Long noncoding RNA GAS5 accelerates diabetic wound healing and promotes lymphangiogenesis via miR-217/Prox1 axis.

BACKGROUND: Diabetes is usually the leading cause of chronic non-healing wounds. LncRNA-GAS5 has been verified to be involved in the regulation of diabetes or high glucose (HG)-stimulated cells. However, its regulatory roles in diabetic wound healing need further investigation. METHOD: GAS5, miR-217 and Prox1 were identified by qRT-PCR. MTT, flow cytometry assay, wound-healing assay and tube formation were used to analyze cell viability, apoptosis, migration and tube formation capacity. Western blotting was carried out to detect the protein expression of c-Myc, CyclinD1, CDK4, Bcl-2, Prox1, VEGFR-3 and LYVE-1. Bioinformatics and luciferase assay were performed to predict and validate the binding sites of miR-217 on GAS5 and Prox1. Immunofluorescence staining detected the expression and distribution of Prox1. The wound healing rate was also assessed by setting up the diabetic mouse model. H&E staining assessed the distribution of inflammatory cells and fibroblasts in the wound tissues. RESULTS: GAS5 was significantly down-regulated whereas miR-217 was obviously up-regulated in diabetic skin, HG-induced lymphatic endothelial cells (LECs) and diabetic mouse model. GAS5 sponged miR-217 to up-regulate Prox1. GAS5 overexpression or miR-217 inhibition rescued the impairments of cell viability, migration and lymphatic vessel formation and the facilitation of apoptosis of LECs caused by HG. Similar impacts were observed on the protein level of VEGFR-3, LYVE-1, and Prox1. GAS5 promoted wound healing and lymphangiogenesis in the diabetic mouse model. CONCLUSION: GAS5 sponged miR-217 to up-regulate Prox1 and promote lymphangiogenesis and diabetic wound healing. This might provide novel therapeutic strategy to improve the efficacy of diabetic wound healing.

Long noncoding RNA-antisense noncoding RNA in the INK4 locus accelerates wound healing in diabetes by promoting lymphangiogenesis via regulating miR-181a/Prox1 axis.

BACKGROUND: Slow lymphangiogenesis is one crucial reason for the impaired wound healing process in diabetes. Accumulative evidence showed that long noncoding RNA-antisense noncoding RNA in the INK4 locus (ANRIL) could influence lymphangiogenesis. Besides, miR-181a has been reported to regulate Prox1 that is essential for lymphangiogenesis. However, the relationship between ANRIL and miR-181a as well as the definitive function of ANRIL in lymphangiogenesis is not clear. METHODS: The diabetic mouse model was set up to assess the wound healing rate in vivo. Quantitative real-time polymerase chain reaction was performed to measure the expressions of ANRIL, miR-181a, and Prox1. Western blot analysis was used to assess the expressions of vascular endothelial growth factor receptor-3, lymphatic vessel hyaluronan receptor-1, Prox1, and epithelial-mesenchymal transition (EMT)-related proteins. Flow cytometry was used to assess the cell apoptosis. Wound healing assay was used to determine the effect of ANRIL on cell migration. Tube-formation assay and immunofluorescence staining were performed to determine tube-formation capacity of human dermal lymphatic endothelial cells (LECs). RESULTS: ANRIL and Prox1 were downregulated, whereas miR-181a was upregulated in the diabetic wound healing mouse model and high glucose (HG)-induced LECs. The wound healing rate and EMT were inhibited during the diabetic wound healing process. Dual-luciferase assay proved that miR-181a could bind Prox1 to repress its expression, whereas ANRIL could sponge miR-181a to recover Prox1 expression. Overexpression of ANRIL or inhibition of miR-181a rescued the impairments of survival, migration, EMT formation, and tube formation of LECs caused by HG. CONCLUSION: ANRIL could promote lymphangiogenesis during the diabetic wound healing process via sponging miR-181a to enhance Prox1 expression, which might help design new therapy to improve the wound healing efficacy for diabetes.

Early laparotomy and timely reconstruction for patients with abdominal electrical injury: Five Case Reports and Literature Review.

INTRODUCTION: High-tension electricity can cause devastating injuries that may result in abdominal wall loss, visceral damage, and sometimes major threat to life. The visceral organ may be exposed after debridement and require flap cover, but the tensile strength of abdominal wall may be lack even if flap transplanted. METHODS: From April 2007 through May 2015, 5 patients with severe abdominal electrical injury were treated at our hospital. Exploratory laparotomy was performed based on their clinical manifestations and debridement findings of abdominal wall at early stage, and decision regarding technique for reconstruction of abdominal wall was based on an assessment of the location and extent of the defect. Medical records were reviewed for these data. RESULTS: Clinical evaluation and debridement findings of the abdomen revealed 4 patients with suspicious visceral damage. Laparotomy was performed in 4 cases, and revealed obvious lesion in 3 cases, including segmental necrosis of small intestine, partial necrosis of diaphragm, left liver and gastric wall, and greater omentum. Five patients underwent abdominal wall reconstruction using island retrograde latissimus dorsi myocutaneous flap or free/island composite anterolateral thigh myocutaneous flap. All flaps survived, abdominal bulging occurred in 3 cases after follow-up of 12 to 36 months. CONCLUSIONS: The clinical manifestations and wound features of abdomen collectively suggest a possible requirement of laparotomy for severe abdominal electrical burns. Retrograde latissimus dorsi myocutaneous flap or composite anterolateral thigh myocutaneous flap is an effective option for reconstruction of abdominal wall loss, the long-term complication of abdominal bulging, however, remains a significant clinical challenge.

WITHDRAWN: Early enteral nutrition versus late enteral nutrition for burns patients: A systematic review and meta-analysis.

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An epidemiological analysis of paediatric burns in urban and rural areas in south central China.

OBJECTIVE: This study aims to analyse the epidemiology of paediatric burns in south central China, illustrate the differences between rural and urban areas, and discern prevention measures to reduce paediatric burns. METHODS: Data were obtained from all paediatric patients admitted to Department of Burns unit of Xiangya Hospital during 2009-2012. A retrospective review was performed, including cause of burn, pre-hospital treatment, place of burn occurrence, anatomical areas involved, extent of burn, date of injury, number of operations, complications, length of hospital stay, hospitalisation cost and cure rate. RESULTS: A total of 278 hospitalised paediatric patients were admitted in this study. The majority (56.47%) were 1-3 years old. Rural patients accounted for 67.99% in total; the ratio of boys to girls was 2.05. Scalding with hot fluids was the most common cause of burns in children (62.59%), followed by flame (17.63), fireworks (9.71%), electricity (5.76%) and other factors such as contact and chemical (4.32%). The living room was the location with the highest frequency of burns in children (53.24%). Burns were more likely to happen in winter and the upper extremities were the most involved anatomic site (53.24%). Total burn surface area (TBSA) ranging from 0% to 9% accounted for 55.4% in total. Rural patients underwent more operations and had longer and costlier hospital stays than urban patients. CONCLUSION: Compared with treatment in urban areas, rural burn patients received less first-aid treatment, underwent more surgery, had more complications and longer and more costly hospital stays. This finding strongly suggests that it is necessary to make more efforts to prevent burns, especially in rural areas.

[Repair of wound on hand or foot due to electrical burn using free lateral upper arm perforator flap].

OBJECTIVE: To observe the effect of free lateral upper arm perforator flap in repairing wound on hand or foot due to electrical burn. METHODS: Six patients with full-thickness wounds on hand or foot resulting from electrical burn were hospitalized from June 2010 to June 2013. The wounds ranged from 6.0 cm ×4.0 cm to 8.5 cm×7.5 cm in area. Free lateral upper arm perforator flaps were used to repair these defects, with flap area ranging from 9 cm ×4 cm to 12 cm × 9 cm. The donor sites in five cases were closed by suturing; the other one donor site was closed by transplantation of full-thickness skin from abdomen. RESULTS: One flap used to repair the wound in middle finger failed due to failure of venous return, and it was repaired with full-thickness skin harvested from abdomen after dressing change. The other five flaps survived resulting in good elasticity and matched appearance of the recipient area without obvious bulkiness. Patients were followed up for 6 to 24 months. The function of the injured hands or feet recovered well, and the results of function evaluation of five hands were excellent in 2 cases, good in 2 cases, and poor in 1 case. Little scar formation with no contraction or function impairment was observed on donor site, and the result was satisfactory. CONCLUSIONS: Free lateral upper arm perforator flap, with long vessel and less adipose tissue, is suitable for repairing small but deep wound on hand or foot due to electrical burn.

[Reconstruction of upper lip defect].

OBJECTIVE: To summarize the clinical experience for the reconstruction of upper lip defects in different degrees. METHODS: Different treatment methods were selected to reconstruct the upper lip defects according to the subunit, size or location of defects on the upper lip. The therapeutic results were analyzed and compared. RESULTS: From Jan. 1998 to Apr. 2012, 243 cases with upper lip defects were treated, including 85 cases of traumatic defects, 110 cases of defects secondary to lip cleft and 48 cases of defects after tumor resection. 48 cases were treated with direct closure, 69 cases with cross-lip flaps, 5 Cases with orbicularis oris muscle flaps, 53 cases with unilateral labial flaps, 42 cases with bilateral labial flaps, 22 cases with lower lip flaps, and 1 case with forehead flap, 1 case with forearm flap and 2 with nasolabial fold flaps. The patients were followed up for 3-18 months with satisfactory results. CONCLUSIONS: Satisfactory results could be achieved for upper lip defects with appropriate treatment methods according to the defects degree and characters.

[Anatomic basis of the accompanying pattern of the superficial temporal arterial branches and veins and its clinical application].

OBJECTIVE: To study the course of branches of the superficial temporal artery (BSTA) and the accompanying pattern of their veins in order to provide anatomic basis for flap design. METHODS: Head and facial part of ten adult corpses (19 sides) were dissected and photographed. The coordinate system was set up with external auditory foramen as the point of origin, aided by the graph analysis software Digimizer. The course of the frontal branch and parietal branch of the superficial temporal artery (STA), and the accompanying pattern between the BSTA and the veins were measured and analyzed. The STA and its branches were located by Ultrasonic Doppler, and the corresponding branches of the superficial temporal vein (BSTV) were disclosed by bowing patient's head with breath holding or cerclaging the basement of the patient's skull. And then 10 sides of transposition (fascia) flaps with axis at the angular bisector between BSTA and BSTV were devised to repair wounds of 9 patients that hospitalized from February 2008 to December 2009. Data were processed with test of variance homogeneity. RESULTS: Frontal BSTV absence was found in 6 head sides, and the maximum distance between artery and vein was (2.1 ± 1.2) cm. Parietal BSTV absence was found in 3 head sides, and the maximum distance was (1.4 ± 0.7) cm. The distance between frontal BSTA and BSTV was larger than that between parietal BSTA and BSTV, and the dispersion degree of the former was higher than that of the latter (F = 0.0404, P = 0.0475). All the (fascia) flaps survived without congestion or necrosis. CONCLUSIONS: When branch of the superficial temporal vessel was selected as the axial vessel of flap, the flap design shall depend on the accompanying pattern of BSTV to avoid the flap necrosis due to poor venous return after surgery. The superficial temporal vein and its branches can be well disclosed by bowing head or cerclaging skull. The approach is simple, useful, safe, and reliable.

Preliminary proteomic analysis of circulating polymorphonuclear neutrophils from rabbits experiencing scald injury and Staphylococcus aureus sepsis.

BACKGROUND: The aim of this study is to identify the differentially expressed proteins in circulating polymorphonuclear neutrophils (PMN) from scalded bacteremia rabbits infected with Staphylococcus aureus to provide a basis to reveal the pathogenesis of burns and sepsis. METHODS: Rabbits were subjected to sham burn (A), A + bacterial challenge (B), 30% scald injury (C), or C + bacterial challenge (D). Bacterial challenge was inflicted as an injection of 2.0 x 10(8) cfu S. aureus 18 h after burn procedure. Animals were sacrificed 24 h after burn. PMN were isolated, and the differential proteins in the PMN from these animals were identified by two-dimensional electrophoresis coupled with MALDI-TOF-MS; two proteins were confirmed by Western blotting. RESULTS: Twenty-one differential protein spots were found, and seven differential proteins were identified. Among the identified proteins, the expression levels of protein disulfide-isomerase and thiol-specific antioxidant protein were down-regulated in groups C and D, and two protein spots of annexin I were identified, one of which was down-regulated and another up-regulated in groups C and D. CONCLUSIONS: Preliminary proteome changes in PMN from rabbits experiencing scald injury and S. aureus sepsis were revealed, which possibly play an important role in the inflammation and pathogenesis of sepsis after scald injury.

Proteomic change of peripheral lymphocytes from scald injury and Pseudomonas aeruginosa sepsis in rabbits.

BACKGROUND: Increased susceptibility to infection has been related to impairment of lymphocyte-regulated immune responses after severe burn. The aim of this study is to identify the differential expression of proteins in circulating lymphocytes from scald injury and Pseudomonas aeruginosa sepsis in rabbits to provide a basis for pathogenesis of burns and sepsis. METHODS: Rabbits were subjected to sham burn (A), 30% scald (B), A+bacterial challenge (C) or B+bacterial challenge (D). Bacterial challenge was inflicted by an injection of 2.0x10(8) CFU P. aeruginosa (ATCC27853) in the auricular vein 22 h after the burn procedure. The animals were sacrificed 24 h later. Lymphocytes were isolated, and the differential proteins in the lymphocytes from the experimental and control animals were identified by two-dimensional electrophoresis (2-DE) coupled with matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), two of which were confirmed by Western blotting. RESULTS: Nineteen differential protein spots were found by 2-DE and 12 spots (11 proteins) were identified. Differential expression of peroxiredoxin and annexin I was validated by Western blotting. Among the identified proteins, the expression levels of cofilin, cyclophilin A, ubiquitin, nucleoside diphosphate kinase, glutamate dehydrogenase and annexin I were down-regulated in group B, excessively down-regulated in group D, but mildly in group C, and peroxiredoxin was up-regulated in groups B and D. CONCLUSIONS: Proteome changes in lymphocytes from P. aeruginosa sepsis in the scalded rabbits were revealed, which are related to immune suppression and the pathogenesis of sepsis after scald injury.

[Proteomic change in lymphocytes of scalded rabbits caused by Staphylococcus aureus invasion].

OBJECTIVE: To study the proteomic change in lymphocytes of rabbits with scald injury and Staphylococcus aureus (SA) invasion. METHODS: Twenty-four rabbits were divided into four groups as follows: control group, scald group, scald with SA invasion 2 hs group, and scald with SA invasion 6 hs group, according to random number table, with 6 rabbits in each group. Except for rabbits in control group (sham scald at 37 degrees C), rabbits in the other 3 groups were subjected to 30% TBSA full-thickness scald. Rabbits in SA invasion 2 and 6 hs groups were injected with 2 mL (1.0 x 10(8) CFU/mL) SA suspension, which was in the log growth phase, via auricle vein 18 hs and 22 hs after injury. Whole blood samples were collected from carotid artery of rabbits in 4 groups 24 hs after scald. Lymphocytes were isolated and its extracted proteins were analyzed by two-dimensional gel electrophoresis coupled with mass spectroscopy. RESULTS: About 1030 protein spots of lymphocytes were detected in each group. Compared with that of control group, 19 protein spots were found to be differentially expressed in the other 3 groups, and 11 spots (10 proteins) were identified. Expression levels of cofilin, cyclophilin A, ubiquitin, nucleoside diphosphate kinase, glutamate dehydrogenase and annexin 1 were down-regulated, but expression level of peroxiredoxin was up-regulated obviously. CONCLUSIONS: There is obvious proteomic change in lymphocytes of scalded rabbits or of scalded rabbits invaded by SA, and it may relate to immune suppression and sepsis after injury.

[Proteomic study of peripheral blood lymphocytes of rabbits with severe burn and Pseudomonas aeruginosa sepsis].

OBJECTIVE: To study the effect of severe burn and Pseudomonas aeruginosa sepsis on the proteomics of lymphocytes (LCs) of rabbits. METHODS: Twenty-four rabbits were divided into four groups, i.e. control, severe scald, severe scald and 2-hour sepsis, severe scald and 6-hour sepsis (6 rabbits in each group). The scald in rabbits was third degree in depth involving 30% of total body surface area (TBSA). The sepsis model was reproduced by intravenous injection of a suspension of Pseudomonas aeruginosa (ATCC 27853, 6 x 10(12)cfu/L) 1 ml/kg 24 hours after scald. The rabbits in control group were treated with warm water of 37 centigrade. Peripheral blood was obtained from the carotid artery 24 hours after scald, or 2 hours after sepsis, or 6 hours after sepsis. The LCs in each blood sample were separated, disrupted and the total proteins of LCs were extracted. The proteins were separated by two dimensional gel electrophoresis. The gels were stained with Coomassie brilliant blue and then were scanned. The images were analyzed by PD quest software. The protein spots of discrepant expression were sieved and then analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). The peptide mass finger printing (PMFs) were obtained and were input into the data bank of proteins for identification of the proteins. RESULTS: The average spots of 6 gels were 1 051+/-21 (control), 1 026+/-30 (severe scald), 1 078+/- 36 (2-hour sepsis) and 1 065+/-31 (6-hour sepsis), and the average matching rate were 91% (control), 89% (severe scald), 92% (2-hour sepsis) and 94% (6-hour sepsis), respectively. No difference was found in the protein expression of LCs between 2-hour sepsis group and 6-hour sepsis group, but the protein expression of LCs in severe scald group, 2-hour sepsis group and 6-hour sepsis group differed when compared with control group. Nineteen protein spots expressed discrepancy were sieved and their PMFs were obtained. Twelve protein spots (including 11 proteins) were identified, including Cofilin, peptidyl- prolyl cis-trans isomerase cyclophilin A, ubiquitin, nucleoside diphosphate kinase, glutamate dehydrogenase, selenium binding protein I, beta-actin, peroxiredoxin-6, annexin I, actin-3, cellular retinoic-acid binding protein. CONCLUSION: The proteomics of peripheral blood LCs alters in rabbits with severe burn and Pseudomonas aeruginosa sepsis. The proteins with discrepant expression included 11 proteins, which are related with the folding, assembling, transportation and degradation of proteins, signal transmission, inflammation, immunization, energy metabolism, the proliferation, differentiation and apoptosis of cells. These proteins might be associated with the pathogenesis of severe burn and Pseudomonas aeruginosa sepsis.

[Application of latissimus dorsi flap in different forms in repair of skin and soft tissue defects in lower extremities].

OBJECTIVE: To explore repair methods of skin and soft tissue defects in lower extremities with free latissimus dorsi flaps. METHODS: Forty-two patients with wounds and soft tissue defects in lower extremities, including 4 cases on knee, 22 cases on leg, 15 cases on ankle and foot, 1 case with extensive avulsion from knee to dorsum of foot, were hospitalized in our unit from February 1996 to February 2008. Wounds or soft tissue defects were respectively repaired with latissimus dorsi musculocutaneous flaps, latissimus dorsi muscle flaps, latissimus dorsi perforator flaps with preserved vascular sleeves, 2 double-leaf segmental latissimus dorsi compound flaps after debridement. The flaps ranged from 18 cm x 8 cm to 40 cm x 18 cm in size. The donor sites were covered by skin grafting in 19 cases. RESULTS: All wounds were healed primarily except vascular crisis occurred in 3 cases, partial necrosis of skin at donor site in 2 cases, and graft site (1 case). Follow-up for 3 to 24 months of 31 patients showed: six cases received two-stage plastic operation on account of bulkiness with trouble in wearing shoes, and mild contraction of muscular flap in 3 cases. CONCLUSIONS: Latissimus dorsi flap in various forms can be satisfactory for repair of large skin and soft tissue defects in lower extremities.

[Repair of nose and adjacent tissue defect deformities after burn].

OBJECTIVE: To look for the best method of repairing nose and adjacent tissue defect after burn and observe the effect. METHODS: Twelve patients with post-burn nose and adjacent tissue defect deformities hospitalized from January 1999 to December 2008 were repaired with expanded forehead flap, pedicled upper-arm flap, axial post-auricular reversed flow island flap, and nasolabial groove flap. Among them, 4 cases with total nasal defect, 8 cases with partial nasal defect; and 3 cases were accompanied with scars on cheek, 5 cases accompanied with scars on forehead, 5 cases accompanied with upper lip ectropion and subtotal upper lip defect. The skin flap size ranged from 3.0 cm x 1.5 cm to 10.0 cm x 8.0 cm. RESULTS: Five cases were repaired with expanded forehead flap, 3 cases with pedicled upper-arm flap, 1 case with axial post-auricular reversed flow island flap, and 3 cases with nasolabial groove flap respectively. All the 12 flaps survived. Patients were followed up for 1 to 7 years, and nasal function and appearance were obviously improved. CONCLUSIONS: Optimal repairing method shall be chosen to repair nasal defect after burn according to its extent, and forehead flap is preferred. Pedicled upper-arm flap and reversed flow axial post-auricular island flap can be employed if local flap and ortho-position skin flap are unavailable when obvious scar is present on face as a result of severe burn.

[Repair of claw hand deformity after burn].

OBJECTIVE: To summarize methods for repair of claw hand deformity after burn. METHODS: Ninety-seven patients with 136 claw hands after burn hospitalized from May 1992 to May 2007 were repaired with skin grafting (104 hands) and transposition of skin flap (32 hands), among which 21 hands were minor-grade, 92 hands moderate, 23 hands severe. The metacarpophalangeal joint was repaired after scar release in dorsum of hand with manual extraction reduction, release of collateral ligament and joint capsula, separation of adhesion in joint, tendon lengthening for obvious contracture. Restitution of finger flexion deformity, lysis of adhesion and grafting among first web and finger webs, repair of central slip extensor tendon or phalangeal arthrodesis were performed according to the abnormal condition after lysis of dorsal scar of hand. The metacarpophalangeal joint from 31 patients were not repaired with above methods for severe finger flexion deformity, their palmar scar were loosened and transplanted firstly, then scar in dorsum of hand were loosened, metacarpophalangeal joint were repaired, flap or skin were transferred or transplanted. General rehabilitation were performed routinely after operation. RESULTS: The ending of flaps (4 hands) due to the scar were necrosis after transposition and healed through dressing change, other skins or flaps all survived. Most articular deformities were corrected completely or basically. Functions including palmar opposition, grasp were also recovered with satisfactory results. CONCLUSION: Skin transplantation and transferring of skin flap with overall planning and individual isatin are the key points for repair of claw hand after burn.

[Repair of large defects in upper extremities with pedicled combined flap].

OBJECTIVE: To explore the flap transposition for repairing large defects in upper extremities. METHODS: 12 cases with large defects in elbow, forearm, wrist or palm caused by high-voltage electricity, hot-pressure or crush, were treated. 4 cases were treated with latissimus dorsal myocutaneous flaps combined iliolumbar flaps. 2 cases were treated with latissimus dorsal myocutaneous flaps combined lateral thoracic flaps. 6 cases were treated with large combined thoracic-abdomen flaps. RESULTS: All the flaps survived except for one flap with 2 cm distal necrosis and sub-flap infection. Satisfactory results were achieved. CONCLUSIONS: Early debridement and reconstruction with pedicled combined flaps are feasible and reliable for large defects in the upper extremities.

[Expression of myeloid cell triggering receptor-1 in monocytes at early post-burn stage].

OBJECTIVE: To investigate the expression of triggering receptor expressed on myeloid cells (TREM-1) in monocytes of burn patients at early post-burn stage, and its significance. METHODS: The monocytes of 8 healthy volunteers (A group), 29 patients with mild and moderate burn (B group), and 9 patients with severe and very serious burns (C group) were isolated from the blood, and the THEM-1 mRNA and protein expression were determined by semi-quantitative RT-PCR and flow cytometry, respectively. The plasma levels of TNF-alpha, IL-1beta were determined by ELISA method. RESULTS: The value of TREM-1 mRNA expression in A, B and C groups were 0.74 +/- 0.13, 1.24 +/- 0.09, and 1.46 +/-0.07, respectively, and the expression rates on cell surface in the 3 groups were (9 +/- 4)%, (51 +/- 6)%, and (71 +/- 7)%, respectively, and there were significant differences among the three groups (P = 0.000). the plasma levels of TNF-alpha and IL-1beta in B and C groups were obviously higher than that in A group (P = 0.000), and they were positively correlated to TREM-1 expression (rs = 0.68, 0.72, P = 0.000). CONCLUSION: Increased expression of TREM-1 in monocytes of burn patients at early post-burn stage is correlated with the release of inflammatory factors, indicating that TREM-1 might contribute to the onset and development of acute inflammatory response after burns.

[Inhibitory effect of epidermal growth factor on apoptosis in HaCaT keratinocytes induced by TNF-alpha].

OBJECTIVE: To explore the effect of epidermal growth factor (EGF) on apoptosis induced by TNF-alpha and the expression of PPARbeta in HaCaT keratinocytes. METHODS: HaCaT keratinocytes were cultured and randomly divided into A (normal control), B (with treatment of 10 ng/ml TNF-alpha for 24 hours), C (with treatment of 20 ng/ml TNF-alpha for 24 hours), D (with treatment of 10 ng/ml TNF-alpha after 20 ng/ml EGF treatment for 4 hours), E (with treatment of 20 ng/ml TNF-alpha after 20 ng/ml EGF treatment for 4 hours) groups. The apoptosis of HaCaT keratinocytes was observed by flow cytometry. The proliferative activity of HaCaT keratinocytes was evaluated by MTT method. The activity of caspase-3 was analyzed with caspase colorimetric assay Kit. The changes in the mRNA and protein expression of PPARbeta in HaCaT keratinocytes were observed by RT-PCR and western-blotting after treatment with different concentrations (5, 10, 20, 40 ng/ml) of EGF for 4 or 24 hrs. RESULTS: Compared with A and B groups [(32 +/- 6)%, (57 +/- 6)%], the apoptosis of HaCaT keratinocytes in D and E groups were significantly increased [(20 +/- 3)%, (28 +/- 4)%, respectively, P < 0.01], while the survival rate of HaCaT keratinocytes in D and E groups increased, and the caspase-3 activity were decreased (P < 0.01). The expression of PPARbeta mRNA and protein in HaCaT keratinocytes reached the peak with the treatment of 20 ng/ml EGF. CONCLUSION: EGF can inhibit the apoptosis of HaCaT keratinocytes induced by TNF-alpha, and it can also increase the expression of PPARbeta.

[The role of PPARbeta in the apoptotic HaCaT keratinocytes induced by TNF-alpha].

OBJECTIVE: To explore the change of transcription activity and expression of PPARbeta in the apoptotic HaCaT keratinocytes induced by TNF-alpha. METHODS: HaCaT keratinocytes were exposed to different concentration TNF-alpha for 24 hours. Apoptotic morphological changes and percentage of apoptotic nuclei were assayed with Hoechst 33258 staining. Activities of Caspase-3 were analyzed with Caspase Colorimetric Assay Kit after HaCaT keratinocytes were exposed to TNF-alpha (10 and 20 ng/ml) for indicated durations. The expression of PPARbeta in HaCaT keratinocytes treated with TNF-alpha was observed by Western-blot and RT-PCR. Electrophoretic mobility shift assays demonstrated a impermanency increase in PPARbeta binding activity with DNA. Furthermore, luciferase assay system were employed to analyze PPARbeta transcription activity. RESULTS: The apoptosis of HaCaT keratinocytes treated with different concentration TNF-alpha for 24 hours was increased by Hoechst 33258 stained, and fluorescent microscopy showed apoptotic cells with condensed chromatin. The nuclear apoptotic percentage were (12 +/- 3)%, (32 +/- 4)%, (57 +/- 5)%, respectively, in HaCaT keratinocytes exposed to TNF-alpha (5, 10, 20 ng/ml) for 24 hours. The activation of Caspase-3 were enhanced in HaCaT keratinocytes treated with TNF-alpha (10 or 20 ng/ml) for indicated durations (P < 0.01). The expression of PPARbeta protein significantly increased in HaCaT keratinocytes treated with TNF-alpha (10 ng/ml) for 12 and 24 hours. After exposure to different concentration of TNF-alpha for 24 hours, Western-blot analysis demonstrated to augment the expression of PPARbeta in HaCaT keratinocytes. RT-PCR testified the expression of PPARbeta mRNA is markedly increased in HaCaT keratinocytes treated with TNF-alpha (10,20 ng/ml) for 3 hours and 6 hours. PPARbeta-DNA binding was assessed by EMSA using a PPARbeta response element (PPRE) and nuclear extracts prepared from HaCaT keratinocytes treated for 30 minutes and 60 minutes with 10 ng/ml of TNF-alpha demerstrated TNF-alpha enhanced PPARbeta DNA binding activity. Furthermore, luciferase assay system obtained TNF-alpha increased PDK1 activity through an PPARbeta-dependent pathway. CONCLUSION: TNF-alpha could increase the expression and transcription activity of PPARbeta in HaCaT keratinocytes.

[Effect of dexamethasone on spontaneously apoptosis of polymorphonuclear neutrophils from postburn rabbits].

OBJECTIVE: To study the effect of dexamethasone on spontaneously apoptosis, bcl-2, and neuclear facor kappa (NFkappaB) expressions of polymorphonuclear neutrophil (PMN) from postburn rabbits. METHODS: PMN were isolated from 8 rabbits on 24 postburn hours and cultured with normal serum (NS), burn serum (BS), normal serum plus dexamethasone (ND), and burn serum plus dexamethasone (BD) for 24 hours, respectively. The quantification of apoptosis was analyzed by acridine orange + ethidium bromide fluorescent staining and flow cytometry , and the contents of bcl-2 and NFkappaB protein detected by immunohistochemical method. RESULTS: In the BS group, the percentage of apoptotic PMN decreased (compared with NS group, 6.18 +/- 0.96 vs. 21.77 +/- 2.32, P<0.05), and the contents of bcl-2 and NFkappaB protein increased (compared with NS group, 83.27 +/- 5.45 vs. 49.95 +/- 2.67, P<0.05). When compared with BS group, the apoptotic percentage of BD group increased (12.67 +/- 0.71 vs. 6.18 +/- 0.96, P<0.05), and the content of NFkappaB protein reduced (0. 1031 +/- 0.0154 vs. 0.1802 +/- 0.0130, P<0.05), but no significant difference between ND and NS group was found. CONCLUSION: Dexamethasone decreases the inhibition of PMN apoptosis by bumn serum, which may be associated with the down-regulation of NFKB expression.