| BackgroudThe population of patients with diabetes mellitus increased rapidly with the improvement of living standards, and diabetic comlications greatly affect Iheir quality of life. About 25% people with diabetes develops a diabetic foot sore in their lifetime, and impaired diabetic wound healing is one major complication of diabetic foot disease, which affects 15% of the 200 million patients with diabetes worldwide. With their increased morbidity, mortality and health expenditure, diabetic chronic wounds have brought heavy load to the patients and their family. Thus, the underlying pathogenetic mechanisms have been a hot spot of research in related academic areas. It has been proved that oxidative stress played a pivotal role in the diabetic chronic wounds.The hallmark of cells under oxidative stress is accumulation of intracellular reactive oxygen species (ROS). Benavente reported that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-dependent ROS production induces apoptosis of human keratinocytes. The NOX pathway and mitochondrial respiratory cycle are two main sources of cellular ROS. Specifically, NOX4 has been reported to play a central role in the ROS-dependent activation in human keratinocytes. However, mitochondria are vulnerable to ROS attack. ROS cause oxidative damage to mitochondrial membrane integrity and DNA, leading to loss of mitochondrial membrane potential (MMP) and activation of DNA repair proteins. ROS also mediate the mitogen-activated protein kinase (MAPK) pathway and induce cell apoptosis. Mitochondrial damage and MAPK pathway both can trigger cell apoptosis.Recent studies have proposed that oxidative stress can disrupt normal wound microenvironment by stimulating the production of oxidative products that are distributed all around the body. In patients with diabetes, laboratory examinations have detected the accumulation of many oxidative products, including advanced oxidation protein products (AOPPs). They participate, by inducing cell apoptosis, in the pathogenesis of many diabetic microvascular and macrovascular complications, such as diabetic cardiovascular comlications, diabetic retinopathy, and diabetic nephropathy. Diwesh Chawla and Omur Tabak demonstrated that the levels of AOPPs in patients with diabetic microvascular complications were two times than normal people and one and a half times higher than diabetic patients without complications. But the relations of AOPPs and diabetic chronic wounds have not been fully elucidated.Our preliminary experiments tested the AOPPs levels in the skin tissue at wound margin. The results indicated that the AOPPs levels in the skin tissue at diabetic chronic wound margin were 2.75 times higher than normal skin tissue. We first introduced AOPPs in the research of diabetic chronic wounds, and to further investigate AOPPs’effects, we used human keratinocytes as research subjects. Our purpose was to find the effects and machnisms of AOPPs in keratinocytes, so that these would set foundations for our following research about AOPPs in diabetic wounds healing.To explore the effects of AOPPs on keratinocytes, in vitro experiments were conducted on human immortalized keratinocyte (HaCaT) cells as the substitute of human keratinocytes. HaCaT is a spontaneously transformed immortalized human epithelial cell line from adult skin. These cells are widely used in skin-related studies because they maintain complete differentiation capacity of epidermal cells, reflect the characteristics of primary cultured skin keratinocytes in vitro, and remain nontumorigenic. In this study, HaCaT cells were treated with extracellular AOPPs at different concentrations or for different time durations. The rate of cell apoptosis and apoptosis-related biomarkers were evaluated. Also, this study determined the effects of AOPPs on HaCaT cell apoptosis and investigated the cellular mechanism underlying the proapoptotic effect of AOPPs.Matedals and methods1. AOPP-HSA Preparation and DeterminationAOPPs-Human Serum Albumin (HSA) was prepared in vitro by incubation of HSA (Sigma, USA) with HOC1 as described. Briefly, HSA was diluted in endoxin-free phosphate-buffered saline (PBS) at the concentration of 20mg/mL. Then, HSA solution was exposed to 40mmol/L HOC1 for 30min at room temperature. Prepared samples were dialyzed against PBS for 24 h to remove free HOC1 and passed through a Detoxi-Gel column (Pierce, USA) to remove contaminated endotoxin. Using chloramine-T as standard control, AOPPs contents in the preparations were determined through measuring the absorbance under acid condition at 340nm by SpectraMax M5 Multifunctional Microplate Reader (Molecular Devices, USA).2. HaCaT cells cultureHuman immortalized keratinocytes (HaCaT, purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; 4.5 g/L glucose) containing 10% fetal calf serum, and 1% penicillin/streptomycin at 37 ℃ in a 5% CO2 atmosphere. Medium was refreshed every 2-3 days and cells were subcultured when they reached 90% confluence. All materials for cell culturing were from Invitrogen Gibco.3. Measurement of cell viabilityTo determine the cell viability after AOPP-HSA treatment, we used the MTT viability assay (Thiazolyl Blue Tetrazolium Bromide; Sigma, USA). HaCaT cells were cultured with AOPP-HSA at the concentration of 50,100,200, or 400ng/mL for 24h and replaced the medium with control medium containing MTT solution (5mg/ml) in an amount equal to 10% of the culture volume. After incubation for 4h, removed cultures and dissolved the resulting MTT formazan with MTT solvent. The absorbance was spectrophotometrically measured at a wavelength of 490 nm.4. Apoptosis assessment by flow cytometry (FACS)HaCaT cells were seeded at 25%confluence and treated with AOPP-HSA at increasing concentrations for 24 h or 100μg/mL AOPP-HSA for increasing time durations the following day. After treatment cells were collected and resuspended in 500mL binding buffer containing 5μL Annexin V-FITC and 3mL PI according to the manufacturer’s instructions (BD Bioscience,USA) and incubated for 10 min in the dark. Quantification of Annexin V-FITC and PI staining was done by FACSCanto II (BD Biosciences, USA) using channels FL-1 (Annexin V-FITC) and FL-3 (PI).5. Mitochondrial membrane potential (MMP) alteration observationThe change of mitochondrial membrane potential (MMP,△ψm) caused by AOPPs was assayed using a membrane-permeant JC-1 dye (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazol-carbocyanine iodide; Keygen Biotech). JC-1 dye accumulates in mitochondria in a potential-dependent manner. After 24 h of AOPP-HSA treatment,5μg/mL of JC-1 dye was added to cells with incubation at 37 ℃ for 20 min, and cells were washed with incubation buffer. The alteration in HaCaT cells was observed with Olympus FluoView FV10i self-contained confocal laser scanning microscope system (Olympus America Inc., USA).6. Determination of ROS generationIntracellular reactive oxygen species (ROS) levels were assessed using an oxidation sensitive fluorescent probe 2’,7’-dichloro-fluorescin diacetate (DCFH-DA; Sigma, USA). First, HaCaT cells seeded at 50% confluence in a 96-well plate were labeled with 10μM probe diluted in serum-free DMEM for 30 min in dark, where afterwards ROS was induced with AOPP-HSA at increasing concentration or 200μg/ml AOPP-HSA for increasing time. Medium was replaced with PBS and fluorescence was measured in SpectraMax M5 Multifunctional Microplate Reader (Molecular Devices, USA) using an excitation wavelength 488 nm and an emission wavelength 525 nm. Analysis was performed in triplicate for each condition. Then, the cells cultured in confocal dishes were incubated with 10μM DCFH-DA probe for 20 min in the dark and treated with AOPP-HSA as described earlier. After washing for three times, the cellular fluorescent images were obtained using Olympus FluoView FV10i self-contained confocal laser scanning microscope system (Olympus America Inc., USA).7. Western blottingCultured cells were lysed in RIPA lysis buffer (Beyotime, China) and protein was collected after centrifugation and mixed with sodium dodecyl sulfate (SDS) sample buffer. The samples were separated by SDS-polyacrylamide gel electrophoresis (PAGE) using 12% acrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Pall Life Science, USA). After incubation with primary and secondary antibodies, the protein bands were detected with chemiluminescence detection reagents (Millipore, USA). The following antibodies (Abs) were used:rabbit anti-GADPH pAb and rabbit anti-NOX4 mAb were from Abeam (USA); rabbit anti-PARP pAb, anti-Bcl-2 pAb, anti-Bax pAb, anti-caspase3 pAb, anti-cleaved caspase 3 pAb, anti-cleaved caspase 9 pAb, anti-p38 pAb, anti-p-p38 pAb, anti-ERKl/2 pAb, anti-p-ERK1/2 pAb, and mouse anti-caspase9 pAb were from Cell Signaling Technology (CST, USA); goat anti-mouse and goat anti-rabbit IgG-horseradish peroxidase (HRP) were purchased from Boster (Wuhan, China).8. Statistical analysisAll experiments were repeated at least three times. Continuous variables are expressed as mean±standard deviation (S.D.). For multiple comparisons within a data set, one-way analysis of variance with least significant difference or Bonferroni’s test was performed. A two-tailed P-value of 0.05 was considered statistically significant. Statistical analyses were performed with SPSS 13.0 software (SPSS Inc., USA).Results:1. AOPPs induced HaCaT cell apoptosisThe MTT assay showed that 400 μg/mL AOPP-HSA caused significant HaCaT cell death (cell viability was 46.72 ± 0.64%) while incubation in 0-200 μg/mL AOPP-HSA had minimal effects on cell fatality (cell viability was above 85%). To eliminate the fatal effects of AOPPs, HaCaT cell cultures were subjected to increasing concentrations of AOPP-HSA (0,50,100, and 200 μg/mL) for 24 h and 100μg/mL AOPP-HSA for increasing time durations (0,15min,30min, 1h,2h,6h, 12h, and 24h). Quantitative fluorescence-activated cell sorting analysis of FITC-annexin V/PI staining indicated that AOPP-HSA induced HaCaT cell apoptosis in a concentration-dependent manner compared with cells treated with unmodified HSA and those cultured in control medium. The HaCaT cell apoptosis rate was 2.45±0.47% for NC group,2.45±0.47% for HSA group,18.59±3.52% for 50μg/mL AOPP-HSA group,20.81±3.14% for 100μg/mL AOPP-HSA group, and 22.92±4.14% for 200μg/mL AOPP-HSA group. On the other hand, FACS results indicated that AOPP-HSA significantly increased HaCaT cell apoptosis after incubation for 12h and 24h, with 24h (apoptosis rate 11.77+3.80%) much higher than 12h (apoptosis rate 20.88+2.93%).2. Mitochondrial membrane potential (MMP) declined in AOPPs-treated HaCaT cellsMMP loss was assessed through JC-1 staining. In healthy cells with a high MMP, JC-1 accumulates in the mitochondria as J-aggregates with red fluorescence. As the MMP declines in apoptotic cells, JC-1 stays in the cytosol as monomers with green fluorescence. Camparing to NC group and HSA group, AOPP-HSA incubated HaCaT cells showed reduced red fluorescence and enhanced green fluorescence. The results also suggested a dose-dependent reduction of red fluorescence and concomitantly enhanced green fluorescence, which indicated the existence of MMP loss in AOPP-treated HaCaT cells.3. Caspase cascade and PARP-1 were activated in AOPPs-treated HaCaT cellsThe Bcl-2 family is a key mediator in mitochondria-related cell apoptosis, and activation of caspase cascade and PARP-1 leads to substantial damage at the subcellular level. To demonstrate whether caspase cascade and PARP-1 participate in AOPPs-induced HaCaT cell apoptosis, their expression was detected using the Western blot analysis. As shown in the results, PARP-1 increased from 30 min after AOPPs treatment and reached the highest level at 2 h (9.67-fold compared with t=0, P< 0.05). The expression of proapoptotic Bax increased from 15 min, with a decrease in antiapoptotic Bcl-2 at the same time, and the highest Bax/Bcl-2 ratio was 16.71 ± 1.42 at 24 h (P< 0.05). After 2h AOPPs treatment, pro-caspase 9 and pro-caspase 3 significantly diminished, accompanied by an overexpression of cleaved caspase 9 and cleaved caspase 3 (P< 0.05). HaCaT cells were also incubated in AOPP-HSA (50,100, or 200 μg/mL), unmodified HSA, or control medium. The results showed that PARP-1, caspase 9, caspase 3, and Bax were activated in a concentration-dependent manner, while Bcl-2 was downregulated (P< 0.05).4. AOPPs stimulated intracellular ROS generation through NOXPrevious studies demonstrated that intracellular ROS mediate HaCaT cell apoptosis under oxidative stress. To determine whether AOPPs as oxidative products can cause redundant ROS accumulation in HaCaT cells, intracellular ROS levels were examined in AOPPs-treated HaCaT cells. Results indicated that ROS production increased in HaCaT cells cultured with AOPP-HSA in a dose-and time-dependent manner (P< 0.05).To elucidate the role of NADPH oxidases in ROS generation, the intracellular ROS levels were then measured in the presence of ROS scavenger N-acetylcysteine (NAC) and NOX inhibitors diphenylene iodonium (DPI) and apocynin. AOPP-induced ROS generation was significantly decreased in HaCaT cells that were pretreated with NAC, DPI, or apocynin separately (P< 0.05).5. AOPPs-induced HaCaT cell apoptosis was mediated via NOX4-ROS-MAPK-caspase cascade-PARP-1 pathwayWestern blot showed that in AOPPs-treated HaCaT cells, the expression of NOX4 (one part of NOX family) increased after 30 min and reached the highest level at 2 h (10.42-fold against t= 0, P< 0.05), while p-ERKl/2 and p-p38 MAPK consecutively increased from 1 to 24 h. Their activation was also mediated by AOPPs in a dose-dependent manner (P< 0.05).To further elucidate the roles of NOX4, ROS, ERK1/2, p38 MAPK, caspase cascade, and PARP in AOPPs-induced apoptosis, HaCaT cells were incubated with an ERK upstream inhibitor (U0126), a p38 MAPK inhibitor (SB203580), the broad-spectrum caspase inhibitor Z-VAD-fink, NAC, DPI, or Apocynin before AOPP-HSA treatment. HaCaT cell apoptosis was found to be significantly abolished under the protection of these inhibitors (P< 0.05) and PARP-1 activation was suppressed by these inhibitors (P< 0.05).Conclusions:1. AOPPs induced HaCaT cell apoptosis.2. AOPPs increased intracellular ROS production through NOX pathway in HaCaT cells.3. AOPPs induced HaCaT cell apoptosis via NOX4—ROS—MAPK—caspase cascade—PARP-1 signal pathway. |
PDF Full Text Request