Thiobarbituric acid

Reactive oxygen species (ROS) in the prostate tissue was dyed with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; MedChemExpress, NJ, USA) probe and visualized under a fluorescence microscope (Olympus, Tokyo, Japan). To detect the oxidative stress degree, the content of lipid peroxidation product malondialdehyde (MDA) and the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were measured with the thiobarbituric acid (TBA) method, xanthine oxidase (XO) method, and ammonium molybdate colorimetry method, respectively (all purchased from Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The tissue iron assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was applied to determine the iron content of prostate lysates according to kit instruction. The protein concentration was detected to normalize the data.

Minimum essential medium (MEM), penicillin-streptomycin (PS), trypsin-EDTA, and fetal bovine serum (FBS) were purchased from Gibco (Gibco, NY, USA). MitoTracker Deep Red FM was obtained from Invitrogen (CA, USA), Hoechst 33342 and rhodamine 123 fluorescent probes were obtained from Sigma-Aldrich (MO, USA), and poly-d-lysine was acquired from BBI (Shanghai, China). HEK293 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Details of the three positive control medicines and the names of the 18 Chinese HMPs are shown in Table 3. The growth of cultured HEK293 cell was significantly inhibited by 20 μg/mL AA, 5–50 μM CsA, and 10–50 μM CDDP (Bai et al., 2014) [20 (link), 39 (link)–41 (link)]. In the present study, dose-response effects of the positive control medicines were tested at concentrations of 0.01–100 μM. The 18 Chinese HMPs were serially diluted to 0.01 μM in MEM. NaCl (0.9%) solution and 40% formaldehyde (diluted to 10% in ultrapure water) were purchased from YiFang S&T (Tianjin, China). Serum creatinine (CRE), blood urea nitrogen (BUN), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), malondialdehyde (MDA), thiobarbituric acid, and trace protein detection assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Malondialdehyde (MDA) level was utilized to quantify the lipid peroxidation in tissues as previously described [21 (link)]. Briefly, following lung homogenate preparation, MDA content was based on the reaction of MDA with thiobarbituric acid (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) at 95°C. Samples were then heated for 1 h at 95°C and centrifuged at 3000 g for 10 min. The absorbance of the supernatant was measured by spectrophotometry at 532 nm using 1,1,3,3-tetramethoxypropane as an external standard. MDA was expressed in micromole per g weight of wet tissue.
Glutathione peroxidase (GSH-Px) activity in lung supernatants was determined spectrophotometrically with minor modification of methods previously described [22 (link)]. The rate of GSSG formation was assessed by measuring the reduction of GSH (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). The unit of GSH-Px activity was defined as μmpl/L reduced GSH · g protein⁻¹ · min⁻¹.