Abstract
Reactive oxygen species (ROS) are crucial in all wound‐healing processes. Raftlin also plays an important role in the induction of the autoimmune response and the vascular inflammatory response. Inflammatory mediators induce continuous synthesis and secretion. To the best of our knowledge, although there are studies in the literature on antioxidant enzyme levels (superoxide dismutase [SOD], catalase [CAT]) and oxidative stress markers, there are no studies on the comparison of these levels in wound patients with the activities of Raftlin, which is known to play a role in the vascular endothelial response. The aim of this study was to compare the levels of oxidative stress and antioxidant response between wound patients and a control group and to compare the levels of Raftlin between the two groups, which is a new biomarker in inflammatory diseases. Between January 2018 and September 2018, 30 healthy control patients and 30 patients with wounds were enrolled in the study as volunteers. Tissue samples were collected and were sent to the biochemistry laboratory to determine the levels of oxidative stress, antioxidant enzymes, and Raftlin, which play an important role in wound healing. The following were evaluated: SOD and CAT levels (as a measure of antioxidant enzymes); malondialdehyde (MDA) levels (as a measure of free oxygen radicals); and Raftlin, which is a lipid raft protein used in determining the level of inflammatory and autoimmune response. The analyses determined a statistically significant correlation between MDA, SOD, CAT, and Raftlin values in wound patients (p<0.05). Raftlin was a considerable parameter in determining the prognostic process of wound healing. The levels of tissue Raftlin were significantly higher in wounded patients. A significant increase in MDA, SOD, and CAT activities of the wounded patients also suggested that the oxidant and antioxidant effect was balanced and that external antioxidant supplementation was not required.
Keywords: oxidative stress, Raftlin, wound
1. INTRODUCTION
Wound healing is a biological process consisting of three consecutive interrelated phases: haemostasis and inflammation, proliferation, and remodelling. In this process, redox signals and increased oxidative stress play an important role in regulating normal wound healing by facilitating haemostasis, inflammation, angiogenesis, granulation tissue formation, wound closure, and maturation of the extracellular matrix.1, 2
Reactive oxygen species (ROS) are crucial in all wound‐healing processes. The low concentrations of ROS formation regulate the signal transmission necessary for cell survival while fighting against invasive microorganisms.2, 3
ROS are primarily produced during normal metabolic events by NADPH oxidase, an enzyme complex system. Hydrogen peroxide (H2O2) from these products is not a radical, but it can result in severe cell damage by forming hydroxyl radicals in the presence of iron and copper ions. In particular, severe inflammatory infiltration in chronic wounds and elevated ROS indicate the presence of oxidative stress. High quantities of ROS cause cytotoxicity and result in delayed wound healing. Therefore, the elimination of ROS is an important condition, especially in the healing of chronic wounds.4
Excessive and uncontrolled oxidative stress causes prolongation and deterioration of the inflammation process, which plays a key role in the pathogenesis of chronic non‐healing wounds. Lipid peroxidase, stimulated by ROS, is one of the most important indicators of oxidative stress, and malondialdehyde (MDA) is a marker of the increase in lipid peroxidation (LPO) in various diseases.4, 5
In chronic wounds, as well as in acute wounds, the activity of enzymatic antioxidants (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase) decreases because of high oxidative stress that occurs when these antioxidants are released in large quantities. In addition, high oxidative stress leads to the depletion of non‐enzymatic antioxidants (vitamins E and C, glutathione). This effect is more potent in chronic wounds than in acute wounds. Thus, supplementation of antioxidants helps to prevent the oxidative damage of cells and improve recovery.6
Raftlin can be defined as the main lipid raft protein found in B cells. It is responsible for regulating the signal transmission of the B cell antigen receptor (BCR). Raftlin also plays an important role in the induction of autoimmune response and vascular inflammatory response. Inflammatory mediators induce continuous synthesis and secretion.7, 8
To the best of our knowledge, although there are studies in the literature on antioxidant enzyme levels (SOD, CAT) and oxidative stress markers, there are no studies in wound patients on the comparison of these factors with the activities of Raftlin, which is known to play a role in vascular endothelial response.
The aims of the study were to compare the levels of oxidative stress and antioxidant response between wound patients and a control group and to compare the levels of Raftlin, which is a new biomarker in inflammatory diseases.
2. MATERIAL AND METHOD
This study was carried out with the approval of the ethics committee of Kahramanmaraş Sütçü İmam University Faculty of Medicine Scientific and Clinical Research (2018/11‐05), and written informed consent was obtained from all patients.
Between January 2018 and September 2018, 30 healthy patients, as a control group, and 30 wounded patients were enrolled in the study as volunteers. Tissue samples (1 × 1 cm) were taken from the wound edges of patients and placed in Eppendorf tubes containing physiological saline and stored at −80°C. The included healthy volunteers were patients undergoing elective surgery, such as abdominoplasty and reduction mammoplasty. The samples for the control group were taken from the specimens of the removed tissues, 1 × 1 cm in size, without disrupting the integrity and quality and were stored in the same manner.
The wounds were determined to be caused by diabetes, vascular pathologies, trauma, and pressure. The mean age of the wounded patients was 42 years (24‐62 years). The mean age of the control group patients was 40 years (25‐58 years). The wounded patients group consisted of 20 male patients and 10 female patients. There were 19 men and 11 women in the control group. The mean time of occurrence for all wounds was over 3 weeks, and all were chronic wounds. Wound formation was observed to occur between 1 and 3 months. Tissue biopsy cultures and blood samples were obtained from all patients before treatment. According to the infection parameters (white blood cell, erythrocyte sedimentation rate, C‐reactive protein, and procalcitonin) and deep tissue biopsy culture results, appropriate antibiotherapy was initiated, and appropriate treatment modalities were carried out depending on the condition of each wound.
Tissue samples were sent to the biochemistry laboratory to determine the levels of oxidative stress, antioxidant enzyme, and Raftlin, which play an important role in wound healing. SOD and CAT levels (as a measure of antioxidant enzymes), MDA levels (as a measure of free oxygen radicals), and Raftlin (a lipid raft protein used in determining the level of inflammatory and autoimmune response) were evaluated.
2.1. Preparation of homogenate
The tissues were homogenised with three volumes of ice‐cold 1.15% potassium chyloride. The activities of antioxidant enzymes, MDA, and Raftlin levels were measured in the supernatant obtained after centrifugation at 14.000g.
2.2. Biochemical analysis
SOD activity was measured in the tissue samples according to the method described by Fridovich.9 This method uses xanthine and xanthine oxidase to generate superoxide radicals that react with p‐iodonitrotetrazlium violet (INT) to form a red formazan dye, which was measured at 505 nm. The assay medium consisted of 0.01 M phosphate buffer, CAPS (3‐cyclohexilamino‐1‐propanesulfonicacid) buffer solution (50 mM CAPS, 0.94 mM Ethylenediaminetetraacetic acid (EDTA), saturated NaOH) with pH 10.2, solution of substrate (0.05 mM xanthine, 0.025 mM INT), and 80 UL xanthine oxidase. SOD activity was expressed as U/mg protein.
CAT activity was determined by measuring the decrease in hydrogen peroxide concentration at 230 nm using the method of Beutler.10 The assay medium consisted of 1 M Tris HCI, 5 mM Na2EDTA buffer solution (pH 8.0), 10 mM H2O2, and a tissue sample in a final volume of 1.0 mL. CAT activity was expressed as U/mg protein.
MDA level in the tissue samples was measured using the thiobarbituric acid (TBA) test (Ohkawa method).11 The reaction mixture contained 0.1 mL of sample, 0.2 mL of 8.1% sodium dodecyl sulphate (SDS), 1.5 mL of 20 % acetic acid, and 1.5 mL of 0.8 % aqueous solution of TBA. The pH of the mixture was adjusted to 3.5, the volume was increased to 4.0 mL with distilled water, and 5.0 mL of a mixture of n‐butanol and pyridine (15:1, v/v) was then added. The mixture was shaken vigorously. After centrifugation at 4000g for 10 min, the absorbance of the organic layer was measured at 532 nm.
Raftlin levels in the tissue samples were measured with Human Raftlin (RFTN 1), an enzyme‐linked immunosorbent assay (ELISA) reader, using commercial kits (SunRed, Human (RFTN1) ELISA Kit, Shanghai, China).
2.3. Statistical analysis
The data were analysed using SPSS v 22.0 software (SPSS, Chicago, Illinois). Thirty patients and 30 healthy subjects were included in the study. A Mann–Whitney U test was used to compare the data. The median, mean value ± SD, and minimum and maximum values were used in the comparisons. Receiver Operating Characteristic (ROC) curves were used to determine the distinguishing performance of the method applied for the enzyme activity. P < .05 was considered statistically significant.
3. RESULTS
A total of 60 subjects were included in the study; 30 were in the control group and 30 in the patient group. The mean age was 42.6 ± 6.02 years (min: 24 max: 62) in the wound group and 40.4 ± 8.36 years in the control group (min: 25 max: 58). The wound group consisted of 20 men and 10 women, and the control group consisted of 19 men and 11 women. There was no statistically significant difference between the groups in terms of age and gender (P < .05). For 10 patients (33.3%), the wound cause was diabetes mellitus; for 5 patients (16.7%), it was pressure; for 12 patients (40%), it was trauma; and in 3 patients (10%), it was a vascular compromise. MDA, CAT, SOD, and Raftlin levels were found to be significantly higher in the patient group compared with the control group (P < .05) (Table 1).
Table 1.
Evaluation of Raftlin, MDA, CAT, and SOD activities in patient and control groups
| Group | Mean | SD | Median | Min | Max | P |
|---|---|---|---|---|---|---|
| MDA (patient; nmol/mL) | 2.75 | 10.27 | 2.32 | 1.86 | 40.11 | <.05a |
| MDA (healthy; nmol/mL) | 1.26 | 0.33 | 1.16 | 0.89 | 10.09 | |
| SOD (patient; U/mg) | 11.59 | 10.27 | 6.8 | 1.07 | 5.62 | <.05a |
| SOD (healthy; U/mg) | 4.44 | 2.76 | 3.4 | 0.88 | 2.25 | |
| CAT (patient; U/mg) | 19.76 | 10.95 | 16.61 | 8.11 | 43.57 | <.05a |
| CAT (healthy; U/mg) | 4.87 | 1.42 | 4.68 | 3.25 | 8.47 | |
| RAFTLIN (patient; ng/mL) | 1048.75 | 155.83 | 1056 | 825 | 1326 | <.05a |
| RAFTLIN (healthy; ng/mL) | 813 | 179.75 | 829 | 543 | 1024 |
Note: Mann–Whitney U test.
Abbreviations: CAT, catalase; Max, maximum; MDA, malondialdehyde; Min: minimum; SOD, superoxide dismutase.
Difference is statistically significant.
In the study, it was found that Raftlin levels of the wounded patients were higher than the control group, and this difference between the groups was statistically significant (P < .05) (Table 1). The ROC curve was drawn for diagnostic measurements of Raftlin. As a result of the analysis, the area under the ROC curve (AUC) was found to be 0.84 (Table 2).
Table 2.
Raftlin, MDA, CAT, and SOD activities and differentiation power of the test
| AUC | SD | P‐value | %95 statistical power | ||
|---|---|---|---|---|---|
| Minimum | Maximum | ||||
| Raftlin | 0.840 | 0.062 | .000a | 0.719 | 0.961 |
| MDA | 0.878 | 0.055 | .000a | 0.770 | 0.985 |
| CAT | 0.998 | 0.004 | .000a | 0.989 | 1.000 |
| SOD | 0.753 | 0.077 | .006a | 0.603 | 0.902 |
ROC curve; a:0.05.
AUC, area under the curve; CAT, catalase; MDA, malondialdehyde; SOD, superoxide dismutase.
Statistically significant.
It was observed that Raftlin had sufficient qualification in diagnostic evaluation. The probability of Raftlin levels being significantly higher was greater than 84% in wounded patients compared with healthy individuals. Thus, the diagnostic value of Raftlin was found to be statistically valid for wounded patients (P < .05) (Figure 1).
Figure 1.
ROC curve for Raftlin area under the curve (AUC) value of 0.84
The ROC curve was drawn for diagnostic measurements of MDA. The AUC was found to be 0.87 (Table 2). It was observed that MDA had good qualification in diagnostic evaluation. The probability of the MDA variable being higher was 87% in wounded patients compared with healthy individuals. The diagnostic value of MDA for wounded patients was found to be statistically significant (P < .05) (Figure 2).
Figure 2.
ROC curve for malondialdehyde (MDA) area under the curve (AUC) value of 0.87
The ROC curve was drawn for the diagnostic measurements of CAT. The area under the ROC curve (AUC) was found to be 0.99 (Table 2). It was observed that CAT had excellent competence in diagnostic evaluation. The probability of the CAT variable being higher was 99% in wounded patients compared with healthy individuals. The diagnostic value of CAT for wounded patients was found to be statistically significant (P < .05) (Figure 3).
Figure 3.
ROC curve for CAT area under the curve (AUC) value of 0.99
The ROC curve was drawn for the diagnostic measurements of SOD. The AUC was found to be 0.75 (Table 2). It was observed that SOD had moderate qualification in diagnostic evaluation. The probability of the SOD variable being higher was 75% in wounded patients compared with the healthy individuals. The diagnostic value of SOD for wounded patients was found to be statistically significant (P < .05) (Figure 4).
Figure 4.
ROC curve for SOD area under the curve (AUC) value of 0.75
The confidence intervals of the MDA, CAT, SOD, and Raftlin protein activity and the differentiation powers of the tests are shown in Table 2. The analyses determined there was a statistically significant correlation between MDA, SOD, CAT, and Raftlin values in wound patients (P < .05).
It is thought that these variables, which have a significant impact on the wound‐healing phase, will be beneficial in the prognosis evaluation of wounds. The excessive increase of free oxygen radicals and the inability of compensation by the antioxidants will cause the wound to become chronic and impair healing. High levels of Raftlin, the new lipid raft protein, also suggest an excessive increase in inflammatory mediators that may adversely affect wound healing by reason of inflammatory response. Therefore, it seems that Raftlin is a considerable parameter in determining the prognostic process of the wound.
4. DISCUSSION
Complex molecular and cellular processes of wound healing occur in overlapping phases, including inflammation, granulation tissue formation, extracellular matrix synthesis, angiogenesis, reepithelisation, and remodelling.1, 2, 12
Oxygen is necessary both to protect wounds against infection and to provide the energy required to heal. In addition, an oxygen (O2)‐dependent redox signal is crucial for wound repair. Physiologically, H2O2 and superoxide act as intracellular messengers that stimulate the basic stages of wound healing, including cellular chemotaxis, cytokine production, and angiogenesis.13
The physiological levels of ROS are tasked with reducing local blood flow by contributing to thrombocyte and inflammatory cell release, providing vasoconstriction, thrombocyte aggregation, and thrombus formation. In addition, the release of ROS in the tissue stimulates the diapedesis of adherent leukocytes along the vascular wall, helping to kill microorganisms in the wound region. High levels of superoxide and H2O2 are produced by neutrophils and macrophages via Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which acts as the primary mechanism for killing bacteria and preventing wound infection. ROS also plays a role in fibroblast proliferation, collagen and fibronectin production, extracellular matrix formation, angiogenesis, and epithelisation by secreting growth factors and cytokines.14, 15
Chronic wound pathogenesis is difficult to understand because of the complexity of wound‐healing phases and the heterogeneity of chronic wounds. In chronic wounds, there are several pathological causes of prolonged and incomplete healing. Local ischaemia and reperfusion injury, type 2 diabetes, chronic inflammation, and advanced age are the most important factors leading to chronic wound formation.16, 17
Chronic inflammation appears in most wounds, caused by these factors. Inflammation is associated with persistent macrophages that limit and delay proliferation. Chronic inflammation also triggers cell aging, which is considered a critical pathophysiological process in the formation of chronic wounds. A chronic wound environment is mainly characterised by increased proteases such as matrix metalloproteinase (MMP), decreased protease inhibitors such as tissue inhibitor of mMMP (TIMPs), excessive amounts of ROS, pro‐inflammatory cytokines, proteolytic enzymes, and the inflammatory cells secreting them.16, 17
The combination of these factors leads to a reduction in extracellular matrix and growth factors, impaired inflammatory response control, inhibition of cellular proliferation, inadequate vascularisation, and accumulation of necrotic tissues because of ischaemia. On the other hand, these effects may cause bacterial colonisation and prolonged inflammatory response by inhibiting wound repair.2, 4, 16, 17
As discussed above, the importance of a delicate balance between the positive role of ROS and its adverse effects is substantial for proper wound healing. While the production of ROS is necessary to initiate wound repair, an excessive amount of ROS formation is detrimental to wound healing.
The ongoing oxidative stress associated with LPO, protein modification, and DNA damage has been demonstrated to disrupt wound‐healing processes, with increased cellular aging and apoptosis. Clinical trials show that unhealed wounds are maintained in a highly oxidising environment that leads to deteriorating wound repair. Clinical conditions such as tissue hypoxia and hyperglycaemia are typically associated with highly oxidising environments.18
Raftlin is a novel lipid raft protein identified in Raji B cells, which are required to provide signal transduction of the BCR. It is also involved in the nucleocapture complex inducing autoimmune response and the activation of Toll‐like receptor 3 (TLR3). Raftlin is a new parameter that can be used in the pathophysiology of vascular inflammatory response to characterise the diagnosis of inflammatory diseases and the immune response.7, 19, 20 Raftlin, mostly investigated for determining sepsis and septic shock, was evaluated in wounded patients in our study and was found to be significantly more abundant in wounded patients compared with healthy volunteers.
Although the conventional treatment options for chronic wound patients include standard wound care applications such as surgical debridement, antibiotic therapy, and moist dressing, recent developments have focused on solving the specific deficiencies in the local wound environment by topical applications such as growth factors, the incorporation of bone marrow‐derived endothelial and epithelial cells, and collagen‐based tissue engineering grafts. As a different concept, the strict control of ROS levels with antioxidants and antioxidative enzyme systems may reduce cellular damage because of oxidative stress.21
5. CONCLUSION
The levels of tissue Raftlin were significantly higher in wounded patients. In addition, a significant increase in MDA, SOD, and CAT activities of the wounded patients suggests that the oxidant and antioxidant effect was balanced, and external antioxidant supplementation was not required. However, in order to determine the sensitivity and specificity of Raftlin in wounded patients, wider groups with larger populations and subgroup studies regarding the wound stages is necessary.
AUTHOR CONTRIBUTIONS
Fatma Bilgen contributed to the collection of data and article writing; Alper Ural contributed to the collection of data and statistical analysis; Ergul Belge Kurutas contributed to the biochemistry examination; Mehmet Bekerecioğlu contributed to the examination of the subject of study and data.
Bilgen F, Ural A, Kurutas EB, Bekerecioglu M. The effect of oxidative stress and Raftlin levels on wound healing. Int Wound J. 2019;16:1178–1184. 10.1111/iwj.13177
Presented at the 40th Congress of National Turkish Plastic Reconstructive and Aesthetic surgery.
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