Chromium (Cr) and its compounds have become a serious public health issue, causing environmental pollution [1
] and threatening human health. The health hazards associated with exposure to Cr are dependent on its oxidation state [3
], with hexavalent chromium Cr(VI) being the most toxic component. Throughout the world, human exposure occurs mainly via industrial uses, such as leather tanning and steel manufacturing, as well as in food additives and tobacco [5
]. Another source of contact is drinking water contaminated with Cr(VI). In vivo and in vitro studies have demonstrated that Cr(VI) can cause a wide range of toxic effects, including hepatotoxicity, in animals and humans [1
]. It has been reported that the liver shows the highest accumulation following oral exposure to Cr(VI) [7
]. The liver is the primary organ involved in xenobiotic metabolism and, for this reason, is particularly susceptible to injury. However, the liver as a target organ for Cr(VI) after oral exposure in humans remains controversial. For instance, Proctor et al. reported that Cr(VI) is not carcinogenic to humans via the oral route of exposure at permissible drinking water concentrations [10
]. Several other studies have suggested that Cr(VI) could induce liver injury [1
] and may cause primary cancer or increase the risk of liver cancer [12
]. The increasing incidence of Cr(VI)-induced hepatotoxicity has emphasized the importance of elucidating the intoxication mechanism and identifying useful antidotes for Cr(VI) toxic effects on the liver.
Cr(VI) can easily enter cells through anion channels, and, once inside, it is reduced to its intermediate metabolites, Cr(IV), Cr(V), and the more stable form Cr(III), by enzymatic and non-enzymatic reductants [15
]. Reactive oxygen species (ROS) are generated in the oxidation–reduction process and play a critical role in the mechanism of Cr(VI)-induced cytotoxicity. ROS accumulation, for instance, is known to cause the collapse of mitochondrial membrane potential and the opening of the mitochondrial permeability transition pore (MPTP) [16
]. Cr(VI) induces cell apoptosis through intrinsic and extrinsic pathways involving the release of cytochrome c (Cyt c) from the mitochondrial intermembrane space. The release of Cyt c from the mitochondrial intermembrane space is regulated by B-cell lymphoma-2 (Bcl-2) family proteins, including anti-apoptotic (such as Bcl-2 and Bcl-xl) and pro-apoptotic proteins (such as Bcl-xs, Bax, and Bid) [17
]. It has been postulated that the ratio of anti- and pro-apoptotic Bcl proteins regulates the function of MPTP within the mitochondria. Cr(VI)-induced ROS accumulation might cause an imbalance in Bcl-2 family proteins, swelling of the mitochondrial membrane, opening of the MPTP, release of Cyt c into the cytoplasm, and activation of caspase-9 and -3 [17
], which would eventually trigger cell apoptosis.
Coenzyme Q (CoQ10) is an essential endogenous molecule in cell respiration and metabolism. It functions as a mitochondrial antioxidant, inhibiting lipid peroxidation and scavenging free radicals, as well as maintaining genome stability [19
]. Moreover, CoQ10 possesses an independent anti-apoptosis function that regulates MPTP [20
]. CoQ10 biosynthesis occurs in the mitochondrial matrix through the mevalonate pathway [21
]. Evidence suggests that mutations in the genes involved in the biosynthesis of CoQ10 could cause primary and secondary CoQ10 deficiencies. They have also been linked to various clinical mitochondrial diseases [22
]. CoQ10 deficiency could disturb mitochondrial bioenergetics and oxidative stress, as demonstrated by decreased ATP generation, increased ROS production, and cell death [24
]. In general, secondary CoQ10 deficiency may be induced by dietary insufficiency or exposure to certain xenobiotics [26
]. Several studies have shown that CoQ10 is susceptible to environmental toxins, which cause CoQ10 deficiency at both the cellular and in vivo levels [28
]. Considering the pivotal role played by CoQ10 in mitochondrial function, this study aimed to investigate whether Cr(VI) can induce changes in the level of CoQ10, and whether CoQ10 treatment is effective against Cr(VI)-induced hepatotoxicity.
In the present study, we demonstrated that Cr(VI)decreased the level of endogenous CoQ10 by disturbing the CoQ10 synthesis pathway, and that the pretreatment with CoQ10 maintained the level of endogenous CoQ10. These findings led us to investigate the role of CoQ10 in the mechanism of Cr(VI)-induced hepatotoxicity and its possible role as a hepatoprotective agent against Cr(VI)-induced hepatocyte damage.
In order to achieve these goals, we examined the genes involved both directly and indirectly in the CoQ10 biosynthetic pathway. Cr(VI) changed the expression of many genes, including ETFDH
, which exhibited the strongest downregulation. ETFDH
is indirectly involved in the biosynthesis of CoQ10 and encodes a component of the electron-transfer system in mitochondria. Gempel et al. reported that mutations in the ETFDH
gene cause pure myopathy, as evidenced in seven patients from five different families with severely decreased activities of respiratory chain complexes I and II + III and CoQ10 deficiency [30
]. We also previously demonstrated that Cr(VI) induces mitochondrial dysfunction by disturbing electron transport and inhibiting the respiratory chain complexes. As a link to our present study, we tentatively propose that Cr(VI) may cause secondary deficiency of CoQ10 in L-02 hepatocytes, resulting in mitochondrial dysfunction and cell apoptosis. This result provides a new perspective on the mechanism of Cr(VI)-induced hepatotoxicity.
In addition, Cr(VI) downregulated the expression of PDSS2
, and COQ9
, which are directly involved in the CoQ10 biosynthesis pathway. Defects in these genes are a cause of CoQ10 deficiency [31
catalyzes the only C-methylation step in the CoQ10 biosynthesis pathway in yeast. Chen et al. demonstrated that an uncoupling chemical in CoQ10 dose deficiency downregulates the mature form of COQ5
. They also showed that knockdown of the COQ5
gene reduces CoQ10 levels, indicating that COQ5
plays a critical role in the biosynthesis of CoQ10 [32
]. In the present study, Cr(VI) suppressed COQ5
gene expression, but not strongly. It is possible that Cr(VI) induced ETFDH
inhibition, causing mitochondrial dysfunction to disturb the expression of genes in CoQ10 biosynthesis. This is supported by Hsiu-Chuan’s study, which reported the suppressive effect of FCCP on COQ5
levels in association with decreased mitochondrial membrane potential, mitochondrial ATP production, and CoQ10 levels [33
]. Interestingly, ADCK3
was upregulated after exposure to Cr(VI), although not significantly. ADCK3
is required for the biosynthesis of CoQ10, and mutation of ADCK3
has been associated with CoQ10 deficiency in humans [34
also functions in an electron-transferring membrane protein complex in the respiratory chain. Tumor suppressor p53 can induce ADCK3
expression, and in response to DNA damage, inhibition of ADCK3
expression partially suppresses p53-induced apoptosis [35
]. We believe that CoQ10 maintains homeostasis only when gene expression is maintained at normal levels. However, little is currently known about the regulation of CoQ10 gene expression. Here, we present preliminary data that mitochondrial CoQ10 deficiency may represent a potential biomarker of Cr(VI) toxicity. However, to better understand the mechanism of Cr(VI)-induced CoQ10 deficiency, more robust evidence is needed.
Mitochondrial loss has been indicated to play a prominent role in mitochondrial dysfunction. Our results showed that Cr(VI) exposure led to mitochondrial loss in hepatocytes, as reflected by a decrease in the mitochondrial mass, mtDNA copy number, and inhibition of expression of components of the mitochondrial respiratory chain [36
]. The observed Cr(VI)-induced decrease in the level of mtDNA copy number supports a link between CoQ10 deficiency and mtDNA depletion [37
]. Moreover, mtDNA is vulnerable to ROS due to the lack of protection from histones and a self-repair mechanism. Our results also sowed Cr(VI)-induced CoQ10 deficiency and ROS accumulation. It is reported that CoQ10 deficiency may cause mitochondrial dysfunction, thus triggering ROS generation. The capability of ROS scavenging is weakened, possibly further aggravating ROS accumulation due to CoQ10 deficiency [39
]. Supplementation with CoQ10 could increase the mitochondrial mass, mtDNA copy number, and mitochondrial electron transport chain activity, as demonstrated by our study and Duberley’s research [40
]. Previous investigations have also shown that CoQ10 protects against neuron apoptosis induced by iron by reducing ROS accumulation and inhibiting lipid peroxidation [41
]. Consistent with previous studies, we found that pretreatment with CoQ10 eliminated excessive ROS and O2−
, reduced lipid peroxidation, and maintained SOD content. SOD plays an important role in the protection of cell membranes against oxidative stress by catalyzing the dismutation of O2−
and to the less reactive species H2
], corroborating the findings that CoQ10 plays a critical role in Cr(VI)-induced mitochondrial dysfunction.
Mitochondria are the factory of ATP production via oxidative phosphorylation [43
]. In return, the primary function of mitochondria and homeostasis are maintained by adequate ATP levels. CoQ10 is an essential cofactor of oxidative phosphorylation to permit ATP biosynthesis [44
]. Our data indicated that Cr(VI) causes CoQ10 deficiency and markedly reduces cellular ATP. ATP production is also related to Ca2+
and ROS generation. Calcium overload has been proposed to play a crucial role in ROS generation [45
], which could aggravate mitochondrial damage in hepatocytes. Furthermore, Ca2+
induces Cyt c release from mitochondria by enhancing Cyt c dislocation, competing for cardiolipin-binding sites in the mitochondrial inner membrane, or activating MPTP opening, resulting in the disturbance of electron transfer and the Q cycle. As a consequence, there is an upsurge in ROS accumulation [46
]. Therefore, eliminating Ca2+
overload and ROS accumulation are conducive to promoting ATP synthesis and repairing damaged mitochondria. In this study, we have demonstrated that CoQ10 could attenuate Cr(VI)-induced adverse effects by preventing Ca2+
overload and scavenging excessive ROS.
Apoptosis is an energy-consuming process that is regulated by the activation of caspases [47
]. Cr(VI) induced the release of Cyt c from mitochondria, accompanied by activation of caspase-3 and 9, which are associated with significantly reduced Bcl-2 expression and enhanced Bax expression. Previous studies have confirmed that, after Cyt c is released from the mitochondrial intermembrane space into the cytosol, it forms a complex with Apaf-1 and pro-caspase, thereby triggering caspase-9 activation and the consequent initiation of the caspase cascade and induction of cell apoptosis [48
]. In addition, cell apoptosis is regulated by the balance of pro- and anti- apoptotic proteins. It is generally believed that the anti-apoptotic protein Bcl-2 counterbalances oxidative damage and maintains the structural and functional integrity of the mitochondrial membrane by preventing Cyt c release. The Bax protein exerts an important effect in cell apoptosis. High Bax expression levels and the formation of homo- or heterodimers with Bcl-2 lead to cell death [49
]. We also saw that pretreatment with CoQ10 could block the release of Cyt c, inhibit the activation of caspase-3 and caspase-9, and regulate the expression of Bcl-2/Bax to reach equilibrium and prevent subsequent apoptosis in L-02 hepatocytes. These results are compatible with the findings of other studies using CoQ10 against iron-induced neuronal toxicity [28
], statin toxicity in hepatocytes [50
], or ethanol-induced apoptosis in corneal fibroblasts [51
As mentioned above, CoQ10 could alleviate Cr(VI)-induced hepatotoxicity. However, the mechanism of the protective effects of CoQ10 is not fully understood. Possible reasons for the protective effects of CoQ10 in Cr(VI)-induced toxicity include the fact that Cr(VI) is a water-soluble compound that can be easily reduced by water-soluble antioxidants [52
]. Therefore, Cr(VI) might be reduced to Cr(III) before entering the cell when co-incubated with CoQ10. However, considering that Cr(VI) can dissolve in the medium, CoQ10, being a lipid-soluble quinone, may fail to interact with Cr(VI) outside the cell. On the other hand, since the cells were pretreated with CoQ10 for 2 h prior to exposure to Cr(VI), it is possible that CoQ10 may have entered the cell, and CoQ10 was distributed on membranes to block the opening of anion channels to prevent the entrance of Cr(VI) into the cell. This is supported by several studies showing that quinones modulate MPTP through a common binding site rather than through redox reactions [53
]. This study also demonstrated that CoQ10 could decrease the MPTP opening degree to antagonize the mitochondrial toxicity induced by Cr(VI). Moreover, considering that exogenous CoQ10 enhanced the level of CoQ10 in mitochondria, we propose that the protective effect of CoQ10 might be associated with its role as a mobile electron transporter. CoQ10 can correct the disorder of electron transfer and improve the Q cycle, thus attenuating Ca2+
overload and Cyt c release. We have shown that CoQ10 can prevent cell apoptosis, but the results did not indicate how CoQ10 plays a protective role against Cr(VI)-induced hepatotoxicity. The detoxification role of CoQ10 against Cr(VI)-induced hepatotoxicity should be more comprehensively investigated.
4. Materials and Methods
Potassium dichromate (K2Cr2Q7), coenzyme Q10, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO, USA). RPMI-1640 culture medium, fetal bovine serum (FBS), trypsin, and penicillin-streptomycin were provided by Dingguo Changsheng Biotechnology Co. LTD (Beijing, China). All solvents and chemicals were analytical grade.
4.2. Cell Culture
The normal liver L-02 cell line (Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) was derived from adult human normal liver, immortalized by stable transfection with the hTERT [55
], and was reported to be liver-specific [56
]. L-02 hepatocytes were maintained in 1640 RPMI medium containing 10% fetal bovine serum and a 1% mixture of penicillin and streptomycin in a 5% CO2
humidified atmosphere at 37 °C. The medium was changed every two days, and the cells were passaged using trypsin.
4.3. Treatment of Cells with Cr(VI) and CoQ10
Cells were treated with a final concentration of 1–4 μM K2Cr2Q7 for 24 h in a complete medium. Cells were pretreated for 2 h prior to K2Cr2Q7 exposure and were co-treated for the 24-h K2Cr2Q7 exposure period at a final concentration of 2.5 μM CoQ10. Control samples were exposed to an equivalent concentration of DMSO as the solvent control.
4.4. Cell Viability Assay
MTT was used to evaluate cell viability. Cells were seeded in 96-well plates (1 × 104/well) and cultured in the presence of 0, 0.5, 1, 2, 4, or 8 μM Cr(VI) or 0, 1.25, 2.5, 5, 10, or 20 μM CoQ10 for 24 h. After 24 h of incubation, 10 μL of MTT solution (stock solution of 5 mg/mL in PBS) was added to each well of the 96-well plates, and the plates were incubated for an additional 4 h at 37 °C. The MTT-reducing activity of the cells was measured by treatment with DMSO prior to reading at 490 nm with an automatic microplate reader.
4.5. Preparation of Mitochondria
The treated cells were washed with PBS and then centrifuged at 600 g for 5 min. The supernatant was discarded, and the pellets were suspended in ice-cold lysis buffer (250 mM sucrose, 20 mM N
′-(2-ethanesulfonic acid (HEPES), pH 7.4, 10 mM KCl, 1.5 mM MgCl2
, 1 mM each of EGTA, EDTA, DTT, and PMSF, and 10 μg/mL each of leupeptin, aprotinin, and pepstatin A) and incubated for 20 min. The cells were homogenized up and down 20 times, on ice, at 1000 r.p.m., transferred to a new tube, and centrifuged at 600× g
for 10 min at 4 °C The supernatant was then centrifuged at 12,000× g
for 10 min at 4 °C. Afterwards, the supernatants were collected and centrifuged at 12,000× g
for 15 min at 4 °C for preparation of the cytosolic fraction. The precipitated pellets were resuspended in the lysis buffer and were used as the mitochondrial fraction after centrifugation at 12,000× g
for 10 min [57
4.6. Extraction and Quantification of CoQ10
Extraction was performed as described previously. Briefly, 250 μL of samples and 750 μL of hexane:ethanol (5:2, v
) were mixed together for 1 min using a vortex mixer. The mixture was centrifuged for 3 min at 4000× g
, and 450 μL of the hexane layer was collected, dried under a stream of nitrogen, and dissolved in 100 μL of ethanol (1:1, v
]. Quantification of CoQ10 was performed by HPLC according to Lass and Sohal [59
]. A 10-μL aliquot of the extract was chromatographed on a reverse-phase C180 HPLC column (150 mm × 4.6 mm, 5 μM; Thermo Hypersil), using a mobile phase consisting of ethanol: methanol (1:1, v
) at a flow rate of 0.8 mL/min. The eluent was monitored with a UV detector at 275 nm.
4.7. Gene Chip ANALYSIS
Total RNA was isolated using Trigo (Sigma), and first-strand cDNA was synthesized using RevertAid M-MuL V Reverse Transcriptase (Thermo, Waltham, MA, USA). A Genechip 30 IVT Express Kit was used to synthesize double-stranded cDNA for in vitro transcription (IVT, standard Affymetrix procedure, Santa Clara, CA, USA). During the synthesis of the amplified RNA (aRNA), a biotinylated nucleotide analog was incorporated as a label for the message, and the aRNA was purified with magnetic beads. A 15-μg quantity of aRNA was fragmented with a fragmentation buffer according to the manufacturer’s instructions. Next, 15 μg of fragmented aRNA were hybridized with Affymetrix Human Genome U133 plus 2.0 arrays, according to the manufacturer’s instructions. The chips were heated in a GeneChip Hybridization Oven-645 for 16 h at 60 rpm and 45 °C. The chips were washed and stained using a Genechip Fluidics Station-450 with the Affymetrix HWS kit. Chip scanning was performed with an Affymetrix Gene-Chip Scanner-3000-7G, and the normalized data were extracted using Affymetrix GCOS software (1.0, Santa Clara, CA, USA). The normalized spot intensities were transformed to gene expression log 2 ratios, and comparisons between control and treated groups were conducted using a t-test. A p-value ≤ 0.05 and a fold change value ≥2 indicated statistically significant regulation. A two-fold change was indicated at log2(Ratio) ≥ 1.0 or log2(Ratio) ≤ −1.
4.8. Real-Time PCR
Total RNA was isolated using Trigo (Sigma), and the first-strand cDNA was synthesized using RevertAid M-MuL V Reverse Transcriptase (Thermo). The cDNA was amplified in 20-μL reactions using SYBR premix Dye I (TAKARA BIO, Shiga, Japan) in a Thermal Cycler Dice Real-Time System (TAKARA BIO). The mRNA expression was normalized to the expression of the standard reference gene GAPDH. The primer sequences are shown in the Table 2
4.9. Determination of Reactive Oxygen Species (ROS)
Cells were washed three times with a serum-free medium, and thereafter CellROX® Green Reagent (Thermo Fisher, Waltham, MA, USA) was added to the cells at 5 μM final concentration, followed by incubation for 30 min at 37 °C. The cells were then washed twice with PBS and harvested. One hundred microliters of resuspended sample were added to 96-well plates, and ROS levels were assessed by fluorescence spectrophotometry with excitation at 485 nm and emission at 520 nm. Cells on the coverslip were observed under a confocal microscope.
4.10. Measurement of Superoxide Anion (O2−)
The treated cells were incubated with the cellular O2−-sensitive fluorescent indicator dihydroethidium (DHE) at a final concentration of 5 μM for 30 min at 37 °C, and protected from light. They were washed three times with PBS, collected, resuspended in 2 mL of PBS, and finally examined by fluorescence spectrophotometry with excitation at 535 nm and emission at 610 nm.
4.11. Evaluation of Methane Dicarboxylic Aldehyde (MDA) and Superoxide Dismutase (SOD) Levels
MDA levels were determined using the trace MDA detection kit and a microplate reader at 530 nm. SOD levels were measured using the total superoxide dismutase activity assay, which involves the inhibition of superoxide-induced chromogen chemiluminescence by SOD, according to the manufacturer’s instructions. The absorbance of the wells was read using a microplate reader at a primary wavelength of 550 nm.
4.12. Measurement of the Mitochondrial Mass
The mitochondrial mass was evaluated using the fluorescent probe 10-N
-nonyl acridine orange (NAO) as previously described [30
]. Treated cells were incubated in a medium containing 5 μM NAO for 30 min at 37 °C and protected from light. The NAO fluorescence intensity was determined using a microplate reader (Gemini EM, Molecular Devices, Sunnyvale, CA, USA). The emission and excitation wavelengths were 530 and 485 nm, respectively.
4.13. Measurement of the Mitochondrial Transmembrane Potential (MMP, Δψm) in Cells
The cells were loaded with JC-1 for 20 min at 37 °C. Depolarization of Δψm was assessed by measuring the fluorescence intensities at excitation and emission wavelengths of 490 and 539 nm, respectively, to measure JC-1 monomers. An excitation wavelength of 525 nm and an emission wavelength of 590 nm were used to measure JC-1 aggregates using a fluorescence microplate reader. During the measurements, the cells were maintained at 4 °C and protected from light. All fluorescence measurements were corrected by autofluorescence, which was determined using cells not loaded with JC-1.
4.14. Measurement of the MPTP Opening Degree
The cells were washed three times with PBS, and calcein-AM was added, followed by incubation for 20 min at 37 °C. Next, the cells were washed twice with GENMED cleaning liquid, harvested, resuspended in a cleaning liquid, and finally examined by fluorescence spectrophotometry with excitation at 488 nm and emission at 505 nm.
4.15. Measurement of Intracellular ATP Levels
The level of ATP was examined using the ATP assay kit (S0026, Beyotime, Shanghai, China), which utilized the catalysis of firefly luciferase to generate fluorescence requiring ATP to develop an ATP quantified method. The measurement was performed following the manufacturer’s instructions. The absorbance was detected using a Luminometer.
4.16. Measurement of the Cellular Calcium Concentration (Ca2+)
Cultured cells were incubated with the cellular Ca2+-sensitive fluorescence indicator Fluo-3AM at a final concentration of 2.5 μM for 30 min at 37 °C and protected from light. The cells were subsequently washed three times with PBS, collected, and resuspended in 2 mL of PBS for posterior examination by fluorescence spectrophotometry with excitation at 488 nm and emission at 525 nm.
4.17. Caspase Activity Assay
Caspase levels were measured with caspase-3 and caspase-9 activity assay kits according to the manufacturer’s instructions (Beyotime). The absorbance was measured on a microplate reader at 405 nm, and the caspase activities were subsequently calculated based on the absorbance.
4.18. Western Blot Analysis
For Western blot analysis, cytosolic and mitochondrial fractions were prepared as reported previously. Samples containing an equal amount of concentrated proteins were separated on 10% gradient SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane by electroblotting for 90 min at 100 V and 4 °C. Non-specific membrane binding sites were blocked with blocking solution (PBS, 0.5% Tween-20, pH 7.4), containing 5% non-fat dry milk for 1 h at 4 °C. The membrane was incubated with primary mouse anti-Cyt c monoclonal antibody (Abcam, Cambridge, UK) diluted 1:1500 or goat anti-rabbit Bcl-2 (1:500, Santa Cruz, Santa Cruz, CA, USA) and Bax antibody (1:500, Santa Cruz) in blocking solution overnight at 4 °C. The membrane was washed thoroughly with PBS-T and then incubated for 1 h with horseradish peroxidase-conjugated anti-mouse (1:4000; Santa Cruz) or anti-rabbit IgG antibody (1:6000; Santa Cruz) in blocking solution, detected by chemiluminescence reagent plus, and exposed to film.
Cyt c translocation from mitochondria to cytoplasm was analyzed by immunofluorescence. Treated cells were incubated with Mito-Tracker Red (Thermo Fisher) at 500 nM for 45 min, washed twice with PBS, and with 2% paraformaldehyde for 20 min at room temperature. The cells were blocked with blocking solution (1% BSA, 0.15% saponin and 10% goat serum in PBS) for 30 min at room temperature, incubated with primary antibody specific for Cyt c (1:200) overnight at 4 °C, and then incubated with secondary antibody for 1 h at room temperature. The cells were washed three times with PBS, and then the nuclei were stained with Hoechst for 5 min. Images were captured with a confocal microscope.
4.20. FITC Annexin V/propidium Iodide (PI) Staining for Apoptotic Cells
Cells were washed three times with PBS before suspension in binding buffer, and 10 μL of Annexin V-FITC were mixed with 200 μL of cell suspension containing 106 cells. The cells were incubated at room temperature for 30 min and shielded from light. Then, 10 μL of PI solution were added to the cells and they were incubated for 10 min on ice. The scatter parameters of the cells were analyzed using a flow cytometer. Usually, four cell populations are identified by the flow cytometer. The viable population is displayed in the lower-left quadrant; the early apoptotic population and late apoptotic population are presented in the lower-right quadrant and in the upper-right quadrant, respectively. Signals in the upper-left quadrant present a necrotic population.
4.21. Protein Assay
All protein assays in this study were measured using a Q5000 UV-Vis spectrophotometer (Quawell, Sunnyvale, CA, USA).
4.22. Statistical Analysis
All data were expressed as the group mean ± SD. Comparisons between control and treated groups were conducted using one-way ANOVA, as appropriate, followed by LSD. A value of p < 0.05 was considered to indicate statistical significance. Statistical analysis of the data was performed using SPSS18.0. All experiments were performed three times.