OBJECTIVE: To investigate the effectiveness and preliminary mechanisms of icariin (ICA) in enhancing the reparative effects of adipose-derived stem cells (ADSCs) on skin radiation damagies in rats. METHODS: Twelve SPF-grade Sprague Dawley rats [body weight (220±10) g] were subjected to a single dose of 10 Gy X-ray irradiation on a 1.5 cm×1.5 cm area of their dorsal skin, with a dose rate of 200 cGy/min to make skin radiation damage model. After successful modelling, the rats were randomly divided into 4 groups ( n=3), and on day 2, the corresponding cells were injected subcutaneously into the irradiated wounds: group A received 0.1 mL of rat ADSCs (1×10 ⁷cells/mL), group B received 0.1 mL of rat ADSCs (1×10 ⁷cells/mL)+1 μmol/L ICA (0.1 mL), group C received 0.1 mL of rat ADSCs (1×10 ⁷cells/mL) pretreated with a hypoxia-inducible factor 2α (HIF-2α) inhibitor+1 μmol/L ICA (0.1 mL), and group D received 0.1 mL of rat ADSCs (1×10 ⁷cells/mL) pretreated with a Notch1 inhibitor+1 μmol/L ICA (0.1 mL). All treatments were administered as single doses. The skin injury in the irradiated areas of the rats was observed continuously from day 1 to day 7 after modelling. On day 28, the rats were sacrificed, and skin tissues from the irradiated areas were harvested for histological examination (HE staining and Masson staining) to assess the repair status and for quantitative collagen content detection. Immunohistochemical staining was performed to detect CD31 expression, while Western blot and real-time fluorescence quantitative PCR (qRT-PCR) were used to measure the protein and mRNA relative expression levels of vascular endothelial growth factor (VEGF), platelet-derived growth factor BB (PDGF-BB), fibroblast growth factor 2 (FGF-2), interleukin 10 (IL-10), transforming growth factor β (TGF-β), HIF-2α, and Notch1, 2, and 3. RESULTS: All groups exhibited skin ulcers and redness after irradiation. On day 3, exudation of tissue fluid was observed in all groups. On day 7, group B showed significantly smaller skin injury areas compared to the other 3 groups. On day 28, histological examination revealed that the epidermis was thickened and the dermal fibers were slightly disordered with occasional inflammatory cell aggregation in group A. In group B, the epidermis appeared more normal, the dermal fibers were more orderly, and there was an increase in new blood vessels without significant inflammatory cell aggregation. In contrast, groups C and D showed significantly increased epidermal thickness, disordered and disrupted dermal fibers. Group B had higher collagen fiber content than the other 3 groups, and group D had lower content than group A, with significant differences ( P<0.05). Immunohistochemical staining showed that group B had significantly higher CD31 expression than the other 3 groups, while groups C and D had lower expression than group A, with significant differences ( P<0.05). Western blot and qRT-PCR results indicated that group B had significantly higher relative expression levels of VEGF, PDGF-BB, FGF-2, IL-10, TGF-β, HIF-2α, and Notch1, 2, and 3 proteins and mRNAs compared to the other 3 groups ( P<0.05). CONCLUSION ICA may enhance the reparative effects of ADSCs on rat skin radiation damage by promoting angiogenesis and reducing inflammatory responses through the HIF-2α-VEGF-Notch signaling pathway.
Animals ; Rats, Sprague-Dawley ; Skin/pathology* ; Rats ; Vascular Endothelial Growth Factor A/genetics* ; Basic Helix-Loop-Helix Transcription Factors/genetics* ; Signal Transduction ; Flavonoids/pharmacology* ; Adipose Tissue/cytology* ; Stem Cells/cytology* ; Receptors, Notch/metabolism* ; Radiation Injuries, Experimental/metabolism* ; Wound Healing/drug effects* ; Male

Objective To investigate the effects of cucurbitacin B (CucB) on alleviating skin lesions and inflammation in psoriasis mice via the cGAS-STING signaling pathway. Methods The expression of genes associated with the cGAS-STING signaling pathway in psoriatic lesions and non-lesional skin was analyzed, and hallmark gene set enrichment analysis was performed. The cytotoxicity of CucB on BMDMs was evaluated using the CCK-8 assay. The expression levels of genes and proteins related to the cGAS-STING signaling pathway, along with the secretion of inflammatory cytokines, were measured at different concentrations of CucB using quantitative PCR, Western blotting, and ELISA. Imiquimod-induced psoriasis BALB/c mice were divided into four groups: normal group, model group, low-dose CucB group [0.1 mg/ (kg.d)], and high-dose CucB group [0.4 mg/ (kg.d)], with five mice per group. PASI scoring was performed to assess the severity of psoriasis after 6 days of treatment, and HE staining was conducted to observe pathological damage. Meanwhile, the mRNA levels of inflammatory cytokines and their secretion were detected by qPCR and ELISA. Results Most cGAS-STING signaling-related genes were upregulated in lesional skin of psoriasis patients, and the hallmark gene set enrichment analysis revealed that the most significantly upregulated genes were primarily associated with immune response signaling pathways. CucB inhibited dsDNA-induced phosphorylation of interferon regulatory factor 3 (IRF3) and STING proteins in both bone-marrow derived macrophages(BMDMs) and THP-1 cells. CucB also suppressed dsDNA-induced mRNA expression of IFNB1, TNF, IFIT1, CXCL10, ISG15, and reduced the secretion of cytokines such as IFN-β, IL-1β, and TNF-α in THP-1 cells. In the imiquimod-induced psoriasis mouse model, CucB treatment reduced psoriatic symptoms, alleviated skin lesions, and attenuated inflammation. ELISA and qPCR results showed that CucB significantly reduced serum secretion levels of IL-6, TNF-α, and IL-1β, as well as the mRNA levels of IL23A, IL1B, IL6, TNF, and IFNB1. Conclusion CucB inhibits cytoplasmic DNA-induced activationc of the GAS-STING pathway. CucB significantly attenuates skin lesions and inflammation in IMQ-induced psoriatic mice, and the potential molecular mechanism may be related to the down-regulation of the cGAS-STING pathway.
Animals ; Psoriasis/pathology* ; Signal Transduction/drug effects* ; Membrane Proteins/genetics* ; Mice ; Nucleotidyltransferases/genetics* ; Disease Models, Animal ; Mice, Inbred BALB C ; Skin/metabolism* ; Triterpenes/therapeutic use* ; Humans ; Cytokines/metabolism* ; Inflammation/drug therapy* ; Male

OBJECTIVESTo investigate the hair growth-promoting effect of Miscanthus sinensis var. purpurascens (MSP) flower extracton on in vitro and in vivo models.

METHODSMSP flower extract was extracted in 99.9% methanol and applied to examine the proliferation of human dermal papilla cells (hDPCs) in vitro at the dose of 3.92-62.50 μg/mL and hair growth of C57BL/6 mice in vivo at the dose of 1000 μg/mL. The expression of transforming growth factor β1 (TGF-β1), hepatocyte growth factor (HGF), β-catenin, substance P was measured by relative quantitative realtime polymerase chain reaction. Histopathological and immunohistochemical analysis were performed.

RESULTSMSP (7.81 μg/mL) down-regulated TGF-β1 and up-regulated HGF and β-catenin in hDPCs (P<0.01). MSP (1000 μg/mL)-treated mice showed the earlier transition of hair follicles from the telogen to the anagen phase. The number of mast cells was lower in the MSP-treated mice than in other groups (P<0.05 vs. NCS group). Substance P and TGF-β1 were expressed in hair follicles and skin of the MSP group lower than that in negative control. Stem cell factor in hair follicles was up-regulated in the MSP-treated mice (P<0.01).

CONCLUSIONSThe MSP flower extract may have hair growth-promotion activities.

Animals ; Antioxidants ; pharmacology ; Cell Count ; Cell Proliferation ; drug effects ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Female ; Flowers ; chemistry ; Hair Follicle ; cytology ; drug effects ; growth & development ; Hepatocyte Growth Factor ; metabolism ; Humans ; Mast Cells ; cytology ; Mice, Inbred C57BL ; Phosphorylation ; drug effects ; Plant Extracts ; pharmacology ; Poaceae ; chemistry ; RNA, Messenger ; genetics ; metabolism ; Skin ; metabolism ; Stem Cell Factor ; metabolism ; Stress, Psychological ; pathology ; Substance P ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; beta Catenin ; metabolism

Recent studies have shown that the chemokine receptor CXCR3 and its ligand CXCL10 in the dorsal root ganglion mediate itch in experimental allergic contact dermatitis (ACD). CXCR3 in the spinal cord also contributes to the maintenance of neuropathic pain. However, whether spinal CXCR3 is involved in acute or chronic itch remains unclear. Here, we report that Cxcr3 mice showed normal scratching in acute itch models but reduced scratching in chronic itch models of dry skin and ACD. In contrast, both formalin-induced acute pain and complete Freund's adjuvant-induced chronic inflammatory pain were reduced in Cxcr3 mice. In addition, the expression of CXCR3 and CXCL10 was increased in the spinal cord in the dry skin model induced by acetone and diethyl ether followed by water (AEW). Intrathecal injection of a CXCR3 antagonist alleviated AEW-induced itch. Furthermore, touch-elicited itch (alloknesis) after compound 48/80 or AEW treatment was suppressed in Cxcr3 mice. Finally, AEW-induced astrocyte activation was inhibited in Cxcr3 mice. Taken together, these data suggest that spinal CXCR3 mediates chronic itch and alloknesis, and targeting CXCR3 may provide effective treatment for chronic pruritus.
Acetamides ; therapeutic use ; Animals ; Chemokine CXCL10 ; metabolism ; Chloroquine ; toxicity ; Chronic Disease ; Cyclopropanes ; adverse effects ; Dehydration ; complications ; Dinitrofluorobenzene ; adverse effects ; Disease Models, Animal ; Formaldehyde ; toxicity ; Freund's Adjuvant ; toxicity ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Motor Activity ; drug effects ; Pain ; chemically induced ; Pruritus ; chemically induced ; pathology ; Pyrimidines ; therapeutic use ; Receptors, CXCR3 ; antagonists & inhibitors ; genetics ; metabolism ; Skin ; pathology ; Spinal Cord ; drug effects ; metabolism ; pathology ; Time Factors ; p-Methoxy-N-methylphenethylamine ; toxicity

Increasing evidence suggests that cytokines and chemokines play crucial roles in chronic itch. In the present study, we evaluated the roles of tumor necrosis factor-alpha (TNF-α) and its receptors TNF receptor subtype-1 (TNFR1) and TNFR2 in acute and chronic itch in mice. Compared to wild-type (WT) mice, TNFR1-knockout (TNFR1-KO) and TNFR1/R2 double-KO (DKO), but not TNFR2-KO mice, exhibited reduced acute itch induced by compound 48/80 and chloroquine (CQ). Application of the TNF-synthesis inhibitor thalidomide and the TNF-α antagonist etanercept dose-dependently suppressed acute itch. Intradermal injection of TNF-α was not sufficient to evoke scratching, but potentiated itch induced by compound 48/80, but not CQ. In addition, compound 48/80 induced TNF-α mRNA expression in the skin, while CQ induced its expression in the dorsal root ganglia (DRG) and spinal cord. Furthermore, chronic itch induced by dry skin was reduced by administration of thalidomide and etanercept and in TNFR1/R2 DKO mice. Dry skin induced TNF-α expression in the skin, DRG, and spinal cord and TNFR1 expression only in the spinal cord. Thus, our findings suggest that TNF-α/TNFR1 signaling is required for the full expression of acute and chronic itch via peripheral and central mechanisms, and targeting TNFR1 may be beneficial for chronic itch treatment.
Animals ; Chloroquine ; toxicity ; Disease Models, Animal ; Dose-Response Relationship, Drug ; Etanercept ; therapeutic use ; Ganglia, Spinal ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Pruritus ; chemically induced ; drug therapy ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Receptors, Tumor Necrosis Factor, Type I ; deficiency ; genetics ; Receptors, Tumor Necrosis Factor, Type II ; deficiency ; genetics ; Signal Transduction ; drug effects ; Skin ; drug effects ; metabolism ; Spinal Cord ; drug effects ; metabolism ; Thalidomide ; therapeutic use ; Time Factors ; Tumor Necrosis Factor-alpha ; adverse effects ; genetics ; metabolism ; p-Methoxy-N-methylphenethylamine ; toxicity

PURPOSE: The purpose of this study was to evaluate the effects of essential oil on oxidative stress, immunity, and skin condition in atopic dermatitis (AD) induced mice. METHODS: This study was a 3x3 factorial design. Factors were oil type (Lavender, Thyme, and 2:1 mixture of lavender and thyme oil [blending oil]) and treatment period (0 day, 7 days, and 21 days). The samples were 45 mice with AD and randomly assigned to nine groups of five mice per group. The dependent variables such as superoxide radical, IgE, degranulated mast cells, and epidermal thickness were measured. Data were collected from February to April in 2014. Descriptive statistics, One-way ANOVA, Two-way ANOVA, and Tukey's HSD test were performed using the SPSS WIN 20.0 program. RESULTS: Dependent variables were not statistically significantly different by the three oil types (p >.05). Essential oils such as lavender, thyme, and blending oil were all effective in reducing AD symptoms and especially 2:1 blending oil were most effective. There were statistically significant differences by the three treatment periods in all dependent variables (p <.001). There were statistically significant interactions between oil types and treatment periods in all dependent variables (p <.01). For decreasing superoxide radical, degranulated mast cells, and epidermal thickness, 2:1 mixed oil should be applied for at least 21 days. Otherwise to reduce IgE, 2:1 mixed oil should be used for at least 7 days. CONCLUSION: These findings provide bases for developing effective interventions for AD patients to manage their AD symptoms.
Animals ; Dermatitis, Atopic/chemically induced/*drug therapy/pathology ; Disease Models, Animal ; *Immunity/drug effects ; Immunoglobulin E/blood ; Lavandula/*chemistry/metabolism ; Mast Cells/cytology/metabolism ; Mice ; Oils, Volatile/chemistry/pharmacology/therapeutic use ; *Oxidative Stress/drug effects ; Picryl Chloride/toxicity ; Plant Oils/chemistry/pharmacology/*therapeutic use ; Singlet Oxygen/metabolism ; Skin/drug effects/pathology ; Thymus Plant/*chemistry/metabolism

PURPOSE: Cutaneous lymphocyte-associated antigen (CLA)-expressing CD8+T cells have been known to play an important role in the pathogenesis of atopic dermatitis (AD). However, the mechanisms underlying the loss of self-tolerance remain unclear. Regulatory T cells (Tregs) play a key role in the development of homeostasis in the immune system. We, therefore, hypothesized that a reduced ability of Tregs to inhibit autologous CD8+CLA+T cells might be underlying mechanism in AD. MATERIALS AND METHODS: CD8+CLA+T cells and Tregs were obtained from the peripheral blood of AD patients and control volunteers. The frequencies of CD8+CLA+T cells were evaluated. The proliferative responses of CD8+CLA+T cells were assessed by flow cytometry, and the levels of transforming growth factor-beta1 (TGF-beta1) and interleukin-10 (IL-10) in culture supernatants were detected by enzyme-linked immunosorbent assay. RESULTS: Our results revealed higher frequency and increased expression of perforin and granzyme-B in peripheral CD8+CLA+T cells in AD, and lower inhibitory ability of Tregs on proliferation of CD8+CLA+T cells in AD. Meanwhile, the levels of TGF-beta1 produced by Tregs were significantly lower in AD, and anti-TGF-beta1 abolished such suppression. CONCLUSION: The attenuated inhibitory ability of Tregs on hyper-activated autologous CD8+CLA+T cells, mediated by TGF-beta1, plays an important role in the pathogenesis of AD.
Adult ; Aged ; CD8-Positive T-Lymphocytes/drug effects/*immunology ; Case-Control Studies ; Cell Proliferation ; Cell Separation ; Dermatitis, Atopic/*immunology/pathology ; Female ; Granzymes/metabolism ; Humans ; Interleukin-10/metabolism ; Lymphocyte Count ; Male ; Perforin/metabolism ; Skin/*immunology/pathology ; T-Lymphocytes, Cytotoxic/drug effects/immunology ; T-Lymphocytes, Regulatory/drug effects/*immunology ; Transforming Growth Factor beta1/pharmacology

Uncontrolled fibrosis of skin and internal organs is the main characteristic of scleroderma, and collagen is a major extracellular matrix protein that deposits in the fibrotic organs. As the chaperone of collagen, heat shock protein 47 (HSP47) is closely related with the development of fibrosis. To explore the potential function of HSP47 in the pathogenesis of scleroderma, the clinical, in vivo and in vitro studies were performed. In clinical, the increased mRNA level of HSP47 was observed in the skin fibroblasts and PBMC from scleroderma patients, and the enhanced protein level of HSP47 was also detected in the skin biopsy and plasma of the above patients. Unexpectedly, the enhanced levels of HSP47 were positively correlated with the presence of anti-centromere antibody in scleroderma patients. Moreover, a high expression of HSP47 was found in the skin lesion of BLM-induced scleroderma mouse model. Further in vitro studies demonstrated that HSP47 knockdown could block the intracellular and extracellular collagen over-productions induced by exogenous TGF-β. Therefore, the results in this study provide direct evidence that HSP47 is involved in the pathogenesis of scleroderma. The high expression of HSP47 can be detected in the circulatory system of scleroderma patients, indicating that HSP47 may become a pathological marker to assess the progression of scleroderma, and also explain the systemic fibrosis of scleroderma. Meanwhile, collagen over-expression is blocked by HSP47 knockdown, suggesting the possibility that HSP47 can be a potential therapeutic target for scleroderma.
Adolescent ; Adult ; Animals ; Biopsy ; Blotting, Western ; Cells, Cultured ; Collagen ; metabolism ; Female ; Fibroblasts ; drug effects ; metabolism ; Fibrosis ; HSP47 Heat-Shock Proteins ; blood ; genetics ; metabolism ; Humans ; Leukocytes, Mononuclear ; metabolism ; Male ; Mice ; Mice, Inbred C3H ; Middle Aged ; NIH 3T3 Cells ; Protein Binding ; RNA Interference ; Reverse Transcriptase Polymerase Chain Reaction ; Scleroderma, Systemic ; blood ; genetics ; metabolism ; Skin ; metabolism ; pathology ; Transforming Growth Factor beta ; pharmacology ; Young Adult

Redox-regulating molecule, recombinant human thioredoxin (rhTRX) which shows anti-inflammatory, and anti-oxidative effects against lipopolysaccharide (LPS)-stimulated inflammation and regulate protein expression levels. LPS-induced reactive oxygen intermediates (ROI) and NO production were inhibited by exogenous rhTRX. We identified up/downregulated intracellular proteins under the LPS-treated condition in exogenous rhTRX-treated A375 cells compared with non-LPS-treated cells via 2-DE proteomic analysis. Also, we quantitatively measured cytokines of in vivo mouse inflammation models using cytometry bead array. Exogenous rhTRX inhibited LPS-stimulated production of ROI and NO levels. TIP47 and ATP synthase may influence the inflammation-related lipid accumulation by affecting lipid metabolism. The modulation of skin redox environments during inflammation is most likely to prevent alterations in lipid metabolism through upregulation of TIP47 and ATP synthase and downregulation of inflammatory cytokines. Our results demonstrate that exogenous rhTRX has anti-inflammatory properties and intracellular regulatory activity in vivo and in vitro. Monitoring of LPS-stimulated pro-inflammatory conditions treated with rhTRX in A375 cells could be useful for diagnosis and follow-up of inflammation reduction related with candidate proteins. These results have a therapeutic role in skin inflammation therapy.
Animals ; Antioxidants/*pharmacology ; Cell Line, Tumor ; Humans ; Inflammation/metabolism ; *Lipid Metabolism ; Lipopolysaccharides/pharmacology ; Mice ; Mice, Inbred C57BL ; Nitric Oxide/metabolism ; Proteome/genetics/metabolism ; Skin/drug effects/metabolism/pathology ; Thioredoxins/*pharmacology

OBJECTIVETo study the effect of Shengji Huayu Recipe (SHR)on the expression of MMP-3 and TIMP-1 in the skin ulcer tissue of diabetic rats.

METHODSThe skin ulcer model was established in diabetic mice. Different compatibility proportions of SHR [the ratio of Shengji Recipe (SJR) to Huayu Recipe (HYR) = 2:1, 1:1, and 1:2, respectively] were used to intervene. The expression of MMP-3 protein in the skin ulcer of diabetic rats was detected by Western blot method,and TIMP-1 protein was detected by immunohistochemical assay.

RESULTSAt each time point, there was no statistical difference in the blood glucose level among groups (P > 0.05). But all of them increased significantly,when compared with those of the normal wound group (P < 0.01). As for the difference between after would area treatment and before would area treatment, better effect was obtained in the SHR No. 3 group and the normal ulcer group than in the diabetic ulcer model group (P < 0.05). Results of Western blot showed that the MMP-3 protein expression was higher in the SHR No. 2 group than in the SHR No.3 group (P < 0.05). Immunohistochemical results showed that TIMP-1 protein expression was lower in the SHR No. 2 group than in the SHR No. 3 group and the diabetic ulcer model group (P < 0.05). TIMP-1 protein expression was higherin the SHR No. 3 group than in the SHR No. 2 group (P < 0.01).

CONCLUSIONUsing SHR No.3 was conducive to the promotion of wound healing in early wound repair stage, and using SHR No. 2 might be conducive to inhibiting the formation of pathological scar.

Animals ; Diabetes Mellitus, Experimental ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Male ; Matrix Metalloproteinase 3 ; metabolism ; Phytotherapy ; Rats ; Rats, Sprague-Dawley ; Skin ; drug effects ; pathology ; Skin Ulcer ; drug therapy ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism