WS1 Cells Research Articles

Upregulation of PRRX2 by silencing Marveld3 as a protective mechanism against radiation-induced ferroptosis in skin cells.

Radiation-induced skin injury (RISI) represents a significant complication in patients receiving radiotherapy and individuals exposed to nuclear accidents, characterized by a protracted wound-healing process relative to injuries from other etiologies. Current preventive and management approaches remain inadequate. Consequently, investigating efficacious intervention strategies that target the disease's progression characteristics holds significant practical importance. Small interfering RNA (siRNA) and overexpression plasmid were used to modulate the expression of Marvel domain containing 3 (Marveld3) and paired related homeobox 2 (PRRX2). Protein and mRNA levels were estimated by Western Blot and real-time PCR, respectively. Intracellular levels of Malondialdehyde (MDA), a terminal product of lipid peroxidation, were measured following the manufacturer's protocol for MDA assay kit. Similarly, intracellular levels of ferrous iron (Fe2+) and reactive oxygen species (ROS) were determined using their respective assay kits. Lipid peroxidation status within the cells was evaluated via BODIPY staining. Immunohistochemistry was conducted to ascertain the expression of PRRX2 in skin tissues collected at various time points following irradiation of rats. The H-score method was used to evaluate the percentage of positively stained cells and staining intensity. RNA sequencing, Gene Ontology (GO) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted by OE Biotech Company. In this study, our findings indicated that Marveld3 suppression could effectively inhibit lipid peroxidation levels in irradiated skin cells, concomitantly reducing intracellular Fe2+ content. Additionally, the silencing of Marveld3 effectively abrogated the impact of a ferroptosis agonist on cellular viability, resulting in the upregulation of 66 and 178 genes, as well as the downregulation of 188 and 31 genes in irradiated HaCaT and WS1 cells, respectively. Among the differentially expressed genes, the PRRX2 which was found to be involved in the process of ferroptosis, exhibited statistically significant upregulation. And the upregulation of PRRX2 expression may attenuate radiation-induced lipid peroxidation in skin cells, thereby functioning as a potential stress-responsive mechanism to counteract radiation effects. This study elucidates the role of Marveld3 in radiation-induced ferroptosis in skin cells. Inhibition of Marveld3 led to the upregulation of PRRX2, which subsequently resulted in a reduction of Fe2+ and ROS levels, as well as the suppression of lipid peroxidation. These effects collectively mitigated the occurrence of ferroptosis.

Phytochemical Characterization of the Aerial Parts of Teucrium nuchense K. Koch: An Endemic Species of Caucasian Flora

Background: The genus Teucrium L. is frequently employed in traditional medicine, primarily for addressing disorders of the digestive and respiratory systems. Biological investigations focused on essential oils have substantiated their antibacterial, antioxidant, anti-tumor, anti-diabetic, and anti-rheumatic properties. Objective: The aim of the study was to assess the chemical composition and biological activities of the aerial parts of the endemic species Teucrium nuchense K. Koch. Methods: The chemical composition of the essential oil was analyzed using gas chromatography-mass spectrometry (GC-MS). The antioxidant activity was evaluated through the Oxygen Radical Absorbance Capacity (ORAC) assay and tested on normal skin fibroblasts (WS-1). Additionally, the antiinflammatory efficacy was assessed by monitoring nitric oxide production. The total phenolic content in the aqueous, methanolic, and chloroformic extracts was quantified using the Folin-Ciocalteu reagent. Individual compounds were isolated using preparative high-performance liquid chromatography. Their identification was accomplished using high-resolution mass spectrometry (HRMS) with an ESI-Q-TOF detector in conjunction with nuclear magnetic resonance (NMR) spectroscopy. Results: The essential oil yield was 0.32 ml/kg. Caryophyllene oxide, constituting 15.19 %, and 1- octen-3-ol, constituting 8.75%, emerged as the primary components in the essential oil. Oxygenated sesquiterpenes dominated the terpenic compounds, representing 25.1 % of the essential oil. The aqueous and methanolic extracts displayed a notable phenolic content, with percentages of 27 ± 2 % and 35 ± 0.1 %, respectively. Both the aqueous and methanolic extracts demonstrated robust antioxidant activity in the ORAC test and in vitro assessments using WS-1 cells. The methanolic extract, at a concentration of 160 μg/ml, inhibited NO formation by 90 %, without toxicity in RAW 264.7. Individual compounds, teucrioside from the phenylethanoid glycoside class, two neo-clerodane diterpenes, teucrin A, and 6-alpha-hydroxyteuscordine, were isolated. Conclusion: The essential oil extracted from the aerial parts of Teucrium nuchense is characterized by the presence of caryophyllene oxide and 1-octen-3-ol. The methanolic extract exhibits notable antioxidant as well as anti-inflammatory properties, whereas the aqueous extract demonstrates high antioxidant activity. Additionally, three compounds of teucrioside, teucrin A, and 6-α- hydroxyteuscordine have been isolated and characterized.

Rubus urticifolius Compounds with Antioxidant Activity, and Inhibition Potential against Tyrosinase, Melanin, Hyaluronidase, Elastase, and Collagenase.

In our study, using chromatographic techniques, we isolated three bioactive compounds, which were structurally elucidated as (E)-2-(3-(3,4-dimethoxyphenyl)acrylamido)-N-methylbenzamide (1), 4-Hydroxyquinoline-2-carboxylic acid (2), and (E)-2-Cyano-3-(4-hydroxyphenyl)acrylic acid (3), using spectroscopic methods. The anti-melanogenic, anti-inflammatory, antioxidant, and anti-aging properties were evaluated in vitro by measuring the activity of pharmacological targets including tyrosinase, melanin, NF-κB, hyaluronidase, elastase, collagenase, and Nrf2. Our results show that compound 1 is the most active with IC50 values of 14.19 μM (tyrosinase inhibition), 22.24 μM (melanin inhibition), 9.82-12.72 μM (NF-κB inhibition), 79.71 μM (hyaluronidase inhibition), 80.13 μM (elastase inhibition), 76.59 μM (collagenase inhibition), and 116-385 nM (Nrf2 activation) in the THP-1, HEK001, WS1, and HMCB cells. These findings underscore the promising profiles of the aqueous extract of R. urticifolius at safe cytotoxic concentrations. Additionally, we report, for the first time, the isolation and characterisation of these nitrogenous compounds in the R. urticifolius species. Finally, compound 1, isolated from R. urticifolius, is a promising candidate for the development of more effective and safer compounds for diseases related to skin pigmentation, protection against inflammation, and oxidative stress.

The Ethyl Acetate Extract of Caulerpa microphysa Promotes Collagen Homeostasis and Inhibits Inflammation in the Skin.

Inflammation and collagen-degrading enzymes' overexpression promote collagen decomposition, which affects the structural integrity of the extracellular matrix. The polysaccharide and peptide extracts of the green alga Caulerpa microphysa (C. microphysa) have been proven to have anti-inflammatory, wound healing, and antioxidant effects in vivo and in vitro. However, the biological properties of the non-water-soluble components of C. microphysa are still unknown. In the present study, we demonstrated the higher effective anti-inflammatory functions of C. microphysa ethyl acetate (EA) extract than water extract up to 16-30% in LPS-induced HaCaT cells, including reducing the production of interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α (TNF-α). Furthermore, the excellent collagen homeostasis effects from C. microphysa were proven by suppressing the matrix metalloproteinase-1 (MMP-1) secretion, enhancing type 1 procollagen and collagen expressions dose-dependently in WS1 cells. Moreover, using UHPLC-QTOF-MS analysis, four terpenoids, siphonaxanthin, caulerpenyne, caulerpal A, and caulerpal B, were identified and may be involved in the superior collagen homeostasis and anti-inflammatory effects of the C. microphysa EA extract.

Erb‐(IL10)2 ameliorates radiation‐induced skin injury through eliminate oxygen free radicals

AbstractObjectiveRadiation‐induced skin injury (RISI) remains a serious concern during radiotherapy. IL‐10 is considered as an immune suppressive cytokine by inhibiting the secretion of the proinflammatory cytokines in cells. The aim of this study was to evaluate the protective role of Erb (IL10) 2 against ionizing radiation.MethodsWe fused Interleukin 10 (IL‐10) dimer onto an anti‐epidermal growth factor receptor antibody Cetuximab (Erbitux) to form a new bispecific protein Erb‐(IL10)2. The protective effect and biological activity of Erb‐(IL10)2 was measured in model of RISI.ResultsUnder the condition of 20 Gy irradiation, surviving cells in the IR group decreased significantly compared with the non‐IR group (p = 0.0021). The survival rates of HaCaT (p = 0.0038) and WS1 (p = 0.0003) cells were significantly increased after IL‐10 treatment. The apoptosis rates of HaCaT (p = 0.0048) and WS1 (p = 0.0074) cells in the IL‐10 group were significantly lower compared to the NC group. Under 20 Gy irradiation conditions, IL‐10 fusion protein reduced the level of reactive oxygen in HaCaT (p = 0.0046) and WS1 (p<0.0001) cells compared to the control group. Relatively normal granular mitochondrial morphology was observed in the IL‐10 group after 20 Gy X‐ray irradiation compared with the NC group. Ater 35 Gy electron radiation, the levels of reactive oxygen species in the skin tissue of C57/B6 mice injected with IL‐10 fusion protein were significantly lower than those in the PBS group (p = 0.001). Compared with the PBS group and the other IL‐10 groups, the group treated with 0.2 mg/kg IL‐10 showed a significant decrease in MDA level (p = 0.0024). Compared with the PBS group, the thickness of the stratum corneum in groups treated with 0.05, 0.1 and 0.2 mg/kg IL‐10 decreased, and the skin appendages were well‐preserved. In the group treated with 0.2 mg/kg IL‐10, the skin tissue structure was still relatively intact, and the masson staining area was smaller than that of the PBS group.ConclusionIL‐10 plays a role in inhibiting radioactive fibrosis in radioactive skin injury. IL‐10 has a protective effect on skin cell damage after ionizing radiation irradiation both in vitro and in vivo. Moreover, IL‐10 plays a role in inhibiting radioactive fibrosis in radioactive skin injury.

Ultrasonic irradiation enhanced the efficacy of antimicrobial photodynamic therapy against methicillin-resistant Staphylococcus aureus biofilm

Antimicrobial photodynamic therapy (aPDT) is a non-pharmacological antimicrobial regimen based on light, photosensitizer and oxygen. It has become a potential method to inactivate multidrug-resistant bacteria. However, limited by the delivery of photosensitizer (PS) in biofilm, eradicating biofilm-associated infections by aPDT remains challenging. This study aimed to explore the feasibility of combining ultrasonic irradiation with aPDT to enhance the efficacy of aPDT against methicillin-resistant staphylococcus aureus (MRSA) biofilm. A cationic benzylidene cyclopentanone photosensitizer with much higher selectivity to bacterial cells than mammalian cells were applied at the concentration of 10 μM. 532 nm laser (40 mW/cm2, 10 min) and 1 MHz ultrasound (500 mW/cm2, 10 min, simultaneously with aPDT) were employed against MRSA biofilms in vitro. In addition to combined with ultrasonic irradiation and aPDT, MRSA biofilms were treated with laser irradiation only, photosensitizer only, ultrasonic irradiation only, ultrasonic irradiation and photosensitizer, and aPDT respectively. The antibacterial efficacy was determined by XTT assay, and the penetration depth of PS in biofilm was observed using a photoluminescence spectrometer and a confocal laser scanning microscopy (CLSM). In addition, the viability of human dermal fibroblasts (WS-1 cells) after the same treatments mentioned above and the uptake of P3 by WS-1 cells after ultrasonic irradiation were detected by CCK-8 and CLSM in vitro. Results showed that the percent decrease in metabolic activity resulting from the US + aPDT group (75.76%) was higher than the sum of the aPDT group (44.14%) and the US group (9.88%), suggesting synergistic effects. Meanwhile, the diffusion of PS in the biofilm of MRSA was significantly increased by 1 MHz ultrasonic irradiation. Ultrasonic irradiation neither induced the PS uptake by WS-1 cells nor reduced the viability of WS-1 cells. These results suggested that 1 MHz ultrasonic irradiation significantly enhanced the efficacy of aPDT against MRSA biofilm by increasing the penetration depth of PS. In addition, the antibacterial efficacy of aPDT can be enhanced by ultrasonic irradiation, the US + aPDT treatment demonstrated encouraging in vivo antibacterial efficacy (1.73 log10 CFU/mL reduction). In conclusion, the combination of aPDT and 1 MHz ultrasound is a potential and promising strategy to eradicate biofilm-associated infections of MRSA.

Discrepant Activation Pattern of Inflammation and Pyroptosis Induced in Dermal Fibroblasts in Response to Dengue Virus Serotypes 1 and 2 and Nonstructural Protein 1.

Four serotypes of dengue virus (DENV-1 to DENV-4) cause mild to severe disease in humans through infected mosquito bites. Dermal fibroblasts were found to be susceptible to DENV, and this may play a critical role in establishing the initial infection stage. However, the cellular response induced by the different DENV serotypes in dermal fibroblasts during the early stage of infection remains unclear. To determine this, normal human dermal fibroblast WS1 cells were infected with DENV-1 or DENV-2. Compared with the response elicited by DENV-1 infection, DENV-2 induced a stronger innate inflammatory response and cell death in the WS1 cells. However, DENV-1 activated a higher level of pyroptosis signaling than did DENV-2, which was associated with higher virion production. Caspase-1 inhibitor Ac-YVAD-cmk and imipramine, an antidepressant drug, reduced DENV-mediated caspase-1 and interleukin 1β (IL-β) cleavage in the pyroptosis pathway. Ac-YVAD-cmk and imipramine downregulated DENV virion production in WS1 cells. Furthermore, DENV-1 and DENV-2 NS1 proteins promoted diverse activation levels of cell death, inflammatory response, and activation of caspase-1 and IL-β in dermal fibroblasts at different time points. Collectively, these data suggest that DENV-1, DENV-2, and their nonstructural protein 1 (NS1) induce discrepant activation patterns of inflammation and pyroptosis in dermal fibroblasts. The pyroptosis caused by virus and NS1 may facilitate DENV replication in dermal fibroblasts. IMPORTANCE Skin fibroblasts are the primary cells of DENV infection through mosquito bites. Establishing a successful infection in dermal fibroblasts might be critical for dengue disease. However, the cellular response induced by DENV in dermal fibroblasts remains unclear. In this in vitro study, we found that DENV-2 and DENV-1 showed different time course patterns of virus replication and inflammation in dermal fibroblasts. We demonstrated that DENV-1 and DNEV-2 and their viral protein NS1 activate the cellular pyroptosis response to regulate virus replication in dermal fibroblasts. This finding suggests that pyroptosis activation in the DENV primary inoculation site plays a role in the establishment of a DENV infection.

The role of human antigen R (HuR) in modulating proliferation, senescence and radiosensitivity of skin cells.

The skin is the largest outermost organ of the human body. It is vulnerable to various damages, such as ionizing radiation. Exploration of proliferation, senescence and radiosensitivity of skin cells contributes to the development of medical and cosmetic countermeasures against skin aging and toward injury protection. Human antigen R (HuR) is one of the most widely studied RNA-binding proteins and serves an important role in stabilization of mRNA and regulation of the expression of the target genes. To investigate the role of HuR in modulating proliferation, senescence and radiosensitivity of skin cells, the present study performed an in vitro study using lentivirus-mediated overexpression or silencing of HuR in human keratinocyte HaCaT cells and human skin fibroblast WS1 cells. The results indicated that overexpression of HuR promoted proliferation, whereas downregulation of HuR inhibited proliferation of HaCaT and WS1 cells. Overexpression of HuR reduced apoptosis and senescence in skin cells. RNA-Seq of skin cells with HuR overexpression or knockdown identified 77 mRNAs positively or negatively correlated with HuR expression levels. In addition, silencing of HuR induced a significant increase in radiogenic reactive oxygen species after irradiation. Overexpression of HuR increased radiotolerance of HaCaT and WS1 cells. RNA immunoprecipitation coupled with RNA-Seq identified 14 mRNAs interacting with HuR upon radiation exposure. Overall, the findings of the present study illustrated the key role of HuR in modulating proliferation, senescence and radiosensitivity of skin cells providing a new therapeutic strategy for cosmetic treatments and to combat skin injury.

Fenofibrate Attenuates Radiation-Induced Oxidative Damage to the Skin through Fatty Acid Binding Protein 4 (FABP4).

Radiation facilities and radioactive materials have been widely used in military, industry, medicine, science and nuclear facilities, which has significantly increased the potential of large-scale, uncontrolled exposure to radiation. The skin is one of the radiosensitive organ systems and radiation-induced skin injury remains a serious concern after ionizing radiation exposure. Our previous report indicates the involvement of the peroxisome proliferator-activated receptor pathway in the response of skin tissues to ionizing radiation. PPARα is a member of the PPAR nuclear hormone receptor superfamily, which can be activated by fibrate ligands. However, the protection of fenofibrate against ionizing radiation in skin keratinocytes and fibroblasts has not been described. The PPARα mRNA levels in irradiated and nonirradiated skin tissues of rats were determined by real-time assay. The expression of PPARα, and FABP4 were evaluated by western blot and IHC assay. The cell proliferation was detected by colony formation. The γH2AX foci and ROS levels in irradiated WS1 cells with FABP4 overexpression than in control cells were performed by Immunofluorescence assay. We found that PPARα expression was lower in the irradiated skin tissues of mouse, rat, monkey, and human patients than in their nonirradiated counterparts. PPARα fenofibrate significantly decreased radiation-induced ROS and apoptosis in a dose-dependent manner in human keratinocyte HaCaT and skin fibroblast WS1 cells. Moreover, fenofibrate significantly decreased radiation-induced ROS and malondialdehyde (MDA) levels in electron beam irradiated skin tissues of rats. Mechanistically, the proximal promoter of fatty acid binding protein 4 (FABP4) harbored three binding sites of PPARα and fenofibrate stimulated the transcription of FABP4 in skin cells. FABP4 overexpression decreased radiation-induced ROS and γH2AX foci. FABP4 inhibitor BMS309403 abrogated the ROS-eliminating activity as well as the lipid-accumulating role of fenofibrate, indicating that FABP4 mediates the radioprotective role of fenofibrate. In addition, FABP4 overexpression significantly decreased radiation-induced oxidative damage in vivo. These results confirm that fenofibrate attenuated radiation-induced oxidative damage to the skin by stimulating FABP4.

Proteomic and miRNA profiling of radon-induced skin damage in mice: FASN regulated by miRNAs.

Radon is a naturally occurring radioactive gas and considered as a serious carcinogen to humans. Continuous radioactive decay of this gas emits high-energy alpha particles. Long-term radon exposure induces oxidative stress and inflammatory response, which results in chronic lung diseases. However, biological effects after radon exposure in other organs have been rarely reported. As the outermost organ of the human body, the skin suffers from environmental damage to agents such as air pollution. Epidemiological studies indicated that areas with high level of radon had a high incidence of skin cancer. However, whether radon exposure induces skin damage has not been reported yet. In this study, we established a radon-exposed mouse model and found that radon exposure affected the structure of skin tissues, which was manifested by inflammatory cell infiltration and skin atrophy. Using proteomic approach, we found 45 preferentially expressed proteins in 60 Working Level Months (WLM) group and 314 preferentially expressed proteins in 120 WLM group from radon-exposed skin tissues. Through microRNA (miRNA) sequencing profiling analysis, 57 dysregulated miRNAs were screened between the control and radon-treated mouse skin. By integrating the dysregulated proteins and miRNAs, radon-induced fatty acid synthase (FASN) was investigated in greater detail. Results showed that FASN was regulated by miR-206-3p and miR-378a-3p and involved in the pathogenesis of radon-induced skin damage. Overexpression of FASN inhibited the proliferation, and induced in WS1 cells. Our present findings illustrate the molecular change during radon-induced skin damage and the potential role of FASN during this process.

Multi-pin contact drawing enables production of anisotropic collagen fiber substrates for alignment of fibroblasts and monocytes

Type I collagen is the most abundant protein in the human body and is known to play important roles in numerous biological processes including tissue morphogenesis and wound healing. As such, it is one of the most frequently used substrates for cell culture, and there have been considerable efforts to develop collagen-based cell culture substrates that mimic the structural organization of collagen as it is found in native tissues, i.e., collagen fibers. However, producing collagen fibers from extracted collagen has been notoriously difficult, with existing methods providing only low throughput production of collagen fibers. In this study, we prepared collagen fibers using a highly efficient, bio-friendly, and cost-effective approach termed contact drawing, which uses an entangled polymer fluid to aid in fiber formation. Contact drawing technology has been demonstrated previously for collagen using highly concentrated dextran solutions with low concentrations of collagen. Here, we show that by replacing dextran with polyethylene oxide (PEO), high collagen content fibers may be readily formed from mixtures of soluble collagen and PEO, a polymer that readily forms fibers by contact drawing at concentrations as low as 0.5%wt. The presence of collagen and the formation of well-ordered collagen structures in the resulting fibers were characterized by attenuated total reflectance Fourier-transform infrared spectromicroscopy, Raman spectromicroscopy, and fluorescence microscopy. Corresponding to well-ordered collagen, the mechanical properties of the PEO-collagen fibers approximated those observed for native collagen fibers. Growth of cells on aligned PEO-collagen fibers attached to a polydimethyl siloxane support was examined for human dermal fibroblast (WS1) and human peripheral leukemia blood monocyte (THP-1) cell lines. WS1 and THP-1 cells readily attached, displayed alignment through migration and spreading, and proliferated on the collagen fiber substrate over the course of several days. We also demonstrated the retrieval of viable cells from the PEO-collagen fiber substrates through enzymatic digestion of the collagen substrate with collagenase IV.

RXRα agonist bexarotene attenuates radiation-induced skin injury by relieving oxidative stress

ObjectiveTo investigate the protective effect of bexarotene in radiation-induced skin injury and elucidate underlying mechanism. MethodsIrradiated cellular and animal models were established using an X-ray linear accelerator. Cell viability and apoptosis were evaluated by CCK8 and flow cytometry. In vivo protective effect of bexarotene was measured in irradiated SD rats. The antioxidant capacity of bexarotene was validated by DCF-DA method. The signaling pathways involved in bexarotene-mediated skin repair were enriched by RNA sequencing. ResultsBexarotene could significantly restore the proliferation and inhibit the apoptosis of WS1 cells with radiation damage (P ​< ​0.05). Moreover, bexarotene could effectively shorten the process of skin damage and promote skin repair in a rat model of radiation-induced skin injury (P ​< ​0.05). Mechanistically, bexarotene effectively reduced the expression of RXRα (P ​< ​0.05), thus leading to an early decrease in reactive oxygen species (ROS) after radiation exposure. Furthermore, a transcriptome analysis indicated that bexarotene-mediated recovery of radiation damage involves redox signaling, immune regulation, lipid metabolism and autophagy. ConclusionBexarotene is a promising therapeutic agent in the treatment of radiation-induced skin injury.

Highly Selective Fluorescent Probe With an Ideal pH Profile for the Rapid and Unambiguous Determination of Subcellular Labile Iron (III) Pools in Human Cells

A convenient tool for detecting iron ions in subcellular structures is desired for better understanding its roles in biological systems. In this work, a new Fe3+ sensor, 2-(2-((1-(6-acetylpyridin-2-yl)ethylidene)amino)ethyl)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthen]-3-one (RhPK), which operates across the entire cellular pH range and is capable of unambiguously detecting Fe3+ ion in live human cells at subcellular resolution, is reported. The sensor exhibits high selectivity and sensitivity toward Fe3+ with a rapid fluorescence response and a 12-fold increase in intensity upon the addition of 1 equivalent Fe3+ at pH 7.3. RhPK forms a 1:1 complex with Fe3+ with an apparent binding constant 1.54 × 107 M−1 and a detection limit of 50 nM. The sensor is stable between pH 4.2 and 9.0 and operates across the whole cellular pH range. Cell imaging demonstrates the ability of the sensor to unambiguously detect basal level Fe3+ as well as its dynamic changes in real-time in live cells at subcellular resolution, with one labile Fe3+ pool identified in mitochondria in human primary fibroblast (ws1) cells for the first time and two Fe3 + pools confirmed in mitochondria and endo/lysosomes in human SH-SY5Y neuroblastoma cells, suggesting different cell types have distinctive Fe3+ storage in subcellular compartments. The RhPK probe is powerful for rapid and sensitive bioimaging of Fe3+ at subcellular level, enabling the unambiguous detection of labile Fe3+ pools at the entire cellular pH range, which is of great significance to understand the biological chemistry of Fe3+ and its roles in physiological processes and diseases.

Role of AUF1 in modulating the proliferation, migration and senescence of skin cells.

AU-rich element RNA-binding factor 1 (AUF1) is a classical RNA-binding protein. AUF1 influences the process of development, apoptosis and tumorigenesis by interacting with adenylate-uridylate rich element-bearing mRNAs. Human skin is the largest organ of the body and acts as a protective barrier against pathogens and injuries. The aim of the present study was to explore the function and potential molecular pathways of AUF1 in human skin cells. AUF1 was overexpressed in human keratinocyte HaCaT cells and human skin fibroblast WS1 cells using adenoviruses and silenced using lentiviruses. AUF1 overexpression facilitated cell proliferation, whereas AUF1 knockdown induced the opposite effect. AUF1 reduced apoptosis but did not affect cell cycle progression. Forced AUF1 expression promoted the migration of human skin cells, as demonstrated by a scratch wound healing assay. Cell senescence was alleviated in AUF1-overexpressing skin cells, while AUF1 knockdown increased cell senescence. WS1 cells with AUF1 overexpression and silencing were used for RNA-sequencing and Kyoto Encyclopedia of Genes and Genomes-based pathway analysis to identify AUF1-affected mRNAs. A total of 18 mRNAs (eight mRNAs with positive associations and 10 mRNAs with negative associations) revealed consistent associations with both AUF1 overexpression and silencing. Enriched pathways associated with AUF1 expression included ‘MAPK’, ‘cell adhesion molecules’, ‘proteasome’, ‘cellular senescence’ and ‘TGF-β signaling’, indicating a complex regulatory network. Overall, the results of the present study revealed that AUF1 is involved in the proliferation, migration and senescence of skin cells in vitro and may be a potential target for cosmetic and disease treatment of skin.

Transplantation of the Stromal Vascular Fraction (SVF) Mitigates Severe Radiation-Induced Skin Injury.

Severe radiation-induced skin injury is a complication of tumor radiotherapy and nuclear accidents. Cell therapy is a potential treatment for radiation-induced skin injury. The stromal vascular fraction (SVF) is a newer material in stem cell therapy that is made up of stem cells harvested from adipose tissue, which has been shown to promote the healing of refractory wounds of different causes. In this study, SVF was isolated from patients with radiation-induced skin injury. Adipose-derived stem cells (ADSCs) accounted for approximately 10% of the SVF by flow cytometry. Compared with the control group of rats, administration with SVF attenuated the skin injury induced by electron beam radiation. The effect of SVF on the human skin fibroblast microenvironment was determined by proteomic profiling of secreted proteins in SVF-co-cultured human skin fibroblast WS1 cells. Results revealed 293 upregulated and 1,481 downregulated proteins in the supernatant of SVF-co-cultured WS1 cells. WS1 co-culture with SVF induced secretion of multiple proteins including collagen and MMP-1. In the clinic, five patients with radiation-induced skin injury were recruited to receive SVF transfer-based therapy, either alone or combined with flap transplantation. Autogenous SVF was isolated and introduced into a multi-needle precision electronic injection device, which automatically and aseptically distributed the SVF to the exact layer of the wound in an accurate amount. After SVF transfer, wound healing clearly improved and pain was significantly relieved. The patients' skin showed satisfactory texture and shape with no further wound recurrence. Our findings suggest that transplantation of SVF could be an effective countermeasure against severe radiation-induced skin injury.

Active Tumoral/Tumor Environmental Dual-Targeting by Non-Covalently Arming with Trispecific Antibodies or Dual-Bispecific Antibodies on Docetaxel-Loaded mPEGylated Nanocarriers to Enhance Chemotherapeutic Efficacy and Minimize Systemic Toxicity.

PurposeThis study was aimed at developing the trispecific antibodies (anti-EGFR/anti-FAP/anti-mPEG, TsAb) or dual bispecific antibodies (anti-EGFR/anti-mPEG and anti-FAP/anti-mPEG) docetaxel (DTX)-loaded mPEGylated lecithin-stabilized micelles (mPEG-lsbPMs) for improving the targeting efficiency and therapeutic efficacy.MethodsmPEG-lsbPMs were simply prepared via thin film method. The trispecific antibodies or bispecific antibodies bound the mPEG-lsbPMs by anti-mPEG Fab fragment. The formulations were characterized by DLS and TEM; in vitro and in vivo studies were also conducted to evaluate the cellular uptake, cell cytotoxicity and therapeutic efficacy.ResultsThe particle sizes of mPEG-lsbPMs with or without the antibodies were around 100 nm; the formulations showed high encapsulation efficiencies of 97.12%. The TsAb and dual bispecific antibodies were fabricated and demonstrated their targeting ability. Two EGFR-overexpressed cell lines (HT-29 and MIA PaCa-2) were co-cultured with FAP-overexpressed WS1 cells (HT-29/WS1; MIA PaCa-2/WS1) to mimic a tumor coexisting in the tumor microenvironment. Cellular binding study revealed that the binding of anti-FAP micelles to three co-culture ratios (4:1, 1:1, and 1:4) of HT-29/EGFR to WS1/FAP was significantly higher than that for TsAb micelles and dual (1:1) micelles, and the binding of those targeting antibodies to WS1/FAP and MIA PaCa-2/EGFR was equally efficacious resulting in a similar binding amount of the TsAb and dual BsAbs (1:1) with the co-culture of MIA PaCa-2/EGFR and WS1/FAP at a 1:1 ratio. Antitumor efficacy study showed that treatment with DTX-loaded mPEG-lsbPMs modified with or without BsAbs, dual BsAbs (1:1), and TsAbs was enhanced in inhibiting tumor growth compared with that for Tynen® while showing fewer signs of adverse effects.ConclusionActive targeting of both tumors and TAF-specific antigens was able to increase the affinity of DTX-loaded mPEG-lsbPMs toward tumor cells and TAFs leading to successive uptake by tumor cells or TAFs which enhanced their chemotherapeutic efficacy against antigen-positive cancer cells.

Activated Beta-Catenin Signaling Ameliorates Radiation-Induced Skin Injury by Suppressing Marvel D3 Expression.

Radiation-induced skin injury remains a serious concern for cancer radiotherapy, radiation accidents and occupational exposure, and the damage mainly occurs due to apoptosis and reactive oxygen species (ROS) generation. There is currently no effective treatment for this disorder. The β-catenin signaling pathway is involved in the repair and regeneration of injured tissues. However, the role of the β-catenin signaling pathway in radiation-induced skin injury has not been reported. In this study, we demonstrated that the β-catenin signaling pathway was activated in response to radiation and that its activation by Wnt3a, a ligand-protein involved in the β-catenin signaling pathway, inhibited apoptosis and the production of ROS in irradiated human keratinocyte HaCaT cells and skin fibroblast WS1 cells. Additionally, Wnt3a promoted cell migration after irradiation. In a mouse model of full-thickness skin wounds combined with total-body irradiation, Wnt3a was shown to facilitate skin wound healing. The results from RNA-Seq revealed that 24 genes were upregulated and 154 were downregulated in Wnt3a-treated irradiated skin cells, and these dysregulated genes were mainly enriched in the tight junction pathway. Among them, Marvel D3 showed the most obvious difference. We further found that the activated β-catenin signaling pathway stimulated the phosphorylation of JNK by silencing Marvel D3. Treatment of irradiated cells with SP600125, a JNK inhibitor, augmented ROS production and impeded cell migration. Furthermore, treatment with Wnt3a or transfection with Marvel D3-specific siRNAs could reverse the above effects. Taken together, these findings illustrate that activated β-catenin signaling stimulates the activation of JNK by negatively regulating Marvel D3 to ameliorate radiation-induced skin injury.

Interferon-α inducible protein 6 (IFI6) confers protection against ionizing radiation in skin cells

BackgroundRadiation-induced skin injury is one of the main adverse effects and a dose-limiting factor of radiotherapy without feasible treatment. The underlying mechanism of this disease is still limited. ObjectiveTo investigate the potential molecular pathways and mechanisms of radiation-induced skin injury. MethodsmRNA expression profiles were determined by Affymetrix Human HTA2.0 microarray.IFI6 overexpression and knockdown were mediated by lentivirus. The functional changes of skin cells were measured by flow cytometry, ROS probe and Edu probe. Protein distribution was detected by immunofluorescence experiment, and IFI6-interacting proteins were detected by immunoprecipitation (IP) combined with mass spectrometry. The global gene changes in IFI6-overexpressed skin cells after irradiation were detected by RNA-seq. ResultsmRNA expression profiling showed 50 upregulated and 13 down regulated genes and interferon alpha inducible protein 6 (IFI6) was top upregulated. Overexpression of IFI6 promoted cell proliferation and reduced cell apoptosis as well as ROS production following radiation, and conversely, increased the radiosensitivity of HaCaT and human skin fibroblast (WS1). IFI6 was translocated into nucleus in irradiated skin cells and the interacting relationship with mitochondrial single-stranded DNA-binding protein 1 (SSBP1), which could enhance the transcriptional activity of heat shock transcription factor 1 (HSF1).IFI6 augmented HSF1 activity following radiation in HaCaT and WS1 cells. RNA-seq analysis showed IFI6 modulated virus infection and cellular response to stress pathways, which may help to further explore how IFI6 regulate the transcriptional activity of HSF1. ConclusionThis study reveals that IFI6 is induced by ionizing radiation and confers radioprotection in skin cells.

Selective Laser Efficiency of Green-Synthesized Silver Nanoparticles by Aloe arborescens and Its Wound Healing Activities in Normal Wounded and Diabetic Wounded Fibroblast Cells: In vitro Studies.

IntroductionSilver nanoparticles (AgNPs) have been extensively used in wound healing applications owing to their valuable physicochemical and biological properties. The main objective of this study was to evaluate the combined effects of green-synthesized silver nanoparticles (G-AgNPs) and photobiomodulation (PBM; laser irradiation at 830 nm with 5 J/cm2) in normal wounded and diabetic wounded fibroblast cells (WS1).MethodsThe combined effect of G-AgNPs and PBM was studied by various in vitro wound healing studies including cell morphology, cell migration rate and percentage wound closure, cell viability, cell proliferation, and filamentous (F)-actin and nuclear morphology staining.ResultsCell viability results revealed good cellular compatibility of G-AgNPs to WS1 cells. The combined therapy of G-AgNPs and PBM demonstrated promising results to achieve progressive migration and wound closure in both normal wounded and diabetic wounded cell models. G-AgNPs alone and in combination with PBM had no negative effect on cell viability and proliferation, and there was an increase in cell migration.ConclusionOverall, these findings demonstrate that the combined treatment of G-AgNPs and PBM does not display any adverse effects on wound healing processes in both normal wounded and diabetic wounded cell models.

In Vitro Hormetic Effect Investigation of Thymol on Human Fibroblast and Gastric Adenocarcinoma Cells.

The concept of hormesis includes a biphasic cellular dose-response to a xenobiotic stimulus defined by low dose beneficial and high dose inhibitory or toxic effects. In the present study, an attempt has been made to help elucidate the beneficial and detrimental effects of thymol on different cell types by evaluating and comparing the impact of various thymol doses on cancerous (AGS) and healthy (WS-1) cells. Cytotoxic, genotoxic, and apoptotic effects, as well as levels of reactive oxygen species and glutathione were studied in both cell lines exposed to thymol (0–600 µM) for 24 h. The results showed significant differences in cell viability of AGS compared to WS-1 cells exposed to thymol. The differences observed were statistically significant at all doses applied (P ≤ 0.001) and revealed hormetic thymol effects on WS-1 cells, whereas toxic effects on AGS cells were detectable at all thymol concentrations. Thymol at low concentrations provides antioxidative protection to WS-1 cells in vitro while already inducing toxic effects in AGS cells. In that sense, the findings of the present study suggest that thymol exerts a dose-dependent hormetic impact on different cell types, thereby providing crucial information for future in vivo studies investigating the therapeutic potential of thymol.