3D skin bioprinting as promising therapeutic strategy for radiation-associated skin injuries.

Both cutaneous radiation injury and radiation combined injury (RCI) could have serious skin traumas, which are collectively referred to as radiation-associated skin injuries in this paper. These two types of skin injuries require special managements of wounds, and the therapeutic effects still need to be further improved. Cutaneous radiation injuries are common in both radiotherapy patients and victims of radioactive source accidents, which could lead to skin necrosis and ulcers in serious conditions. At present, there are still many challenges in management of cutaneous radiation injuries including early diagnosis, lesion assessment, and treatment prognosis. Radiation combined injuries are special and important issues in severe nuclear accidents, which often accompanied by serious skin traumas. Mass victims of RCI would be the focus of public health concern. Three-dimensional (3D) bioprinting, as a versatile and favourable technique, offers effective approaches to fabricate biomimetic architectures with bioactivity, which provides potentials for resolve the challenges in treating radiation-associated skin injuries. Combining with the cutting-edge advances in 3D skin bioprinting, the authors analyse the damage characteristics of skin wounds in both cutaneous radiation injury and RCI and look forward to the potential value of 3D skin bioprinting for the treatments of radiation-associated skin injuries.

Ethylmalonic encephalopathy 1 initiates overactive autophagy in depleted uranium-induced cytotoxicity in the human embryonic kidney 293 cells.

The kidney is the target of the acute toxicity of depleted uranium (DU). However, the mechanism of DU-induced cytotoxicity is not clear. The study was to demonstrate the role of autophagy in DU-induced cytotoxicity and to determine the potential mechanism. We confirmed that after a 4-h exposure to DU, the autophagic vacuoles and the autophagy marker light chain 3-II in the human embryonic kidney 293 cells (HEK293) increased, and cytotoxicity decreased by abrogation of excessive autophagy using autophagy inhibitor. We also found activation of nucleus p53 and inhibiting mTOR pathways in DU-treated HEK293 cells. Meanwhile, ethylmalonic encephalopathy 1 (ETHE1) decreased as the exposure dose of DU increased, with increasing autophagy flux. We suggested that by reducing ETHE1, activation of the p53 pathway, and inhibiting mTOR pathways, DU might induce overactive autophagy, which affected the cytotoxicity. This study will provide a novel therapeutic target for the treatment of DU-induced cytotoxicity.

MiR-21 ameliorates age-associated skin wound healing defects in mice.

BACKGROUND: The cellular and molecular mechanisms responsible for the age-associated delay of cutaneous wound healing are still not well understood. Previous studies have shown that miR-21 plays key roles during skin wound healing. We presumed that dysregulation of miR-21 may be involved in age-associated defects in wound healing and that miR-21 may be one potential therapeutic target by which to ameliorate wound defects in elderly subjects. METHODS: Circular full thickness excisional wounds were made on the dorsal skin of young (2-month-old) and aged (12-month-old) female mice. The wound healing rates were quantified and compared between wild-type and miR-21 knock-in mice. Both histologic and morphometric analyses of the wounds were evaluated. Furthermore, the expression patterns of miR-21 during wound healing in both young and aged mice were assessed by in situ hybridization. The effects of topical miR-21 overexpression on wound healing in aged mice were estimated by both wound closure quantification and histological analyses. RESULTS: Aged miR-21 knock-in female mice showed significantly improved wound healing compared to their wild-type counterparts with respect to mature granulation tissue, smaller wound width and thinner epidermis. The expression patterns of miR-21 showed that miR-21 levels were insufficient for repairing granulation tissue in aged mice. Intradermal injection of miR-21 plasmid around wounds could upregulate miR-21 levels during wound healing and ameliorate age-associated skin wound defects. CONCLUSIONS: The results of the present study reveal that the upregulation of miR-21 levels could improve wound repair in aged mice, which suggests that a therapeutic strategy targeting miR-21 expression in age-associated wound healing may be feasible.

Downregulation of miR-205 in migrating epithelial tongue facilitates skin wound re-epithelialization by derepressing ITGA5.

Keratinocyte migration is essential for re-epithelialization during skin wound healing, but the molecular mechanisms regulating this cellular response remain to be completely clarified. Here we show that keratinocyte-specific miR-205 is significantly downregulated in the leading edge of the migrating epithelial tongue after skin injury in mice. In HaCaT keratinocytes, miR-205 could be downregulated by TGF-ß1 stimulation. And similar to the effect of TGF-ß1, miR-205 knockdown could promote keratinocyte migration in wound scratch model in vitro. Furthermore, topical inhibition of miR-205 by administrating Pluronic gel containing antagomir-205 could accelerate re-epithelialization in mouse skin wound model in vivo. Moreover, we identified integrin alpha 5 (ITGA5) as one key functional miR-205 target in the re-epithelialization process and epidermal downregulation of miR-205 may desilence ITGA5 to promote keratinocyte migration. And knockdown of ITGA5 would abolish the pro-migratory effects of miR-205 inhibition in vitro. What's more, we found dysregulation of miR-205 and its target ITGA5 in epidermis of clinical chronic wound samples with persistence of high level miR-205 and absence of ITGA5. Our findings indicate that downregulation of miR-205 in the leading migrating keratinocytes is critical for re-epithelialization and miR-205 may be a potential therapeutic target for chronic wounds.

Cell Density-Dependent Upregulation of PDCD4 in Keratinocytes and Its Implications for Epidermal Homeostasis and Repair.

Programmed cell death 4 (PDCD4) is one multi-functional tumor suppressor inhibiting neoplastic transformation and tumor invasion. The role of PDCD4 in tumorigenesis has attracted more attention and has been systematically elucidated in cutaneous tumors. However, the normal biological function of PDCD4 in skin is still unclear. In this study, for the first time, we find that tumor suppressor PDCD4 is uniquely induced in a cell density-dependent manner in keratinocytes. To determine the potential role of PDCD4 in keratinocyte cell biology, we show that knockdown of PDCD4 by siRNAs can promote cell proliferation in lower cell density and partially impair contact inhibition in confluent HaCaT cells, indicating that PDCD4 serves as an important regulator of keratinocytes proliferation and contact inhibition in vitro. Further, knockdown of PDCD4 can induce upregulation of cyclin D1, one key regulator of the cell cycle. Furthermore, the expression patterns of PDCD4 in normal skin, different hair cycles and the process of wound healing are described in detail in vivo, which suggest a steady-state regulatory role of PDCD4 in epidermal homeostasis and wound healing. These findings provide a novel molecular mechanism for keratinocytes' biology and indicate that PDCD4 plays a role in epidermal homeostasis.

Enhancement of antiviral activity of human alpha-defensin 5 against herpes simplex virus 2 by arginine mutagenesis at adaptive evolution sites.

Herpes simplex virus 2 (HSV-2) infection is still one of the common causes of sexually transmitted diseases worldwide. The prevalence of HSV strains resistant to traditional nucleoside antiviral agents has led to the development of novel antiviral drugs. Human alpha-defensin 5 (HD5), a kind of endogenous antimicrobial peptide expressed in the epithelia of the small intestine and urogenital tract, displays natural antiviral activity. Based on arginine-rich features and adaptive evolution characteristics of vertebrate defensins, we conducted a screen for HD5 derivatives with enhanced anti-HSV-2 activity by a single arginine substitution at the adaptive evolution sites. Cell protection assay and temporal antiviral studies showed that HD5 and its mutants displayed affirmatory but differential anti-HSV-2 effects in vitro by inhibiting viral adhesion and entry. Inspiringly, the E21R-HD5 mutant had significantly higher antiviral activity than natural HD5, which is possibly attributed to the stronger binding affinity of the E21R-HD5 mutant with HSV-2 capsid protein gD, indicating that E21R mutation can increase the anti-HSV-2 potency of HD5. In a mouse model of lethal HSV-2 infection, prophylactic and/or therapeutic treatment with E21R-HD5 via intravaginal instillation remarkably alleviated the symptoms and delayed disease progress and resulted in about a 1.5-fold-higher survival rate than in the HD5 group. Furthermore, the E21R variant exhibited a 2-fold-higher antiviral potency against HIV-1 over parental HD5 in vitro. This study demonstrates that arginine mutagenesis at appropriate evolution sites may significantly enhance the antiviral activity of HD5, which also paves a facile way to search for potent antiviral drugs based on natural antimicrobial peptides.

miR-21 regulates skin wound healing by targeting multiple aspects of the healing process.

With the clarification of the important roles of microRNAs (miRNAs) in diverse physiologic and pathologic processes, the effects of miRNAs in wound healing have attracted more attention recently. However, the global pattern of miRNA expression in wound tissue is still unknown. In the present study, we depicted the miRNA profile and identified at least 54 miRNAs, including miR-21, changed for more than twofold at the stage of granulation formation during wound healing. These miRNAs were closely related to the major events of wound healing, including cell migration and proliferation, angiogenesis, and matrix remolding. Furthermore, we found that miR-21 was up-regulated after skin injury, mainly in activated and migrating epithelial cells of epidermis and mesenchymal cells of dermis. Locally antagonizing miR-21 by directly injecting antagomir to wound edge caused significant delay of wound closure with impaired collagen deposition. Unexpectedly, we found wounds treated with miR-21 antagomir had an obvious defect in wound contraction at an early stage of wound healing. The significant role of miR-21 in wound contraction was further confirmed by in vivo gain-of-function and in vitro loss-of-function experiments. In conclusion, the present study has for the first time depicted miRNA profiling of wound healing and demonstrated the involvement of miR-21 in regulating the wound contraction and collagen deposition. These results suggest that miR-21 may be a new medical target in skin wound manipulation.

Rational and efficient preparation of a chimeric protein containing a tandem dimer of thrombopoietin mimetic peptide fused to human growth hormone in Escherichia coli.

The 14-mer thrombopoietin mimetic peptide (TMP), especially in the form of dimer, displayed potent megakaryocytopoiesis activity in vitro. However, it is difficult to prepare such short peptide with high bioactivity through gene-engineering approaches. In this study, a chimeric protein containing a tandem dimer of TMP (dTMP) fused to human growth hormone (hGH), a kind of hematopoietic growth factor that activates the same signal pathways as thrombopoietin, was produced in Escherichia coli by soluble expression. By rational utilization of the XmnI and EcoRV restriction sites, a PCR fragment encoding dTMP-GH was inserted into the plasmid vector pMAL-p2X at the position right after Xa factor cleavage site, in frame with maltose-binding protein (MBP) gene. Under optimized conditions, a high-level expression of soluble MBP-dTMP-GH fusion protein was obtained. By application of amylose resin chromatography, Xa factor digestion, hydrophobic chromatography followed by gel filtration, the dTMP-GH fusion protein was separated. Finally, a relatively high yield of dTMP-GH fusion protein with high purity (>98%) and without redundant amino acid was achieved, as identified by high-performance liquid chromatography, mass spectrometry, and amino acid sequencing. The functional assays showed that dTMP-GH could promote the proliferation of megakaryoblast cells and maturation of murine megakaryocytes derived from bone marrow, in a dose-dependent manner. Moreover, an enhanced effect of dTMP-GH on megakaryocytopoiesis was found as compared with equimolar concentration of dTMP and rhGH. This work provides a new avenue to generate thrombopoietic agents based on TMP.

Phosphate Metabolic Inhibition Contributes to Irradiation-Induced Myelosuppression through Dampening Hematopoietic Stem Cell Survival.

Myelosuppression is a common and intractable side effect of cancer therapies including radiotherapy and chemotherapy, while the underlying mechanism remains incompletely understood. Here, using a mouse model of radiotherapy-induced myelosuppression, we show that inorganic phosphate (Pi) metabolism is acutely inhibited in hematopoietic stem cells (HSCs) during irradiation-induced myelosuppression, and closely correlated with the severity and prognosis of myelosuppression. Mechanistically, the acute Pi metabolic inhibition in HSCs results from extrinsic Pi loss in the bone marrow niche and the intrinsic transcriptional suppression of soluble carrier family 20 member 1 (SLC20A1)-mediated Pi uptake by p53. Meanwhile, Pi metabolic inhibition blunts irradiation-induced Akt hyperactivation in HSCs, thereby weakening its ability to counteract p53-mediated Pi metabolic inhibition and the apoptosis of HSCs and consequently contributing to myelosuppression progression. Conversely, the modulation of the Pi metabolism in HSCs via a high Pi diet or renal Klotho deficiency protects against irradiation-induced myelosuppression. These findings reveal that Pi metabolism and HSC survival are causally linked by the Akt/p53-SLC20A1 axis during myelosuppression and provide valuable insights into the pathogenesis and management of myelosuppression.

Induced endothelial differentiation of cells from a murine embryonic mesenchymal cell line C3H/10T1/2 by angiogenic factors in vitro.

A murine embryonic mesenchymal cell line C3H/10T1/2 possesses the potential to differentiate into multiple cell phenotypes and has been recognized as multipotent mesenchymal stem cells, but no in vitro model of its endothelial differentiation has been established and the effect of angiogenic factors on the differentiation is unknown. The aim of the present study was to evaluate the role of angiogenic factors in inducing endothelial differentiation of C3H/10T1/2 cells in vitro. C3H/10T1/2 cells were treated with angiogenic factors, VEGF (10 ng/mL) and bFGF (5 ng/mL). At specified time points, cells were subjected to morphological study, immunofluorescence staining, RT-PCR, LDL-uptake tests and 3-D culture for the examination of the structural and functional characteristics of endothelial cells. Classic cobblestone-like growth pattern appeared at 6 day of the induced differentiation. Immunofluorescence staining and RT-PCR analyses revealed that the induced cells exhibited endothelial cell-specific markers such as CD31, von Willebrand factor, Flk1, Flt1, VE-cadherin, Tie2, EphrinB2 and Vezf1 at 9 day. The induced C3H/10T1/2 cells exhibited functional characteristics of the mature endothelial phenotype, such as uptake of acetylated low-density lipoproteins (Ac-LDL) and formation of capillary-like structures in three-dimensional culture. At 9 day, Weibel-Palade bodies were observed under a transmission electron microscope. This study demonstrates, for the first time, endothelial differentiation of C3H/10T1/2 cells induced by angiogenic factors, VEGF and bFGF, and confirms the multipotential differentiation ability. This in vitro model is useful for investigating the molecular events in endothelial differentiation of mesenchymal stem cells.

Detection of CD4+ CD25+ FOXP3+ regulatory T cells in peripheral blood of patients with chronic autoimmune urticaria.

BACKGROUND/OBJECTIVES: Compelling evidence indicates a significant role for a population of CD4(+) T regulatory cells in suppressing immune responses and in maintaining immunological homeostasis. This study aims to investigate the potential role of CD4(+) CD25(HIGH) FOXP3(+) T regulatory cells in patients with chronic autoimmune urticaria and to define the characteristics of CD4(+) CD25(HIGH) FOXP3(+) cells in chronic urticaria. METHODS: We used flow cytometry to assess the expression of CD4(+) CD25(HIGH) FOXP3(+) cells in the peripheral blood mononuclear cells of patients with chronic autoimmune urticaria. RESULTS: In this study, we found that patients with chronic autoimmune urticaria have a significantly reduced frequency of CD4(+) CD25(HIGH) FOXP3(+) cells (1.39 ± 0.27% vs 2.09 ± 0.34%; P = 0.001) in their peripheral blood, accompanied by a decreased intensity of FOXP3 expression (50.13 ± 9.79 vs 68.19 ± 6.40; P < 0.001). Notably, although patients with chronic idiopathic urticaria had a reduced frequency of CD4(+) CD25(HIGH) FOXP3(+) cells (1.85 ± 0.46% vs 3.64 ± 0.48%; P < 0.001), their FOXP3 expression levels did not differ from those in healthy controls. CONCLUSIONS: Patients with chronic autoimmune urticaria displayed a reduced percentage of CD4(+) CD25(+) FOXP3(+) regulatory T cells. The results imply CD4(+) CD25(+) FOXP3(+) regulatory T cells may contribute to the autoimmune pathological process of chronic autoimmune urticaria.

Chronic oral depleted uranium leads to reproductive damage in male rats through the ROS-hnRNP A2/B1-COX-2 signaling pathway.

Depleted uranium (DU) is widely used in civil and military activities. The testis is one of the target organs of DU chronic toxicity. In this study, male SD rats were chronically exposed to DU by 3, 30, 300 mg U/kg through oral intake. After 6 months and 12 months of exposure, it was found that DU could lead to increased oxidative stress levels, decreased glutathione S-transferases (GSTs) expression, resulting in testicular injury and decreased serum testosterone (T) level in rats. Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) expression increases with the increase of DU exposure dose. After upregulation of hnRNP A2/B1 expression, the GC-1 cell injury caused by DU is aggravated, suggesting that hnRNP A2/B1 may play an important role in the reproductive toxicity of DU. At the same time, 12 months after chronic oral exposure to DU, the expression level of cyclooxygenase-2 (COX-2) and proinflammatory factor prostaglandin E2 (PGE2) in testicular tissue were increased, and the level of hnRNP A2/B1 caused by DU was decreased by reactive oxygen scavenger N-acetylcysteine (NAC). As hnRNP A2/B1 is a COX-2 regulator, DU may lead to the upregulation of hnRNP A2/B1 expression through the increase of oxidative stress level in germ cells, which in turn leads to the increase of COX-2 and PGE2 level, and ultimately result in the reproductive toxicity. In this study, the regulation mechanism of the ROS-hnRNP A2/B1-COX-2 pathway on DU-induced reproductive damage in male rats was hypothesized, providing a new target for the prevention and treatment of chronic poisoning of DU.

Neutrophils-derived Spink7 as one safeguard against experimental murine colitis.

The uncontrolled abnormal intestinal immune responses play important role in eliciting inflammatory bowel disease (IBD), yet the molecular events regulating intestinal inflammation during IBD remain poorly understood. Here, we describe an endogenous, homeostatic pattern that controls inflammatory responses in experimental murine colitis. We show that Spink7 (serine peptidase inhibitor, kazal type 7), the ortholog of human SPINK7, is significantly upregulated in dextran sodium sulfate (DSS)-induced murine colitis model. Spink7-deficient mice showed highly susceptible to experimental colitis characterized by enhanced weight loss, shorter colon length, higher disease activity index and increased colonic tissue destruction. Bone marrow reconstitution experiments demonstrated that expression of Spink7 in the immune compartment makes main contribution to its protective role in colitis. What's more, neutrophils are the primary sources of Spink7 in experimental murine colitis. Loss of Spink7 leads to augmented productions of multiple chemokines and cytokines in colitis. In summary, this study identifies neutrophils-derived endogenous Spink7-mediated control of chemokines/cytokines production as a molecular mechanism contributing to inflammation resolution during colitis.

Bone marrow derivation of interstitial cells of cajal in small intestine following intestinal injury.

Interstitial cells of Cajal (ICCs) in gastrointestinal tract are specialized cells serving as pacemaker cells. The origin of ICCs is currently not fully characterized. In this work, we aimed to study whether bone marrow-derived cells (BMDCs) could contribute to the origin of ICCs in the muscular plexus of small intestine using GFP-C57BL/6 chimeric mice.Engraftment of BMDCs in the intestine was investigated for GFP expression. GFP positive bone marrow mononuclear cells reached a proportion of 95.65% +/- 3.72% at different times in chimerism. Donor-derived cells distributed widely in all the layers of the gastrointestinal tract. There were GFP positive BMDCs in the myenteric plexus, which resembled characteristics of ICCs, including myenteric location, c-Kit positive staining, and ramified morphology. Donor-derived ICCs in the myenteric plexus contributed to a percentage ranging 9.25% +/- 4.9% of all the ICCs in the myenteric plexus. In conclusion, here we described that donor-derived BMDCs might differentiate into gastrointestinal ICCs after radiation injury, which provided an alternative source for the origin of the ICCs in the muscular plexus of adult intestine. These results further identified the plasticity of BMDCs and indicated therapeutic implications of BMDCs for the gastrointestinal dysmotility caused by ICCs disorders.

Long-term repopulation effects of donor BMDCs on intestinal epithelium.

BACKGROUND: Bone marrow-derived cells (BMDCs) have the ability to differentiate into intestinal epithelial cells after transplantation and participate in the regeneration process of damaged epithelium. AIMS: To investigate whether BMDCs could differentiate into intestinal epithelium long term in chimeric mice after transplantation and without special treatment. METHODS: Forty irradiated C57BL/6 mice were used. Thirty of them (group A) received transplantation of BMDCs from GFP transgenic mice, and ten (group B) received PBS. The chimeric percentage at the 14th month was examined by flow cytometry. Engraftment of BMDCs was detected by immunohistochemistry in intestinal epithelium. Immunofluorescence observation was used to detect coexpression of PCK, CD45 and Chromogranin A with GFP. BMDCs in the epithelium were observed by an immune electron microscope. The percent of GFP(+) epithelial cells was also determined by flow cytometry. RESULTS: Mice in group A had a survival rate of 93.3% 1 week after transplantation. BMDCs could engraft into recipients' intestinal epithelium. These cells expressed epithelial cell marker PCK, but could not express CD45. Some of them differentiated into enteroendocrine cells expressing Chromogranin A. GFP(+) villous epithelial cells ranged from 9.41 to 16.07% in different subgroups of group A. BMDCs in epithelium developed the characteristics of enterocytes and goblet cells. GFP(+)/PCK(+) epithelial cells at the 6th month made up a proportion of 16.11% among all the isolated epithelial cells. CONCLUSIONS: Long term, BMDCs could repopulate recipient's intestinal epithelium even without any special treatment, which suggests a novel insight into the maintenance of the intestinal epithelial constitution.

High efficiency preparation of bioactive human alpha-defensin 6 in Escherichia coli Origami(DE3)pLysS by soluble fusion expression.

Human alpha-defensin 6 (HD(6)), a small cysteine-rich cationic peptide specially expressed in epithelial cells of digestive tract, may play a crucial role in mucosal immunity. This is the first report on efficient production of bioactive HD(6) through a gene-engineering approach in Escherichia coli. The recombinant plasmid pET32a-omHD(6) was primarily constructed by inserting a PCR fragment encoding mature HD(6) peptide (mHD(6)) preceded by an enterokinase recognition sequence into the expression vector pET32a(+), in frame with the upstream thioredoxin (TrxA) gene. Under optimized expression conditions, a high percentage (>60%) of soluble TrxA-omHD(6) fusion protein was obtained with a yield of about 1.69 g/l, and the theoretical productivity of recombinant mHD(6) (rmHD(6)) reached 0.38 g/l. A feasible three-step purification strategy involving nickel-sepharose chromatography, enterokinase-cleavage and cation exchange chromatography was developed to purify rmHD(6), followed by characteristic identifications by Western blot, mass spectrometry and sequencing. About 102 mg/l of rmHD(6) with its intact N-terminal amino acid sequence was finally achieved. The in vitro experiments showed that rmHD(6) possesses high potency to inhibit herpes simplex virus-2 infection. This work settles substantial foundation for further functional study of HD(6).

A review of biological effects and treatments of inhaled depleted uranium aerosol.

Depleted uranium (DU) is primarily used for DU bombs and DU tanks in the military. Aerosol inhalation is considered the primary route of DU exposure. Although laboratory tests have confirmed that inhalation of DU aerosol can cause lung, kidney, and other organ damage, epidemiological studies have found no conclusive evidence that persons in areas with prolonged exposure to DU-containing bombs are affected. After the body inhaled DU aerosols, we first clear the insoluble DU through whole-lung lavage (WLL). Then we eliminate the soluble uranium by the chelating agent. Besides, reducing DU damage to tissues and cells through drugs is also an important treatment method. In future research, emphasis should be placed on the damage mechanism of DU aerosol, the laboratory and clinical research of DU chelating agents, the research on the combination of DU chelating agent and WLL, and the research and development of new drugs to prevent DU damage.

MicroRNA-34a deficiency leads to impaired wound closure by augmented inflammation in mice.

BACKGROUND: Proper inflammation resolution is critical for cutaneous wound healing and disordered inflammation resolution results in chronic nonhealing wounds. However, the cellular and molecular mechanisms for resolution of inflammation during skin wound healing are not well understood. MicroRNA-34a is regarded as one tumor suppressor with complexed immune regulatory effects, yet its role during skin wound repair is still unclear. METHODS: Circular full thickness excisional wounds were made on the dorsal skin of C57 mice and miR-34a expression pattern was examined by real time RT-PCR and in situ hybridization. The wound healing rates and histologic morphometric analysis were quantified and compared between wounds treated with antagomir-34a and autologous control antagomir-NC wounds, as well as wounds between miR-34a knockout (KO) and wild type (WT) mice. Immunohistochemistry (IHC) for both MPO and F4/80 were performed to examine the infiltrative neutrophils and macrophages in wounds from miR-34a KO and WT mice. Cytokines including IL-1ß, IL-6, TNF-α and IL-10, were detected and analyzed by real time RT-PCR during wound healing. IHC for IL-6 and p-STAT3 were quantified, and WB for p-STAT3 and IL-6R were examined in wounds of miR-34a KO and WT mice. RESULTS: We found miR-34a was significantly downregulated in the inflammatory phase and back to normal levels in the proliferative phase. Both topical knockdown wounds miR-34a levels by antagomir gel and systematic knockout miR-34a using KO mice resulted in impaired wound healing with delayed re-epithelialization and augmented inflammation. IHC results indicated that there were more residual infiltrative inflammatory cells in the proliferative phase. Moreover, over-activated IL-6/STAT3 signal pathway was identified in the wounds of miR-34a KO mice. CONCLUSIONS: Our findings reveal that miR-34a deficiency augments skin wound inflammation response and leads to impaired wound healing, which suggest that targeted inhibition of miR-34a for tissue repair/regeneration should be with serious consideration.

DNA synthesis of rat bone marrow mesenchymal stem cells through alpha1-adrenergic receptors.

Multipotential bone marrow mesenchymal stem cells (BMSCs) are important in maintaining the microenvironment of the bone marrow (BM). Sympathetic nerves histologically innervate the BM; however, their role remains unclear. In this study, the effects of norepinephrine on DNA synthesis and the related signaling molecules involved in rBMSCs were examined. mRNA levels of the alpha1-adrenergic receptor subtypes increased following norepinephrine stimulation (10(-5) M for 30 min). DNA synthesis increased in dose- and time-dependent manners as determined by [(3)H]thymidine incorporation. Intracellular Ca(2+) concentration and translocation of protein kinase C from the cytosol to the membrane were also found to be elevated in rBMSCs. Phentolamine was able to suppress translocation of PKC. Norepinephrine also induced phosphorylation of ERK1/2, which was prevented by staurosporine treatment. Pretreatment with PD98059 inhibited ERK1/2 phosphorylation and DNA synthesis in rBMSCs. These findings indicate that norepinephrine stimulates DNA synthesis via alpha1-adrenergic receptors and downstream Ca(2+)/PKC and ERK1/2 activation in rBMSCs.

Protective effect of an extract from Periplaneta americana on hematopoiesis in irradiated rats.

PURPOSE: To investigate the protective effect of W(11)-a(12), an extract from Periplaneta americana, on hematopoiesis in irradiated rats. MATERIALS AND METHODS: Wistar rats receiving total body irradiation of (60)Co gamma-rays alone or with combined radiation and skin wound injury were used in this study. W(11)-a(12) was applied either topically into the skin wounds or systemically by intraperitoneal injection. The numbers of white blood cells in peripheral blood, the nucleated cells and the colony-forming unit of granulocyte/macrophage progenitors (CFU-GM) in bone marrow were measured, respectively. RESULTS: Topical application of W(11)-a(12) into skin wounds in rats with combined 6 Gy total body irradiation and skin wound injury could increase the neutrophils and macrophages in the wounded area and the nucleated cells in bone marrow at 24 h and 48 h, while the peripheral white blood cells did not show significant change. However, in rats with 4 Gy total body irradiation alone, the peripheral white blood cells, bone marrow nucleated cells and the number of colony-forming unit of granulocyte-macrophage progenitors were all significantly higher in the treatment groups by intraperitoneal injection of W(11)-a(12) than those in the control groups by injection of normal saline at days 3 and days 5 after radiation. CONCLUSIONS: W(11)-a(12) showed a protective effect on hematopoiesis after total body irradiation and could increase the inflammatory cells in wounded tissues at the initiation stage after irradiation, which will benefit the management of combined radiation and skin wound injury.