Computational Drug Discovery in Diaphragm dysfunction via Text Mining and Biomedical Database.

The diaphragm, which is crucial for ventilation, is the primary muscle responsible for inspiration. Patients with severe burns who experience diaphragmatic dysfunction have an increased risk of mortality. Unfortunately, there are currently no effective medications available to prevent or treat this condition. The objective of our study is to utilize bioinformatics to identify potential genes and drugs associated with diaphragmatic dysfunction. In this study, text mining techniques were utilized to identify genes associated with diaphragmatic dysfunction and recovery. Common genes were then analyzed using GO and KEGG pathway analysis, as well as protein-protein interaction (PPI) network analysis. The obtained hub genes were processed using Cytoscape software, and their expression levels in diaphragmatic dysfunction were validated using quantitative real-time polymerase chain reaction (qRT-PCR) in severe burn rats. Genes that were confirmed were then examined in drug-gene interaction databases to identify potential drugs associated with these genes. Our analysis revealed 96 genes that were common to both the "Diaphragm dysfunction" and "Functional Recovery" text mining concepts. Gene enrichment analysis identified 19 genes representing ten pathways. qRT-PCR showed a significant increase in expression levels of 13 genes, including CCL2, CCL3, CD4, EGF, HGF, IFNG, IGF1, IL17A, IL6, LEP, PTGS2, TGFB1, and TNF, in samples with diaphragmatic dysfunction. Additionally, we found that a total of 56 drugs targeted 5 potential genes. These findings provide new insights into the development of more effective drugs for treating diaphragmatic dysfunction, and also present substantial opportunities for researching new target pharmacology and promoting drugs in the pharmaceutical industry.

[New research direction of organ dysfunction caused by hemorrhagic shock: mechanisms of mitochondrial quality control].

Hemorrhagic shock (HS) is one of the leading causes of death among young adults worldwide. Multiple organ dysfunction in HS is caused by an imbalance between tissue oxygen supply and demand, which is closely related to the poor prognosis of patient. Mitochondrial dysfunction is one of the key mechanisms contributing to multiple organ dysfunction in HS, while mitochondrial quality control regulates mitochondrial function through a series of processes, including mitochondrial biogenesis, mitochondrial dynamics, mitophagy, mitochondrial-derived vesicles, and mitochondrial protein homeostasis. Modulating mitochondrial quality control can improve organ dysfunction. This review aims to summarize the effects of mitochondrial dysfunction on organ function in HS and discuss the potential mechanisms of mitochondrial quality control, providing insights into the injury mechanisms underlying HS and guiding clinical management.

Respiratory Muscle Contraction Characteristics and Potential Mechanisms in Severely Burned Rats.

Post-burn hypermetabolism remains an important clinical problem. During this phase, there is a significant loss of diaphragmatic proteins. Better understanding of respiratory muscle dynamics and potential mechanisms affecting respiratory muscle function is necessary for the development of effective therapeutic approaches. Male wistar rats were subjected to 50% total body surface area (TBSA) burns and sham injuries, and respiratory muscle function was assessed with 0, 1, 4, 7, and 14 d post-injury, including Pulmonary function, blood gas analysis, transdiaphragmatic pressure, diaphragm ultrasonography, isolated diaphragm contractility, and fatigue index. protein oxidative stress content, and ATP levels. Burned Rats had significantly reduced inspiratory time, expiratory time and tidal volume, and significantly increased respiratory rate and minute ventilation. At the same time, the isolated diaphragm contractility, specific force during fatigue, and fatigue index were significantly decreased in the burned rats. Pdi, Pdimax, diaphragm thickness, diaphragm thickening fraction, and diaphragm excursion also decreased significantly post-burn, whereas the Pdi/Pdimax ratio increased significantly. Finally, the content of protein carbonyls and lactic acid of burned rats were increased, and ATP levels of burned rats was decreased. The present study demonstrates the dynamic changes in diaphragm contractile properties post-burn from both in vivo and in vitro perspectives, while cursorily exploring the possibility that protein oxidative stress and reduced ATP production may be the cause of diaphragm dysfunction. This understanding contributes to the development of methods to mitigate the extent of diaphragmatic function loss after severe burns.

Epidemiological and Clinical Characteristics of 471 Elderly Burn Patients in China: A Burn Center-based Study.

Burns are characterized by difficult treatment, poor prognosis, and high mortality especially in elderly patients. The aim of this paper is to study the epidemiological and clinical characteristics of elderly burn patients admitted to a major burn center from 2016 to 2020. The data of 471 elderly burn patients admitted to our burn center from January 2016 to December 2020 were retrospectively analyzed. Demographic and clinical variables of different age groups were statistically analyzed, and variables related to length of stay (LOS) and hospitalization costs were analyzed through linear regression models. The mean age of included patients was 69.03 years. Scald injuries accounted for 42.7% of all burns with the extremities being the most commonly affected anatomic site (46.5%). Approximately 67.5% and 5.9% of all patients suffered from full-thickness burns and inhalation injuries, respectively. These patients required more surgical procedures, longer operation durations, and higher costs compared with other burn patients. The mean LOS was 17 days with a mortality rate of 2.1%. There were statistically significant differences in the etiological characteristics and clinical manifestations of burn patients in different age groups. Individualized targeted prevention and treatment strategies should be performed according to the clinical characteristics and relevant risk factors of each patient.

Establishment of a Bama miniature pig burn model with different burn depths.

Background: The skin morphological characteristics of the Bama miniature pig are very similar to those of humans; thus, the Bama miniature pig is an ideal choice for establishing a skin burn model. Methods: In this study, 6 ordinary, male, Bama miniature pigs (weight: 23-28 kg and length: 71-75 cm) were used to establish burn models. A mixture of 1 mg of Ketamine and Sumianxin II was used for Bama miniature pigs anesthetizing, and 1 mg of Pentobarbital sodium was added as necessary. The different burn depths were made using a continuous pressure of 1 kg and contact times of 0 s, 10 s, 15 s, 20 s, 25 s, 30 s, 35 s, 40 s, and 45 s by the newly invented electronic burn instrument. The burned tissues were collected and examined with hematoxylin and eosin (H&E) and Masson staining. Results: Burning for 10-15 s caused a first-degree burn; the blood vessels in the superficial dermis were dilated and congested, and necrosis occurred above the basal layer of the epidermis. Burning for 20-25 s caused a superficial partial-thickness burn; the whole epidermal layer was necrotic, and the collagen fibers were slightly deformed. Burning for 30-35 s caused a deep partial-thickness burn; the whole epidermal layer and dermal layers were necrotic with leukocyte infiltration zones, and the collagen fibers were disordered, degenerated, and necrotized. Burning for 40-45 s caused a third-degree burn; the skin layers and adipose tissues were necrotic, and the thick blood vessels in the skin adipose tissues were full of disintegrated and agglutinated red blood cells. Conclusions: Stable burn depth models of Bama miniature pigs were constructed using a new and innovative electronic burn instrument. Our findings provide a basis for further research on the burn mechanism and evaluations of therapeutic drugs.

Vacuum sealing drainage with instillation in the treatment of necrotising soft-tissue infection: a retrospective analysis.

OBJECTIVE: Necrotising soft-tissue infection is a rare but life-threatening infectious disease with high morbidity and mortality. It is typically caused by toxin-producing bacteria and characterised clinically by a very rapid progression of the disease with significant local tissue destruction. In this study, we intend to explore effective wound management to control the invasive infection and to decrease the high mortality. METHOD: This retrospective analysis explored the wound management and mortality in patients with necrotising soft-tissue infection. Extensive debridement, vacuum sealing drainage (VSD) with normal saline instillation combined with broad-spectrum or sensitive antibiotics, and supportive therapies were used. RESULTS: All 17 patients included in the analysis survived. The microbiology of 11 patients was found to be polymicrobial. Of the patients, 14 were discharged with completely healed wounds and three were transferred to a local hospital after the systemic and invasive wound infection was controlled. CONCLUSION: Our experiences revealed the outstanding effect of VSD with instillation in removing the debris of necrotising tissue on the wound bed, in the continual and complete drainage of wound exudates, and in prompting wound healing.

Down-Regulation of miR-301a-3p Reduces Burn-Induced Vascular Endothelial Apoptosis by potentiating hMSC-Secreted IGF-1 and PI3K/Akt/FOXO3a Pathway.

Vascular endothelium dysfunction plays a pivotal role in the initiation and progression of multiple organ dysfunction. The mesenchymal stem cell (MSC) maintains vascular endothelial barrier survival via secreting bioactive factors. However, the mechanism of human umbilical cord MSC (hMSC) in protecting endothelial survival remains unclear. Here, we found IGF-1 secreted by hMSC suppressed severe burn-induced apoptosis of human umbilical vein endothelial cells (HUVECs) and alleviated the dysfunction of vascular endothelial barrier and multiple organs in severely burned rats. Severe burn repressed miR-301a-3p expression, which directly regulated IGF-1 synthesis and secretion in hMSC. Down-regulation of miR-301a-3p decreased HUVECs apoptosis, stabilized endothelial barrier permeability, and subsequently protected against multiple organ dysfunction in vivo. Additionally, miR-301a-3p negatively regulated PI3K/Akt/FOXO3 signaling through IGF-1. Taken together, our study highlights the protective function of IGF-1 against the dysfunction of multiple organs negatively regulated by miR-301a-3p, which may provide the theoretical foundation for further clinical application of hMSC.

Is Mitochondrial Oxidative Stress the Key Contributor to Diaphragm Atrophy and Dysfunction in Critically Ill Patients?

Diaphragm dysfunction is prevalent in the progress of respiratory dysfunction in various critical illnesses. Respiratory muscle weakness may result in insufficient ventilation, coughing reflection suppression, pulmonary infection, and difficulty in weaning off respirators. All of these further induce respiratory dysfunction and even threaten the patients' survival. The potential mechanisms of diaphragm atrophy and dysfunction include impairment of myofiber protein anabolism, enhancement of myofiber protein degradation, release of inflammatory mediators, imbalance of metabolic hormones, myonuclear apoptosis, autophagy, and oxidative stress. Among these contributors, mitochondrial oxidative stress is strongly implicated to play a key role in the process as it modulates diaphragm protein synthesis and degradation, induces protein oxidation and functional alteration, enhances apoptosis and autophagy, reduces mitochondrial energy supply, and is regulated by inflammatory cytokines via related signaling molecules. This review aims to provide a concise overview of pathological mechanisms of diaphragmatic dysfunction in critically ill patients, with special emphasis on the role and modulating mechanisms of mitochondrial oxidative stress.

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.

Apoptosis and death receptor signaling in diaphragm of burnt rats.

BACKGROUND: Respiratory dysfunction is a frequent complication after severe burn injury. Respiratory muscle atrophy may induce respiratory dysfunction due to insufficient inspiratory motive power. Accumulated evidence suggests that apoptosis is very important in skeletal muscle atrophy in multiple pathologic conditions. Therefore, we hypothesize that myonuclear apoptosis contributes to diaphragm atrophy induced by burn injury, and death receptor signaling activation plays a role in this process. METHODS: Wistar rats in the burn-injured group were subjected to a full-thickness scald injury around 40% of total body surface area. Diaphragm samples were examined for myonuclear apoptosis by transmission electron microscope, terminal deoxynucleotidyl transferase-mediated nick end labeling assay, and immunohistochemistry for caspase-3. Serum level of apoptotic ligands were assessed by ELISA. Activation of death receptor signaling was examined by Western blotting. RESULTS: Burn injury resulted in significant reductions of diaphragm muscle mass and myofiber cross-section area. Apoptosis in diaphragm appeared from day 1 and peaked on day 4 after injury. The level of soluble TNF-related apoptosis-inducing ligand and the ratio of Fas ligand to soluble Fas in serum significantly increased after burn injury. In diaphragm of burnt animals, the expressions of proapoptotic proteins, such as cleaved caspase-8, cleaved caspase-3, and Bax-to-Bcl-2 ratio were upregulated, whereas expression of pAkt, an antiapoptotic protein, was downregulated. Immunohistochemistry revealed that the most of the caspase-3 was expressed in myofiber nuclei and their surrounding cytoplasm area in tissue sections. CONCLUSIONS: Severe burn injury induces myonuclear apoptosis in diaphragm, which could be a contributor to diaphragm muscle atrophy. Activation of death receptor signaling may be a mechanism of apoptosis in diaphragm.

Autocrine interferon-γ may affect malignant behavior and sensitivity to tamoxifen of MCF-7 via estrogen receptor ß subtype.

Mitogenic actions of estrogens are mediated by two distinct estrogen receptors (ERs), which are critical in the progression and therapeutic response of breast cancer. ER expression is a dynamic phenomenon that is regulated by numerous factors, including cytokines, in the tumor microenvironment. Recently, studies have shown that autocrine production of IL-4 promotes cancer cell growth and there is negative correlation between tumor IL-4 and hormone receptor levels, suggesting that there is crosstalk between cytokine receptors and ER. Thus, we evaluated for interaction between the two ERs and the cytokines IL-4 and IFN-γ, and if this interaction modulates malignant behavior. We identified that ERß exerts protective activity in the progression of breast cancer cell line MCF-7, which co-expresses ERα and ERß. IFN-γ and IL-4 have the opposite effects on malignant biological behavior. Furthermore, we found positive correlation between IFN-γ and ERß expression in MCF-7. We also determined that autocrine IFN-γ in MCF-7 increases mRNA expression of ERß resulting in enhanced sensitivity to tamoxifen (TAM). These results indicate that ERß and autocrine IFN-γ represent two putative targets for breast cancer therapy.

Comparison of systemic inflammation response and vital organ damage induced by severe burns in different area.

BACKGROUND: In this study, we will establish a stable and optimized rat model that can meet strictly diagnosed criteria and serve as a tool to investigate the potential of novel therapeutics in this preclinical model through comparative analysis of systemic alterations, levels of pro-inflammatory cytokines in serum and infiltrated numbers of inflammatory cells in distant organ between 30% and 50% TBSA with a full-thickness burn. MATERIALS AND METHODS: The adult male Wistar rats were randomly divided into the following groups: control group, 30% TBSA with a full-thickness burn group, and 50% TBSA with a full-thickness burn group. The blood and serum samples in the 3 groups were collected and detected by blood routine examination and biochemical detection at 6 h, 12 h, 24 h and 48 h post burn. The levels of TNF-α, IL-1ß and IL-6 in serum were detected by ELISA. The sections of lung, renal, liver and heart were analyzed by H&E and immunohistochemical staining detection. RESULTS: Our results showed that temperature in 50% TBSA with a full-thickness burn group was always hypothermia, and lower than 36°C at defined timepoints post burn, that was in 30% TBSA with a full-thickness burn group was lower than 36°C only at 48 h post burn. The levels of TNF-α, IL-1ß and IL-6 were significantly increased in 30% and 50% groups at 6 h, 12 h, 24 h and 48 h post burn. The apoptosis in distant organs and the biochemical parameters such as ALT, AST, troponin, CK, CK-MB, LDH, urea and creatinine in 30% and 50% groups were also increased at different degrees at defined timepoints after burn, but changes in 50% group were more obvious than that in 30% group. CONCLUSION: We choose 50% TBSA with a full-thickness burn to establish a stable and optimized rat model that can meet strictly diagnosed criteria and serve as a tool to investigate the potential of novel therapeutics in this preclinical model.

Development of an Animal Model for Burn-Blast Combined Injury and Cardiopulmonary System Changes in the Early Shock Stage.

The purposes of this study were to establish an animal model for burn-blast combined injury research and elaborate cardiopulmonary system changes in the early shock stage. In this study, royal demolition explosive or RDX (hexagon, ring trimethylene nitramine) was used as an explosive source, and the injury conditions of the canine test subjects at various distances to the explosion (30, 50, and 70 cm) were observed by gross anatomy and pathology to determine a larger animal model of moderate blast injury. The canines were then subjected to a 35 % total body surface area (TBSA) full-thickness flame injury using napalm, which completed the development of a burn-blast combined injury model. Based on this model, the hemodynamic changes and arterial blood gas analysis after the burn-blast combined injury were measured to identify the cardiopulmonary system characteristics. In this research, RDX explosion and flame injury were used to develop a severe burn-blast injury animal model that was stable, close to reality, and easily controllable. The hemodynamic and arterial blood gas changes in the canine subjects after burn-blast injury changed distinctly from the burn and blast injuries. Blood pressure and cardiac output fluctuated, and the preload was significantly reduced, whereas the afterload significantly increased. Meanwhile, the oxygen saturation (SO2) decreased markedly with carbon dioxide partial pressure (PCO2), and lactic acid (Lac) rose, and oxygen partial pressure (PO2) reduced. These changes suggested that immediate clinical treatment is important during burn-blast injury both to stabilize cardiac function and supply blood volume and to reduce the vascular permeability, thereby preventing acute pneumonedema or other complications.

[Advances in the research on the relationship between brown adipose tissue and metabolism in burn and trauma].

Hypermetabolism and insulin resistance are prominent features of trauma including burn injury, surgery, and infection. Hypermetabolism results in insufficiency in energy supply, which induces organ function lesion, immune suppression, high infection rate, and wound healing delay, thus exerting a strong impact on patients' quality of life and prognosis. The molecular mechanism in the occurrence and development of hypermetabolism is very complicated, and it has not been fully elucidated. Recently, brown adipose tissue (BAT) was found to be present not only in rodents but also in humans, and its activity was associated with resting metabolic rate. BAT may become the new target of research in prevention and control of metabolic disorder.

miR-194 Promotes burn-induced hyperglycemia via attenuating IGF-IR expression.

Hyperglycemia is one of the most important clinical features of burn patients. Previous reports had demonstrated that miRNA was involved in regulating glucose metabolism in various diseases such as diabetes and obesity. Our current study discovered the relationship between miR-194 and hyperglycemia in burn rats via suppressing insulin-like growth factor 1 receptor (IGF-IR). We found that the fasting blood glucose was significantly increased in rats of the burn group, and protein expression of IGF-IR was attenuated in response to burn injury. Similar to the results of animal experiments, miR-194 expression was significantly elevated and IGF-IR protein level was suppressed in L6 cells treated with serum from burn rats compared with those treated by serum from sham rats. However, IGF-IR mRNA level was comparable between burn and sham rats, suggesting that IGF-IR may be downregulated at the translation level. Further experiments revealed that miR-194 was significantly increased in burn rats compared with sham rats using miRNA array and real-time polymerase chain reaction (PCR) assay. And IGF-IR protein expression was reduced in L6 cells transfected with miR-194 plasmid. Insulin-like growth factor 1 receptor expression was also repressed and fasting blood glucose was increased in rats injected with miR-194 plasmid. In general, we have identified a novel function of miR-194 in modulating burn-induced hyperglycemia via suppressing the expression of IGF-IR.

[Effects of endotoxin/lipopolysaccharide on proliferation and apoptosis of human umbilical cord mesenchymal stem cells].

OBJECTIVE: To investigate the effects of different concentrations of lipopolysaccharide (LPS) on proliferation and apoptosis of human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro, and to explore their possible mechanism. METHODS: hUCMSCs from umbilical cord tissue of full-term healthy fetus delivered by caesarean section were isolated and cultured in vitro using tissue attachment method. The 3rd passage hUCMSCs were used in the study. Cells were divided into groups A, B, C, D, and E, which were treated with DMEM/F12 medium containing 0, 0.1, 1.0, 10.0, and 100.0 µg/mL of LPS respectively. In groups B, C, D, and E, methyl-thiazole-tetrazolium assay was used to detect proliferative activity of hUCMSCs at post treatment hour (PTH) 12, 24, and 48 (denoted as absorption value), with 5 samples in each group at each time point; apoptosis of hUCMSCs at PBH 24 was identified with acridine orange-ethidium bromide (AO-EB) staining, with 4 samples in each group; apoptotic rate of hUCMSCs was determined by flow cytometer, with 5 samples in each group. Above-mentioned indexes were determined in group A at the same time points. Data were processed with analysis of variance and LSD- t test. RESULTS: (1) There was no statistically significant difference in proliferative activity of hUCMSCs at PTH 12 among groups A, B, C, D, and E (with t values from -1.67 to 1.33, P values above 0.05). Compared with that of group A, proliferative activity of hUCMSCs was increased in groups B, C, and D at PTH 24 and 48 (with t values from -13.42 to 17.34, P < 0.05 or P < 0.01), especially so in group C. Proliferative activity of hUCMSCs was lower in group E at PTH 24 and 48 than in group A (with t values respectively 8.64 and 17.34, P values below 0.01). (2) Obvious apoptosis of hUCMSCs was observed in group E but not in the other 4 groups with AO-EB staining. (3) Apoptosis rates of hUCMSCs in groups A, B, C, D, and E were respectively (3.1 ± 0.6)%, (2.6 ± 0.7)%, (2.9 ± 0.8)%, (3.1 ± 0.4)%, (25.1 ± 2.7)% (F = 272.19, P < 0.01). Apoptotic rate of hUCMSCs in group B, C, or D was respectively close to that in group A (with t values respectively 1.22, 0.57, -0.14, P values above 0.05), but it was higher in group E than in group A (t = -17.63, P < 0.01). CONCLUSIONS: hUCMSCs proliferation may be promoted by low concentration of LPS. hUCMSCs proliferation is inhibited or induced to apoptosis along with the increase in concentration of LPS, and it may be related to activation of different major molecular signaling pathways by different concentrations of LPS.

[Effects of lipopolysaccharide pretreatment on endotoxin tolerance of human umbilical cord mesenchymal stem cells].

OBJECTIVE: To explore the effects of lipopolysaccharide (LPS) pretreatment on endotoxin tolerance of human umbilical cord mesenchymal stem cells (hUCMSCs) and its possible mechanism. METHODS: hUCMSCs (1×10(4) cells/well) were exposed to 0, 0.1, 1.0, 10.0, 20.0, 30.0, 40.0, 50.0 µg/ml LPS for 24 h respectively. And the cell viability of hUCMSCs was detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). 1 µg/ml and 50.0 µg/ml LPS were used as pretreatment and apoptosis induction concentrations respectively. Pyrrolidine dithiocarbamate (PDTC) (20 µmol/L, pretreatment for 20 min) was used as a specific inhibitor of nuclear transcription factor NF-κB. hUCMSCs were randomly divided by Stata software into 7 groups: control (A), LPS induction (B), pretreatment + LPS induction (C), PDTC (D), PDTC+ pretreatment + LPS induction (E), pretreatment (F) and PDTC + pretreatment (G). The apoptosis of hUCMSCs was measured by Hoechst 33258 staining and flow cytometry (FCM). The expressions of NF-κB p65 and cellular FLICE-inhibitory protein (c-FLIP) were measured by Western blot. RESULTS: The cell viability of 0, 0.1, 1.0, 10.0, 20.0, 30.0, 40.0, 50.0 µg/ml LPS groups were 100%, (117.0 ± 8.8)%, (134.7 ± 6.9)%, (105.3 ± 8.3)%, (99.2 ± 8.3)%, (84.2 ± 9.3)%, (66.4 ± 6.6)% and (59.2 ± 8.0)% respectively. In comparison with 0 µg/ml LPS group, the cell viability of 1.0 µg/ml LPS group increased significantly (P = 0.004) while decreased in 40 and 50 µg/ml LPS groups (P = 0.005, 0.002). Hoechst 33258 staining indicated that chromatin of hUCMSCs was distributed evenly in group A; the apoptotic cell in group B dramatically increased; and the apoptotic cell in group C significantly decreased in comparison with that in group B. Apoptotic rates of groups A, B, C, D and E were (2.8 ± 0.8)%, (29.7 ± 3.4)%, (17.8 ± 3.0)%, (2.9 ± 0.4)% and (23.2 ± 2.6)% respectively. Compared with group A, apoptosis rate significantly increased in group B (P < 0.001). The apoptotic rate in group C significantly decreased than that in group B (P < 0.001) while group E was higher than group C (P = 0.015). The levels of NF-κB p65 and c-FLIP in group F (0.851 ± 0.031, 0.534 ± 0.053) was higher than that in group A (0.220 ± 0.021, 0.049 ± 0.009) (both P < 0.001), G (0.418 ± 0.007, 0.299 ± 0.061) (P < 0.001, P = 0.007). CONCLUSIONS: LPS pretreatment can resist LPS-induced hUCMSCs apoptosis and enhance the ability of endotoxin tolerance. And the mechanism may be related with activating the NF-κB signaling pathway and up-regulating the expression of c-FLIP.

[Effect and mechanism of dantrolene on skeletal muscle of rats with severe scald injury].

OBJECTIVE: To explore the effect and mechanism of ryanodine receptor antagonist dantrolene on skeletal muscle of rats with severe scald injury. METHODS: A total of 56 Wistar rats were divided into control, scald and dantrolene treatment groups according to a random digital table. Rats in scald and dantrolene treatment groups were subject to 50% total body surface area (TBSA) full-thickness scald by a 12-second immersion of back and a 6-second immersion of abdomen in 94 °C water and then received an intraperitoneal injection of Ringer's solution. At the same time, the rats in scald group received 5% mannitol through caudal vein while those in dantrolene treatment group received dantrolene 2 mg/kg (dissolved in 5% mannitol). Rats in control group were sham-injured through an immersion of back and abdomen into 37 °C warm water. Tibialis anterior muscle samples were harvested at Days 1, 4 and 7 post-scalding. Changes of skeletal muscle ultrastructure were observed by transmission electron microscope, subcellular calcium ion (Ca(2+)) contents of skeletal muscle (including cytoplasm, mitochondria & sarcoplasm reticulum) were detected by electron probe X-ray microanalysis (EPMA) and the levels of calpain-1 and calpain-2 protein were determined by Western blot. And the activities of calpain were detected by enzyme-linked immunosorbent assay. RESULTS: In scald group, assorted arrangement appeared immediately at Day 1 post-injury and partial disappearance of Z lines at Day 7 post-injury. There were no significant ultrastructure changes in dantrolene treatment group at Day 1 and 4 post-injury. Curled filament and mild fracture occurred merely in dantrolene treatment group at Day 7 post-injury. The cytoplasmic contents of Ca(2+) were significantly higher in scald group than those in control group at Day 1 and 4 ((0.964 ± 0.060), (0.639 ± 0.067) vs (0.266 ± 0.029) µmol/L respectively, all P < 0.05) while the contents of Ca(2+) within sarcoplasm reticulum were obviously lower in scald group than those in control group at Day 1 and 4 ((0.368 ± 0.060), (0.814 ± 0.089) vs (1.337 ± 0.112) µmol/L respectively, all P < 0.05). However, those subcellular regions in dantrolene treatment group ((0.310 ± 0.069), (0.490 ± 0.039) and (1.241 ± 0.073), (1.161 ± 0.094) µmol/L) had no significant difference with control group (all P > 0.05). Calpain-1 and calpain-2 protein levels in scald group increased significantly at Day 1 and 4 post-injury versus control group (1.371 ± 0.034, 1.214 ± 0.030 vs 0.838 ± 0.017 & 1.464 ± 0.015, 1.390 ± 0.023 vs 0.806 ± 0.026 respectively, all P < 0.05), whereas calpain-1 and calpain-2 protein levels in dantrolene treatment (0.984 ± 0.031, 0.935 ± 0.023 and 0.836 ± 0.014, 0.741 ± 0.020) obviously were lower than those in scald group respectively (all P < 0.05). The activities of calpain in scald and dantrolene treatment groups at Day 1, 4 and 7 post-injury were (8.33 ± 0.21), (9.33 ± 0.21), (10.59 ± 0.18) and (7.76 ± 0.28), (7.86 ± 0.20), (7.91 ± 0.22) µmol/L respectively while the activity of calpain in control group was (7.62 ± 0.19) µmol/L. The activities of calpain in scald group were significantly higher than those in dantrolene treatment and control groups (all P < 0.05) whereas the activities of calpain in dantrolene treatment group had no obvious change versus control group (all P > 0.05). CONCLUSIONS: Dantrolene offers significant protection from skeletal muscle tissue damage and minimizes the ultrastructural change of tibialis anterior muscle induced by severe scald injury. The mechanism is probably through inhibiting an excessive release of Ca(2+) within sarcoplasm reticulum and down-regulated cytoplasmic expression and activity of calpain-1 and calpain-2.

Human umbilical cord mesenchymal stem cells transplantation promotes cutaneous wound healing of severe burned rats.

BACKGROUND: Severe burns are a common and highly lethal trauma. The key step for severe burn therapy is to promote the wound healing as early as possible, and reports indicate that mesenchymal stem cell (MSC) therapy contributes to facilitate wound healing. In this study, we investigated effect of human umbilical cord MSCs (hUC-MSCs) could on wound healing in a rat model of severe burn and its potential mechanism. METHODS: Adult male Wistar rats were randomly divided into sham, burn, and burn transplanted hUC-MSCs. GFP labeled hUC-MSCs or PBS was intravenous injected into respective groups. The rate of wound closure was evaluated by Image Pro Plus. GFP-labeled hUC-MSCs were tracked by in vivo bioluminescence imaging (BLI), and human-specific DNA expression in wounds was detected by PCR. Inflammatory cells, neutrophils, macrophages, capillaries and collagen types I/III in wounds were evaluated by histochemical staining. Wound blood flow was evaluated by laser Doppler blood flow meter. The levels of proinflammatory and anti-inflammatory factors, VEGF, collagen types I/III in wounds were analyzed using an ELISA. RESULTS: We found that wound healing was significantly accelerated in the hUC-MSC therapy group. The hUC-MSCs migrated into wound and remarkably decreased the quantity of infiltrated inflammatory cells and levels of IL-1, IL-6, TNF-α and increased levels of IL-10 and TSG-6 in wounds. Additionally, the neovascularization and levels of VEGF in wounds in the hUC-MSC therapy group were markedly higher than those in other control groups. The ratio of collagen types I and III in the hUC-MSC therapy group were markedly higher than that in the burn group at indicated time after transplantation. CONCLUSION: The study suggests that hUC-MSCs transplantation can effectively improve wound healing in severe burned rat model. Moreover, these data might provide the theoretical foundation for the further clinical application of hUC-MSC in burn areas.