Biochemical assay kit

Total ROS Assay kit 520 nm (No. 88-5930-74) was purchased from Thermo Fisher, USA. Biochemical assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Apoptosis Kit (No. 556547) was purchased from Becton Dickinson Company (Franklin Lakes, USA). Antibodies against Nod-like receptor protein 3(NLRP3) and nuclear factor-κB (NF-κB) were obtained from Sagon Biotech (Shanghai, China). The antibody against Gasdermin D was obtained from Thermo Fisher (USA) and β-actin from Bioss (Beijing, China). Caspase-1 p20 antibody was purchased from Boster Biological Technology Co., Ltd (Wuhan, Hubei, China). Ageratina adenophora was collected from Xichang, Sichuan Province, Southwest China. The collected leaves were cleaned, ground and screened at room temperature to make dry powder. The powder was stored in shade condition with an ambient temperature of 20 ± 2°C. For the preparation of 10%, 20% and 30% diet pellet, A. adenophora and mice feed were homogenized in water solution by the ratio of 1 : 9, 1 : 8 and 1 : 7, respectively. Then the diet was cast in the form of cylinders and dried at 27°C for 48 h.

Rat kidney tissue, serum, and urine were collected. The right kidney tissues of rats were supplemented with appropriate amount of normal saline and then grind into tissue homogenates with a tissue grinder. Then, the homogenates were centrifuged (4°C, 3000 r/min for 10 min), and the supernatant was collected. Also, the blood was centrifuged (3500 rpm for 15 min) to collect serum. In the light of the instructions of biochemical assay kits (Nanjing Jiancheng Bioengineering Institute, China), serum, urine, and supernatant of the tissue homogenates were diluted into gradient solutions with different concentration, respectively. Subsequently, a biochemical automatic analyzer was utilized to determine the levels of blood urea nitrogen (BUN), creatinine (Cr), and 24 h urinary protein (24-up) in serum, oxalate (Abcam, USA) in urine, and malondialdehyde (MDA), superoxide dismutase (SOD), lactate dehydrogenase (LDH), and reactive oxygen species (ROS) in tissue supernatant [20 (link)].

MDA content and MPO activity in kidney homogenate were measured by biochemical assay kits (Nanjing Jiancheng, Nanjing, China) according to the manufacturer’s instructions (Ye et al., 2015 (link)).

Body weight and food intake of the animals were measured weekly. At the end of the study, mice were sacrificed by CO₂ inhalation after overnight fasting. The body weights of the mice were counted after fasting, and the liver tissue and epididymal adipose tissue were collected and counted separately. Fat to body weight is the ratio of the absolute weight of epididymal fat to body weight. Blood was collected from the heart, and serum was obtained by centrifugation at 3000 rpm for 20 min for subsequent analysis. Serum total cholesterol (TCHO), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), alanine amioTransferase (ALT) and aspartate Transaminase (AST) were measured by using biochemical assay kits provided by the Jiancheng Bioengineering Institute (Nanjing, China). All assays were performed according to the instructions supplied by the manufacturer.

At 12 h postlight exposure, HeLa cells were irradiated by 4 Gy X-rays with X-RAD 320iX machine (Precision X-ray, Inc., USA), at 24 h postirradiation, the cells were homogenized using homogenate medium (pH 7.4, 0.01 M Tris-HCl, 0.001 M EDTA-2Na, 0.01 M saccharose, and 0.8% NaCl) (Nanjing Jiancheng Bioengineering Institute, China), and protein concentrations were determined. Na⁺-K⁺ and Ca²⁺-Mg²⁺ ATPases and ATP were measured using biochemical assay kits (Nanjing Jiancheng Bioengineering Institute, China) and a spectrophotometer (Beckman, USA) with 636 nm excitation wavelengths. There were 4 replicate wells per group, and the experiment was performed in triplicate.

Hepatic tissue samples kept at −80°C were homogenized with ice-cold normal saline (100 mg: 900 μL), and the supernatant samples were prepared for biochemical assays by centrifuging (1000 g, 4°C for 15 minutes). Biochemical assay kits were purchased form Nanjing Jiancheng Bioengineering Institute (Nanjing, China): MDA (malondialdehyde, A003-1-1), SOD (superoxide dismutase, A001-3-1), CAT (catalase, A007-1-1), and GSH-Px (glutathione peroxidase, A005-1-2). The levels of MDA and activities of SOD, CAT, and GSH-Px in hepatic tissue were assayed following the manufacturer's protocols.