Pparγ

Incomplete Freund's adjuvant (IFA) and Bacillus Calmette-Guérin (BCG) were purchased from Rebio Scientific (Shanghai, China). TNF-α, IL-1β, IL-10, and IL-6 ELISA kits were the products of Multi Science (Hangzhou, China). Biochemical quantification kits for the determination of malondialdehyde (MDA), reduced glutathione (GSH), total superoxide dismutase activity (SOD), total antioxidant capacity (T-AOC), nitric oxide (NO), nitric oxide synthase (inducer) (iNOS), and carbonic oxide (CO) were brought from Jiancheng Bioengineering Institute (Jiangsu, China). The primary antibodies used in immunoblotting and immunohistochemical experiments including anti-MMP3, COX-2, TLR4, SIRT1, PPAR-γ, p65, p-p65, and β-actin antibodies together with secondary antibodies were provided by ABclonal Technology (Wuhan, China). Fluorescein-tagged anti-CD3, CD4, and IL-17α antibodies for the use of flow cytometry analysis were supplied by Beyotime Biotech (Nantong, Jiangsu, China). Rat peripheral blood mononuclear cell (PBMC) isolation kit, TaqMan RT-qPCR kit, and cDNA synthesis kit were purchased from Solarbio Technologies (Beijing, China). All the solvents were of analytical grade and supplied by Merck Chemicals (Shanghai, China). High-purity compounds (>98%) kaempferol, paeoniflorin, matrine, sophocarpine, and sinomenine were all obtained from Yuanye Bio-Technology (Shanghai, China).

In our study, 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS solution), 2,2-diphenyl-1-picrylhydrazyl (DPPH), N,N,N′,N′-tetramethylethylenediamine (TEMED), acrylamide/bisacrylamide, ammonium persulfate (APS), ascorbic acid (vitamin C), bovine serum albumin (BSA), ethanol (96%), glycine, methanol, Ponceau S, resazurin sodium salt (RES), sodium dodecyl sulfate (SDS), Trolox, trypsin-EDTA solution, Tris-HCl, Tris-Base, and Tween-20 were purchased from Merck KGaA (Darmstadt, Germany). The PVDF membrane with 0.45 µm-pore size and primary antibodies against SOD1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The primary antibodies against GAPDH, NF-κB, IκBα, SRC, GLUT4, PCNA, PGC-1α, PPARγ, p-ERK1/2, and ERK1/2 were purchased from ABClonal (Woburn, MA, USA). The primary antibodies against NLRP3, anti-mouse- and anti-rabbit-HRP-conjugated antibodies were purchased from ThermoFisher (Waltham, MA, USA). Potassium persulfate (Warchem, Zakręt, Poland), antibiotics (Penicillin-Streptomycin, Life Technologies, Bleiswijk, The Netherlands), DMEM (Dulbecco’s Modification of Eagle’s Medium, Biological Industries, Beit Haemek, Israel), FBS (Fetal Bovine Serum, Biological Industries, Genos, Lodz, Poland), and phosphate buffered saline (PBS, pH 7.00 ± 0.05, ChemPur, Piekary Ślaskie, Poland) were also used during the analyses.

Total protein from the cell was collected using the ProteinExt^®Mammalian Total Protein Extraction Kit (TransGen Biotech, Beijing, China), following the manufacturer’s protocol. The concentration of the protein was measured using the Bradford protein assay kit (Novoprotein, Shanghai, China). The protein was resolved on 10% SDS-PAGE and then transferred to a PVDF membrane, followed by sealing of the sealing fluid. The membranes were incubated with the corresponding primary antibodies for 8 h at 4 °C and subsequently incubated with the secondary anti-bodies (Goat Anti-Rabbit IgG H&L (HRP), Zen bioscience, Chengdu, China) for 2 h. Then the bands were washed three times, and the hypersensitive chromogenic solution was developed using chemiluminescence in Touch Imager Pro, e-BLOT Life Science (Shanghai) Co., Ltd. (Shanghai, China). The primary antibodies PCNA, CDK2, and CDK4 were purchased from Zen bioscience (Chengdu, China). The primary antibodies PPARγ, CEBP/α, FABP4, SREBP1, and RAD51 were purchased from the Abclonal (Wuhan, China).

The samples were cleaned using PBS, sectioned, sterilized by adding 3% H₂O₂, and incubated for 15 min at room temperature. By supplementing goat serum (Beyotime, C0265, China), the sections were blocked for 60 min at room temperature, taken out, and dried. A primary antibody was provided and incubated at 4°C overnight (ki67, A2094, 1:200, ABclonal, China; PPARγ, A0270, 1:200, ABclonal, China). The specimens were washed thrice using PBS for 5 min. A secondary antibody working solution was used for incubation at room temperature for 1.5 h and washed again as previously described. DAB (ZSGB-BIO, ZLI-9019, China) was applied for development for 10 min (in the dark). Hematoxylin was used for restaining 5 min, followed by 1% hydrochloric acid ethanol differentiation for 5 s, and 95% ethanol dehydration for 2 min. Fresh 95% ethanol was employed for dehydration for another 2 min, and xylene was employed for transparency for 5 min. Fresh xylene was used for transparency for an additional 5 min, and the slides were fixed with neutral resin. The sections were photographed by an inverted Mshot MF53 microscope produced by Guangzhou Mingmei Optoelectronic Technology Co. Ltd.