Mda detection kit

Perfluorooctanesulfonic acid potassium salt (PFOS), Dimethylsulfoxide (DMSO), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). PFOS was dissolved in DMSO and stored at 4°C [19] . Dulbecco's modified Eagle's medium (DMEM), Fetal bovine serum (FBS) were purchased from Hyclone Company (USA). Antioxidant enzyme detection kits (SOD, GSH-PX), ROS and MDA detection kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Annexin V-FITC apoptosis detection kit was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Agar, cholesterol and other chemicals were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).

The gut tissues were homogenized (1:9 w/v) with a glass Teflon homogenizer (Potter-Elvehjem 64792-10) in a 0.9% normal saline buffer. Subsequently, the samples were centrifuged at 3,000 g for 10 min at 4°C, and the supernatant was collected to detect the activities of oxidative index and the secretion of IL-6 and IL-2 and stored at 4°C.
The activity of the SOD, CAT, GSH-Px, and MDA concentration in the gut tissues was measured by a spectrophotometric method following the instructions of the SOD, CAT, GSH-Px, and MDA detection kits, respectively (Nanjing Jiancheng Bioengineering Institute, China). The level of pro-inflammatory cytokines IL-2 and IL-6 in the gut tissues was measured by the Goat IL-2 ELISA Kit and Goat IL-6 ELISA Kit according to the manufacturer's instructions (Jiangsu Yutong Biological Technology Co., Ltd., China). All of the experiments were carried out in triplicate.

Kidney tissue homogenates (0.01 M PBS buffer to produce a 10% tissue lysate) were used to measure kidney MDA levels by the thiobarbituric acid (TBA) method. The MDA-TBA adduct produced during the reaction of MDA in samples with TBA was detected spectrophotometrically at 535 nm (UV-2401PC, Shimadzu, Japan). The result was expressed as nmol per milligram of protein (nmol/mg.prot). MDA detection kits were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Bergenin (purity of 95%) was obtained from Meilun Biological Products Co. Ltd. (Dalian, China). The chemical structure of bergenin is shown in Figure 6. The cell culture products were from Gibco BRL (Grand Island, NY, USA). Injectable cyclophosphamide was obtained from Jiangsu Hengrui Medicine Co. (Lianyungang, Jiangsu, China). A HE staining kit was purchased from Beyotime (Jiangsu, China). Concanavalin A (Con A), lipopolysaccharide (LPS) and neutral red (N7005) were provided by Sigma-Aldrich (St. Louis, MO, USA). The Ig kit and cytokines ELISA kits were purchased from Lianke Biotechnology Co. Ltd. (Hangzhou, China). SOD, CAT, GSH-Px and MDA detection kits were provided by Nanjing Jiancheng Bio-engineering Institute (Nanjing, China). 2-(2-Methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, mono- sodium salt] (WST-8) was purchased from Amresco Co. (Solon, OH, USA) Anti-CD4⁺ (APC) and anti-CD8⁺ (PE) antibodies were provided by Bio Legend, Inc. (San Diego, CA, USA).

Atrial tissue was collected from each group to detect malondialdehyde (MDA), superoxide dismutase (SOD) and reactive oxygen species (ROS) to observe the effect of oxidative stress. After the tissue homogenate was completed, the levels of MDA, SOD and ROS were detected by using the respective assay kits according to the instructions. ROS detection kits were purchased from Beyotime Biotechnology (Shanghai, China). MDA detection kits and SOD activity assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The levels of the NOX4 protein were determined by ELISA. The ELISA kits for the protein Nox4 were purchased from Westang Biotechnology (Shanghai, China).

The pH of water for plant growth during 0 h, 6 h, 12 h and 24 h was detected by Precision pH meter (S20 Mettler Toledo Instrument Co., LTD). SOD (superoxide dismutase), POD (peroxidase), Pro (proline) and MDA (malondialdehyde) contents in leaves, stems and roots were measured by SOD kits (Nanjing Jiancheng Bioengineering Research Institute), POD kits (Nanjing Jiancheng Bioengineering Research Institute), Pro kits (Nanjing Jiancheng Bioengineering Research Institute) and MDA detection kits (Nanjing Jiancheng Bioengineering Research Institute). The absorbance of SOD, POD, Pro and MDA at 450 nm, 420 nm, 520 nm and 532 nm for 0 h, 6 h, 12 h and 24 h were recorded with three replications for each sample by Ultraviolet–visible spectrophotometer (NanoDrop2000 Seymour Flight).