Identification of compounds with regenerative effects in the mucus of the garden snail Cornu aspersum

Kigelia africana (Lam.) Benth. (Bignoniaceae) syn. Kigelia pinnata (Jacq. DC) is a tropical plant that is native to tropical Africa. The aim of this study was to determine if a methanolic extract prepared from Kigelia africana (KAE) can promote wound healing in treated human normal epidermal keratinocyte (HaCaT) cells and human normal foreskin fibroblast cell line (BJ) cells compared with untreated cells. Experimental steps included: the methanolic extraction of the leaf and fruit of the Kigelia africana plant; the preparation of HaCaT and BJ cell lines; cell culture with a stable tetrazolium salt-based proliferation assay; and the evaluation of the wound healing effect of KAE (2μg/ml) in BJ and HaCaT cells. The phytochemical contents of KAE were determined using liquid chromatography quadrupole time-of-flight mass spectrometry. The following molecules were identified as being present in the KAE, among others: cholesterol sulfate; lignoceric acid; embelin; isostearic acid; linoleic acid; dioctyl phthalate; arg-pro-thr; 15-methyl-15(S)-PGE1; sucrose; benzododecinium (Ajatin); and 9-Octadecenamide (oleamide). KAE effected faster wound healing in treated cells compared with untreated cells for both cell lines. HaCaT cells that had been mechanically injured and treated with KAE healed completely in 48 hours compared with 72 hours for untreated HaCaT cells. Treated BJ cells healed completely in 72 hours compared with 96 hours for untreated BJ cells. Concentrations of KAE up to 300μg/ml had a very low cytotoxic effect on treated BJ and HaCaT cells. The experimental data in this study support the potential of KAE-based wound healing treatment to accelerate wound healing.

This study aimed to investigate the antioxidant and in vitro wound healing activities of fulvic acid (FA) purified from Hüsamlar leonardite in Muğla/Turkey. Purified FA was analyzed with FT/IR spectrophotometer and flame photometer for determining functional groups and cation impurities, respectively. 1, 5, 10, 20, 30, 50, 100 μg mL-1 concentrations of FA were tested for antioxidant activity (by using DPPH radical scavenging and H2O2 scavenging assays). Also, effect of FA on BJ human foreskin fibroblast and HaCaT spontaneously immortalized non-tumorigenic human keratinocyte cells proliferation was tested by in vitro MTT and WST-8 assays and on cell migration by wound healing assay (scratch assay). The results of the study show that the FA has a low DPPH radical scavenging activity, but it exhibited high H2O2 scavenging activity at low concentrations. The effect of FA on the proliferation of BJ and HaCaT cells varied according to the cell type, FA concentration and treatment time. However, it accelerated wound healing by increasing cell migration, especially in HaCaT cells. 1 and 10 μg mL-1 FA had a significant wound healing effect on BJ cells, and all concentrations of FA had a significant wound healing effect on HaCaT cells at the end of the 24 h treatments.

Background/Objectives: The treatment of diabetic foot ulcers (DFU) is a challenging medical problem of extreme clinical and social importance, as a consequence of the emerging antibiotic resistance and decreased quality of life of diabetic patients due to impaired wound healing. One of the current trends in world science is the search for biologically active substances derived from living organisms. Biologically active peptides from snail mucus attract considerable scientific interest because of their pleiotropic pharmacological properties. The aim of our study was to evaluate the activity of a combination between a snail mucus protein fraction (MW > 20 kDa) obtained from the garden snail Cornu aspersum and the clinically applied antibacterial chemotherapeutic ciprofloxacin on pathogenic bacterial strains isolated from DFU. Results: The test bacterial strains were characterized as multidrug resistant. The combination between ciprofloxacin and the snail mucus fraction of interest led to additive or synergistic effects depending on the test strain. The mucus fraction exerted a well-pronounced wound-healing effect and no cytotoxicity on normal human fibroblasts and keratinocytes. Methods: The snail mucus was obtained by a patented technology (BG Utility model 2097/2015) and its electrophoretic profile was presented by SDS-PAGE. The bacterial strains were identified and tested for antimicrobial susceptibility (BD Phoenix M50 and Kirby-Bauer assay). The in vitro cytotoxicity of the mucus was evaluated by ISO 10995-5. The antimicrobial activity and combination effects were tested through ISO 20776/1 and the Checkerboard assay. Conclusions: The obtained results are promising and open new horizons for the development of novel combination treatment schemas for healing of infected DFU.

BackgroundAlthough chlorpyrifos (CPS) has been banned in many developed countries, it still remains one of the best-selling pesticides in the world. Widespread environmental and occupational exposure to CPS pose a serious risk to human health. Another environmental factor that can adversely affect human health is ultraviolet radiation B (UVB, 280–315 nm wave length). Here we attempt determine if exposure to CPS can modify toxic effects of UVB. Such situation might be a common phenomenon in agriculture workers, where exposure to both factors takes place.MethodsTwo skin cell lines; namely human immortalized keratinocytes HaCaT and BJ human fibroblasts were used in this study. Cytotoxicity was investigated using a cell membrane damage detection assay (LDH Cytotoxicity Assay), a DNA damage detection assay (Comet Assay), an apoptosis induction detection assay (Apo-ONE Homogeneous Caspase-3/7 Assay) and a cell reactive oxygen species detection assay (ROS-Glo H2O2 assay). Cytokine IL-6 production was also measured in cells using an ELISA IL-6 Assay.ResultsPre-incubation of skin cells with CPS significantly increased UVB-induced toxicity at the highest UVB doses (15 and 20 mJ/cm2). Also pre-exposure of BJ cells to CPS significantly increased the level of DNA damage, except for 20 mJ/cm2 UVB. In contrast, pre-exposure of HaCaT cells, to CPS prior to UVB radiation did not cause any significant changes. A decrease in caspase 3/7 activity was observed in HaCaT cells pre-exposed to 250 µM CPS and 5 mJ/cm2 UVB. Meanwhile, no statistically significant changes were observed in fibroblasts. In HaCaT cells, pre-exposure to CPS resulted in a statistically significant increase in ROS production. Also, in BJ cells, similar results were obtained except for 20 mJ/cm2. Interestingly, CPS seems to inhibited IL-6 production in HaCaT and BJ cells exposed to UVB (in the case of HaCaT cells for all UVB doses, while for BJ cells only at 15 and 20 mJ/cm2).ConclusionsIn conclusion, the present study indicates that CPS may contribute to the increased UVB-induced toxicity in skin cells, which was likely due to the induction of ROS formation along with the generation of DNA damage. However, further studies are required to gain better understanding of the mechanisms involved.

Schisandrol A-loaded mesenchymal stem cell exosomes modulate FTO/miR-21/WNT5A axis to promote skin healing.

Impaired wound healing that occurs in diabetics can result in many life-threatening complications associated with excessive expression of matrix metalloproteinases (MMPs), which mediate the proteolysis of major matrix constituents. In this study, the dendrimer polyamidoamine (PAMAM) and the polysaccharide hyaluronic acid (HA) were connected through the substrate polypeptide (Gly-PLGLAG-Cys) of MMP-2 to obtain the MMP-2-responsive nanocarrier HA-pep-PAMAM. Insoluble astragaloside (ASI) was encapsulated in this nanocarrier to achieve controlled release at the site of intractable wounds. The HA-pep-PAMAM-ASI was successfully prepared with an average diameter of 142.3 ± 28.9 nm. Immunohistochemical staining of the skin revealed that the hard-to-heal wounds of diabetic mice showed stronger expression of MMP-2 than the wounds of normal mice. HA-pep-PAMAM-ASI achieved 73.9% release in the presence of MMP-2, but only 13.5% in PBS. A dose-dependent effect of H₂O₂ on the proliferation of BJ and HaCaT cells was observed, and HA-pep-PAMAM-ASI treatment had the best antioxidant capacity with MMP-2 pretreatment. HA-pep-PAMAM-ASI significantly increased GSH levels and reduced reactive oxygen species (ROS) levels to achieve antioxidant effects. The MMP-2-pretreated HA-pep-PAMAM-ASI group showed more improved cell proliferation and migration abilities. Compared with ASI group, the expression of all wound-repair-related genes in the group of HA-pep-PAMAM-ASI was significantly increased, and HA-pep-PAMAM-ASI showed a pronounced in vivo therapeutic effect. Therefore, our results revealed that enzyme-responsive MMP-2-loaded PAMAM nanoparticles could promote wound healing in diabetes and may be a promising biomaterial for treatment.

Long-term exposure to arsenic has been linked to a variety of cancers, among which skin cancer is the most prevalent form. However, the mechanism underlying arsenic carcinogenesis is unclear, and there is still limited information on the role of miRNAs in arsenic-induced skin cancer. This study aims to explore the role of miR-96-5p in the arsenite-induced proliferation and malignant transformation of human HaCaT keratinocytes. The GEO database (accession numbers GSE97303, GSE97305, and GSE97306) was used to extract mRNA and miRNA expression profiles of HaCaT cells treated with or without 0.1 μmol/L sodium arsenite for 3 and 7 weeks. In this paper, according to the CCK8 assay result, HaCaT cells exposed to 0.1 μmol/L sodium arsenite for 48 h were finalized. CCK8, MTT, EdU incorporation, and colony formation assays were used to determine the viability and proliferation of HaCaT cells and transformed HaCaT (T-HaCaT) cells. The subcellular localization and relative expression levels of DTL, as well as miR-96-5p in HaCaT cells induced by arsenite, were determined via immunofluorescence, RT-qPCR, and Western blot. Dual-luciferase reporter assay was performed to identify miR-96-5p bound directly to DTL. Transfection of miR-96-5p mimics or DTL siRNA was conducted to verify the arsenite-induced viability of HaCaT cells and T-HaCaT cells. T-HaCaT cells and nude mice were used to construct arsenite-induced malignant transformation and an in vivo xenograft model to demonstrate the over-expressed effect of miR-96-5p. The results showed that DTL was the target gene of miR-96-5p. Meanwhile, we also found that 0.1 μmol/L sodium arsenite upregulated DTL by decreasing the miR-96-5p level, leading to the proliferation and malignant transformation of HaCaT cells. MiR-96-5p agomir treatment slowed the growth of transplanted HaCaT cells transformed by arsenite in a manner associated with DTL downregulation in the nude mice xenograft model. Taken together, we confirmed that miR-96-5p, as a potent regulator of DTL, suppressed arsenite-induced HaCaT cell proliferation and malignant transformation, which might provide a novel therapeutic target for the treatment of arsenic-induced skin cancer.

Highly-expressed micoRNA-21 in adipose derived stem cell exosomes can enhance the migration and proliferation of the HaCaT cells by increasing the MMP-9 expression through the PI3K/AKT pathway

Quercetin and phenylpropanoids are well known chemoprotective compounds identified in many plants. This study was aimed at determining their effects on activation of Nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response element (Nrf2-ARE) signalling pathway and expression of its important downstream effector phase II detoxification enzyme glutathione-S-transferase P1 (GSTP1) in BJ foreskin fibroblasts and skin HaCaT keratinocytes. Cell lines and their corresponding Nrf2-ARE luciferase reporter cells were treated by ginger phenylpropanoids and quercetin for 10 h and the level of Nrf2 activity was subsequently determined. Both, ginger phenylpropanoids and quercetin, significantly increased the level of Nrf2 activity. Subsequent western blot analyses of proteins showed the increased expression level of glutathione-S-transferase P1 (GSTP1) in BJ cells but not in HaCaT cells. Such phenomenon of unresponsive downstream target expression in HaCaT cells was consistent with previous studies showing a constitutive expression of their GSTP1. Thus, while both ginger phenylpropanoids and quercetin have the property of increasing the level of Nrf2 both in HaCaT and in BJ cells, their effects on its downstream signalling were mediated only in BJ cells.

BackgroundSchizophyllan (SPG), a bioactive polysaccharide from Schizophyllum commune, possesses significant anti-inflammatory, antioxidant, and immunomodulatory properties. The molecular weight of polysaccharides significantly impacts their structural properties and biological functions. However, the functional characteristics of low molecular weight polysaccharides derived from Schizophyllum commune remain inadequately explored.MethodsThis study developed an ultrasound-assisted hydrogen peroxide (H2O2) degradation method to produce low-molecular-weight SPG with enhanced bioactivity. The process was optimized using response surface methodology, focusing on ultrasound duration, ultrasonic power, and H2O2 concentration. This approach effectively reduced the molecular weight of SPG from 4,409,608 Da to 257,500 Da, yielding three distinct variants: SPG-a (257,500 Da), SPG-b (429,300 Da), and SPG-c (364,800 Da). The bioactivity of these variants was assessed through in vitro cell proliferation and migration assays using BJ and HaCaT cells, as well as an in vivo zebrafish larval caudal fin regeneration model.ResultsIn vitro, SPG-b significantly promoted cell proliferation, increasing BJ and HaCaT cells growth by 53.69% and 14.59%, respectively, at a concentration of 300 μg/mL (p < 0.05), compared to undegraded SPG. Additionally, scratch assays revealed that SPG-a enhanced BJ cells migration by 24.13% (p < 0.05), while SPG-b exhibited most pronounced effect on HaCaT cells migration (17.12%, p < 0.05), compared to the undegraded SPG. In vivo, SPG-c (3.125 mg/mL) significantly improved fin regeneration rates by 6.97% (p < 0.05) in zebrafish larvae, compared to the undegraded SPG.ConclusionThis study demonstrates that ultrasound-assisted H2O2 degradation effectively reduces SPG molecular weight while enhancing its functional properties. These findings provide a foundation for the further development of SPG in pharmaceutical and cosmetic applications, highlighting its potential for broader utilization.

Hemigraphis colorata, a prostrate herb, is used in traditional medicine for the treatment of wounds. Hexane, ethyl acetate and methanol extracts of leaves were analyzed for anti-oxidant and free radical scavenging abilities, of which ethyl acetate extract showed maximum activity. In addition, ethyl acetate extract also displayed high amounts of phenolic and flavonoid contents. 5-LOX, COX-1 and COX-2, enzymes involved in inflammatory processes, were also inhibited by the ethyl acetate extract. Lipopolysaccharide induced expression of pro-inflammatory genes (TNF-α, COX-2 and IL-1β) was inhibited in a dose dependent manner in human keratinocyte cell line (HaCaT cells) treated with the ethyl acetate extract. Further, the ethyl acetate extract induced the proliferation of HaCaT cells at lower concentrations with no alteration of morphology. Scratch wound healing model confirmed the wound healing capabilities of the ethyl acetate extract. This study highlights the potential of the ethyl acetate extract of H. colorata leaves as an anti-inflammatory and wound healing agent.

Psoriasis is a chronic cutaneous disease, affecting a significant portion of the global population. Topoisomerase II alpha (TOP2A) is upregulated in psoriasis samples, but the precise molecular mechanism remains unclear. We aimed to clarify the biological contribution of TOP2A in psoriasis progression. An in vitro psoriasis model was established on M5-induced keratinocytes (HaCaT cells) to simulate the psoriasis-like alterations. Following TOP2A knockdown without or with c terminal binding protein 1 (CTBP1) overexpression, CCK-8 and EDU staining were employed to analyze the viability and proliferation of HaCaT cells under M5 conditions. The capacities of HaCaT cell migration and invasion were examined with wound healing- and transwell assays. RT-qPCR and immunoblotting were adopted for evaluation of the inflammation and differentiation of M5-stimualted HaCaT cells. Additionally, the binding between TOP2A and CTBP1 was predicated using bioinformatics tools and detected by Co-IP. Finally, the expression of proteins in Wnt/β-catenin signaling was analyzed with the application of immunoblotting. Results suggested that TOP2A was upregulated in psoriasis skin lesions and M5-induced HaCaT cells. Interference with TOP2A attenuated the proliferation, migration, invasion, and inflammatory response in M5-treated HaCaT cells. In particular, TOP2A bound to CTBP1 and upregulated CTBP1 expression in HaCaT cells. Remarkably, CTBP1 upregulation blocked the impacts of TOP2A depletion on the biological behaviors of M5-treated HaCaT cells. Besides, TOP2A deficiency upregulated DKK1 expression as well as downregulated Wnt1, β-catenin, and c-Myc expression in HaCaT cells exposed to M5, which was restored by further CTBP1 overexpression. In summary, TOP2A binds CTBP1 to activate Wnt/β-catenin signaling, thereby promoting the progression of psoriasis.

Snail mucus has shown promise in promoting wound healing; however, its active components and their mechanisms of action are poorly understood. In the present study snail mucus was isolated and hydrolyzed using trypsin to obtain snail mucus active peptides (SMAPs). SMAPs were analyzed using liquid chromatography–mass spectrometry, and bioinformatics screening. Among the screened peptides, an active 12-amino-acid peptide, EK-12 (molecular weight: 1366.2 Da), was identified and synthesized using a solid-phase peptide synthesis approach. In vitro functional verification showed that EK-12 significantly promoted endothelial cell proliferation, migration, and tube formation. An in vivo experiment demonstrated that EK-12 significantly accelerated wound healing in mouse models. Pathological examination revealed a significantly upregulated expression of CD31 and vascular endothelial growth factors in wound tissues, suggesting that this is the mechanism by which the active peptide promotes angiogenesis and wound healing. Thus, snail mucus-derived peptides hold strong potential for development as therapeutic agents for wound healing.

We demonstrated that glyphosate possesses tumor promoting potential in mouse skin carcinogenesis and SOD 1, calcyclin (S100A6), and calgranulin B (S100A9) have been associated with this potential, although the mechanism is unclear. We aimed to clarify whether imbalance in between [Ca2+]i levels and oxidative stress is associated with glyphosate-induced proliferation in human keratinocytes HaCaT cells. The [Ca2+]i levels, ROS generation, and expressions of G1/S cyclins, IP3R1, S100A6, S100A9, and SOD 1, and apoptosis-related proteins were investigated upon glyphosate exposure in HaCaT cells. Glyphosate (0.1 mM) significantly induced proliferation, decreases [Ca2+]i, and increases ROS generation in HaCaT cells, whereas antioxidant N-acetyl-L-cysteine (NAC) pretreatment reverts these effects which directly indicated that glyphosate induced cell proliferation by lowering [Ca2+]i levels via ROS generation. Glyphosate also enhanced the expression of G1/S cyclins associated with a sharp decrease in G0/G1 and a corresponding increase in S-phases. Additionally, glyphosate also triggers S100A6/S100A9 expression and decreases IP3R1 and SOD 1 expressions in HaCaT cells. Notably, Ca2+ suppression also prevented apoptotic related events including Bax/Bcl-2 ratio and caspases activation. This study highlights that glyphosate promotes proliferation in HaCaT cells probably by disrupting the balance in between [Ca2+]i levels and oxidative stress which in turn facilitated the downregulation of mitochondrial apoptotic signaling pathways.

Objective To estimate the effect of glycyrrhetinic acid on epidermal growth factor (EGF)-induced proliferation of HaCaT cells,and to investigate its possible mechanism.Methods Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the proliferation of HaCaT cells treated with different concentrations of EGF (0,1,5,10,25,50,100 μg/L) and glycyrrhetinic acid (0,0.1,1.0,10,25,50,100μmol/L) alone,or the combination of 25 μg/L EGF with 25 μ mol/L glycyrrhetinic acid or 10 μ mol/L U0126 (an inhibitor of MEK1/2).Western blot was carried out to measure the protein expression of proliferating cell nuclear antigen (PCNA),Notch-1,ERK 1/2 and phosphorylated ERK 1/2 in HaCaT cells treated with 25 μg/L EGF,10 μmol/L U0126,25μmol/L glycyrrhetinic acid alone or in combination.Data were statistically analyzed by using t test,analysis of variance and correlation analysis with SPSS 17.0 software.Results EGF of 0-100 μg/L promoted the proliferation of HaCaT cells in a dose-dependent manner (r =0.798,P ＜ 0.05),and there was a linear correlation between the effect and concentration within the concentration range 0-50 μg/L (r =0.859,P ＜ 0.05).However,glycyrrhetinic acid of 10-100 μmol/L inhibited the proliferation of HaCaT cells in a dose-dependent manner (r =-0.945,P ＜0.01),and 10 μmol/L glycyrrhetinic acid could suppress the EGF (25 μg/L)-induced proliferation and phosphorylation of ERK1/2 in HaCaT cells.Also,both 25 μmol/L glycyrrhetinic acid and 10 μmol/L U0126 could attenuate the increase in PCNA and Notch-1 expression in HaCaT cells induced by 25 μg/L EGF.Conclusion Glycyrrhetinic acid can inhibit the EGF-induced proliferation of HaCaT cells,likely by suppressing the activation of ERK1/2 signaling pathway.