Citrus pectin protects mice from burn injury by modulating intestinal microbiota, GLP-1 secretion and immune response.

Water-soluble rhamnogalacturonan-I enriched citrus pectin (WRP) has promising effect on antimicrobial defense. We aim to determine whether the modified acidic (A) or neutral (B) WRP solutions can improve intestinal microbial dysbiosis in burn-injured mice. Male Balb/c mice were gavaged with WRPs at 80, 160, 320 mg/kg. Body weight daily for 21 days before exposed to thermal injury of 15 % total body surface area and mortality was monitored. Mice with 80 mg/kg WRPs were also subjected to fecal DNAs and T cell metabonomics analysis, intestinal and plasma glucagon-like peptide 1 (GLP-1) detection, plasma defensin, immunoglobin and intestinal barrier examinations at 1 and 3d postburn (p.b.). Burn-induced mortality was only improved by low dose WRP-A (P = 0.039). Both WRPs could prevent the dysbiosis of gut microbiota in burn injury by reducing the expansion of inflammation-promoting bacteria. Both WRPs suppressed ileum GLP-1 production at 1d p.b. (P = 0.002) and plasma GLP-1 levels at 3d p.b. (P = 0.013). Plasma GLP-1 level correlated closely with ileum GLP-1 production (P = 0.019) but negatively with microbiota diversity at 1d p.b. (P = 0.003). Intestinal T cell number was increased by both WRPs in jejunum at 3d p.b. However, the exaggerated splenic T cell metabolism in burn injury was reversed by both WRPs at 1d p.b. The burn-increased plasma defensin ß1 level was only reduced by WRP-B. Similarly, the intestinal barrier permeability was only rescued by WRP-B at 1d p.b. WRP-A rather than WRP-B could reduce burn-induced mortality in mice by suppressing intestinal GLP-1 secretion, restoring gut microbiota dysbiosis and improving adaptive immune response.

Effects of Blast Wave-induced Biomechanical Changes on Lung Injury in Rats.

OBJECTIVE: To observe the dynamic impacts of shock waves on the severity of lung injury in rats with different injury distances. METHODS: Simulate open-field shock waves; detect the biomechanical effects of explosion sources at distances of 40, 44, and 48 cm from rats; and examine the changes in the gross anatomy of the lungs, lung wet/dry weight ratio, hemoglobin concentration, blood gas analysis, and pathology. RESULTS: Biomechanical parameters such as the overpressure peak and impulse were gradually attenuated with an increase in the injury distance. The lung tissue hemorrhage, edema, oxygenation index, and pathology changed more significantly for the 40 cm group than for the 44 and 48 cm groups. The overpressure peak and impulse were significantly higher for the 40 cm group than for the 44 and 48 cm groups ( P < 0.05 or P < 0.01). The animal mortality was significantly higher for the 40 cm group than for the other two groups (41.2% vs. 17.8% and 10.0%, P < 0.05). The healing time of injured lung tissues for the 40 cm group was longer than those for the 44 and 48 cm groups. CONCLUSIONS: The effects of simulated open-field shock waves on the severity of lung injuries in rats were correlated with the injury distances, the peak overpressure, and the overpressure impulse.

Usage of density analysis based on micro-CT for studying lung injury associated with burn-blast combined injury.

BACKGROUND: Burn-blast combined injury is a kind of injury caused by heat and blast at the same time. The lung injury after burn-blast combined injuries is of primary importance, and investigation of lung injury is needed in the clinical care of patients. Computed tomography (CT) is one of the standard tools used to observe the anatomical basis and pathophysiology of acute lung injury. METHODS: We applied a method of fast 3D (three-dimensional) reconstruction to calculate the density value of the lung injury by CT analysis. Blast-injury group (BL group), burn-injury group (B group), burn-blast combined injury group (BBL group), and sham control group (C group) were established. Each group had 16 rats. The three-dimensional images of the lung tissue were obtained at 6h, 24h, and 48h according to the CT value. The average density of the whole lung, left lung, and right lung were measured. The lung tissues were paraffin-embedded and HE stained. Smith scoring was performed according to the pathological findings. RESULTS: In the BBL group, the density of the lung tissue was higher than those of the BL group and B group (P<0.01). The lung tissue density values at 24h after injury were higher than those at 6h and 48h after injury (P<0.01). Pathological results confirmed the changes of density analysis of the lung tissue. CONCLUSION: The results have indicated that density analysis through a CT scan can be used as a way to evaluate lung injury in a burn-blast injury.

Transdermal permeation of drugs with differing lipophilicity: Effect of penetration enhancer camphor.

The aim of the present study was to investigate the potential application of (+)-camphor as a penetration enhancer for the transdermal delivery of drugs with differing lipophilicity. The skin irritation of camphor was evaluated by in vitro cytotoxicity assays and in vivo transdermal water loss (TEWL) measurements. A series of model drugs with a wide span of lipophilicity (logP value ranging from 3.80 to -0.95), namely indometacin, lidocaine, aspirin, antipyrine, tegafur and 5-fluorouracil, were tested using in vitro transdermal permeation experiments to assess the penetration-enhancing profile of camphor. Meanwhile, the in vivo skin microdialysis was carried out to further investigate the enhancing effect of camphor on the lipophilic and hydrophilic model drugs (i.e. lidocaine and tegafur). SC (stratum corneum)/vehicle partition coefficient and Fourier transform infrared spectroscopy (FTIR) were performed to probe the regulation action of camphor in the skin permeability barrier. It was found that camphor produced a relatively low skin irritation, compared with the frequently-used and standard penetration enhancer laurocapram. In vitro skin permeation studies showed that camphor could significantly facilitate the transdermal absorption of model drugs with differing lipophilicity, and the penetration-enhancing activities were in a parabola curve going downwards with the drug logP values, which displayed the optimal penetration-enhancing efficiency for the weak lipophilic or hydrophilic drugs (an estimated logP value of 0). In vivo skin microdialysis showed that camphor had a similar penetration behavior on transdermal absorption of model drugs. Meanwhile, the partition of lipophilic drugs into SC was increased after treatment with camphor, and camphor also produced a shift of CH2 vibration of SC lipid to higher wavenumbers and decreased the peak area of the CH2 vibration, probably resulting in the alteration of the skin permeability barrier. This suggests that camphor might be a safe and effective penetration enhancer for transdermal drug delivery.

Lipopolysaccharide pretreatment inhibits LPS-induced human umbilical cord mesenchymal stem cell apoptosis via upregulating the expression of cellular FLICE-inhibitory protein.

Mesenchymal stem cell (MSC)-based regenerative therapy is currently regarded as a novel approach with which to repair damaged tissues. However, the efficiency of MSC transplantation is limited due to the low survival rate of engrafted MSCs. Lipopolysaccharide (LPS) production is increased in numerous diseases and serves an essential function in the regulation of apoptosis in a variety of cell types. Previous studies have indicated that low-dose LPS pretreatment contributes to cytoprotection. In the current study, LPS was demonstrated to induce apoptosis in human umbilical cord mesenchymal stem cells (hUCMSCs) via the activation of caspase, in a dose-dependent manner. Low-dose LPS pretreatment may protect hUCMSCs against apoptosis induced by high-dose LPS, by upregulating the expression of cellular FADD-like IL-1ß-converting enzyme-inhibitory protein (c-FLIP). The results of the present study indicate that pretreatment with an appropriate concentration of LPS may alleviate high-dose LPS-induced apoptosis.

[Comparison of different methods for the isolation of human umbilical cord mesenchymal stem cells].

OBJECTIVE: To explore the most appropriate method for the isolation of human umbilical cord mesenchymal stem cells (MSCs) through a comparison of different methods. METHODS: Fifteen umbilical cord specimens from full-term healthy fetus with caesarean birth were completely rinsed with phosphate buffer saline (PBS) and sliced into 1 mm(3) tissue blocks after removal of umbilical vessels and external membrane. These tissue blocks were averagely divided into 4 groups after washing and centrifuge. Then four methods for the isolation of human umbilical cord MSCs were compared: an explant culture and three enzymatic methods of collagenaseII, collagenaseII/trypsin and collagenaseII/hyaluronidase. The count of living cells was evaluated by trypan blue dye exclusion test. Cell morphology was observed under inverted microscope. The expressions of cell surface markers CD105, CD90, CD73, CD31, CD44, CD45, human leukocyte antigen-I (HLA-I) and human leukocyte antigen class IImolecules (HLA-DR) were detected by immunofluorescent staining. Cell proliferation was assayed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). RESULTS: The human umbilical cord MSCs were successfully isolated by four isolated methods. However the isolation method used profoundly altered the cell number and proliferation capacity of isolated cells. Isolated cells using four methods were counted at (5.44 ± 0.21)×10(5), (4.03 ± 0.24)×10(5), (4.91 ± 0.33)×10(5) and (5.94 ± 0.40)×10(5) respectively. More cells were obtained with collagenaseII/hyaluronidase than other three methods (all P < 0.05). Cells out of tissue blocks were observed at Day 9-11 and cells were observed at Day 2 with three types of enzyme digestion. The fusion time of cells were (18.5 ± 3.5), (8.0 ± 1.0), (7.5 ± 1.5) and (3.5 ± 0.5) days respectively. The fusion time of cells obtained with collagenaseII/hyaluronidase was lower than other methods (all P < 0.05). Cell morphology: polygonal, irregular and of large volume for explant culture; relatively short and small for collagenaseII and collagenaseII/trypsin methods; thin spindle for collagenaseII/hyaluronidase method. Immunofluorescent staining revealed that CD105, CD73, CD90 and CD44 were expressed in all groups while there was no expression of CD31, CD45 or HLA-DR. And the cells obtained with collagenaseII/hyaluronidase method were in a higher cell proliferation rate and activity compared to other methods. CONCLUSION: The collagenaseII/hyaluronidase method is optimal for the isolation of human umbilical cord MSCs than other methods.

Basic fibroblast growth factor/vascular endothelial growth factor in the serum from severe burn patients stimulates the proliferation of cultured human umbilical cord mesenchymal stem cells via activation of Notch signaling pathways.

BACKGROUND: Mesenchymal stem cells (MSCs) are the leading cellular constituents used in regenerative medicine. MSCs repair and reconstruct wounds of acute traumata and radiation-induced burns through proliferation, differentiation, and trophic activity. However, repair effect of MSCs on severe burn wounds remain to be clarified because severe burns are much more complex traumata than radiation-induced burns. Survival and proliferation of MSCs in microenvironments affected by severe burns are very important for improving wound repair/regeneration. This study aimed to elucidate the survival and proliferation effects and the potential proliferation mechanism of serum from severe burn patients (BPS) on human umbilical cord MSCs (hUCMSCs) in vitro. METHODS: The hUCMSCs were isolated, cultured, and identified. Next, we evaluated the effects of BPS on cell numbers, cell cycle progression, cyclin D expression, and key proteins and genes of the Notch signaling pathway. Putative mechanisms underlying the proliferation of hUCMSCs were investigated. RESULTS: BPS markedly increased the number of hUCMSCs, and the results of the cell cycle studies indicated that BPS induced cell cycle progression into the M phase. Cyclin D expression was higher with BPS than in the control group. Moreover, Notch-1, a key determinant of hUCMSC activation and proliferation, and its target gene Hes-1 were overexpressed after BPS treatment. Proliferation numbers of hUCMSC, rate of proliferation period (G2/M+S), and the expression of cyclin D, Notch-1, and Hes-1 were markedly decreased by Notch signaling inhibitors (DAPT/GSI). In the case of BPS, basic fibroblast growth factor and vascular endothelial growth factor were the key factors that promoted hUCMSC proliferation. CONCLUSION: This study provides novel evidence for the role of BPS in the survival and rapid proliferation of hUCMSCs and suggests that these cells could be used for cell therapy-based clinical applications for treating severe burns. Furthermore, hUCMSC proliferation was induced by basic fibroblast growth factor/vascular endothelial growth factor in BPS through activation of Notch signal.

[Effects on collagen I and III after transplantation of porcine acellular dermal matrix with hyaluronic acid].

OBJECTIVE: To investigate the changes of collagens I and III after the addition of hyaluronic acid in the transplantation of porcine acellular dermal matrix. METHODS: Full-thickness skin defects were created on the dorsa of Japanese white rabbits. And the rabbits were divided randomly into 3 groups: Group A (hyaluronic acid, porcine acellular dermal matrix plus thin skin autografts), Group B (porcine acellular dermal matrix plus thin skin autografts) and Group C (skin autografts). Skin biopsies were performed at Day 50 post-grafting to detect the contents of collagens I and III by histological examinations, immunohistochemistry method and Western blot. RESULTS: The areas of skin graft were (13.3 ± 1.2), (9.5 ± 0.9) and (10.0 ± 1.4) cm(2) in Groups A, B and C respectively. Group A was larger than Groups B and C(all P < 0. 01). There was no statistical difference between Groups B and C (P > 0.05). The expressions of collagen I were 1894 ± 164, 515 ± 38 and 395 ± 43 in Groups A, B and C respectively. Group A was higher than Groups B and C (P < 0.01). And the expressions of collagen III were 5411 ± 435, 874 ± 70 and 2078 ± 175 in Groups A, B and C respectively. Group C was higher than Group B and yet lower than Group A (all P < 0.01). The ratios of collagen I and collagen III in Group A (0.39) and Group B (0.59) were higher than that of Group C (0.19) (all P < 0.01). CONCLUSION: The addition of hyaluronic acid may boost the expression of collagens I and III and decrease the ratio of collagen I/collagen III. Thus it facilitates wound healing and basilar membrane remodeling and alleviates the contraction of skin transplant.

[Growth and migration of umbilical cord mesenchymal stem cells on polycarbonate membrane with different pore sizes].

OBJECTIVE: To observe the growth and migration of human umbilical cord mesenchymal stem cells (hUCMSCs) on polycarbonate membrane with different pore sizes and explore the criteria of selecting optimal Transwell insert for indirect co-culture to induce the differentiation of hUCMSCs. METHODS: hUCMSCs were isolated in vitro and then expanded in culture medium. After the treatment of mitomycin C, the cells were seeded on porous membranes of 6-well-dish Transwell inserts with different pore sizes of 0.4, 3.0 and 8.0 µm respectively. After culturing for 7 days, the cells were observed and counted on the bottom of each porous membrane. Then the calculation of migration ratio was performed. The growth and migration of hUCMSCs on porous membranes were also examined under scanning electron microscope (SEM). RESULTS: The migration ratios of hUCMSCs on membranes of 0.4, 3.0 and 8.0 µm pore sizes were 0, 1.8% and 8.0% respectively. The migration ratio of cells on 0.4 µm pore size membrane was statistically different from that of the other two pore size groups (P < 0.01). Under SEM, a small portion of cells were growing on the bottoms of membranes and moving through the pores. But there was no cell movement through 0.4 µm pore size membrane. CONCLUSIONS: hUCMSCs can migrate through the polycarbonate membranes of 3.0 µm and 8.0 µm pore sizes but not through the 0.4 µm one. Thus both sides of polycarbonate membrane of 0.4 µm pore size may be used for close indirect co-culture to induce the differentiation of hUCMSCs.

[Influence of Aloe polysaccharide on proliferation and hyaluronic acid and hydroxyproline secretion of human fibroblasts in vitro].

OBJECTIVE: To investigate the effect of Aloe polysaccharide on proliferation and hyaluronic acid and hydroxyproline secretion of human fibroblasts in vitro. METHODS: The fibroblasts were treated with different doses of polysaccharide (0, 25, 50, 100, 200, 400 mg/L). Subsequently, cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, cell cycle by flow cytometry, evaluation of the Aloe polysaccharide toxic effect by acridine orange-ethidium bromide staining, evaluation of the cell injury by lactate dehydrogenase (LDH) assay, and the collagen synthesis by (3)H-proline incorporation. In addition, hyaluronic acid and hydroxyproline levels in the supernatants of cultured fibroblasts were measured by enzyme-linked immunosorbent assay. RESULTS: The proliferation of fibroblasts was induced with polysaccharide in a dose-dependent manner, reaching its highest level on 5th day. Meanwhile, the percentage of cells at phase G(0)/G(1) was decreased, while that at phases G(2)/M and S was increased significantly in Aloe polysaccharide-treated groups as compared with those in the control group (P<0.05). Additionally, the apoptosis of the fibroblasts showed no differences among all groups. The collagen synthesis was increased and cell injury decreased in polysaccharide-treated groups as compared with those in control group (P<0.05), while the levels of hyaluronic acid and hydroxyproline in the supernatants of fibroblasts treated with polysaccharide were significantly higher than those in the control group (P<0.05). CONCLUSION: The Aloe polysaccharide promotes both the proliferation of fibroblasts and the production of hyaluronic acid and hydroxyproline in fibroblasts. This indicates that the Aloe polysaccharide may play an important role in the extracellular matrix remodeling during wound healing.