MiR-211 regulates cutaneous wound healing through inhibiting inflammatory reactions and oxidative stress by binding SOX11
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Introduction: Loss of skin integrity due to a wound or disease can lead to severe disability or even life threat. The highly expressed microRNAs in the skin are of great significance for skin development. The investigation purposed to explore the effect and mechanism of miR-211 on inflammation, oxidative stress and migration in keratinocytes. Methods: The HaCaT keratinocytes were treated with hydrogen peroxide (H2O2) to establish a wound-healing model. The expression of miR-211 was examined by quantitative real-time PCR (qRT-PCR). The cell function was reflected in proliferative ability, migration, apoptosis, and inflammation, which were evaluated by Cell Counting Kit-8 (CCK-8) assay, Transwell test, flow cytometry technique, and enzyme-linked immunosorbent assay (ELISA). The target of miR-211 was verified by luciferase luminescence measurements. Results: H2O2 inhibited HaCaT cell proliferation, migration, and promoted cell apoptosis, accompanied with the downregulation of miR-211. H2O2 led to inflammatory response and oxidative damage to HaCaT. MiR-211 promoted proliferation and migration, but improved cell apoptosis of HaCaT. The role of H2O2 on inflammatory response and oxidative stress was alleviated by miR-211. SRY-box transcription factor 11 (SOX11) was a targeted mediator of miR-211. SOX11 reversed the influence of miR-211 on cell proliferation, migration, apoptosis, inflammatory response and oxidative stress. Conclusion: MiR-211 regulated proliferation, migration, apoptosis, inflammation and oxidative stress of keratinocytes by mediating SOX11, thus participating in cutaneous wound healing.Keywords:
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Hepatocyte growth factor-like protein (HGFl) and its receptor, Recepteur d'Origine Nantais (RON), have been implicated in the development of wound chronicity. HGFl and RON expression was detected in acute wound tissue, chronic wound tissue and in normal skin using quantitative polymerase chain reaction (Q-PCR). HGFl and RON expression was also assessed in chronic healing and chronic non-healing wound tissues using Q-PCR and immunohistochemical staining. Expression was similarly detected in the HaCaT immortalized human keratinocyte cell line using reverse transcription polymerase chain reaction (RT-PCR). rhHGFl was used to assess the impact of this molecule on HaCaT cell functionality using in vitro growth assays and electric cell-substrate impendence sensing (ECIS) migration assays. HGFl and RON transcript expression were significantly increased in acute wound tissue compared to chronic wound tissue and were also elevated, though non-significantly, in comparison to normal skin. Minimal expression was seen in both healing and non-healing chronic wounds. Treatment of HaCaT cells with rhHGFl had no effect on growth rates but did enhance cell migration. This effect was abolished by the addition of a phospholipase C gamma (PLCγ) small molecule inhibitor. The increased expression of HGFl and RON in acute, healing wounds and the pro-migratory effect of HGFl in an in vitro human keratinocyte model, may indicate a role for HGFl in active wound healing.
HaCaT
Chronic wound
Human skin
Keratinocyte growth factor
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Proliferation and migration of keratinocytes are vital processes for the successful epithelization specifically after wounding. MiR-221 has been identified to play a potential role in promoting wound regeneration by inducing blood vessel formation. However, little is known about the role of miR-221 in the keratinocyte proliferation and migration during wound healing. An in vivo mice wound-healing model was generated; the expression levels of miR-221 were assessed by qRT-PCR and fluorescence in situ hybridization. Initially, we found that miR-221 was upregulated in the proliferative phase of wound healing. Further, in an in vivo wound-healing mice model, targeted delivery of miR-221 mimics accelerated wound healing. Contrastingly, inhibition of miR-221 delayed healing. Additionally, we observed that overexpression of miR-221 promoted cell proliferation and migration, while inhibition of miR-221 had the opposite effects. Moreover, we identified SOCS7 as a direct target of miR-221 in keratinocytes and overexpression of SOCS7 reversed the effects of miR-221 in HaCaT keratinocytes. Finally, we identified that YB-1 regulates the expression of miR-221 in HaCaT keratinocytes. Overall, our experiments suggest that miR-221 is regulated by YB-1 in HaCaT keratinocytes and acts on SOCS7, thereby playing an important role in HaCaT keratinocyte proliferation and migration during wound healing.
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Previous studies have shown that the endogenous electric field (EF) is an overriding cure in guiding cell migration toward the wound center to promote wound healing, but the mechanism underlying is unclear. In this study, we investigated the molecular mechanism of electric field-guided cell migration in human keratinocyte HaCaT cells. Our results showed that HaCaT cells migrate toward the anode under EFs. The phosphorylation levels of p38 MAPK and Akt were obviously elevated in the EF. Knocking down p38 MAPK obviously abolished directed migration of HaCaT cells under the EFs. Inhibiting p38 MAPK by SB203580 impaired the EF-guided cell migration. The electric field may guide HaCaT cell migration through the EGFR/p38 MAPK/Akt pathway.
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Abstract Background Cell proliferation and death are key components of wound healing and tissue repair. Telocytes (TCs) represent a newly discovered cell type that can protect tissue from acute injury via cell–cell communication with adjacent cells. The aim of this study was to use a mouse model of skin wound healing and lipopolysaccharide (LPS)-induced cell injury to evaluate the effects of TCs on skin wound healing in vivo and in vitro. Material/methods Immunohistochemical staining was performed to evaluate the alteration of TCs in tissues from normal and chronic wound patients. Then, a male C57BL/6 mouse wound model of the back was established. The mice were divided randomly into three groups, and wound healing was estimated according to the wound healing rate and histology. An LPS-induced co-culture model of a mouse lung telocyte cell line (TCs) with human keratinocyte (HaCaT), human dermal microvascular endothelial cell (HDMEC) or murine fibroblast (L929) cell lines was established to analyse the effects of TCs on constitutive cell types of the skin. Cell proliferation, migration and apoptosis were examined, and reactive oxygen species (ROS) and inflammatory factors in HaCaT cells, HDMECs, and L929 cells were detected to study the mechanisms involved in TC protection in skin wounds. Results TCs were significantly increased in tissues from chronic wound patients compared with healthy controls. Wound healing was significantly improved in wound mouse models treated with exogenous TCs compared with LPS-induced models. TCs reversed the LPS-induced inhibition of HaCaT cells and HDMECs and reduced the LPS-induced apoptosis of HaCaT cells and the death ratios of HDMECs and L929 cells. TCs reversed LPS-induced ROS in HDMECs and L929 cells and decreased inflammatory factor mRNA levels in HaCaT cells, HDMECs and L929 cells. Conclusions TCs reduce wound healing delay, and inflammatory responses caused by LPS might be mediated by inflammatory inhibition, thus restricting apoptosis and promoting migration of the main component cell types in the skin.
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Wounds may be acute, subacute, or chronic and are the common clinical life entities. It is a complex biochemical process to restore the cell structures that depends upon cell proliferation and migration, mainly by keratinocytes and fibroblast cells. The aim of the present study was to evaluate the effect of the Biofield Energy Treatment (Consciousness Energy Healing Treatment-The Trivedi Effect®) in the HaCaT cell line (Human keratinocytes) and DMEM (Dulbecco’s Modified Eagle Medium) for wound healing action using scratch assay (48 hours) against positive control i.e., recombinant Human Epidermal Growth Factor (Hu-EGF, 30 ng/mL). In vitro scratch assay monitored proliferation and migration of keratinocytes in the scratched wounded area using WimScratch Image analysis software. The results showed that the Biofield Energy Treated DMEM group have significantly higher percentage of keratinocytes migration i.e., 35.4%, while no migration was monitored in the Biofield Energy Treated HaCaT cells as compared with the baseline control group. Additionally, the percentage of scratch area was significantly decreased by 7.3% in the Biofield Energy Treated DMEM, while an increased scratched area was reported by 14% in the Biofield Energy Treated cells. Hence, the results using scratch assay showed a promising scientific approach to differentiate the Biofield Energy Treatment on cells or media for their wound healing potential. The data concluded that The Trivedi Effect® has the significant capability in wound healing activity in DMEM as compared with the HaCaT cell line with respect to the cell migration and reduced wound scratched area. Consciousness Energy Healing Treatment (The Trivedi Effect®) can be used as a complementary and alternative approach to improve the cellular migration and proliferation and can be used in psoriasis, seborrheic dermatitis, skin cancer, rashes from bacterial or fungal infections as anti-wrinkling, skin-whitening, anti-ageing, and rejuvenating agent.
HaCaT
Viability assay
Human skin
MTT assay
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The wound-healing assay is simple, inexpensive, and one of the earliest developed methods to study directional cell migration in vitro. This method mimics cell migration during wound healing in vivo. The basic steps involve creating a "wound" in a cell monolayer, capturing the images at the beginning and at regular intervals during cell migration to close the wound, and comparing the images to quantify the migration rate of the cells. It is particularly suitable for studies on the effects of cell-matrix and cell-cell interactions on cell migration. A variation of this method that tracks the migration of individual cells in the leading edge of the wound is also described in this chapter.
Matrix (chemical analysis)
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Research on treatment alternatives that improve wound healing is an ever-evolving area in medicine, and a wound healing agent that carries minimal pain, discomfort, and scarring for patients with burn wounds, venous and decubitis ulcers, traumatic wounds, and many others is needed. The phases of wound healing include homeostasis, inflammation, migration, proliferation, and maturation. Adeps suillus (axonge) is known as a therapeutic agent for skin diseases and mainly consists of triglycerides.In the current study, the proliferation effect of axonge was determined on human normal epidermal keratinocyte (HaCaT) cells and human normal foreskin fibroblast cell line (BJ) cells.Experimental steps included preparation of HaCaT and BJ cell lines, axonge's stable tetrazolium salt-based proliferation assay, and evaluation of the wound healing effect of axonge on HaCaT and BJ cells.Axonge concentrations of 3.12 µg/mL, 6.25 µg/mL, 12.5 µg/mL, 25 µg/mL, and 50 µg/mL showed no cytotoxic effect on both HaCaT and BJ cells for 24, 48, and 72 hours. Considering the wound area of HaCaT cells, after 6 hours the wound healing effect of the axonge group reached almost 70% and then stopped. According to the results of the study on BJ cells, after 6 hours axonge wound closure was found to be 50% while the control group was only 10%.On the basis of this study, the authors determined that axonge might have potential for use in wound healing.
HaCaT
Foreskin
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Objective To explore the mechanism of Qipiyin on treating psoriasis,and effects on cell cycle and proliferation.Methods The effect of Qipiyin and its components on HaCaT proliferation was measured through MTT method,and flow cytometry analysis was used to detect the content of DNA and cell cycle in various concentrations at different time points.Results Qipiyin could markedly inhibited the proliferation of HaCaT,and the effect was enhanced with concentration.The medicine could also disturbed the distribution of HaCaT cell cycle obviously,which showed that the cell percentage of HaCaT in G0/G1 phase increased and that in S phase decreased.Conclusion The mechanism of Qipiyin inhibiting the proliferation of keratinocyte may be related to the change of cell cycle.
HaCaT
MTT assay
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EPLIN (Epithelial Protein Lost In Neoplasm) is a cytoskeletal associated protein
whose expression is often reduced in cancer cells. It may function as a tumour
suppressor through its effects on cancer cell migration and invasion. To date, its role
in wound healing has not been elucidated. We examine the impact of EPLIN on
keratinocyte migration and its implications in wound healing. A mammalian
expression construct containing the full EPLIN coding sequence was used to
overexpress EPLIN in human keratinocyte cell (HaCaT). Following overexpression
verification, the impact of EPLIN on HaCaT cell migration was assessed using a
conventional scratch wounding assay and an electric cell-substrate impedance sensing
(ECIS) system-based assay. Protein expression was examined using western blot, ICC
and IFC analysis. Transfection of HaCaT cells with the EPLIN expression construct
successfully resulted in enhanced HaCaT EPLIN expression. Enhanced EPLIN levels
were seen to negatively impact on cell migration as determined by both the scratch
wound assay and the ECIS model system with migration rates of HaCaT cells overexpressing
EPLIN being substantially less than the control HaCaT cells.
Overexpression of EPLIN was found to slow keratinocyte migration rates using two
independent assays as well as show convincing association and interaction with two
NWASP and E-Cadherin. These important findings suggest novel routes to positively
manipulate the wound healing process and has significance in further translational
research.
HaCaT
Human skin
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Wound dressings that exert an antimicrobial effect in order to prevent and treat wound infections can be harmful to the wound healing process. Dressings with hydrophobic coatings, however, have been suggested to both reduce the microbial load and promote the healing process. Therefore, the potential effects of a dialkylcarbamoyl chloride (DACC)-coated dressing on fibroblasts and keratinocytes in wound healing were studied using mechanical scratch wounding of confluent cell layers as an in vitro model. Additionally, gene expression analysis by qRT-PCR was used to elucidate the longitudinal effects of the DACC-coated dressing on cell responses, specifically inflammation, growth factor induction and collagen synthesis. DACC promoted cell viability, did not stick to the cell layers, and supported normal wound healing progression in vitro. In contrast, cells became attached to the uncoated reference material, which inhibited scratch closure. Moreover, DACC slightly induced KGF, VEGF, and GM-CSF expression in HaCaT cells and NHDF. Physiological COL1A1 and COL3A1 gene expression by NHDF was observed under DACC treatment with no observable effect on S100A7 and RNASE7 levels in HaCaT cells. Overall, the DACC coating was found to be safe and may positively influence the wound healing outcome. Graphical abstract.
HaCaT
Viability assay
Scratch
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