Proliferation Of HaCaT Cells Research Articles

Rehmannioside A Inhibits TRAF6/MAPK Pathway and Improves Psoriasis by Interfering with the Interaction of HaCaT Cells with IL-17A.

As a common chronic inflammatory skin disease, psoriasis seriously affects the physical health and psychological well-being of patients. Various clinical treatments for psoriasis have their own drawbacks, so it is important to find effective and safe drugs. Rehmannioside A (ReA) has anti-inflammatory properties and is the main active ingredient in Fuzhengzhiyanghefuzhiyang decoction (FZHFZY), an herbal compound for the treatment of psoriasis. But no studies have been conducted to determine whether ReA alone can treat psoriasis. Therefore, this study was designed to investigate the effect of ReA in the treatment of psoriasis and its potential mechanism of action. HaCaT cells were treated with ReA and IL-17A alone for 24 h and 48 h, and the most effective concentrations of ReA and interleukin (IL)-17A were found at 25 μg/mL and 100 ng/mL, respectively. A psoriasis cell model was constructed by stimulating HaCaT cells with IL-17A, followed by intervention with ReA. Cell viability and cell cycle distribution were measured by MTT assay and flow cytometry. The expression levels of keratin family members and chemokines were detected by real-time quantitative PCR (RT-qPCR), the levels of pro-inflammatory cytokines by enzyme-linked immunosorbent assay (ELISA), and key proteins of TRAF6/MAPK signaling pathway by Western blot. ReA weaken cell viability, down-regulate the expression of keratin family members (KRT6 and KRT17), restore cell cycle distribution to normal distribution, inhibit the release of pro-inflammatory cytokines (IL-6, IL-8 and IL-1β) and lower the expression of chemokines (S100A7, S100A9 and CXCL2) by interfering with the interaction between HaCaT cells and IL-17A. Thus, it exerts an anti-psoriatic effect by reducing the inflammatory response and inhibiting abnormal proliferation of HaCaT cells. Mechanistically, ReA inhibited the TRAF6/MAPK signaling pathway activated by IL-17A stimulation in HaCaT cells. ReA has in vitro anti-psoriatic effects and may be a new therapeutic agent for psoriasis.

Human Parathyroid Hormone Analog (3–34/29–34) promotes wound re-epithelialization through inducing keratinocyte migration and epithelial–mesenchymal transition via PTHR1-PI3K/AKT activation

BackgroundRe-epithelialization is important in the process of wound healing. Various methods have been identified to expedite the process, but their clinical application remains limited. While parathyroid hormone (PTH) has shown promising results in wound healing due to its role in promoting collagen deposition and cell migration, application is limited by its potentially inhibitive effects when being continuously and locally administrated. Herein, we developed a novel PTH analog, Human parathyroid hormone (hPTH) (3–34/29–34) (henceforth MY-1), by partially replacing and repeating the amino acid sequences of hPTH (1–34), and evaluated its effect on skin wound re-epithelialization.MethodsCCK-8, colony formation unit assay, and Ki67 immunofluorescent staining were performed to evaluate the effect of MY-1 on HaCaT cell proliferation. Then, wound scratch assay, Transwell assay and lamellipodia staining were carried out to evaluate the effect of MY-1 on cell migration. Moreover, the epithelial–mesenchymal transition (EMT) markers were measured using qPCR and western blot analysis. For in-vivo drug delivery, gelatin methacryloyl (GelMA) hydrogel was employed to load the MY-1, with the physicochemical characteristics evaluated prior to its application in wound models. Then, MY-1’s role in wound healing was determined via acute skin wound models. Finally, the mechanism that MY-1 activated was also detected on HaCaT cells and in-vivo wound models.ResultsIn-vitro, MY-1 accelerated the migration and EMT of HaCaT cells, while having little effect on cell proliferation. GelMA and MY-1-incorporated GelMA hydrogels showed similar physicochemical characteristics and were used in the in-vivo studies, where the results revealed that MY-1 led to a stronger re-epithelialization by inducing basal keratinocyte migration and EMT. Further studies on in-vivo wound models and in-vitro HaCaT cells revealed that MY-1 regulated cell migration and EMT through activating PI3K/AKT signaling. The parathyroid hormone type 1 receptor (PTHR1), the main receptor of PTH, was found to be the upstream of PI3K/AKT signaling, through interfering PTHR1 expression with a small interference RNA following detection of the PI3K/AKT activation.ConclusionCollectively, our study demonstrated that MY-1 accelerates skin wound re-epithelialization by inducing keratinocyte migration and EMT via PTHR1-PI3K/AKT axis activation.8WeJxjiMKQ-SAupiANbiq5Video

PTCH/SMO gene mutations in odontogenic keratocysts and drug interventions.

Odontogenic keratocysts (OKCs) are odontogenic jaw lesions that cause destruction and dysfunction of the jawbone. OKCs can be sporadic or associated with nevoid basic cell carcinoma syndrome (NBCCS). However, the factors that initiate OKCs and the mechanism of cyst formation remain unclear. Here, we investigated the impact of PTCH1 and SMO mutations on disease progression, as well as the effects of sonic hedgehog (SHH) signaling pathway inhibitors GDC-0449 and GANT61 on OKC fibroblasts. Eight sporadic OKC fibroblasts without gene mutations were used as the control, and six NBCCS-related fibroblasts were cultured in vitro. The effect of PTCH1 non-truncated mutation 3499G>A (p.G1167R) and SMO c.2081C>G (p.P694R) mutation on OKC fibroblast proliferation was examined by EdU assay. CCK8 and wound-healing assays detected the effects of OKC fibroblasts carrying PTCH1 c.3499G>A (p.G1167R) and SMO c.2081C>G (p.P694R) mutations on the proliferation and migration of HaCaT cells after co-culture. Quantitative real-time PCR detected the effects of GDC-0449 or GANT61 on the SHH signaling pathway in NBCCS-related OKCs with PTCH1 truncated mutations and PTCH1 c.3499G>A (p.G1167R) and/or SMO c.2081C>G (p.P694R) mutations. PTCH1 c.3499G>A (p.G1167R) and SMO c.2081C>G (p.P694R) promoted the proliferation of OKC fibroblasts. The proliferation and migration of HaCaT cells were affected by NBCCS-related OKC fibroblasts carrying PTCH1 c.3499G>A (p.G1167R) and SMO c.2081C>G (p.P694R) mutations. GDC-0449 significantly inhibited the SHH signaling pathway in NBCCS-related OKC fibroblasts with PTCH1 truncated mutations. An NBCCS-related OKC carrying PTCH1 c.3499G>A (p.G1167R) and SMO c.2081C>G (p.P694R) mutations were resistant to GDC-0449 but inhibited by GANT61. Genetic mutations in OKC fibroblasts may affect the biological behavior of epithelial and stromal cells and cause disease. GDC-0449 could be used to treat OKCs, especially NBCCS-related OKCs with PTCH1 truncated mutations. SMO c.2081C>G (p.P694R) may lead to resistance to GDC-0449; however, GANT61 may be used as an alternative inhibitor.

Semen Ziziphus jujube Saponins Protects HaCaT Cells against UV Damage and Alleviates the Aging of Caenorhabditis elegans.

The role of Semen Ziziphus jujube saponins in sedative and hypnosis has attracted much attention. The study aimed to investigate its possible UV damage protection and anti-aging effects. Total saponins (SZR I) and purified saponins (SZR II) were analyzed and compared by infrared spectroscopy and high-performance liquid chromatography (HPLC). The protective effects of SZR I, SZR II, and their three monomers on HaCaT cells damaged by UV were studied, and their anti-aging activities were observed by Caenorhabditis elegans with paraquat-induced oxidative stress. The results showed that SZR I and SZR II differ in chemical composition but both have the same three monomers. The cell survival rate treated with SZR I and SZR II at a concentration of 400 μg/mL increased by 34.45 and 88.98%, respectively, indicating that they could promote the proliferation of UVB-damaged HaCaT cells. Jujuboside A, Jujuboside B, and spinosin from the saponins exhibited similar effects on UVB-damaged HaCaT cells. SZR I and SZR II had little effect on reproductive performance but could delay the senescence caused by heat and oxidative stress of the C. elegans model. These results provide useful data that Semen Z. jujube saponin is a potential natural product with UV damage protection and anti-aging characteristics.

EGF-expressed human mesenchymal stem cells inhibit collagenase1 expression in keratinocytes

Mesenchymal stem cells (MSCs) repair tissue injury by upregulating the paracrine secretion of cytokines and growth factors. Human MSC has been recognized as a promising therapeutic material for treatment of various human diseases. Even though the effect of epidermal growth factor (EGF) has been well investigated, the synergetic effect of EGF and MSC has not been studied. Therefore, we expect our basic study to contribute to developing new therapeutic reagents for skin diseases or innovative cosmetics. In this study, we examined the effect of human epidermal growth factor-transfected MSCs (hEGF MSCs) on human keratinocyte HaCaT cell proliferation and the mechanisms that regulate matrix metalloproteinase (MMP)-1 expression in HaCaT cells. To identify the hEGF plasmid and its transfection into MSCs, we performed gel electrophoresis and quantitative PCR. Proliferation and migration of HaCaT cells were examined using water Soluble Tetrazolium (WST-1) and wound-healing assays, respectively. Zymography was performed to investigate the correlation between hEGF MSC-conditioned medium (CM)-treated HaCaT cells and MMP-1 expression. We found that cell proliferation and wound-healing rates were increased in hEGF MSC-CM-treated HaCaT cells compared to those in MSC-CM-treated cells, and conversely collagenase activity was decreased. The mRNA and protein levels of MMP-1 were also decreased in hEGF MSC-CM-treated HaCaT cells. 2-DE analysis showed that the expression of carboxypeptidase, which promotes growth factors and wound healing, was increased in hEGF MSC-CM-treated HaCaT cells. Finally, western blot was used to determine whether MMP-1 expression was reduced via the mitogen-activated protein kinase (MAPK) pathway; the results showed that the levels of MAPK pathway-related proteins (pErk, pJNK, and p-p38) and the levels of transcription factors (pCREB, NFκB, and p-c-Fos) were decreased. In addition, pAkt expression was found to be elevated. The results of our study suggest that hEGF MSCs promote cell proliferation and reduce MMP-1 expression via the MAPK pathway in human keratinocyte HaCaT cells.

Preventive and ameliorative effects of potato exosomes on UVB‑induced photodamage in keratinocyte HaCaT cells.

Exosomes isolated from potato (Solanum tuberosum) exhibit the biophysical characteristics of exosomes observed in mammalian cells and microorganisms, as determined by dynamic light scattering analysis and transmission electron microscopy. In the present study, it was shown that potato exosomes (ExoPs) can penetrate keratinocyte HaCaT cells, as determined by confocal microscopy and flow cytometry. In addition, ExoPs can suppress the expression of the collagen‑destroying enzymes MMP1, 2 and 9, and the inflammatory cytokines IL6 and TNF‑α, while inducing the expression of glutathione S‑transferase α 4, a cellular detoxifying enzyme, as revealed by reverse transcription‑quantitative PCR. Furthermore, ExoPs promote HaCaT cell proliferation, exhibit invitro antioxidant activity against the free radical 2,2‑diphenyl‑β‑picrylhydrazyl, and protect cells from hydrogen peroxide‑induced cytotoxicity. ExoPs can also minimize the induction of photodamage initiated by ultraviolet B (UVB) irradiation, and have the tendency to cure the photodamage already incurred on cells by UVB irradiation. ExoPs also prevent collagen degradation as observed in the culture media of UVB‑irradiated HaCaT cells. Collectively, ExoPs may protect and ameliorate photodamage in keratinocyte HaCaT cells.

Self-assembly of chlorogenic acid into hydrogel for accelerating wound healing

Wound healing remains a considerable challenge due to its complex inflammatory microenvironment. Developing novel wound dressing materials with superior wound repair capabilities is highly required. However, conventional dressing hydrogels for wound healing are often limited by their complex cross-linking, high treatment costs, and drug-related side effects. In this study, we report a novel dressing hydrogel constructed only by the self-assembly of chlorogenic acid (CA). Molecular dynamic simulation studies revealed the formation of CA hydrogel was mainly through non-covalent interactions, such as π-π and hydrogen bond. Meanwhile, CA hydrogel exhibited superior self-healing, injectability, and biocompatibility properties, making it a promising candidate for wound treatment. As expected, in vitro experiments demonstrated that CA hydrogel possessed remarkable anti-inflammatory activity, and its ability to promote the generation of microvessels in HUVEC cells, as well as the promotion of microvessel formation in HUVEC cells and proliferation of HaCAT cells. Subsequent in vivo investigation further demonstrated that CA hydrogel accelerated wound healing in rats through regulating macrophage polarization. Mechanistically, the CA hydrogel treatment enhanced the closure rate, collagen deposition, and re-epithelialization while simultaneously suppressing the secretion of pro-inflammatory cytokines and increasing the production of CD31 and VEGF during the wound healing process. Our findings indicate that this multifunctional CA hydrogel is a promising candidate for wound healing, particularly in cases of impaired angiogenesis and inflammatory responses.

Mechanisms of action underlying the effect of Tongsaimai on wound healing based on experimental and network pharmacology.

Tongsaimai (TSM) is a traditional Chinese medicine that has several therapeutic qualities, including anti-inflammatory, anti-oxidative, and anti-vasculitis effects. However, its impacts and underlying mechanisms on wound healing remain unclear. The aim of our study was to evaluate TSM for its pro-healing effect and the relevant mechanisms using both experimental validation and network pharmacology analysis. The components of TSM were detected by high-performance liquid chromatography combined with diode array detector (HPLC-DAD). Skin wounds with a diameter of 4 mm were created on the backs of mice, after which, topical treatments of 2.5-10% TSM were applied onto the lesions once daily for either 2 or 7 days. Then, the wound tissues were collected to determine the impacts of TSM on collagen deposition, epithelial cell proliferation, oxidative stress, inflammation, and angiogenesis. Moreover, the effects of TSM (0.5-2 mg/mL) on the cell viability of HUVECs and HaCaT cells were evaluated. A total of 11 components in TSM were identified by HPLC-DAD. TSM was found to enhance the rate of wound contraction and increase epithelial thickness and collagen deposition during the healing process. In addition, TSM increased SOD activity and downregulated MDA and IL-1β levels in the wound tissues. Immunofluorescence analysis further indicated an increased expression of Ki67, CD31, and VEGF in wound tissues following TSM administration. Results of the network pharmacology analysis revealed that multiple pathways including VEGF, PI3K/Akt, and MAPK pathways were involved in the pharmacological actions of TSM on wound healing. Accordantly, in vitro experiments revealed that TSM promoted the proliferation of HUVECs and HaCaT cells while activating the PI3K/Akt pathway. Our results suggest that TSM may serve as a therapeutic medication to improve wound healing by employing multiple regulatory mechanisms that affect proliferation, angiogenesis, collagen deposition, oxidative stress, and inflammation.

Preliminary Study on Human Adipose Stem Cells Promoting Skin Wound Healing through Notch Signaling Pathway.

Mesenchymal stem cells (MSCs) have been documented as possible candidates for wound healing treatment because their use could reinforce the regenerative capacity of many tissues. Human adipose stem cells (hADSCs) have the advantages of easy access, large quantity and easy operation. They can be fully applied in the treatment of skin wounds. In this study, we aim to explore the roles and potential mechanisms of hADSCs in cutaneous wound healing. hADSCs were obtained from human subcutaneous fat. Adipocytes and osteocytes differentiated from hADSCs were determined by staining with Oil Red O and alkaline phosphatase (ALP), respectively. We assessed the effects of hADSCs and hADSC conditional medium (CM) on wound healing in an injury model of mice. Then, we investigated the biological effects of hADSCs on human keratinocytes HaCAT cells in vitro. The results showed that hADSCs could be successfully differentiated into osteogenic and lipogenic cells. hADSCs and hADSCs-CM significantly promote skin wound healing in vivo. hADSCs significantly promoted HaCAT cell proliferation and migration by activating the Notch signaling pathway and activated the AKT signaling pathway by Rps6kb1 kinase in HaCAT cells. In addition, we found that hADSCs-mediated activation of Rps6kb1/AKT signaling was dependent on the Notch signaling pathway. We demonstrated that hADSCs can promote skin cell-HaCAT cell proliferation and migration via the Notch pathway, suggesting that hADSCs may provide an alternative therapeutic approach for the treatment of skin injury.

Extracts from Seseli mairei Wolff attenuate imiquimod-induced psoriasis-like inflammation by inhibiting Th17 cells

ObjectiveSeseli mairei Wolff extracts (SMWE) are widely used to treat psoriasis as a Chinese medicine, but their effect and mechanism are unclear. This study verified the effect of SMWE on psoriasis by regulating Th17 cells. MethodsHaCaT cells were treated with IL-17A in vitro to evaluate the effect of SMWE on psoriasis. In vivo, the mice psoriasis model was established using imiquimod (IMQ, 62.5 mg/d), and intragastrically treated with the different drugs for six days. The severity of skin inflammation was evaluated with Psoriasis Area and Severity Index (PASI) scores and pathology. The levels of inflammation cytokines were assessed with immunofluorescence, immunochemistry, ELISA, and real-time PCR. The number of Th17 cells was determined with flows. ResultsSMWE inhibited the proliferation of HaCaT cells and reduced the IL-17A-induced IL-6 production in vitro. In vivo, SMWE deduced the levels of IL-1β, IL-6, IL-8, IL-17A, IL-17F, IL-22, IL-23, and TNF-α, while increasing the level of IL-10 compared to the model group. SMWE also inhibited the levels of NF-κB, JAK2, and STAT3 proteins, while declining the expressions of Gr-1, and MPO. Interestingly, SMWE significantly decreased the number of Th17 cells. ConclusionSMWE inhibited the proliferation of HaCaT cells and attenuated the development of psoriasis lesions by inhibiting Th17 cells to regulate the levels of inflammation cytokines.

Punicalagin alleviates the hyperproliferation of keratinocytes in psoriasis through inhibiting SKP2 expression.

Psoriasis is a chronic inflammatory skin disorder characterized by abnormal keratinocytes proliferation and multiple immune cells infiltration in the dermis and epidermis. Although most psoriasis-related researches have been concentrated on the interleukin-23 (IL-23)/interleukin-17 (IL-17) axis, new data suggest that keratinocytes also play a pivotal role in psoriasis. Previously, we found that punicalagin (PUN), a bioactive ellagitannin extracted from Pericarpium Granati (the pericarpium of Punica granatum L.), exerts a therapeutic effect on psoriasis. However, the underlying mechanism, especially its potential modulatory effect on keratinocytes, remains obscure. Our study aims to reveal the potential regulatory effect and its underlying cellular mechanism of PUN on the hyperproliferation of keratinocytes. We used tumor necrosis factor α (TNF-α), IL-17A and interleukin-6 (IL-6) to induce abnormal proliferation of HaCaT cells (Human Keratinocytes Cells) in vitro. Then, we evaluated the effects of PUN through MTT assay, EdU staining and cell cycle detection. Finally, we explored the underlying cellular mechanisms of PUN via RNA-sequencing, WB in vitro and in vivo. Here, we found that PUN can directly and dose-dependently decrease TNF-α, IL-17A and IL-6-induced abnormal proliferation of HaCaT cells in vitro. Mechanically, PUN suppresses the hyperproliferation of keratinocytes through repressing S-phase kinase-associated protein 2 (SKP2) expression in vitro and in vivo. Moreover, overexpression of SKP2 can partly abolish PUN-mediated inhibition of aberrantly proliferative keratinocytes. These results illustrate that PUN can reduce the severity of psoriasis through directly repressing SKP2-mediated abnormal proliferation of keratinocytes, which gives new insight into the therapeutic mechanism of PUN on psoriasis. Moreover, these findings imply that PUN might be a promising drug candidate for the treatment of psoriasis.

RIG-I contributes to keratinocyte proliferation and wound repair by inducing TIMP-1 expression through NF-κB signaling pathway.

Epithelial keratinocyte proliferation is an essential element of wound repair, and chronic wound conditions, such as diabetic foot, are characterized by aberrant re-epithelialization. In this study, we examined the functional role of retinoic acid inducible-gene I (RIG-I),a key regulator of epidermal keratinocyte proliferation, in promoting TIMP-1 expression. We found that RIG-Iis overexpressed in keratinocytes of skin injury and underexpressed in skin wound sites of diabetic foot and streptozotocin-induced diabetic mice. Moreover, mice lacking RIG-I developed an aggravated phenotype when subjected to skin injury. Mechanistically, RIG-I promoted keratinocyte proliferation and wound repair by inducing TIMP-1 via the NF-κB signaling pathway. Indeed, recombinant TIMP-1 directly accelerated HaCaT cell proliferation in vitro and promoted wound healing in Ddx58-/- and diabetic mice in vivo. In summary, we demonstrated that RIG-I is a crucial factor that mediates epidermal keratinocyte proliferation and may be a potential biomarker for skin injury severity, thus making it an attractive locally therapeutic target for the treatment of chronic wounds such as diabetic foot.

Exosomes with overexpressed miR 147a suppress angiogenesis and infammatory injury in an experimental model of atopic dermatitis

Atopic dermatitis is defined as an intensely systemic inflammation among skin diseases. Exosomes derived from adipose-derived stem cells may be a novel cell-free therapeutic strategy for atopic dermatitis treatment. This study aims to elucidate the possible underlying mechanism of adipose-derived stem cells-exosomes harboring microRNA-147a in atopic dermatitis pathogenesis. BALB/c mice treated with Dermatophagoides farinae extract/2,4-dinitrochlorobenzene were defined as a mouse model of atopic dermatitis, either with inflamed HaCaT cells and HUVECs exposed with TNF-α/IFN-γ stimulation were applied for a cell model of atopic dermatitis. The concentrations of IL-1β and TNF-α in the supernatants were examined by ELISA. Cell viability and migration were assessed by MTT and Transwell assay. The apoptosis was examined using flow cytometry and TUNEL staining. The tube formation assay was employed to analyzed angiogenesis. The molecular regulations among miR-147a, MEF2A, TSLP and VEGFA were confirmed using luciferase reporter assay, either with ChIP. microRNA-147a was markedly downregulated in the serum and skin samples of atopic dermatitis mice, of which overexpression remarkably promoted HaCaT cell proliferation, meanwhile inhibiting inflammatory response and cell apoptosis. microRNA-147a in adipose-derived stem cells was subsequently overexpressed, and exosomes (Exos-miR-147a mimics) were collected. Functionally, exos-microRNA-147a mimics attenuated TNF-α/IFN-γ-induced HaCaT cell inflammatory response and apoptosis, and suppressed HUVECs angiogenesis. Encouraging, molecular interaction experiments revealed that exosomal microRNA-147a suppressed TNF-α/IFN-γ-induced HUVECs angiogenesis by targeting VEGFA, and exosomal microRNA-147a repressed HaCaT cells inflammatory injury through the MEF2A-TSLP axis. Mechanistically, exosomal microRNA-147a repressed pathological angiogenesis and inflammatory injury during atopic dermatitis progression by targeting VEGFA and MEF2A-TSLP axis. microRNA-147a-overexpressing adipose-derived stem cells-derived exosomes suppressed pathological angiogenesis and inflammatory injury in atopic dermatitis by targeting VEGFA and MEF2A-TSLP axis.

Overexpression and potential roles of midkine via regulation of vascular endothelial growth factor A in psoriasis.

Midkine plays a critical role in angiogenesis by regulating the vascular endothelial growth factor (VEGF) signalling pathway, which is known to be associated with psoriasis pathogenesis. However, research on midkine-psoriasis relationship remains limited. The objective of this study was to detect midkine expression in psoriasis and investigate its potential role in the disease. Midkine expression was measured using immunohistochemistry and ELISA. Effects of midkine on HaCaT cell proliferation, VEGF-A production and signalling pathways were assessed using CCK8, RT-PCR and WB. Scratch and in vitro tube formation tests were used to evaluate the effects of HaCaT-cell-activated midkine on the migration and tube formation of human dermal microvascular endothelial cells. Murine psoriasiform models were injected with midkine recombinant protein and midkine monoclonal antibody to investigate skin lesions, tissue sections and dermal microvessel density. Levels of midkine significantly increased in both lesions and serum of patients with psoriasis. Serum expression of midkine decreased after treatment and a positive correlation was found between midkine and disease severity. Midkine promoted HaCaT cell proliferation and VEGF-A production. The Notch2/HES1/JAK2-STAT5A pathway expression increased after midkine treatment of HaCaT cells. The supernatant of HaCaT cells treated with midkine promoted HMEC-1 migration and angiogenesis in vitro. Recombinant midkine protein exacerbated psoriasiform lesions with increased expressions of VEGF-A and microvessel density, while midkine monoclonal antibody alleviated psoriasis lesions. Midkine may have a significant impact on psoriasis angiogenesis by regulating VEGF-A expression through the Notch2/HES1/JAK2-STAT5A pathway, highlighting a potential therapeutic target for psoriasis treatment.

Wip1 regulates wound healing by affecting activities of keratinocytes and endothelial cells through ATM-p53 and mTOR signaling

BackgroundAs a p53-regulated gene, Wip1 regulates proliferation, migration, apoptosis, and senescence of several type cells, but its biological functions in keratinocytes and endothelial cells which are involved wound healing are not fully understood. This study aims to reveal the function and underlying mechanism of Wip1 in wound healing using models of transgenic animal, keratinocytes, and endothelial cells. MethodsUsing Wip1 knockout C57 BL/6 mice, we investigated effect of Wip1 deficiency on wound healing and angiogenesis; And using HaCaT and HUVEC as keratinocytes and endothelial cells, combined using primary keratinocytes from Wip1 knockout mice, we studied the effects of Wip1 knockdown/knockout or overexpression on proliferation, migration, and protein expressions of signaling components in ATM-p53 and mTOR pathway. ResultsWip1 deficiency in mice impaired the wound repair and endothelial angiogenesis, reduced the thickness of granulation tissue, and decreased the number of Ki67-positive cells and CD31 positive vessels in granulation tissue. Knockdown of Wip1 by shRNAs suppressed the proliferation and migration of HaCaT and HUVEC cells and induced notably apoptosis in the two cells. In western blot, Wip1 knockdown enriched p53 and ATM proteins, while decreased activated AKT, mTOR and activated S6 ribosomal protein (pS6) levels in HaCaT and HUVEC cells. Ectopic expression of Wip1 decreased the p53 and ATM proteins, while increased activated AKT, mTOR and pS6 levels in HaCaT and HUVEC cells. And in primary keratinocytes from mice tail skin, Wip1 knockout increased p53 and ATM, while decreased activated AKT, mTOR and pS6 protein levels. ConclusionOur study directly supports that Wip1 regulated skin wound healing possibly by affecting bioactivities including proliferation, migration and apoptosis of keratinocytes and endothelial cells at least through by modulating ATM-p53 and mTOR signaling.

Gelatin-based spongy and compressive resistant cryogels with shape recovery ability as ideal scaffolds to support cell adhesion for tissue regeneration

Methacryloyl gelatin (GelMA) was blended with dextran methacrylate (DexMA) to produce polymer networks with interconnected macropores via cryostructuring and radical crosslinking of the polymers at subzero temperatures. The experimental set-up was optimized to allow the formation of monolithic networks characterized by highly uniform structure with interconnected macropores. The total polymers mass, the amount of the gel-forming reagents and particularly the rheological properties of GelMA resulted the most critical factors for the fabrication of homogeneous and not collapsed scaffolds. Indeed, only the use of GelMA with very low gelation temperature resulted in the formation of uniform monolithic cryogels. However, blending with DexMA produced general worsening of the mechanical properties of the scaffolds, due to DexMA interference with secondary structuring of GelMA during the cryogelation process. DexMA also had negative effect on the ability of the cryogel to support growth and proliferation of HaCat cells, bringing to slower cell adhesion to the scaffold.

Glycomacropeptide Protects against Inflammation and Oxidative Stress, and Promotes Wound Healing in an Atopic Dermatitis Model of Human Keratinocytes.

Keratinocytes are actively implicated in the physiopathology of atopic dermatitis (AD), a skin allergy condition widely distributed worldwide. Glycomacropeptide (GMP) is a milk-derived bioactive peptide generated during cheese making processes or gastric digestion. It has antiallergic and skin barrier restoring properties when it is orally administered in experimental AD. This study aimed to evaluate the effect of GMP on the inflammatory, oxidative, proliferative, and migratory responses of HaCaT keratinocytes in an in vitro AD model. GMP protected keratinocytes from death and apoptosis in a dose dependent manner. GMP at 6.3 and 25 mg/mL, respectively, reduced nitric oxide by 50% and 83.2% as well as lipid hydroperoxides by 27.5% and 45.18% in activated HaCaT cells. The gene expression of TSLP, IL33, TARC, MDC, and NGF was significantly downregulated comparably to control by GMP treatment in activated keratinocytes, while that of cGRP was enhanced. Finally, in an AD microenvironment, GMP at 25 mg/mL stimulated HaCaT cell proliferation, while concentrations of 0.01 and 0.1 mg/mL promoted the HaCaT cell migration. Therefore, we demonstrate that GMP has anti-inflammatory and antioxidative properties and stimulates wound closure on an AD model of keratinocytes, which could support its reported bioactivity in vivo.

Multi-omics data integration reveals the molecular network of dysregulation IQGAP2-mTOR promotes cell proliferation.

IQGAP2 as a tumor suppressor gene can influence cell proliferation in multiple tumor cell lines. However, the regulation network of cell proliferation resulting solely from the deficiency of IQGAP2 in cells was still unclear. Here, we integrated transcriptome, proteome, and phosphoproteome analyses to investigate the regulatory network of cell proliferation in IQGAP2 knockdown HaCaT and HEK293 cells. Our findings revealed that the dysregulation of the IQGAP2-mTOR molecular network led to increased cell proliferation. We demonstrated that IQGAP2 knockdown enhanced the phosphorylation levels of AKT and S6K, leading to increased cell proliferation. Additionally, we found that AKT and mTOR inhibitors partially rescued abnormal cell proliferation by reducing hyperphosphorylation. Our data suggest a potential connection between the mTOR signaling pathway and aberrant cell proliferation in IQGAP2 knockdown cells. These findings offer a new therapeutic strategy for patients with IQGAP2 deficiency.

In vitro antioxidant and wound healing activity of Sargassum polycystum hydroethanolic extract in fibroblasts and keratinocytes

Objective: To investigate the in vitro antioxidant and wound healing properties of the hydroethanolic extract of Sargassum polycystum, and elucidate the mechanism of its wound healing activity. Methods: Human dermal fibroblast and HaCaT cells were used to evaluate the proliferation by sulforhodamine B and dsDNA assay after treatment with Sargassum polycystum extracts. Scratch wound healing and phalloidin-rhodamine staining were employed to observe migratory activity and filopodia formation, respectively. Western blot and real-time RT-PCR assays were performed to determine the protein and gene expressions related to wound healing activities. Results: The phytochemical analysis found a higher level of flavonoid than phenolic compound in Sargassum polycystumextracts. In human dermal fibroblast cells, Sargassum polycystum extracts at 50 and 100 µg/mL significantly increased fibroblast proliferation and the gene expressions of hyaluronic acid synthase 1 (HAS1), HAS2, HAS3, collagen type 1 alpha 1 chain (COL1A1), collagen type 3 alpha 1 chain (COL3A1), and elastin. The phosphorylation of Akt, ERK1/2, and p38 MAPK was also significantly upregulated after treatment with Sargassum polycystumextracts. Additionally, 50 and 100 µg/mL of the extracts prominently enhanced the proliferation, migration, and filopodia formation of HaCaT cells, as well as the protein levels of pFAK/FAK, pSrc/Src, pAkt/Akt, pERK1/2/ERK1/2, Rac1 and Cdc42. Conclusions: Sargassum polycystum extracts show promising wound healing activities in human dermal fibroblasts and keratinocytes.

Guanidinylated/PEGylated chitosan in the bioink promotes the formation of multi-layered keratinocytes in a human skin equivalent

The biological differences of skin between rodent and human beings and the strong appeal to replace the experimental animals have led to the development of alternative models with structures similar to the real human skin. Keratinocytes cultured in vitro on conventional dermal scaffolds tend to form monolayer rather than multi-layer epithelial tissue architectures. How to construct human skin or epidermal equivalents with multi-layered keratinocytes similar to real human epidermis remains one of the greatest challenges. Herein, a human skin equivalent with multi-layered keratinocytes was constructed by 3D bioprinting fibroblasts and subsequent culturing epidermal keratinocytes. Biocompatible guanidinylated/PEGylated chitosan (GPCS) was used as the main component of bioink to 3D bioprint tissue-engineered dermis. The function of GPCS to promote HaCat cell proliferation and connection was confirmed at the genetic, cellular, and histological levels. Compared with the skin tissues with mono-layered keratinocytes engineered with collagen and gelatin, adding GPCS in the bioink generated tissue-engineered human skin equivalents with multi-layered keratinocytes. Such human skin equivalents could be alternative models for biomedical, toxicological, and pharmaceutical research.