Gb13138 1

Coronal sections of mouse brains from 3-, 5- and 7-month-old PPT1 KI mice and 7-month-old WT mice were used. All chemicals and partial service were provided by Wuhan Servicebio Technology CO., LTD. The sections were washed in TBS, and the antigen was retrieved in citric acid buffer for 20 min at 70 °C. Then, all brain sections were rinsed in TBS for 8 min before and after incubation in 10%MeOH and 3%H₂O₂ in TBS for 10 min. Sections were then incubated with primary anti-NeuN (diluted 1:500, GB13138-1, Servicebio, Wuhan, China) over night at 4 °C, after incubation in blocking reagent. Sections were rinsed in TBS for 8 min followed by incubation in the secondary antibody (HRP-conjugated Goat Anti-Mouse IgG (H + L), Servicebio, Wuhan, China) diluted 1:400 in Supermix for 1 h. Sections were then incubated in 0.05% DAB (3,30-diaminobenzidine) tetrahydrochloride. Slides were mounted for histology with micro cover slides. Sections were analyzed using case viewer 2.0 (3DHISTECH Ltd. Budapest, Hungary) and ImageJ 1.52d (National Institutes of Health, Bethesda, MD, USA) at 200× multiples in the hippocampal CA1.

On postoperative day 7, the L4–L6 spinal cord was harvested from rats (n = 3 for SCI group), embedded in paraffin, and cut into 20-μm-thick sections. These sections (n = 3 for each sample) were dewaxed, dehydrated by gradient alcohol, and repaired by antigen. Next, the sections were blocked with endogenous peroxidase and 10% donkey serum and then incubated with the following primary antibodies: NLRP3 (1:200; bs-6655R, Bioss), GFAP (1:500; GB12096, Servicebio), IBA-1(1:500; GB12105, Servicebio), and NeuN (1:100; GB13138-1, Servicebio) overnight at 4°C. After the primary antibody incubation, the sections were incubated with the corresponding secondary antibodies conjugated with CY3 and FITC for 1 h in dark conditions at 37°C. Finally, the dorsal horns were observed and photographed under a fluorescence microscope (Olympus, Japan).

Kaempferol was purchased from Chengdu Alfa biotechnology CO., LTD (Chengdu, China). Antibodies against caspase-3 (ab13847), BDNF (ab108319), TrkB (187041), COX-2 (ab15191), CitH3 (ab281584), Galectin-3 (ab76245) and ICAM-1 (ab171123) were purchased from Abcam (Cambridge, UK). Antibodies for β-actin (3700), p-PI3K (4288), PI3K (4257), Bcl-xl (2764T), p-AKT (9271S), AKT (9272S), MyD88 (4283), p-NF-κB (3033), NF-κB (4764), p-JAK1 (3331), JAK1 (88617), p-STAT3 (9145) and STAT3 (9139) were purchased from Cell Signaling Technology (Beverley, CA, USA). Anti-Bax antibody (50500-2-Ig), anti-Bcl-2 antibody (12789-1-AP), anti-ZO-1 antibody (1773-1-AP) and anti-occludin antibody (13409-1-AP) were the products of Proteintech (Wuhan, China). Antibodies for TLR4 (sc-293072) was provided by Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Antibodies against NeuN (GB13138-1), Iba-1 (GB13105-1) and GFAP (GB11096) were provided by Servicebio (Wuhan, China).