐CD206 were purchased from Thermo Fisher Scientific (Waltham, MA).

Protein lysates were prepared from animal orbital tissues and human OFs; orbital tissues from each group were homogenized with PRO-PREP solution (Intron, Gyeonggido, Korea). A total of 20 ug proteins were separated by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis and transferred to the membrane. Membranes transferred from gels were incubated with primary antibodies such as anti-PPARγ (GeneTex, Irvine, CA, USA), C/EBPα (GeneTex), IGF-1R (GeneTex), p-SMAD3 (GeneTex), p-SMAD2 (GeneTex), IL-6 (GeneTex), TNFα (GeneTex), ICAM-1 (Thermo Fisher Scientific), TGFβ1 (GeneTex), and β-actin (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). After washing, second antibodies (GeneTex), horseradish peroxidase-conjugated anti-rabbit or mouse IgG were diluted 1:5000 and were incubated with membranes at room temperature for 2 h. The target protein bands were detected with enhanced chemiluminescence (ECL) solution (Bio-Rad Laboratories, Hercules, CA, USA) using an ImageQuant LAS 4000 (GE Healthcare Life Sciences, Little Chalfont, UK).

Western blot analysis was performed as previously described (22 (link)). Total protein from five mice was extracted from the ipsilateral cortices using ice-cold RIPA buffer. Protein concentrations were detected using BCA Protein Assay Kits (Thermo Fisher). Protein samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes. The relevant proteins were detected by incubating the membranes with primary antibodies overnight (i.e., β-actin, 1:1,000, CST, Cat #: 4970S; TNF-α, 1:10,000, GeneTex, Cat #: GTX110520; IL-1β, 1:1,000, CST, Cat #: 12242S; TLR4, 1:1,000, Abcam, Cat #: ab13556; NF-κB, 1:1,000, CST, Cat #: 8482S; TGF-β, 1:2,000, Torrey Pines Biolabs; ZO-1, 1:1,000, Invitrogen, Cat #: 40-2200; and occludin, 1:1,000, Invitrogen, Cat #: 33-1500) and then with secondary antibodies (i.e., horseradish-peroxidase conjugated goat anti-rabbit or anti-mouse IgG, 1:3,000, Cell Signaling Technology). Immunoblots were visualized using a Millipore ECL Western Blotting Detection System (Millipore, Billerica, MA, USA).

Briefly, the brain tissues from the left hemisphere were washed twice with PBS and disrupted with RIPA lysis buffer. Then, the tissue suspension was centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant was used to determine the concentration of the sample with a BCA protein assay kit (Beyotime, China). Equal amounts of samples were separated by SDS-PAGE and electrotransfered to polyvinylidene difluoride membranes. Subsequently, the membranes were blocked with 5% skim milk for 1 h at room temperature and incubated overnight at 4°C with the indicated primary antibodies against NLRP3 (1 : 500, Bios), caspase 1 (1 : 1000, Abcam), IL-1β (1 : 500, Abcam), IL-6 (1 : 1000, Abcam), TNF-α (1 : 500, Gene Tex), MMP-9 (1 : 500, Bios), vascular endothelial growth factor (VEGF) (1 : 500, Bios), vascular endothelial growth factor receptor 2 (VEGFR2) (1 : 1000, CST), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1 : 2500, Abcam). After rinsing with TBST three times, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (1 : 1000, Abcam) for 1 h at 37°C. Chemiluminescence (ECL) kits were used to visualize the membranes, and a Syngene Tanon 5200 imaging system (Tanon, China) was used for imaging. The optical density of the bands was quantified using ImageJ software and corrected according to the corresponding GAPDH level.