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The Role of Vitamin C in the Gene Expression of Oxidative Stress Markers in Fibroblasts from Burn Patients1

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The Role of Vitamin C in the Gene Expression of Oxidative Stress Markers in Fibroblasts from Burn Patients1 6-Clinical Investigation The role of vitamin C in the gene expression of oxidative stress markers in fibroblasts from burn patients1 Jessica BonucciI, Alfredo GragnaniII, Marcelo Moraes TrincadoIII, Victor VincentinIII, Silvana Aparecida Alves CorreaIV, Lydia Masako FerreiraV IFellow MSc degree, Division of Plastic Surgery, Department of Surgery, Universidade Federal de São Paulo (UNIFESP), Brazil. Conception and design of the study; acquisition, analysis and interpretation of data; technical procedures; manuscript preparation and writing. IIPhD, Associate Professor, Division of Plastic Surgery, Department of Surgery, UNIFESP, Sao Paulo-SP, Brazil. Conception and design of the study; acquisition, analysis and interpretation of data; manuscript writing; critical revision; final approval. IIIGraduate student, UNIFESP, Sao Paulo-SP, Brazil. Acquisition, analysis and interpretation of data. IVPhD, Postdoctoral degree, Division of Plastic Surgery, Department of Surgery, UNIFESP, Sao Paulo-SP, Brazil. Conception and design of the study, technical procedures, analysis and interpretation of data, statistical analysis, critical revision. VHead, Full Professor, Division of Plastic Surgery, UNIFESP, Researcher 1A-CNPq, Director Medicine III-CAPES, Sao Paulo- SP, Brazil. Intellectual, scientific, conception and design of the study; critical revision. Abstract Purpose: To assess the action of vitamin C on the expression of 84 oxidative stress related- genes in cultured skin fibroblasts from burn patients. Methods: Skin samples were obtained from ten burn patients. Human primary fibroblasts were isolated and cultured to be distributed into 2 groups: TF (n = 10, fibroblasts treated with vitamin C) and UF (n = 10, untreated fibroblasts). Gene expression analysis using quantitative polymerase chain reaction array was performed for comparisons between groups. Results: The comparison revealed 10 upregulated genes as follows: arachidonate 12-lipoxygenase (ALOX12), 24-dehydrocholesterol reductase (DHCR24), dual oxidase 1 (DUOX1), glutathione peroxidase 2 (GPX2), glutathione peroxidase 5 (GPX5), microsomal glutathione S-transferase 3 (MGST3), peroxiredoxin 4 (PRDX4), phosphatidylinositol-3,4,5- trisphosphate dependent Rac exchange factor 1 (P-REX1), prostaglandin-endoperoxide synthase 1 (PTGS1), and ring finger protein 7 (RNF7). Conclusion: Cultured fibroblasts obtained from burn patients and treated with vitamin C resulted in 10 differentially expressed genes, all overexpressed, with DUOX1, GPX5, GPX2 and PTGS1 being of most interest. Key words: Burns. Gene Expression. Fibroblasts. Oxidative Stress. Ascorbic Acid. DOI: http://dx.doi.org/10.1590/s0102-865020180080000006 Acta Cir Bras. 2018;33(8):703-712 703 The role of vitamin C in the gene expression of oxidative stress markers Acta Cir Bras. 2018;33(8):703-712 in fibroblasts from burn patients Bonucci J et al. ■ Introduction mechanisms, physical and antioxidant defenses. Enzymatic antioxidant defenses are Burn injuries are a serious worldwide known as superoxide dismutase, glutathione public health problem. More than 300.000 peroxidase (GPX), and catalase. Non-enzymatic deaths are caused by fire-related burn injuries antioxidants are represented by L-ascorbic per year throughout the world1. Disruption acid (vitamin C), alpha-tocopherol (vitamin E), in the skin integrity lead to decreased body glutathione, carotenoids, flavonoids, and other temperature and consequent increased energy antioxidants8. consumption. Burn injuries lead to depression Vitamin C is a water-soluble of immune potential, which may result in micronutrient required for various biological secondary infections2. The metabolic response functions. It acts as a cofactor in enzymatic is immediate and may persist for at least 5 weeks. reactions such as collagen synthesis9, and This hypermetabolic state is characterized in human plasma is an relevant antioxidant, by an increase in body temperature, oxygen eliminating free radicals10. In the first 5 days and glucose consumption, carbon dioxide after a burn injury, patients presented a production, proteolysis, glycogenolysis, and decrease in vitamin C, beta-carotene, lycopene lipolysis3. After a burn trauma, mediators (antioxidant vitamins) and increase in lipid of inflammation released at the wound site peroxidation products11. associated with cellular hydration result in An inflammatory response is extensive edema formation4. triggered by a cutaneous burn with Inflammation is an important factor in consequent production of free radicals, the healing of thermal injuries. The beneficial leading to alterations in gene expression. The effects of local inflammation include cell growth identification of the mechanisms regulating and proliferation, protection from microbial gene expression of oxidative stress markers, agents and cleansing of cell debris. At the in the presence or absence of vitamin C, is same time, a prolonged inflammatory process important to understand the processes that makes healing difficult, once the increasing on influence the recovery of burn patients12. cytokines level promotes collagen degradation, The purpose of the present study dermal cell apoptosis, vascular impairment, was to analyze the action of vitamin C on the and oxygen free radical production5. Free expression of 84 oxidative stress related-genes radicals are constantly produced during the in cultured skin fibroblasts from burn patients. normal functioning of cells, mostly in the form of reactive oxygen species (ROS). Reperfusion ■ Methods of ischemic tissues following thermal injury results in imbalance between ROS and the This single-center, interventional, antioxidant defense system due to excessive analytic, cross-sectional, controlled, in vitro generation of free radicals, known as oxidative study was conducted at the Plastic Surgery stress6. Studies in several organisms and Unit and Burn Treatment Unit, Universidade cellular systems have identified the common Federal de São Paulo. It was approved by involvement of ROS as mediators of the initial the Institutional Ethics Committee (approval inflammatory response to wound healing7. number 269.015) and a written consent was The defense mechanisms against given from all patients enrolled. oxidative stress induced by free radicals Ten hospitalized adult patients, involve prevention mechanisms, repair consisting of 5 men and 5 women, with a 704 Acta Cir Bras. 2018;33(8):703-712 The role of vitamin C in the gene expression of oxidative stress markers in fibroblasts from burn patients Bonucci J et al. mean age of 34.2 years, with second- or third- with 100 IU / ml penicillin (Gibco, Big Island, degree burn injuries covering twenty five to NY, USA) and 100 μg / ml streptomycin (Gibco, fifty percent of the total body surface area and Grand Island, NY, USA), then sent to the requiring surgical procedures were included in laboratory for being handled immediately. this study. Patients with previous skin or superficial Skin fibroblasts lesions (psoriasis and similar diseases), Human primary fibroblasts were infectious conditions, or clinical diseases (e.g., obtained by explant culture and distributed colagenoses, such as lupus erythematosus, into 2 groups: the TF (n = 10, fibroblasts treated scleroderma, dermatopolymyositis, and mixed with vitamin C) and UF (n = 10, untreated connective tissue disease) that could directly fibroblasts) groups. interfere with wound healing were not included Culture flasks with some contamination in the study. or low proliferation rate (less than 80% The “n” was considered adequate confluence), and subsamples with insufficient because it was a lineage of cultured cells, extraction of RNA for analysis or loss of proceeding with the extraction of its genetic extracted material were excluded. material, there being no other variables besides the individual cellular genetic load. Fibroblast culture medium The experiment was performed after the third passage, at which time there is no cellular The culture medium of fibroblast was alteration by systemic signals that could affect composed of DMEM supplemented with the cells in the immediate isolation after antibiotics (penicillin 100 IU/ml - streptomycin/ obtaining the skin fragment. The sample size 100 µg/mL), and 20% fetal bovine serum (FBS; calculation was not performed due to lack of ATCC). data in the literature explaining the number used in in vitro study, therefore, a convenience Isolation and culture sample was used. Tissue cleansing was performed Surgical procedure within a laminar flow to maintain the sterile environment by plunging the skin fragment Debridement is the removal of dead into 8 consecutive tubes containing 30 mL tissue from a wound. It is commonly performed of modified phosphate-buffered saline on the third day postburn in patients with a (Dulbecco’s PBS, without calcium and second- or third-degree burn injury. During magnesium hydrochloride) (Sigma, St. Louis, surgical debridement, performed by the plastic USA) supplemented with 100 IU / ml penicillin surgeon, viable skin surrounding the wound is (Gibco, Grand Island, NY, USA) and 100 μg / ml partially removed since the instrument used streptomycin (Gibco, Grand Island, NY, USA), (Blair knife) does not allow the removal of only shaking each tube vigorously for 45 seconds to the necrotic skin, which has irregular margins. remove blood and other impurities13. These sterile fragments of normal skin For separation of the epidermal sheet, that would be discarded were immediately the skin fragments were cut into
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