Polydatin, a natural polyphenol, protects arterial smooth muscle cells against mitochondrial dysfunction and lysosomal destabilization following hemorrhagic shock.

The main objective of this study was to investigate the activity of polydatin on mitochondrial dysfunction and lysosomal stability of arteriolar smooth muscle cells (ASMCs) in severe shock. The experimental animals (rats) were divided into five groups: control, hemorrhagic shock, shock + CsA, shock + Res, and shock + PD (exposed to cyclosporin A, resveratrol, or polydatin following induction of hemorrhagic shock, respectively). The calcein-Co(2+) technique revealed opening of ASMC mitochondrial permeability transition pores (mPTP) after shock with resulting mitochondrial swelling, decreased mitochondrial membrane potential (ΔΨm), and reduced intracellular ATP levels. These alterations were all inhibited by exposure to PD, which was significantly more effective than CsA and Res. PD also preserved lysosomal stability, suppressed activation of K(ATP) channels, ASMC hyperpolarization, and reduced vasoresponsiveness to norepinephrine that normally follows severe shock. The results demonstrate that exposure to PD after initiation of severe shock effectively preserves ASMC mitochondrial integrity and has a significant therapeutic effect in severe shock. The effects may partially result from lysosomal stabilization against shock-induced oxidative stress and depressed relocation of hydrolytic enzymes and redox-active lysosomal iron that, in turn, may induce mPTP opening.

Basic fibroblast growth factor (bFGF) alleviates the scar of the rabbit ear model in wound healing.

Studies suggest a possible antiscarring effect of basic fibroblast growth factor (bFGF) during wound healing. However, little is known about the precise pathological mechanisms of bFGF. In particular, there is only limited information available about the mechanism of exogenous administration of bFGF to scar formation. To investigate the effect of bFGF on the hypertrophic scar in the rabbit ear model and to clarify the mechanisms of bFGF on treatment for scar in wound healing, the rabbit ear model of wound healing was created and treated topically with bFGF once daily for 3 months; then we examined the changes of macroscopic and histopathological characteristics of scars and the expression of collagen and collagenase-1 (matrix metalloproteinase-1). The results of macroscopic and histologic characteristics revealed a significant difference between scars treated with bFGF and control scars. The expression of collagen in the scars treated with bFGF was decreased, as compared with the scars treated with saline. Further study revealed that bFGF could remarkably enhance expression of matrix metalloproteinase-1. bFGF could improve the quality of wound healing and remarkably alleviate the scar in the rabbit ear model in wound healing, which suggests that bFGF exerted a net negative effecton scar formation in wound healing. The evidence should contribute to a better understanding of the biological activities of bFGF during hypertrophic scar formation.

Effect of bone marrow mesenchymal stem cells on the polarization of macrophages.

Inflammation is a defensive response in the living tissue of the vascular system that acts against damage factors and involves various types of immune cells, including macrophages, neutrophils, endothelial cells and other associated immune molecules. If the release of inflammatory mediators is excessive, systemic inflammatory response syndrome may develop. Sepsis is the most common complication of severe burns and is a systemic inflammatory response syndrome that is caused by infectious factors and is capable of leading to multiple organ dysfunction and potentially death. Research concerning the mechanism and treatment of sepsis is crucial. Macrophages are an important type of immune cell that remove invasive pathogens and are involved in innate and adaptive immune responses. It has been previously reported that bone marrow mesenchymal stem cells (BMSCs) affect macrophages by regulating immunity. The present study aimed to investigate the effect of BMSCs on macrophage polarization in vivo and in vitro, in addition to the potential therapeutic effect of these cells on experimental sepsis. BMSCs and peritoneal macrophages were isolated from Sprague­Dawley rats and co­cultured overnight as a mixed culture or Transwell system, and subsequently stimulated with 100 ng/ml lipopolysaccharide (LPS). After 12 h, the medium was replaced with normal complete medium for various durations and supernatants were collected to extract proteins and cells for ELISA, western blot and flow cytometry analysis to investigate different aspects of macrophages. Sepsis was induced in Sprague­Dawley rats by injection of LPS (5 mg/kg), followed by tail vein injection of BMSCs or PBS 1 h later. After 6, 12, 24 and 48 h, lung tissues were harvested for pathological observation and peritoneal macrophages were collected for flow cytometry analysis to assess the expression of markers, including cluster of differentiation (CD)68 (used for gating), CD11c and CD206. The results demonstrated that, in the culture medium, LPS stimulation increased the expression of CD11c in macrophages, and the levels of tumor necrosis factor­α and inducible nitric oxide synthase were also increased. By contrast, in macrophages treated with BMSCs directly, the expression of CD11c was reduced compared with the LPS­stimulated macrophage alone group. However, the secretion of interleukin­10, transforming growth factor­ß and arginase­1 was increased in the direct co­culture group, compared with the LPS­stimulated macrophage alone group. BMSCs reduced the inflammation in lung tissues and inhibited macrophage expression of CD11c in the rat model of sepsis. The results of the present study demonstrated that BMSCs co­cultured with macrophages directly inhibited macrophage differentiation into the M1 phenotype and reduced inflammation in macrophages stimulated by LPS. In vivo, BMSCs decreased the expression of CD11c in peritoneal macrophages and reduced the pathological inflammatory response in the lungs. The findings of the present study demonstrated that BMSCs may reduce the extent of the systemic inflammatory response, which may contribute to the development for a novel type of treatment for sepsis in the future.

[Establishing a parabiosis animal model of two-way paradigm].

OBJECTIVE: To establish a parabiosis model between allogenic conspecific adult mice to study two-way paradigm. METHODS: Fifty-four female Balb/c mice and 54 male C57BL/6 mice were paired and equally divided into 3 groups, namely group 1 with normal saline (NS) injection, group 2 with injections of spleen cells and cyclophosphamide (CP), and group 3 injected with spleen cells, CP, and cyclosporin A (CsA). The treatments were performed by injecting the spleen cells from one of the mice in a pair into the other via tail vein and vise versa, and two days after the operation, CP (150 mg/kg) was injected intraperitoneally. Intraperitoneal CsA (30 mg/kg daily) injection was given starting from 2 days before till 7 days after the operation. Twelve of the 18 pairs of parabiosis mice in each group were separated after 1 week, and part of the skin were transplanted to each other. The maintenance of parabiosis was observed in the other 6 pairs of parabiosis mice were observed. Mixed lymphocyte reaction (MLR) and delayed-type hypersensitivity (DTH) were observed and studied in the separated mice. RESULT: The duration of parabiosis maintenance and skin survival of the group 3 was significantly longer than those in the other two groups, and group 3 showed suppressed MLR and DTH. CONCLUSION: With the application of immunosuppressants, we have successfully established the two-way paradigm model in mice.

[Application of laminar air flow techniques in burn treatment].

OBJECTIVE: To evaluate the value of laminar flow in the treatment of burns. METHODS: The air in the laminar flow chamber and the wound tissues of the patients were sampled for bacterial detection. The number and stains of bacterial colony from different classes of laminar air flow chambers at different time points were inspected and compared. RESULTS: The bacterial number was 0 in the laminar flow chamber of 1000 grade, which was obviously different from that in the public area. The mortality was obviously decreased in the laminar air flow chamber with shorter treatment time and hospitalization. No wound infection occurred and the wounds healed smoothly in all these patients. CONCLUSION: The application of laminar air flow can be helpful for the treatment of severe burns.

Normal skin and hypertrophic scar fibroblasts differentially regulate collagen and fibronectin expression as well as mitochondrial membrane potential in response to basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) regulates skin wound healing; however, the underlying mechanism remains to be defined. In the present study, we determined the effects of bFGF on the regulation of cell growth as well as collagen and fibronectin expression in fibroblasts from normal human skin and from hypertrophic scars. We then explored the involvement of mitochondria in mediating bFGF-induced effects on the fibroblasts. We isolated and cultivated normal and hypertrophic scar fibroblasts from tissue biopsies of patients who underwent plastic surgery for repairing hypertrophic scars. The fibroblasts were then treated with different concentrations of bFGF (ranging from 0.1 to 1000 ng/mL). The growth of hypertrophic scar fibroblasts became slower with selective inhibition of type I collagen production after exposure to bFGF. However, type III collagen expression was affected in both normal and hypertrophic scar fibroblasts. Moreover, fibronectin expression in the normal fibroblasts was up-regulated after bFGF treatment. bFGF (1000 ng/mL) also induced mitochondrial depolarization in hypertrophic scar fibroblasts (P < 0.01). The cellular ATP level decreased in hypertrophic scar fibroblasts (P < 0.05), while it increased in the normal fibroblasts following treatment with bFGF (P < 0.01). These data suggest that bFGF has differential effects and mechanisms on fibroblasts of the normal skin and hypertrophic scars, indicating that bFGF may play a role in the early phase of skin wound healing and post-burn scar formation.

[Clinical application of low-molecular-weight heparin (Fraxiparine) in rescuing venous crisis of island skin flap].

OBJECTIVE: To evaluate the clinical efficacy of low molecular weight heparin (Fraxiparine) in rescuing venous crisis of island skin flap. METHODS: Of the 73 patients with venous crisis of island skin flap, 47 received subcutaneous injection of low-molecular-weight heparin (group I) and 26 were treated with phlebotomy, local compression and topical application of unfractionated heparin solution gauze (group II). RESULTS: The flap survival ratio was (88.46∓8.64)% in group I and (38.37∓6.53)% in group II (P<0.001). At 0, 2, and 4 h after injection of low-molecular-weight heparin, the activated partial thromboplastin time (APTT) was obviously delayed (24.28∓6.71, 41.35∓7.64 and 32.34∓6.35, respectively, P<0.01), FXa:C level was significantly decreased (152.4∓30.7, 65.8∓24.4 and 83.4∓18.4, respectively, P<0.01), while FIIa:C level underwent no obvious alterations (155.70∓31.61, 143.20∓24.75, and 143.4∓23.35, respectively, P=NS). CONCLUSION: Fraxiparine has good antithrombotic efficacy in rescuing venous crisis of island skin flap without adverse effect on systemic coagulation.

[Establishment of a rat model of bone marrow mesenchymal stem cell transplantation for repairing full-thickness skin defect].

OBJECTIVE: To establish a rat model of full-thickness skin defect to receive bone marrow mesenchymal stem cell transplantation for wound repair. METHODS: A full-thickness skin defect measuring 4 cmx4 cm in 36 F344 rats, which were divided into 3 groups with the wound covered with alloskin graft, acellular dermal matrix, or petrolatum gauze. In vitro cultured BMSCs in the 5th passage were transplanted into the skin defect, and the time of wound dressing dissociation and number of transplanted Brdu-positive cells in the wound were observed 14 days later. RESULTS: The alloskin graft resulted in significantly longer time before dressing dissociation, with greater number of Brdu-positive cells in the wound than the other two wound dressings (P<0.001). The acellular dermal matrix showed better effect than petrolatum gauze in terms of the dressing dissociation time and the viable transplanted cell number in the wound. CONCLUSION: Alloskin graft can be ideal for covering the wound surface to protect the transplanted BMSCs in rats.

Normal skin and hypertrophic scar fibroblasts differentially regulate collagen and fibronectin expression as well as mitochondrial membrane potential in response to basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) regulates skin wound healing; however, the underlying mechanism remains to be defined. In the present study, we determined the effects of bFGF on the regulation of cell growth as well as collagen and fibronectin expression in fibroblasts from normal human skin and from hypertrophic scars. We then explored the involvement of mitochondria in mediating bFGF-inducedeffects on the fibroblasts. We isolated and cultivated normal and hypertrophic scar fibroblasts from tissue biopsies of patients who underwent plastic surgery for repairing hypertrophic scars. The fibroblasts were then treated with different concentrations of bFGF (ranging from 0.1 to 1000 ng/mL). The growth of hypertrophic scar fibroblasts became slower with selective inhibition of type I collagen production after exposure to bFGF. However, type III collagen expression was affected in both normal and hypertrophic scar fibroblasts. Moreover, fibronectin expression in the normal fibroblasts was up-regulated after bFGF treatment. bFGF (1000 ng/mL) also induced mitochondrial depolarization in hypertrophic scar fibroblasts (P < 0.01). The cellular ATP level decreased in hypertrophic scar fibroblasts (P < 0.05), while it increased in the normal fibroblasts following treatment with bFGF (P < 0.01). These data suggest that bFGF has differential effects and mechanisms on fibroblasts of the normal skin and hypertrophic scars, indicating that bFGF may play a role in the early phase of skin wound healing and post-burn scar formation.

[Effects of citrus reticulata blanco extract on fibroblasts from human hypertrophic scar in vitro].

OBJECTIVE: To investigate the effects of citrus reticulata blanco extract on the proliferation and collagen metabolism of fibroblasts from human hypertrophic scar. METHODS: Human hypertrophic scar fibroblasts from two burn patients obtained from plastic surgery were cultured in vitro and divided into experimental group (n = 12, with basic culture medium and 2.5, 5.0, 10.0,25.0 mg/L citrus reticulata blanco extract, respectively, 3 bottles for each concentration of citrus reticulata blanco extract ), control group 1 (n = 3, with basic culture medium) , and control group 2 ( n = 3, with basic culture medium and 5% ethyl alcohol). The cell proliferation in each group was observed with MTT method, then the inhibition rate was calculated. Apoptosis and its index ( AI) in each group were determined after TUNEL staining . The changes in the content of ICTP and PINP in each group were observed by radioimmunity. RESULTS: The inhibition rate in the experimental group with the citrus reticulata blanco extract in concentration of 2. 5, 5.0, 10.0, 25. 0 microg/ ml were (7. 100+/-0.038)% , (8. 100+/- 0. 048)% , (10. 900+/-0. 055)%, (15.900+/-0. 097) %, respectively, which were significantly higher than those in other two groups ( P <0.05 ). The Al (69. 7% , 71.7%, 86.4% , 95.2% ), ICTP [(17.2+/-0.6), (18.3+/-0.6), (19.8+/-0.5), (23.2+/-0.6) microg/L] and PINP [ (101.7+/-1.4) , (107. 8+/-1. 1) , (111.6+/-1.2) , (124. 6+/-1.3) microg/L] in experimental group with the citrus reticulata blanco extract in concentration of 2.5, 5.0, 10.0 , 25.0 mg/L were also obviously higher than other two control groups( P <0.05) ,but these indices in control 1 group were similar to those in control 2 group( P >0. 05). CONCLUSION: The citrus reticulata blanco extract might be beneficial for the management of hypertrophic scar through inhibition of the proliferation of fibroblasts in hypertrophic scar, by promoting apoptosis and collagen degradation.

[Comparison of the effects of rhEGF with rhbFGF on the acceleration of wound healing].

OBJECTIVE: To investigate the mechanism and the accelerating effect of rhEGF and rhbFGF on wound healing. METHODS: Twelve New Zealand rabbits with 72 incised wounds on ventral side of 24 ears were randomly divided into two therapeutic groups (rhEGF of 10 ug/cm(2) and rhbFGF of 100 AU/cm(2)) and a control group (1% silver sulfadiazine cream, SD-Ag). The general conditions of the wound healing was observed grossly. Biopsies were harvested at different time points for the pathomorphological examination, the electron microscopic examination, and for assessment of integrin beta1 mRNA expression by in situ hybridization. RESULTS: The expressions of integrin beta 1 mRNA in two therapeutic groups were significantly higher than that of control group. The quality of the wound healing was improved in therapeutic group with its healing time shortened when compared with that in control group (P < 0.05). There was an obvious difference in the number of fibroblasts and capillary gemmules between the therapeutic and control groups (P < 0.05). CONCLUSION: The wound healing and quality could be improved by both rhEGF and rhbFGF, but rhbFGF seemed better to be employed during the early and middle stages of the wound repair for the growth of granulation tissue, while rhEGF should be applied at the late stage of wound repair to accelerate the re-epithelialization of the wound. Combined application of rhEGF with rhbFGF according to time effect could be more beneficial to the wound repair.

[A preliminary study on the identification and distribution of epidermal stem cells in different degrees of burn wounds in scalded rats].

OBJECTIVE: To investigate the distribution of epidermal stem cells (ESCs) in different degrees of burn wounds in scalded rats. METHODS: Thirty-two Sprague-Dawley (SD) rats were employed in the study. First degree (I), shallow (shallow II) and deep partial thickness (deep II) and full thickness burn wounds (III) were created on the rat skin. Burn wound samples were harvested at 24 postburn hours (PBHs) from all the wounds and were processed to tissue slices. The tissue slices were stained by immunohistochemistry technique. The expression and distribution of ESCs in different degrees of burn wounds were observed with integrins alpha 2 beta 1 and keratin 10 (K10) as first antibodies. RESULTS: K10 positive cells were found to distribute in the strata spinosum, granulosum and lucidum in the first degree burn wound (I) with large amounts of integrins alpha 2 beta 1 positive cells in the residual basal layer and skin appendages (hair follicles) in shallow partial thickness burn wound (shallow II degree), and there were less integrins alpha 2 beta 1 positive cells in the remaining skin appendages in deep dermis in deep partial thickness burn wound (deep II degree). Finally, integrins alpha 2 beta 1 positive cells were sparsely found in the III degree burn wound. CONCLUSION: The distribution of ESCs in burn wounds was closely related to the depth of burn wound. The residual ESCs might be the origin of burn wound regeneration and reepithelization.