Previously, we found that treatment of cutaneous wounds with Atropa belladonna L. (AB) revealed shortened process of acute inflammation as well as increased tensile strength and collagen deposition in healing skin wounds (Gál et al. 2009). To better understand AB effect on skin wound healing male SpragueDawley rats were submitted to one round full thickness skin wound on the back. In two experimental groups two different concentrations of AB extract were daily applied whereas the control group remained untreated. For histological evaluation samples were removed on day 21 after surgery and stained for wide spectrum cytokeratin, collagen III, fibronectin, galectin-1, and vimentin. In addition, in the in vitro study different concentration of AB extract were used to evaluate differences in HaCaT keratinocytes proliferation and differentiation by detection of Ki67 and keratin-19 expressions. Furthermore, to assess ECM formation of human dermal fibroblasts on the in vitro level fibronectin and galectin-1 were visualized. Our study showed that AB induces fibronectin and galectin-1 rich ECM formation in vitro and in vivo. In addition, the proliferation of keratinocytes was also increased. In conclusion, AB is an effective modulator of skin wound healing. Nevertheless, further research is needed to find optimal therapeutic concentration and exact underlying mechanism of action., P. Gál ... [et al.]., and Obsahuje seznam literatury
The glycophenotyping of mammalian cells with plant lectins maps aspects of the glycomic profile and disease-associated alterations. A salient step toward delineating their functional dimension is the detection of endogenous lectins. They can translate sugar-encoded changes into cellular responses. Among them, the members of the lectin family of galectins are emerging regulators of cell adhesion, migration and proliferation. Focusing on galectins-1, -3 and -7, we addressed the issue whether their expression is regulated during wound healing in porcine skin as model. A conspicuous upregulation is detected for galectin-1 in the dermis and a neoexpression in the epidermis, where an increased level of galectin-7 was also found. Applying biotinylated tissue lectins as probes, the signal intensities for accessible binding sites decreased, intimating an interaction of the cell lectin with reactive sites. In contrast, galectin-3 parameters remained rather constant. Of note, epidermal cells in culture also showed an increase in expression/presence of galectin-1, measured on the levels of mRNA and protein, in this case by Western blotting and quantitative immunocytochemistry. Used as matrix, galectin-1 conferred resistance to trypsin treatment to attached human keratinocytes and reduced migration into scratch-wound areas in vitro. This report thus presents new information on endogenous lectins in wound healing and differential regulation among the three tested cases., J. Klíma ... [et al.]., and Obsahuje seznam literatury
Rapid wound closure in extensively burned patients has remained one of the major unresolved issues of medicine. Integra® is the most widely established artificial skin, which is composed of a porous matrix of cross-linked bovine collagen and chondroitin 6-sulphate covered by a semi-permeable silicone layer. We present here a (immuno)histological study of a severely burned patient with a full-thickness burn treated with a tissue-engineered dermal template (Integra®) and split-thickness skin graft-based protocol. Immunohistochemical investigation of the artificial dermis revealed that immune cell infiltration reached its peak on day 10. Tissue immunophenotyping found an increase in CD3+ cells over the course of the study as well as CD4 and CD8 positivity on day 40, indicating remaining T-cell subpopulations. We observed weak/no infiltration of NK cells (CD56+). In conclusion, the use of bi-layer Integra® represents a feasible and safe procedure resulting in formation of non-irritating dermal substitutes.
During the last decades, plant extracts containing phytoestrogens have increasingly been used as an alternative to oestradiol hormone replacement therapy. The aim of the present study was to compare the effects of genistein with those of different phytoestrogen-containing plant extracts (from red clover flowers and soybeans) on the proliferation and differentiation of NIH-3T3, HaCaT and MCF-7 cells. Our results showed poor correlations between direct anti/prooxidant effects and cytotoxicity of the tested samples. In contrast, genistein showed a direct correlation between significant pro-oxidative effects at cytotoxic concentrations and almost no pro-oxidative effects at non-cytotoxic concentrations. Moreover, the tested red clover extract and genistein induced keratin-8 (luminal and prognostic marker in breast cancer) expression only in MCF-7 cells, but this effect was not seen following treatment with the soybean extract. From this point of view, the effect of consumption of phytoestrogens in oestrogen-positive breast cancer remains to be elucidated. In conclusion, our study demonstrates that various phytoestrogen-containing plant extracts and genistein are able to specifically modulate antioxidant properties and differentiation of studied cells. and Corresponding author: Peter Gál or Ivana Šušaníková
Transforming growth factor beta 1 (TGF-β1) is a pro-fibrotic cytokine with a key role in wound repair and regeneration, including induction of fibroblast-to-myofibroblast transition. Genistein is a naturally occurring selective estrogen receptor modulator with promising anti-fibrotic properties. In the present study we aimed to investigate whether genistein modulates TGF-β1 (canonical and non-canonical) signaling in normal dermal fibroblasts at the protein level (Western blot and immunofluorescence). We demonstrated that TGF-β1 induces the myofibroblast-like phenotype in the studied fibroblast signaling via canonical (SMAD) and non-canonical (AKT, ERK1/2, ROCK) pathways. Genistein induced only ERK1/2 expression, whereas the combination of TGF-β1 and genistein attenuated the ERK1/2 and ROCK signaling. Of note, the other studied pathways remained almost unaffected. From this point of view, genistein does not impair conversion of normal fibroblasts to myofibroblast-like cells., Miriam Kaňuchová, Lukáš Urban, Nikola Melegová, Matúš Čoma, Barbora Dvořánková, Karel Smetana Jr., Peter Gál., and Obsahuje bibliografii
It has been shown previously that oestradiol protects the vascular network, leading to increased skin flap viability associated with Bcl-2, VEGF and FGF-2 up-regulation. We have shown that genistein, a natural selective oestrogen receptor modulator, also increases skin flap viability in rats and induces Bcl-2 expression in human umbilical vein endothelial cells. In the present study we aimed to answer the question whether genistein increases expression of Bcl-2, a potent anti-apoptotic protein, in human dermal microvascular endothelial cells (HMVEC-d) as well. Our results showed that administration of genistein induces Bcl-2 expression in a concentration-dependent manner. Cell co-treatment with genistein and anti-ER compounds (MPP, PHTPP, ICI, G-15) diminished the observed positive effect of genistein on Bcl-2 expression. The decrease in Bcl-2 expression in HMVEC-d was most prominent after co-treatment with ICI (nuclear ER antagonist/ GPR30 agonist) and PHTPP (selective ER-β antagonist). In conclusion, genistein increases Bcl-2 expression in HMVEC-d, contributing to its protective effect on the skin flap viability. However, the question whether the mechanism is ER-specific (via ER-β) has to be answered in further studies using a model of gene silencing or genetically modified cells.
Diabetic foot ulcer (DFU) is a serious complication of diabetes and hyperbaric oxygen therapy (HBOT) is also considered in comprehensive treatment. The evidence supporting the use of HBOT in DFU treatment is controversial. The aim of this work was to introduce a DFU model in ZDF rat by creating a wound on the back of an animal and to investigate the effect of HBOT on the defect by macroscopic evaluation, quantitative histological evaluation of collagen (types I and III), evaluation of angiogenesis and determination of interleukin 6 (IL6) levels in the plasma. The study included 10 rats in the control group (CONT) and 10 in the HBOT group, who underwent HBOT in standard clinical regimen. Histological evaluation was performed on the 18th day after induction of defect. The results show that HBOT did not affect the macroscopic size of the defect nor IL6 plasma levels. A volume fraction of type I collagen was slightly increased by HBOT without reaching statistical significance (1.35±0.49 and 1.94±0.67 %, CONT and HBOT, respectively). In contrast, the collagen type III volume fraction was ~120 % higher in HBOT wounds (1.41±0.81 %) than in CONT ones (0.63±0.37 %; p=0.046). In addition, the ratio of the volume fraction of both collagens in the wound ((I+III)w) to the volume fraction of both collagens in the adjacent healthy skin ((I+III)h) was ~65 % higher in rats subjected to HBOT (8.9±3.07 vs. 5.38±1.86 %, HBOT and CONT, respectively; p=0.028). Vessels density (number per 1 mm2 ) was found to be higher in CONT vs. HBOT (206.5±41.8 and 124±28.2, respectively, p<0.001). Our study suggests that HBOT promotes collagen III formation and decreases the number of newly formed vessels at the early phases of healing., Jiří Růžička, Martina Grajciarová, Lucie Vištejnová, Pavel Klein, Filip Tichánek, Zbyněk Tonar, Jiří Dejmek, Jiří Beneš, Lukáš Bolek, Robert Bajgar, Jitka Kuncová., and Obsahuje bibliografii
Chronic wound is a serious medical issue due to its high prevalence and complications; hyperbaric oxygen therapy (HBOT) is also considered in comprehensive treatment. Clinical trials, including large meta-analyses bring inconsistent results about HBOT efficacy. This review is summarizing the possible effect of HBOT on the healing of chronic wound models at the cellular level. HBOT undoubtedly escalates the production of reactive oxygen and nitrogen radicals (ROS and RNS), which underlie both the therapeutic and toxic effects of HBOT on certain tissues. HBOT paradoxically elevates the concentration of Hypoxia inducible factor (HIF) 1 by diverting the HIF-1 degradation to pathways that are independent of the oxygen concentration. Elevated HIF-1 stimulates the production of different growth factors, boosting the healing process. HBOT supports synthesis of Heat shock proteins (HSP), which are serving as chaperones of HIF-1. HBOT has antimicrobial effect, increases the effectiveness of some antibiotics, stimulates fibroblasts growth, collagen synthesis and suppresses the activity of proteolytic enzymes like matrix metalloproteinases. All effects of HBOT were investigated on cell cultures and animal models, the limitation of their translation is discussed at the end of this review