Jiake Chai

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.

Role of ubiquitin-proteasome pathway in skeletal muscle wasting in rats with endotoxemia.

OBJECTIVE To investigate the mechanism of muscle protein breakdown under endotoxemia condition. DESIGN Randomized, controlled, animal experiment in a hospital institute. SETTING Experimental laboratory. INTERVENTION Either saline or endotoxin (Escherichia coli O(55)B(5), 10 mg/kg) were administered into the peritoneal cavity in rats. MEASUREMENTS AND MAIN RESULTS The rate of total protein breakdown was increased by 29% and 61% in extensor digitorum longus muscle at 2 hrs and 6 hrs, whereas the myofibrillar proteolytic rate was increased by 155%, 222%, and 40% at 2 hrs, 6 hrs, and 12 hrs, respectively, in the endotoxin treatment group compared with that of the pair-fed normal control group. Meanwhile, compared with the normal control group, the level of 2.4-kilobase (kb) messenger RNA (mRNA) for ubiquitin in extensor digitorum longus muscle in rats was increased by 153% and 470% at 2 hrs and 6 hrs. There were 87% and 117% increases in 1.2-kb mRNA for E2-14K, and 89% and 168% increase in RC2 mRNA expression in extensor digitorum longus muscle in endotoxemic rats than normal control rats at 2 hrs and 6 hrs after injection of endotoxin peritoneally. The tumor necrosis factor-alpha and interleukin-6 concentrations in rat plasma progressively increased after endotoxin treatment, but tumor necrosis factor-alpha peaked at the 2-hr time point, whereas interleukin-6 peaked at 12 hrs. Endotoxin administration resulted in a marked increase in endotoxin level at 2 hrs and 6 hrs. No significant change was observed in soleus muscle after endotoxin injection. A significantly positive correlation was found between the net release of 3-methylhistidine and respective values of endotoxin, intensity of mRNA expression of 2.-kb ubiquitin, 1.2-kb E2-14K, and subunit RC2 in extensor digitorum longus muscle (r =.9882, .9731, .9653, .9814, p <.05). However, no significant correlation was seen between tumor necrosis factor-alpha or interleukin-6 and respective values of 3-methylhistidine, mRNA expression of 2.4-kb ubiquitin, 1.2-kb E2-14K, and subunit RC2 (r =.3580, .4521, .5277, .4931, p >.05; r =.3950, .1767, .2136, .2519, p >.05, respectively.) in soleus muscle. CONCLUSIONS Endotoxemia can induce enhancement of skeletal muscle protein breakdown, mainly involving myofibrillar protein and white, fast-twitch extensor digitorum longus muscle. Ubiquitin-proteasome proteolytic pathway plays an important and major role in skeletal muscle proteolysis. Endotoxin, tumor necrosis factor-alpha, and interleukin-6 can directly or indirectly regulate muscle protein breakdown.

Effects of CD14-159 C/T polymorphism on CD14 expression and the balance between proinflammatory and anti-inflammatory cytokines in whole blood culture.

CD14 is an important receptor of innate immunity. When CD14 is anchored by ligands to LPS, peptidoglycans, or lipoteichoic acid, it can result in either proinflammatory or anti-inflammatory responses. To determine whether CD14-159 C/T polymorphism is associated with CD14 expression and the balance of proinflammatory and anti-inflammatory responses, we studied 118 healthy ethnic Han Chinese using a whole blood culture model. The CD14-159 C/T polymorphism was determined by polymerase chain reaction and subsequent HaeIII restriction enzyme digestion of the polymerase chain reaction products. Meanwhile, CD14 mRNA expression in leukocytes and the levels of soluble CD14, TNF-&agr;, IL-6, and IL-10 were also determined in the supernatants. Among the 118 individuals, there were 40 TT homozygotes, 62 heterozygotes, and 16 subjects homozygous for C allele. After LPS stimulation, the levels of CD14 mRNA expression in TT and TC genotypes were significantly higher than in CC homozygotes (P = 0.017), and soluble CD14 levels were also higher than in CC genotypes (P = 0.008). In addition, TT homozygotes had the highest LPS-stimulated TNF-&agr;, IL-6 production (P = 0.044, P = 0.004), and the lowest IL-10 release (P = 0.003). In conclusion, CD14-159 C/T polymorphism is correlated with CD14 expression and may thus influence the balance of proinflammatory and anti-inflammatory responses in ethnic Han Chinese. These results suggest that CD14-159 C/T polymorphism might partly explain the difference in predisposition to develop complications of infectious diseases in different patients and may provide a therapeutic target for sepsis intervention strategies.

The relationship between skeletal muscle proteolysis and ubiquitin-proteasome proteolytic pathway in burned rats.

Negative nitrogen balance and accelerated muscle protein breakdown are characteristics of burn injury. The mechanism by which muscle proteolysis occurs may be activation of the ubiquitin-proteasome pathway, but needs to be further elucidated. The aim of this study was to gain more insight into the role of ubiquitin-proteasome pathway in muscle proteolysis, after burn injury in a rat burn injury model. The proteolytic rates and mRNA expression of ubiquitin, E2-14K, and subunit RC2 in extensor digital longus (EDL) and soleus (SOL) muscle were determined by amino acid analyzer and Northern blot, respectively. The results were as follows: the total and myofibrillar proteolytic rate of EDL muscle increased markedly, especially at 12 and 24h post-burn. The levels of 2.4kb mRNA for ubiquitin, 1.2kb mRNA for E2-14K (a rate-limiting and regulated enzyme for conjugation of ubiquitin with protein substrate) and mRNA for subunit RC2 (the largest subunit of 20S proteasome) predominantly increased in EDL muscle after the stimulation of burn injury. No significant changes in proteolytic rate and transcription of ubiquitin, E2-14K, and subunit RC2 in SOL muscle were observed. There was a significantly positive correlation between the proteolytic rate and the levels of 2.4kb mRNA for ubiquitin, 1.2kb mRNA for E2-14K, or mRNA for subunit RC2. The results indicated that muscle wasting after burn injury was mainly due to the accelerated breakdown of myofibrils, and EDL muscle was more sensitive to burn injury than SOL muscle. The activation of ubiquitin-proteasome pathway was one reason for the enhanced protein catabolism in skeletal muscle. This is the first demonstration of upregulated expression of E2-14K and subunit RC2 in muscle, in response to burn injury, and it provides a clue to reduce muscle wasting by specifically inhibiting the specific enzymes or subunits involved in the enhancement of the activity of ubiquitin-proteasome pathway after burn injury.

Exosome Derived From Human Umbilical Cord Mesenchymal Stem Cell Mediates MiR-181c Attenuating Burn-induced Excessive Inflammation

Mesenchymal stem cell (MSC)-derived exosomes have diverse functions in regulating wound healing and inflammation; however, the molecular mechanism of human umbilical cord MSC (hUCMSC)-derived exosomes in regulating burn-induced inflammation is not well understood. We found that burn injury significantly increased the inflammatory reaction of rats or macrophages exposed to lipopolysaccharide (LPS), increased tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) levels and decreased IL-10 levels. hUCMSC-exosome administration successfully reversed this reaction. Further studies showed that miR-181c in the exosomes played a pivotal role in regulating inflammation. Compared to control hUCMSC-exosomes, hUCMSC-exosomes overexpressing miR-181c more effectively suppressed the TLR4 signaling pathway and alleviated inflammation in burned rats. Administration of miR-181c-expressing hUCMSC-exosomes or TLR4 knockdown significantly reduced LPS-induced TLR4 expression by macrophages and the inflammatory reaction. In summary, miR-181c expression in hUCMSC-exosomes reduces burn-induced inflammation by downregulating the TLR4 signaling pathway.

Regulation of signaling pathways downstream of IGF-I/insulin by androgen in skeletal muscle of glucocorticoid-treated rats.

BACKGROUND The mechanisms by which androgens ameliorate glucocorticoid-induced muscle wasting are still under investigation. In the present study, we tested the hypothesis that androgens effects in reversing muscle wasting are related to activating the signaling pathways downstream of insulin-like growth factor-1 (IGF-I)/insulin. METHODS Forty female Sprague-Dawley rats were randomly divided into four groups: control group, dexamethasone (DEX) group, testosterone (TES) group, and TES + DEX group. Each group was injected with saline or DEX (0.1 mg/100 g/d) for 10 days and sesame oil or TES (0.5 mg/100 g/d) for 13 days. Several downstream targets of IGF-I/insulin in skeletal muscle including protein kinase B (Akt), p70 ribosomal protein S6 kinase (p70S6K), and glycogen synthase kinase-3beta (GSK-3beta) that are associated with protein synthesis were examined. Two proteolysis-related ubiquitin E3-ligases, muscle atrophy F-box, and muscle RING finger-1 that are also regulated by IGF-I/insulin were also assessed. RESULTS TES attenuated gastrocnemius muscle atrophy induced by DEX. TES prevented the DEX-induced decrease of IGF-I expression in gastrocnemius muscle, but not in serum. TES ameliorated DEX-induced dephosphorylation of Akt and p70S6K and promoted the phosphorylation of GSK-3beta in gastrocnemius muscle. The total amount of Akt, p70S6K, or GSK-3beta proteins was not changed among these groups. TES did not show any effects on the DEX-induced upregulation of muscle atrophy F-box, and muscle RING finger-1 mRNA in gastrocnemius muscle. CONCLUSION This findings suggest that the effects of TES in reversing DEX-induced muscle atrophy are related to signaling pathways downstream of IGF-I/insulin that are associated with protein synthesis.

Repair and reconstruction of massively damaged burn wounds

OBJECTIVE To report repair and reconstruction of massively damaged burn wounds under unusual condition. METHODS One hundred forty-eight patients with deep burn tissue defects admitted from January 1993 through December 2000 were analyzed, among them 96 patients suffered from electrical injury which constituted 65.3% of all cases, 18 patients with hot press injury, 18 cases with deep burns as a result of CO poisoning or epileptic seizure, accounting for 12.2 and 12.2%, respectively, 6 cases with radiation injury, comprising 4.0% of all cases, 2 cases with explosive injury, 2 cases with chemical burn and 6 cases caused by erosive chemicals and wound infection. One hundred seventy-six flaps were transferred with mostly local flaps to repair deep burn wounds in 148 patients with tissue defects, in which necrotic tendons were replaced by acellular allogeneic tendons simultaneously in seven cases. Sixty-one iliolumbar arterial axial skin flaps for coverage of soft-tissue defects in hands or wrists were transplanted. Technical innovations to repair large soft-tissue defects of temporal region and ear, chin and lip, and dorso-lateral aspect of foot due to deep burn were explored. New technics to define necrotic tissue and vascular damage as a result of electrical injury were developed. RESULTS The biggest dimension of flaps in this group was 22cmx30cm. The survival rate of flap was 96.5%, while necrosis of the tip of flap occurred in 3.5%. The function and configuration were satisfactory after 4 months to 8 years follow-up. The technique of 99Tcm-methylene di-phosphonate (99Tcm-MDP) scintigraphy helped identify necrotic tissue before operation, and with the help of digital subtraction arteriogram (DSA) arterial injury could be identified. B-mode ultrasound was helpful to show the extent of endothelial injury, and Colour Doppler was useful to show luminal blood flow signal and filling defect in the injured artery. CONCLUSIONS Repair and reconstruction of massively damaged burn wound at early stage could be achieved. Techniques which helped define the extent of damage to the soft tissues, including arteries and tendons were essential preoperatively for successful reconstruction. Functional and aesthetic reconstruction, as well as the general condition of the patients, could thus be significantly improved early after the devastating injuries. Further innovations of operative technics would benefit more patients with such injuries.

Effects of glucagon-like peptide 1 on glycemia control and its metabolic consequence after severe thermal injury--studies in an animal model.

BACKGROUND Hyperglycemia with insulin resistance is commonly seen in severely burned patients and tight glycemia control with insulin may be beneficial in this condition. The most potent insulinotropic hormone, glucagon-like peptide 1 (GLP-1), stimulates insulin secretion in a glucose-dependent manner. Because infusion of GLP-1 never reduces glucose levels to below ∼70 mg/dL, the risk of hypoglycemia by using insulin is reduced. In this study we investigated the metabolic effects of GLP-1 infusion after burn injury in an animal model. METHODS Male CD rats were divided in 3 groups: burn injury with saline, burn injury with GLP-1 treatment, and sham burn (SB). Burn injury was full thickness 40% total body surface area. The burn injury with GLP-1 treatment group received GLP-1 infusion via osmotic pump. Fasting blood glucose, plasma insulin, and plasma GLP-1 levels were measured during intraperitoneal glucose tolerance tests. Expressions of caspase 3 and bcl-2 were evaluated in pancreatic islets. In a subset of animals, protein metabolism and total energy expenditure were measured. RESULTS Fasting GLP-1 was reduced in burn injury with saline compared to SB or burn injury with GLP-1 treatment. Burn injury with GLP-1 treatment showed reduced fasting blood glucose, improved intraperitoneal glucose tolerance test results, with increased plasma insulin and GLP-1 responses to glucose. GLP-1 reduced protein breakdown and total energy expenditure in burn injury with GLP-1 treatment versus burn injury with saline, with improved protein balance. Increased expression of caspase 3 and decreased expression of bcl-2 in islet cells by burn injury were ameliorated by GLP-1. CONCLUSION Burn injury reduced plasma GLP-1 in association with insulin resistance. GLP-1 infusion improved glucose tolerance and showed anabolic effects on protein metabolism and reduced total energy expenditure after burn injury, possibly via insulinotropic and non insulinotropic mechanisms.

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

ABSTRACT 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.

Low HLA-DR expression on CD14+ monocytes of burn victims with sepsis, and the effect of carbachol in vitro

This study aimed to investigate changes in the expression of human leukocyte antigen-DR (HLA-DR) on CD14+ monocytes in the peripheral blood of burn victims with delayed resuscitation in relation to the development of sepsis, and the effect of carbachol in vitro. The study population comprised 25 people with burns of at least 30% of total body surface area and delayed resuscitation, and 20 healthy volunteers as controls. Peripheral blood was collected on post-burn days 1, 3, 7, 14 and 28. When 7 participants developed sepsis, their peripheral blood was drawn on 2 consecutive days. Expression of HLA-DR on CD14+ monocytes in peripheral blood of burned participants was lower than that of controls, and fell further with the development of sepsis, when the rate and concentration of tumour necrosis factor-alpha (TNF-alpha) rose above those of controls and burned participants without sepsis. Expression of HLA-DR on CD14+ monocytes was negatively correlated with interleukin-10 (IL-10) levels on post-burn days 1, 7 and 28. In vitro, HLA-DR expression on monocytes also decreased with lipopolysaccharide (LPS) stimulation, but after treatment with carbachol, rose in a concentration-dependent manner. Thus expression of HLA-DR on CD14+ monocytes may be a useful parameter for monitoring the immune function of burn victims with and without sepsis. Carbachol significantly inhibited LPS-induced immunosuppression in human monocytes in vitro.