Gsh px kit

Monarda didymaL. essential oil used in the experiment was originated from Heilongjiang Province, China, and cultivated by our team. Donepezil (Shanghai Yuanye Bio-Technology Co., Ltd., >98%, Shenyang, China); D-galactose (Beijing Dingguo Changsheng Biotech Co., Ltd., >98%, Beijing, China); SOD, MDA, and GSH-Px kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China); All antibodies for western blot were procured from Beijing Boao Biotechnology Co., Ltd.

Selenium-enriched soybeans were purchased from Enshi Se-Run Health Tech Development Co., Ltd. (Enshi city, China), while L-SeMet was obtained from Shanghai Macklin Biochemical Co., Ltd. Na₂SeO₃ was purchased from Sigma Chemical Co. (St. Louis, MO, United States). Xi’an Ruidi Biotechnology Co., Ltd., supplied the honeysuckle extract. The alanine aminotransferase (ALT), aspartate aminotransferase (AST), catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), GSH, and GSH-Px kits were obtained from Nanjing Jiancheng Bioengineering Institute. The IL-1β, IL-6, TNF-α, GSH, and GSH-Px ELISA kits were acquired from Jiangsu Meibiao Biotechnology Co., Ltd., while the BCA protein concentration assay kit was purchased from Beijing Solarbio Science & Technology Co., Ltd. The skim milk powder was supplied by Becton, Dickinson, and Company. Furthermore, the PVDF membrane, TBST, NF-κB p65 rabbit monoclonal antibody, phospho-IκBα (Ser32/36) rabbit polyclonal antibody, α-Tubulin rabbit polyclonal antibody, horseradish peroxidase-labeled goat anti-rabbit IgG (H + L), Western secondary antibody dilution, and BeyoECL Star (extra super-sensitive enhanced chemiluminescence kit) were purchased from Beyotime Biotechnology.

Saline (0.9 % NaCl, No. 121112B43) was purchased from Fuzhou Neptunus Futao Pharmaceuticals Co. Ltd., China. Alcohol (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was diluted with saline (0.9 % NaCl) to 53 % v/v for this study. Honey of lychee flower (FM020204130301) was provided by Fujian Shenfeng Science Development Co. Ltd., China. Positive drug, “RU-21 wake up clear” pills (lot#25727, Spirit Sciences, USA), a commercial dietary supplement for consumers of alcohol, was purchased from local market. For serum MDA and GSH-Px assay, MDA and GSH-Px kits were purchased from Nanjing Jiancheng Bioengineering Institute, China.

Ileum and liver (about 100 mg) were devolved and mixed in a 0.9 mL stroke-physiological saline solution (4 °C, 0.9% NaCl, pH = 7.2–7.4) to obtain 10% tissue homogenate. The activity or content of GSH-Px, T-SOD, CAT, and MDA in the liver and ileum tissues were assessed according to the requirements of the GSH-Px kits (Item No.: A005-1-2), T-SOD kits (Item No.: A001-3-2), CAT kits (Item No.: A007-1-1), and MDA kit (Item No.: A003-1-2) (purchased from Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The absorbance was determined using a UV–visible spectrophotometer (UV1100, MAPADA, Shanghai, China). The total protein content of each sample required in the kit was measured by the BCA Protein Assay Kit (Beyotime, Shanghai, China).

The liver tissues stored at -80°C were homogenized in PBS (pH = 7.4). Homogenates were centrifuged at 3000 × g for 10 min at 4°C [41 ]. Then the suspensions were used for antioxidant indexes analysis. The protein concentration in livers was estimated with BCA protein assay kit (Beijing Solarbio Science & Technology Co., Ltd., China)
To determine the oxidative injury in piglets induced by RV, the content of malonaldehyde (MDA) and activities of antioxidant enzymes (Superoxide dismutase, SOD; glutathione peroxidase, GSH-Px) in serum and liver were evaluated by using the MDA, SOD and GSH-Px kits, respectively, according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, China). The optical densities of each index were recorded using a spectrophotometer (UV-2800A, UNICO, China) at 532nm, 550nm, and 412nm, respectively. The SOD and GSH-Px activities in serum and liver were expressed as U per milligram of protein (U/mg pro).

The activities of the antioxidant enzymes of malondialdehyde (MDA), the superoxide dismutase (SOD), reduced glutathione (GSH), the glutathione peroxidase (GSH-PX) and total antioxidant capacity (T-AOC) within Hip and mPFC tissues were measured with MDA activity assay kit (No. A003-1-2), SOD activity assay kit (No. A001-3-2) and GSH activity assay kit (No. A006-2-1), GSH-PX activity assay kit (No. A005-1-2) and T-AOC assay kit (S0121, Beyotime, China). MDA, SOD, GSH and GSH-PX kits were purchased from Jiancheng Inc. (Nanjing, China). In addition, it was necessary to pre-treat the brain tissues before measuring the above assay kits. Briefly, the tissues were weighted and PBS (pH 7.2–7.4, 0.01 mol/L) was added at a 1:9 proportion (MDA, SOD, GSH and GSH-PX kits) and a 1:5 proportion for T-AOC. Next, the samples were treated with tissue lyser (Jingxin, China), followed  by centrifuging the homogenate at 12,000 rpm for 10 min at 4 °C, and collecting the supernatant to measure.

ASP (Purity ≥ 95%) was purchased from Ci Yuan Biotechnology Co. Ltd. (Xi’an, China) and dissolved in saline at the concentration of 20 g/L and sterilized by ultrafiltration. 5-FU was purchased from First Affiliated Hospital of Chongqing Medical University and dissolved in DMSO to make storage solution at the concentration of 12.5, 25, 50 and 100 mg/L. Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle Medium (DMEM)/High Glucose were purchased from Gibco (Waltham, MA, USA). The SA-β-gal Staining and Reactive Oxygen Species Assay Kits were purchased from the Beyotime Institute of Biotechnology (Shanghai, China). The SOD and GSH-Px kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The antibodies against γ-H2AX were obtained from Cell Signaling Technology (Danvers, MA, USA). The antibodies against SDF-1 were purchased from Boster Biological Technology (Wuhan, China). The antibodies against Cx43 were purchased from Abcam (Cambridge, UK). The Human ELISA Kit for 8-OHdG, RANTES, and SCF were obtained from Neobioscience Biological Technology (Guangdong, China). Lucifer Yellow CH dilithium salt was purchased from Sigma-Aldrich (St Louis, CA, USA).