Role of Autophagy and Apoptosis in Wound Tissue of Deep Second‐degree Burn in Rats

Abstract

The pathogenesis of burn wound progression is poorly understood. Contributing factors include continuous loss of blood perfusion, excessive inflammation, and elevated apoptosis levels in wound tissue. Macroautophagy (here referred to simply as “autophagy”) is associated with many chronic diseases. The authors hypothesized that autophagy is involved in burn wound progression in a rat model of deep second-degree burn. Deep second-degree burns were modeled using a brass rod heated to 100°C applied for 6 seconds to the back skin of Wistar rats. Full-thickness biopsies were obtained from burned and nonburned controls at several times postburn. Western blotting and immunohistochemical (IHC) staining determined expression of the autophagy markers Light Chain 3 (LC3) and beclin-1. Apoptosis was determined by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay and laser Doppler flowmetry (LDF)-measured tissue perfusion. Myeloperoxidase (MPO) activity assay measured inflammation. Hematoxylin and eosin (H&E) and Masson's trichrome staining–determined pathology and wound depth. The LC3 and beclin-1 protein level in burn wounds decreased to one-fourth of normal levels (p < 0.01) over 24 hours and then began to increase but still did not reach their normal level. TUNEL-positive cells in burn wounds were 3.7-fold (p < 0.01) elevated over 48 hours and then decreased slightly, yet still remained higher than in normal skin. The burn wound progressed in depth over 72 hours. In addition, significant decrease in LDF values and upregulation of MPO activity were observed. Enhanced LC3-positive cells were observed in the deep dermal layer of burn wounds as shown by IHC staining. A reduction in autophagy and blood flow and an increase in apoptosis and inflammation were observed in burn wounds early during the course of burn injury progression. This suggests that autophagy, complemented by apoptosis, play important roles in burn progression. Enhanced autophagy in the deep dermis may be a prosurvival mechanism against ischemia and inflammation after burn injury. La patogénesis de la progresión en la lesión por quemadura es poco conocida. Los factores contribuyentes incluyen la pérdida continua de perfusión sanguínea, la excesiva inflamación y los marcadores de apoptosis elevados en el tejido lesionado. La macroautofagia (aquí referida simplemente como “autofagia”) se asocia con muchas enfermedades crónicas. Se generó la hipótesis que la autofagia está implicada en la progresión de la lesión por quemadura en un modelo de quemadura de segundo grado en ratas. Las quemaduras de segundo grado se simularon usando un varilla de metal calentada hasta 100°C y se aplicó durante 6 segundos en la piel del dorso de las ratas Wistar. Se obtuvieron biopsias de todo el grosor de los quemados y de los controles no quemados en diversas ocasiones tras la quemadura. Se determinó la expresión de marcadores de autofagia Light Chain 3 (LC3), y Beclin-1 mediante Wetern blot y tinción inmunohistoquímica. La apoptosis se determinó por técnica TUNEL (TUNEL assay) y la flujometría Doppler láser midió la perfusión del tejido. La técnica de actividad mieloperoxidasa midió la inflamación. Las tinciones con hematoxilina y eosina, y con tricrómico de Masson determinaron la patología y profundidad de la lesión. El valor de proteínas Beclin-1 y LC3 en lesiones por quemadura disminuyó a un cuarto del valor normal (p < 0,01) a las 24 horas, y después empezó a incrementarse pero no alcanzó sus valores normales. Las células positivas TUNEL en las lesiones por quemadura fueron 3,7 veces (p < 0,01) más elevadas a las 48 horas, y después disminuyeron ligeramente, permaneciendo todavía más altas que en la piel normal. La lesión por quemadura progresó en profundidad durante 72 horas. Además, se observaron un descenso significativo en los valores de flujometría Doppler láser y un aumento de la actividad mieloperoxidasa. Se observaron mediante células positivas LC3 aumentadas en la dermis profunda de las lesions por quemadure la tíncion inmunohistoquímica. Se observaron una reducción de la autofagia y del flujo sanguíneo y un incremento en la apoptosis y la inflamación en las heridas por quemadura al inicio del curso de la progresión de la lesión. Esto indica que la autofagia, junto con la apoptosis, juegan papeles importantes en la progresión de la quemadura. El incremento de la autofagia en la dermis profunda puede ser un mecanismo a favor de la supervivencia y que actúa en contra de la isquemia y la inflamación tras la lesión por quemadura. Burn wound progression is a widely acknowledged pathophysiologic process that often occurs within 72 hours after a burn.1 The progression of burn wounds makes treatment more difficult, prolongs hospital stay, increases costs, and increases the likelihood of scarring. For these reasons, it is of great significance to prevent burn wound progression. A number of mechanisms are thought to be involved in the process of burn wound progression, including local tissue hypoperfusion, edema, prolonged inflammation, hypercoagulability, free radical damage, and accumulation of cytotoxic cytokines.1-3 Many attempts have been made to prevent burn wound progression by modulating these mechanisms. However, further study is still needed to better understand this process. Recently studies2 have suggested that cell death may occur as a result of three distinct pathways, including necrosis, apoptosis, and autophagy, all of which may lead to further burn injury progression. Studies have shown that apoptosis and necrosis are both involved in burn wound progression,4, 5 and it has been demonstrated5 that apoptosis is one of the contributing factors of burn wound progression. Inhibiting apoptosis reduces continuous tissue loss and promotes burn wound healing.6, 7 However, to date, little is known about the relationship between autophagy and burn wound progression. Autophagy8, 9 is a highly conserved pathway that delivers intracellular macromolecule waste to lysosomes, where they are degraded into biologically active monomers, such as amino acids, that are subsequently reused to maintain cellular metabolic turnover and homeostasis. Autophagy is linked to apoptosis in many ways, yet their roles in the pathogenesis of burn wounds remain unclear. This study was designed to determine whether there were changes in autophagy and apoptosis in a deep second-degree rodent burn model and to explore the association between these changes and wound progression. This was a laboratory study of burn wound progression in the murine model. The experimental protocol was approved by the Institutional Animal Care & Use Committee of First Hospital Affiliated to PLA General Hospital. Experiments were performed on 56 male Wistar rats weighing 200 to 220 grams. Animals were housed for at least 7 days prior to experiments in a ventilated and temperature-controlled room and had access to water ad libitum. Anesthesia was performed by intramuscular injection of 100 mg/kg ketamine (Gutian Pharmaceutical Co., LTD, Fujian, China) and 25 mg/kg xylazine hydrochloride (Dunhua Animal Pharmaceutical Co., LTD, Jilin, China). The hair on the dorsal skin of the rats was removed by electric clippers. A 1.8-cm-diameter brass rod with a height of 3 cm, and a weight of 200 g was heated to 100°C and applied to the skin for 6 seconds to produce a deep second-degree burn as previously described.10 Only the weight of the rod was used to create the burns; no additional pressure was placed on it. To avoid variations in creating the burns, one person created all burns. Three burns were created on each half of the dorsal skin at 1 cm apart. Six burns (10% total body surface area, approximately) were created on each animal. The temperature of the brass rod was regulated by a temperature controller and varied by <1°C during the process of thermal injury. The burn depth was confirmed by two experienced histopathologists and, by microscopic examination, showed destruction of hair follicles and vessel walls, microthrombi, and neutrophils in the deep dermis, yet a few hair follicles remained. The rats were randomly divided into six time points (eight rats at each time point) to assess postburn endpoints at 1, 6, 12, 24, 48, and 72 hours. Eight healthy adult rats without burns were used as controls. The control group was assumed to be unchanged over the time course of the investigation. The data from the control group were used for multiple comparisons with the experimental groups at each time point. After laser Doppler flowmetry (LDF) values were measured, the rats were euthanized. All skin/wound samples were collected and sorted by the number of rats; in other words, the eight samples in each time point were from eight different rats. The samples were subjected to hematoxylin and eosin (H&E), Masson's trichrome staining (Masson staining), terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay, and immunohistochemical (IHC) staining. The other parts of the specimen were homogenized for myeloperoxidase (MPO) activity assay and Western blot analysis. Western blot analysis was employed to determine the protein levels of two autophagy markers, Light Chain 3 (LC3) and beclin-1. Expression of these markers reflected autophagy levels in burn wounds. The autophagy marker LC3 was originally identified as a subunit of microtubule-associated proteins 1A and 1B and was subsequently found to display similarity to the yeast protein Apg8/Aut7/Cvt5, which is critical for autophagy.11 Cleavage of LC3 at the carboxy terminus immediately following synthesis yields the cytosolic LC3-I form. During autophagy, LC3-I is converted to LC3-II through lipidation (the covalent binding of a lipid group to a peptide chain) by an ubiquitin-like system involving Atg7 and Atg3 that allows LC3 to become associated with autophagic vesicles.12 The ratio of LC3-II/LC3-I has been used as an indicator of autophagy.13 Beclin-1, the mammalian ortholog of the yeast autophagy protein Apg6/Vps30, is one of the proteins critical to autophagy.14 The level of beclin-1 was also an indicator of autophagy.15 The skin/wound samples were cut into small pieces and homogenized (DIAX 900, Heidolph, Schwabach, Germany; 4°C, at 25,000 rpm, for 5 minutes) in lysis buffer containing 50 mmol/L Tris HCl (pH 7.6), 20 mmol/L MgCl2, 150 mmol/L NaCl, 0.5% Triton-X, 5 units/mL aprotinin, 5 μg/mL leupeptin, 5 μg/mL pepstatin, 1 mmol/L benzamidine, and 1 mmol/L phenylmethylsulfonyl fluoride. The debris was removed by centrifugation, and the protein levels in the lysates were determined by bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL). Protein (50 μg) in the lysates was separated using sodium dodecyl sulfate–polyacrylamide 15% gel electrophoresis and electrophoretically transferred to a polyvinylidene difluoride membrane. The membranes were blocked for 2 hours at room temperature in Tris-buffered saline with Tween (TBST) buffer (0.01 mol/L Tris-HCl, pH 7.5, 0.15 mol/L NaCl, and 0.05% Tween 20) containing 5% skimmed milk. They were then incubated with rabbit anti-LC3 antibodies (1:500, Cell Signaling Technology, Boston, MA) and anti-beclin-1 antibodies (1:500, Cell Signaling Technology) diluted in TBST buffer overnight at 4°C. The membranes were washed three times and incubated for 1 hour at room temperature with secondary antibody linked to horseradish peroxidase (1:2000; Invitrogen, Carlsbad, CA). Immunoreactive bands were developed using an enhanced chemiluminescence reagent (Pierce Biotechnology) and exposed onto Kodak film (Eastman Kodak, Rochester, NY). Band densities were normalized using glyceraldehyde-3-phosphate dehydrogenase and quantified using a scanning densitometric analysis Image J software (National Institutes of Health, Bethesda, MD). In addition to Western blot assay, we performed IHC staining of LC3 to be supplemented with visualized images. Samples were fixed in 10% neutralized formalin, embedded in paraffin, and sectioned by a slicing machine (Leica Microsystems, Wetzlar, Germany). Serial sections (5 μm) were deparaffinized, rehydrated, and washed in phosphate-buffered saline (PBS) for 10 minutes and then incubated in hydrogen peroxide block for 10 minutes. This was followed by washing with PBS containing 0.3% Tween for 10 minutes and blocking with 3% milk and 5% fetal bovine serum in 0.01 mol/L PBS for 2 hours. The sections were incubated with rabbit anti-LC3 antibodies (1:100; Cell Signaling Technology) diluted in PBS overnight at 4°C. After being rinsed with PBS, the sections were incubated with goat anti-rabbit secondary antibody (1:500; Boster, Wuhan, China) for 1 hour at room temperature. Since LC3 is expressed in the cytoplasm, cells staining in their and in their were The sections were observed a at The were to the were and from to to were and were obtained by number by The of a was obtained by a from at least six analysis was performed using software and was for analysis. To the apoptosis level of burn we TUNEL staining to the apoptosis level in burn wounds after the burns were The TUNEL staining assay kit and all associated were performed to the for the in cell death samples were fixed in 10% neutralized and embedded in Serial sections were deparaffinized, rehydrated, and incubated for 20 minutes at with μg/mL in 10 mmol/L Tris-HCl, pH After being rinsed with PBS, were incubated in hydrogen peroxide block for 10 and then TUNEL were on the were incubated for minutes at in a in the After being rinsed with PBS, were to 50 of and incubated in a for minutes at and then 50 of was and the were incubated for 10 minutes at After being rinsed with PBS, were a with were The were to the were and from to to were and were obtained by number by The was obtained by the TUNEL-positive cells to all the cells at a A was from at least eight values and the values were subjected to analysis. and Masson staining were performed to the changes of burn wounds. Masson staining was also designed to the of tissue necrosis progressed over The assay were from China). For sections were and After were washed and hematoxylin was for 5 after with HCl was for 10 were washed for 25 0.5% eosin was for 2 and then were with and was for 5 were observed a For Masson sections were deparaffinized, rehydrated, and incubated in hematoxylin for 10 minutes. acid was for 1 and then were washed for 25 minutes. was for minutes and acid for 5 minutes. After were incubated with for 5 and acid was for 1 were then in and and in were observed a Doppler flow values in burn wounds were an indicator of burn wound blood designed the LDF assay to the blood flow changes in burn wounds over time and with the changes of autophagic levels in the process of burn wound progression. For of the rats were using the protocol as in the experimental model. LDF was measured with a LDF was measured to the of the as by the was used to the and rat skin and the was by the wound was and were samples were obtained by number by The of a burn wound was obtained by a from at least six values within the The room temperature was at and the was Myeloperoxidase activity the in the burn performed this assay to determine the changes in of the burn wound over time and with the changes of autophagic levels in the process of burn wound progression. of MPO activity in the burn wound tissue was to a kit and the China). tissue samples were homogenized on in of PBS, pH containing Tween samples were at g at for 10 minutes. were on for analysis. of tissue was incubated with 200 of buffer of of mol/L and of mol/L of 0.3% and of HCl to 50 with A was to changes at over a MPO in samples were determined using a of MPO in the are expressed as of activity of tissue. there was no we performed a study to the values and of the two Western blot of LC3 and TUNEL in each to the of the we that the of autophagy level as determined by Western blot of LC3 at 1, 6, 12, 24, 48, and 72 hours postburn normal level were and and the increases of as determined by TUNEL staining at 6, 24, 48, and 72 hours postburn normal level were and The was then obtained by using software a significance level and a of 1 analysis was using software NY). are as and The data from the control group were used for multiple comparisons with the experimental groups at each time point. of was used and multiple group comparisons were performed by using of < were of LC3 and beclin-1 protein were at a in normal skin tissue and After the burn was levels of these proteins in wound tissue 24 hours after the burn. then slightly, but remained (p < 0.01) than in normal skin analysis showed that autophagy levels decreased to one-fourth of normal level over 24 and then but did not to the normal level during the study found more cells apoptosis in burn wounds than controls. The in burn wounds were at 6 at 24 at 48 and at 72 hours of controls (p < at 6 < 0.01 at 24, 48, and at 72 The TUNEL-positive cells were observed in the of hair follicles blood analysis that TUNEL-positive cells in burn wounds were elevated about 3.7-fold over 48 hours and then decreased slightly, yet remained higher than in normal skin that the burn wounds levels of the skin as time the and time there were with more tissue necrosis, and more than at the time point. Masson staining a indicator of burn wound and the of the the tissue necrosis of of the burn wound injury found that blood as measured by LDF decreased to one-fourth of normal level over the hours after injury and then began to but remained (p < 0.01) the normal level. The MPO activity about over the 48 hours after injury and then decreased slightly, but still remained higher (p < 0.01) than in normal tissue with the determined by Western blot assay, cells LC3 in wound were decreased as to controls not we found LC3-positive cells in the deep dermal layer to the which in this deep second-degree burn model may the of were more than that of controls. In addition, we found that LC3-positive cells were observed in the of hair follicles not analysis significant increases in LC3-positive cells in the deep dermis at 24 hours (p < 48 hours (p < and 72 hours (p < 0.01) postburn Burn wound progression to with a and that burn wounds were by local edema, free radical damage, accumulation of cell and all of which to wound factors are all thought to play an important in burn wound progression, and have with to control burn wound through that these 20 have on the of apoptosis and necrosis in burn wound progression. has been that the of cell apoptosis in deep burn wounds is higher than in normal skin of burn which that apoptosis may be involved in burn wound of cell death in burn wounds dermal tissue loss and wound healing.6, 7 apoptosis an important in cell and in against and in the of However, in of cellular apoptosis was found to damage, apoptosis that the increase of apoptosis the within the deep dermal of burn wounds and in the burn wound progression. Autophagy is a highly conserved pathway that delivers intracellular macromolecule waste to lysosomes, where they are degraded into biologically active monomers, such as amino acids, that are subsequently reused to maintain cellular metabolic turnover and Autophagy an to against including and in the process of autophagy have been to and of autophagic activity has been shown to against the process of and against many are few the relationship between autophagy and burn wound progression. is known that autophagy roles in cell and cell During autophagy is and has a but several autophagy and, distinct cell death be is not be to whether autophagy a prosurvival in the of burn injury progression. In the autophagy level was at and then yet remained that in normal skin. that at the early of burn wound progression the number of cells autophagy is as cellular necrosis the the the tissue necrosis the tissue to was to and and then autophagy to against these as a which for the in the levels of The prosurvival of autophagy is by IHC staining that autophagy in the deep dermis, which in this burn model the of In addition, at the to 72 hours after burn tissue was and inflammation was in with the levels of autophagy, which the prosurvival of autophagy in this burn model. is important to that the of blood flow obtained using LDF in this study are not of this and may be by different other Since the and were used for both the of blood flow between groups is The data obtained be used to between groups and not as point Apoptosis with autophagy in many different ways, which are as as roles in and that autophagy is a for cell that autophagy against apoptosis and In this the determined by TUNEL assay in burn wounds at and then after 48 hours which is to the changes in the levels of may the roles of autophagy and apoptosis in this model of burn wound progression. However, further study is still needed to these and the cellular and mechanisms involved in this process to be study is by small and the study is also to rats and be to was no to the study with endpoints such as and scarring. study is still needed to enhanced decreased autophagy, through apoptosis burn wound progression, and wound study levels of autophagy and blood flow with levels of inflammation and apoptosis during the early course of burn wound progression in a rodent model. that autophagy and apoptosis play roles in the process of burn progression. Enhanced autophagy in the deep dermal may as a prosurvival mechanism against inflammation and