Adenosine diphosphate ribosylation factor 6 inhibition protects burn sepsis induced lung injury through preserving vascular integrity and suppressing ASC inflammasome transmission.

BACKGROUND: Severe burns are devastating injuries with significant immune dysfunction and result in substantial mortality and morbidity due to sepsis induced organ failure. Acute lung injury is the most common type of organ injury in sepsis, however, the mechanisms of which are poorly understood and effective therapeutic measures are limited. This study is aimed to investigate the effect of a small Guanosine triphosphatase (GTPase), Adenosine diphosphate ribosylation factor 6 (ARF6), on burn sepsis induced lung injury, and discuss the possible mechanisms. METHODS: Burn sepsis was established in male C57BL/6 mice. Mice were anesthetised by intramuscular injection of ketamine and xylazine hydrochloride, then 30% TBSA full thickness burn followed by sub-eschar injection of lipopolysaccharide. Animals were treated with intraperitoneal injection of a small molecule inhibitor of ARF6: NAV-2729, or vehicle, right after the burn and sepsis stimuli were inflicted. Lung tissues were harvested for histopathological observation and the acute lung injury scores were calculated. Organ permeability, Vascular Endothelial Cadherin (VE-cadherin) expression, inflammatory cytokine levels and myeloperoxidase activity in lung tissues were detected. Rat pulmonary microvascular endothelial cells (PMVECs) were stimulated by burn sepsis serum with or without 10 µM NAV-2729. The ARF6 activation, VE-cadherin expression, inflammasome activity, adapter protein apoptosis speck-like protein containing a caspase recruiting domain (ASC) specks and cytokines secretion were determined. Student's t test was used for comparison between two groups. Multiple comparisons among groups were performed by using analysis of variance, with Tukey's test for the post hoc test. RESULTS: NAV-2729 treatment attenuated burn sepsis induced lung injury and promoted survival of burn septic mice by preserving VE-cadherin expression in endothelial cell adherent junction and limited vascular hyperpermeability in lung tissues. Moreover, inflammatory cytokine expression and inflammatory injury in lung tissues were alleviated. Mechanistically, NAV-2729 enhanced vascular integrity by inhibiting ARF6 activation and restoring VE-cadherin expression in PMVECs. In addition, NAV-2729 inhibited ARF6-dependent phagocytosis of ASC specks, thus preventing inflammation propagation mediated by cell-to-cell transmission of ASC specks. CONCLUSIONS: ARF6 inhibition preserved vascular integrity by restoring expression of VE-cadherin and suppressed the spread of inflammation by affecting phagocytosis of ASC specks, thus protected against sepsis induced lung injury and improve survival of burn septic animals. The findings of this study implied potential therapeutics by which ARF6 inhibition can protect lung function from septic induced lung injury and improve outcomes in burn sepsis.

Simulated aeromedical evacuation exacerbates burn induced lung injury: targeting mitochondrial DNA for reversal.

BACKGROUND: Aeromedical evacuation of patients with burn trauma is an important transport method in times of peace and war, during which patients are exposed to prolonged periods of hypobaric hypoxia; however, the effects of such exposure on burn injuries, particularly on burn-induced lung injuries, are largely unexplored. This study aimed to determine the effects of hypobaric hypoxia on burn-induced lung injuries and to investigate the underlying mechanism using a rat burn model. METHODS: A total of 40 male Wistar rats were randomly divided into four groups (10 in each group): sham burn (SB) group, burn in normoxia condition (BN) group, burn in hypoxia condition (BH) group, and burn in hypoxia condition with treatment intervention (BHD) group. Rats with 30% total body surface area burns were exposed to hypobaric hypoxia (2000 m altitude simulation) or normoxia conditions for 4 h. Deoxyribonuclease I (DNase I) was administered systemically as a treatment intervention. Systemic inflammatory mediator and mitochondrial deoxyribonucleic acid (mtDNA) levels were determined. A histopathological evaluation was performed and the acute lung injury (ALI) score was determined. Malonaldehyde (MDA) content, myeloperoxidase (MPO) activity, and the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome level were determined in lung tissues. Data among groups were compared using analysis of variance followed by Tukey's test post hoc analysis. RESULTS: Burns resulted in a remarkably higher level of systemic inflammatory cytokines and mtDNA release, which was further heightened by hypobaric hypoxia exposure (P < 0.01). Moreover, hypobaric hypoxia exposure gave rise to increased NLRP3 inflammasome expression, MDA content, and MPO activity in the lung (P < 0.05 or P < 0.01). Burn-induced lung injuries were exacerbated, as shown by the histopathological evaluation and ALI score (P < 0.01). Administration of DNase I markedly reduced mtDNA release and systemic inflammatory cytokine production. Furthermore, the NLRP3 inflammasome level in lung tissues was decreased and burn-induced lung injury was ameliorated (P < 0.01). CONCLUSIONS: Our results suggested that simulated aeromedical evacuation further increased burn-induced mtDNA release and exacerbated burn-induced inflammation and lung injury. DNase I reduced the release of mtDNA, limited mtDNA-induced systemic inflammation, and ameliorated burn-induced ALI. The intervening mtDNA level is thus a potential target to protect from burn-induced lung injury during aeromedical conditions and provides safer air evacuations for severely burned patients.

Long non-coding RNA H19 promotes the proliferation, migration and invasion while inhibits apoptosis of hypertrophic scarring fibroblasts by targeting miR-3187-3p/GAB1 axis.

BACKGROUND: It had been reported that long non-coding RNA (lncRNA) H19 was associated with the proliferation of fibroblasts. However, the regulatory mechanism of H19 remains unclear. Thus, the study was designed to explore the underlying mechanism of H19 in the process of Hypertrophic scarring (HS). METHODS: The expression levels of H19, miR-3187-3p, and growth factor receptor binding 2-associated binding protein 1 (GAB1) in HS tissues and HS fibroblasts were measured by real-time quantitative polymerase chain reaction (RT-qPCR) assay. The biological behaviors of HS fibroblasts, such as cell proliferation, apoptosis, migration, and invasion were assessed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide (MTT), colony formation, flow cytometry, and transwell assays, respectively. The protein expression level was quantified by western blot assay. The interaction association between miR-3187-3p and H19 or GAB1 was predicted by Starbase database analysis and confirmed by dual-luciferase reporter assay, respectively. RESULTS: H19 was significantly increased in HS tissues and HS fibroblasts. Loss-of-functional experiments revealed that knockdown of H19 inhibited the development of HS. Moreover, silencing of H19 impeded the proliferation, migration, and invasion, while enhanced apoptosis of HS fibroblasts by increasing miR-3187-3p expression. In addition, overexpression of GAB1 could abolish miR-3187-3p overexpression-induced effects on cell proliferation, apoptosis, migration, and invasion of HS fibroblasts. Mechanistically, H19 could act as a sponge of miR-3187-3p to upregulate the expression of GAB1 in HS fibroblasts. CONCLUSION: Collectively, our results revealed that H19 promoted the proliferation, migration, and invasion, while impeded apoptosis of HS fibroblasts by targeting miR-3187-3p/GAB1 axis.

miR-628 Promotes Burn-Induced Skeletal Muscle Atrophy via Targeting IRS1.

Skeletal muscle atrophy is a common clinical feature among patients with severe burns. Previous studies have shown that miRNAs play critical roles in the regulation of stress-induced skeletal muscle atrophy. Our previous study showed that burn-induced skeletal muscle atrophy is mediated by miR-628. In this study, compared with sham rats, rats subjected to burn injury exhibited skeletal muscle atrophy, as well as significantly decreased insulin receptor substrate 1 (IRS1) protein expression and significantly increased skeletal muscle cell apoptosis. An miRNA array showed that the levels of miR-628, a potential regulator of IRS1 protein translation, were also clearly elevated. Second, L6 myocyte cell apoptosis increased after induction of miR-628 expression, and IRS1 and p-Akt protein expression decreased significantly. Expression of the cell apoptosis-related proteins FoxO3a and cleaved caspase 3 also increased after induction of miR-628 expression. Finally, forced miR-628 expression in normal rats resulted in increased cell apoptosis and skeletal muscle atrophy, as well as changes in IRS1/Akt/FoxO3a signaling pathway activity consistent with the changes in protein expression described above. Inhibiting cell apoptosis with Z-VAD-FMK resulted in alleviation of burn-induced skeletal muscle atrophy. In general, our results indicate that miR-628 mediates burn-induced skeletal muscle atrophy by regulating the IRS1/Akt/FoxO3a signaling pathway.

TSG-6 secreted by human umbilical cord-MSCs attenuates severe burn-induced excessive inflammation via inhibiting activations of P38 and JNK signaling.

The hMSCs have become a promising approach for inflammation treatment in acute phase. Our previous study has demonstrated that human umbilical cord-MSCs could alleviate the inflammatory reaction of severely burned wound. In this study, we further investigated the potential role and mechanism of the MSCs on severe burn-induced excessive inflammation. Wistar rats were randomly divided into following groups: Sham, Burn, Burn+MSCs, Burn+MAPKs inhibitors, and Burn, Burn+MSCs, Burn+Vehicle, Burn+siTSG-6, Burn+rhTSG-6 in the both experiments. It was found that MSCs could only down-regulate P38 and JNK signaling, but had no effect on ERK in peritoneal macrophages of severe burn rats. Furthermore, suppression of P38 and JNK activations significantly reduced the excessive inflammation induced by severe burn. TSG-6 was secreted by MSCs using different inflammatory mediators. TSG-6 from MSCs and recombinant human (rh)TSG-6 all significantly reduced activations of P38 and JNK signaling induced by severe burn and then attenuated excessive inflammations. On the contrary, knockdown TSG-6 in the cells significantly increased phosphorylation of P38 and JNK signaling and reduced therapeutic effect of the MSCs on excessive inflammation. Taken together, this study suggested TSG-6 from MSCs attenuated severe burn-induced excessive inflammation via inhibiting activation of P38 and JNK signaling.

3,4-Methylenedioxy-ß-Nitrostyrene Ameliorates Experimental Burn Wound Progression by Inhibiting the NLRP3 Inflammasome Activation.

BACKGROUND: Burn wound progression remains a challenging problem in the clinic. Secondary tissue damage caused by unlimited inflammatory response is considered to be one of the key factors contributing to this clinical problem. Nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome has recently been found to play important roles in immune activation and the inflammatory response after burn/trauma. This experimental study aims (1) to observe the expression and distribution of NLRP3 inflammasome in burn wounds of a rat burn model and (2) to study whether inhibiting the NLRP3 inflammasome activation would ameliorate burn wound progression. METHODS: A deep second-degree burn was inflicted on the backs of Wistar rats. The expression of NLRP3 inflammasome components and interleukin-1ß were determined by Western blot and coimmunoprecipitation. The distribution of NLRP3 inflammasome was assessed by immunohistochemical staining and double-labeling immunofluorescence. Neutrophil infiltration, wound perfusion, burn depth, and wound healing time were assessed. RESULTS: Burn induced remarkable NLRP3 inflammasome activation and cleavage of interleukin-1ß. The NLRP3 inflammasome was observed mainly in macrophages of the zone of stasis. 3,4-Methylenedioxy-ß-nitrostyrene significantly inhibited NLRP3 inflammasome activation and inflammatory cytokine production in burn wounds. Consequently, neutrophil infiltration was reduced, wound perfusion was restored, burn wound progression was ameliorated, and wound healing was accelerated. CONCLUSIONS: In this study, the authors demonstrated that burn induced NLRP3 inflammasome activation and inflammatory response in wounds, which may be associated with burn wound progression. Treatment with 3,4-methylenedioxy-ß-nitrostyrene inhibited NLRP3 inflammasome activation, ameliorated burn wound progression, and promoted wound healing.

[Research progress on autophagy regulating excessive inflammation].

Autophagy is a highly conserved cellular self-digestion pathway, by which intracellular damaged proteins or organelles are delivered to lysosomes for degradation, so as to protect from various dangerous stimuli and maintain cellular homeostasis. Inflammation is a defensive response to injury or pathogens, through which various inflammatory mediators coordinate host defense and repair. However, uncontrolled inflammatory responses can lead to secondary damage and pathogenesis of inflammatory disease. Recent studies indicate that autophagy pathway and related proteins may play important roles in regulating immune response and controlling excessive inflammation. This review introduced research progress in the role of autophagy in regulating excessive inflammation and possible mechanisms.

Role of autophagy and apoptosis in wound tissue of deep second-degree burn in rats.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Rapamycin reduces burn wound progression by enhancing autophagy in deep second-degree burn in rats.

Burn wound progression is caused by many mechanisms including local tissue hypoperfusion, prolonged inflammation, free radical damage, apoptosis, and necrosis in burn wounds. Autophagy, a homeostatic process by which cells break down their own components, was found to protect against ischemic injury, inflammatory diseases, and apoptosis in some cases. We tested whether rapamycin, an autophagy inducer, could ameliorate burn wound progression and promote wound healing through autophagy enhancement. Using a previously described deep second-degree burn model, we first tested the effects of rapamycin on autophagic response in burn wound tissue. Autophagy levels in wound tissue of treated rats were increased as compared with controls. Furthermore, we found that laser Doppler flowmetry values and Na/K-ATPase activities were markedly higher in the treated wounds. The content of interleukin-8, methane dicarboxylic aldehyde, and myeloperoxidase activity in the wounds of treated rats were much lower than in controls. The apoptotic rates in treated wounds were much lower than controls as determined by terminal deoxynucleotidyl transferase mediated nick end labeling assay. Finally, histomorphological analysis showed that burn wound progression in the treatment group was ameliorated. The time to wound reepithelialization was shorter in the treated wounds than controls 22.5 ± 1.4 days vs. 24.8 ± 1.3 days (mean ± standard deviation, p < 0.01).