2.1. Materials and reagents
The human lung adenocarcinoma cell line A549 and the mouse cell line LLC-1 were obtained from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Cisplatin (DDP) was acquired from Jiangsu Haoseng Pharmacuetics Co., Ltd. (Lot number 181001; Jiangsu, China). Fetal bovine serum (FBS), high-glucose DMEM, trypsin- EDTA, penicillin and streptomycin were bought from Gibco (Grand Island, NY, USA). Curcumin (Cur) was purchased from Sigma-Aldrich and was stored at JinZhou Medical University of Technology until use. Test kits glutathione (GSH) and malondialdehyde (MDA) as well as the mitochondrial membrane potential assay kit of JC-1 were all bought from Beyotime Institute (Shanghai, China). Annexin V-FITC Apoptosis Detection Kit was obtained from Vazyme Biotech Co.,Ltd. (Nanjing, China).
2.2. Cell lines
The human lung adenocarcinoma cell line A549 and the Lewis lung cancer cell line were cultured in DMEM added 10% FBS, 100 U/mL of penicillin and 100 μg/mL of streptomycin (complete medium). Replace the complete medium every two days.
2.3. Cell viability assay
Cell survival rate was measured through MTT analysis. In simple terms, logarithmic growth A549 and LLC-1 cells were slaked with 0.25% trypsin, added to DMEM containing 10% fetal bovine serum and eventually plated on 96-well plates at a density of 0.8 × 104 cells/well. After hatch for 24 h, DDP was combined with different concentrations of Cur. Next, MTT (5 mg/mL, 10 μL) reagent was joined and incubated at 37 °C for 4 h. Then, the supernatant was abandoned, and dimethyl sulfoxide (DMSO, 150 μL) was joined to each well. The OD value was obtained at 490 nm by Spectra Max 190 micrometer. The OD (optical density) of each hole was in comparison with the normal group treated with DMSO alone. The measurements should be made three times.
2.4. Annexin -V/PI staining
The Annexin V-FITC Apoptosis Assay Kit was used to assess the percentage of apoptotic/necrotic cells. The cells were suspended in 110 μL of 1× Binding Buffer containing FITC-labeled Annexin and propidium iodide (kit provided). After incubation for 10 min, 400 μL of 1 × Binding Buffer was added to the samples, which were analyzed in a BD FACSCalibur™ cytometer using the FL1-H (FITC) and FL3-H (PI) channels. Appropriate staining controls were used to calibrate the blood cell counter. FLOWJO® software was used to allocate and quantize living cells (Annexin V-/PI-), early apoptotic cells (Annexin V+/PI-), late apoptotic ells (Annexin V+/PI+) and necrotic cells (Annexin V-/PI+).
2.5. Analysis of ΔΨm
The ΔΨm was tested by a ΔΨm detection kit (Beyotime). Under normal conditions, JC-1 aggregates in intact chondriosome and emits a red fluorescent light; when ΔΨm is lower, the red fluorescence goes down and green fluorescence goes up. Therefore, ΔΨm transformation can be mirrored by the ratio of green to red fluorescence. The green/red fluorescence ratio was used for estimating chondriosome unpolarizing.
2.6. Lung tumor-bearing model in mouse and its treatments
Forty-eight C57BL/6 male mice, weighing about 20 ± 2 g, were bought from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (Certificate number: SCXK 2006-0006, Beijing, China). They were housed at a regulated humidity of 55 ± 5% and temperature of 23 ± 2 °C with a light/dark cycle of 12 h. After they were cultivated in medium at about 4 d, LLC-1 cells were adjusted to a density of 1 × 107 cell/mL with sterile PBS to product a cell suspension. Then, the cell suspension was subcutaneously injected to the right armpits of forty-eight healthy mice at a dose of 0.2 mL each mouse. After tumors growing about 4 d, tumors with a dimension of approximately 0.5 cm were deemed a sign of a successfully established model. Mice in the normal group were treated with saline, DDP (5 mg/kg), DDP plus Cur L (5 mg/kg + 50 mg/kg), DDP plus Cur M (5 mg/kg + 100 mg/kg), DDP plus Cur H (5 mg/kg + 200 mg/kg) or DDP plus SB203580 (5 mg/kg + 4 mg/kg). Cur was administered by intragastric administration once a day, while DDP were given through intraperitoneal every two days and SB203580 were given through intraperitoneal every three days; treatment with a daily injection began immediately and lasted 14 days when the models were built successfully. The solid tumor, spleen, thymus, liver and kidney were immediately removed after mice were sacrificed, then washed them with precooled sterile saline (4 °C), dried and weighed. Serum was isolated at 1700 g about 10 min and deposited in −80 °C.
2.7. Biochemical indicators and enzyme-linked immunosorbent assay
The GSH and MDA were assessed using an ELISA kit. Once the eyeballs were enucleated from the mice, peripheral blood was collected in condensation tubes and anticoagulation tubes 24 h after the last administration; a Beckman Coulter Hematology Analyzer was used to automatically analyze and calculate the amount of RBCs, WBCs, HGB and PLTs, Scr, and BUN.
2.8. Western blotting analysis
Kidney tissues were homogenized in cold RIPA lysis buffer (Beyotime Inc., Nanjing, China) for immunoblotting analysis. The total protein concentration was quantified by a BCA protein assay kit (Applygen, China). The proteins (100 μg) were separated by electrophoresis and transferred onto PVDF membranes (Thermo Fisher Scientific, USA). After closing for 1 h at 37 °C in confining liquid, the membranes were incubated overnight at 4 °C with different antibodies. Then, membranes were incubated with horseradish peroxidase (HRP)-labeled antibody. Actin (1:3000, ab8227, Abcam) served as a reference gene.
2.9. Histology staining
After fixation in formaldehyde solution for 48 h, the tissues were embedded in paraffin, and sectioned into slices (5 μm thick). The slices were stained with hematoxylin-eosin (H&E) (Phygene Life Sciences Co., Ltd, Fuzhou, China), next observation under a microscope at 400× magnification.
2.10. Statistical analyses
After three independent experiments were conducted, the data were appeared as the means ± SD (standard deviation). Statistical analyses were carried out with Student’s t-test by a software package (SPSS 23.0, Chicago, IL, USA). P < 0.05 was considered statistically significant.