Cell line, chemicals and reagents
Hepa 1-6 cell lines and Dulbecco’s modified Eagle’s medium (DMEM) were both purchased from Procell Life Science&Technology Co,.Ltd. (Wuhan, China). Dodecanoic acid (DDA) was provided by Macklin Biochemical Co.,Ltd (Shanghai, China). Fetal bovine serum (FBS) and fatty acid free-bovine serum albumin (FAF-BSA) were obtained by CellMax Co.,Ltd (Beijing, China). CCK8 was purchased from Biosharp (Hefei, China). SOD assay kit, GSH assay kit, ROS assay kit, ATP detection assay kit and mitochondrial membrane potential assay kit with JC-1 were obtained from Nanjing Jiancheng Bio-Engineering Institute (Nanjing, Jiancheng, China). BCA detection assay kit, Annexin V-FITC/PI apoptosis detection kit and DNA content assay kit were obtained from Solarbio Co. Ltd. (Beijing, China). The primary antibodies for Caspase 3, Bcl-2 and Bax were obtained from Bioworld (Wuhan, China). The secondary antibodies (goat anti-rabbit, goat anti-mouse) were obtained from Bioworld and EpiZyme, respectively.
Cell culturing
The Hepa 1-6 cell lines were firstly cultivated in DMEM medium containing 10% FBS after resuscitation. When reaching 70-80% confluence, the cells were digested with 0.25% trypsin and passaged routinely. The passaged cells were then seeded in the cell culture plate for DDA treatment or in the cell flask for passage under a humidified atmosphere at 37℃ in 5% CO2.
Grouping and dodecanoic acid treatment
Hepa 1-6 cells in logarithmic growth phase were trypsinized, harvested and seeded into 96-well plates at a concentration of 2×105 cells/mL with 100 μL (each well) DMEM supplemented with 10% FBS. Following incubation of 24h, the culturing medium was replaced with 100 μL DMEM containing freshly prepared dodecanoic acid (4 mol/L), and then conjugated 0.4% FAF-BSA to final concentrations of 0.1, 0.3, 0.5, 1, 2 and 4 mM, followed by incubation for 24, 48 and 72 h to find out the optimal concentration of dodecanoic acid by CCK-8 assay. The cells treated with 0 mM dodecanoic acid were used as the control. All experimental studies were undertaken in triplicate.
CCK-8 assay
CCK-8 (Cell-Counting Kit-8) is now widely used in detecting cell viability on the base of WST-8, a MTT-like compound being reduced into yellow formazan dye by the dehydrogenases located in mitochondria [21]. The amount of produced formazan dye is directly proportional to the number of living cells. Briefly, after 24 h, 48 h and 72 h of administration of various doses of DDA, the cells were harvested as described above, and washed with D-PBS twice, then followed by adding 100 μL of 0.4% FAF-BSA-supplemented DMEM and 10 μL of CCK-8 per well. After 2h of incubation, a microplate reader was used to measure the absorbance value at 450 nm.
Microscopic observation of cell morphology
The cells were treated with the indicated concentration of DDA for 24 h, 48 h and 72 h, respectively. The morphological changes of the cells were observed under a convert microscope (SOPTOP, Japan).
Cell cycle analysis
The cells were seeded in a 6-well plate at a concentration of 2.5×105 cells/mL and cultured routinely overnight. After washing in DMEM containing 0.4% FAF-BSA twice, the cells was incubated in DMEM medium containing an indicated concentration of DDA for 24 h, 48 h and 72 h. The cells were digested, harvested and fixed in pre-cooled 70% ethanol overnight, then treated with RNase A solution at 37℃ for 30 min, followed by PI staining in dark for 30 min at 4℃ using a DNA content assay Kit (Solarbio, Beijing). Finally, the distribution of cell cycle was analyzed using a flow cytometry (Beckman, USA).
Apoptosis detection
Culturing, grouping and treatment of the cells were done as described above. Cell apoptosis was assessed by Annexin V-FTIC/PI staining based on the instruction of Annexin V-FITC apoptosis detection kit (Solarbio, Beijing). In brief, the harvested cells were washed with pre-cooled PBS twice and re-suspended with 1× Binding buffer. After adjusting the cell density to 1×106 cells/mL, 1×105 cells in a volume of 100 μL were collected and mixed with 5 μL Annexin V-FITC and 10 μL PI. The cells were cultivated with the dyes for 15 min in darkness at room temperature. Thereafter, 500 μL PBS buffer was added for re-suspending the cells. After mixing, the samples were analyzed using flow cytometry for cell apoptosis rate.
Western blot analysis
Immunoblot analysis was carried out for assessing cell apoptosis-related proteins Bcl-2, Bax and Caspase-3. Hepa 1-6 cells were administrated with the indicated concentration of DDA for 24 h, 48 h and 72 h, respectively. After incubation, the cells were collected and lysed in RIPA solution on ice for 30 min, then centrifuged at 12000 rpm for 5 min. The supernatant containing total protein of cell extracts was collected and quantified by the BCA method at 595 nm using a microtiter plate reader. Meanwhile, the equal amounts of protein from the control or treated cells was loaded onto a 12.5% SDS-PAGE gel and transferred to PVDF membrane. Protein blocking was performed by 5% TBST-nonfat dry milk for 2 h. Subsequently, the membranes were probed with primary antibodies against Bcl-2 rabbit antibody, Bax rabbit Ab, caspase-3 rabbit antibody (diluted 1:1000) and incubated overnight at 4℃. β-actin (diluted 1:8000) was used as a internal control. After incubated with the secondary antibody, the protein signals were detected using ECL substrate. The experiment was repeated in triplicates.
Determination of biomarkers of oxidative stress
After treatment with the indicated concentration of DDA for 24 h, 48 h and 72 h at 37℃, the cells were collected to detect the related biomarkers of oxidative stress as follows:
Briefly, the harvested cells were centrifuged at 1000 rpm for 20 min. The supernatant was discarded, and the pellet was re-suspended in PBS buffer. After incubation at 37℃ for 20 min, SOD activity was determined using a microplate reader (Tecan Spark, Tecan Trading AG, Switzerland) at 450 nm, and calculated according to the instruction of SOD assay kits (Nanjing, Jiancheng, China).
- Intracellular ROS analysis
The cells were trypsinized and centrifuged at 1000 rpm for 10 min, then re-suspended by D-PBS buffer. The cellular production of ROS was measured in accordance with the instructions of ROS assay kit as follows: the cells were incubated with a final concentration of 10 μM DCFH-DA reagent (Nanjing Jiancheng, China) at 37℃ for 1 h, and centrifuged at 1000 rpm for 5 min. The obtained pellets were re-suspended in D-PBS, and 200 μL of the sample was added into a white microplate (Jing'an Hi-Tech, Jiangxi, China) and the fluorescent intensity of DCF was measured (Ex at 500 nm; Em at 525 nm) using multimode microplate reader. The intensity of fluorescence was proportional to ROS content, so fluorescent intensity can be used to indicate the level of cellular ROS.
Cell harvesting and rupture were undertaken as above described. The GSH level in cell extracts was determined by a GSH assay kit (Jiancheng Bioengineering Co. Ltd., Nanjing, China) following the manufacturer’s guideline. In brief, 100 μL buffer solution and 25 μL chromogenic reagent were added into 100 μL cell supernatant and then sufficiently mixed, finally the reaction was monitored at 405nm using a microplate reader. The GSH content was expressed in terms of µmol/gprot.
Mitochondrial membrane potential (MMP) assay
MMP was determined using a MMP assay kit (Nanjing, Jiancheng, China). In brief, Hepa 1-6 cells were administrated with the indicated concentration of DDA for 24h, 48 h and 72h, washed with pre-cooled PBS buffer, and stained using JC-1 working solution (a 1:500 dilution) for 20 min at 37℃, followed by centrifugation at 2000rpm for 5 min at room temperature. The supernatant was discarded, and cell pellets were washed and re-suspended using pre-cooled 1×incubation buffer for subsequent flow cytometric analysis. Normal mitochondria containing red JC-1 aggregates were detected with PI channel, and apoptotic cells containing green JC-1 monomer were detected with FITC channel.
Cellular ATP level determination
After treatment with the indicated concentration of DDA for different time at 37℃ as mentioned above, the cells were harvested, ruptured by boiling and spinning at 4000 rpm for 5 min. The precipitate was removed, and supernatant extract was quantitatively analyzed for mitochondrial ATP content using a microplate reader at 636nm according to the protocol of ATP assay kit (Nanjing, Jiancheng, China). Total protein was determined by the BCA method. Cellular ATP level was expressed as a term of µmol/gprot.
Statistical analysis
Data are expressed as the mean ± standard deviation (SD) and all experiments were independently repeated three times. The data analysis was performed using SPSS 17.0 sofware. One-way ANOVA test was applied for analyzing multiple comparisons. P-value<0.05 was considered statistically significantly different.