Western blot assays

The total proteins in different groups were extracted using a total protein extraction kit according to the manufacturer’s instructions (Cal. No. WLA019, Wanleibio, China). GAPDH was used as an internal reference. The protein concentration in each sample was determined using the BCA method. A 20-μL aliquot of protein (40 μg) was separated by electrophoresis on a 10% sodium dodecylsulfate polyacrylamide gel. Following separation, the targeted proteins were transferred onto polyvinylidene difluoride (PVDF; BD, San Jose, CA, USA) sheets, which were then washed with TTBS for 5 min prior to being incubated in a powdered skim milk solution for 1 h. Primary antibodies against NLRP3 (1:400, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Notch1 (1:1500, Santa Cruz Biotechnology Santa Cruz, CA, USA), Bcl-2 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bcl-XL (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), caspase-1 (1:2000, Abcam, Cambridge, MA, USA), caspase-3 (1:800, Abcam, Cambridge, MA, USA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:4000, Abcam; Cambridge, MA, USA) were incubated with the membranes at 4 °C overnight. Following incubation, the membranes were washed four times with TTBS, after which secondary HRP goat anti-rabbit antibodies (1:20,000, Cat. No. BA1054, Wuhan, China) were added to the mixture and incubated with the membranes for 45 min at 37 °C. After an additional six washes with TTBS, the blots were developed using Beyo ECL Plus reagent, and the results were detected with a gel imaging system. The relative levels of BDNF expression in the different samples were calculated using Gel-Pro-Analyzer (Media Cybernetics, Rockville, MD, USA).