Ao-Xue Wang

MicroRNA-132 enhances transition from inflammation to proliferation during wound healing

Wound healing is a complex process that is characterized by an initial inflammatory phase followed by a proliferative phase. This transition is a critical regulatory point; however, the factors that mediate this process are not fully understood. Here, we evaluated microRNAs (miRs) in skin wound healing and characterized the dynamic change of the miRNome in human skin wounds. miR-132 was highly upregulated during the inflammatory phase of wound repair, predominantly expressed in epidermal keratinocytes, and peaked in the subsequent proliferative phase. TGF-β1 and TGF-β2 induced miR-132 expression in keratinocytes, and transcriptome analysis of these cells revealed that miR-132 regulates a large number of immune response- and cell cycle-related genes. In keratinocytes, miR-132 decreased the production of chemokines and the capability to attract leukocytes by suppressing the NF-κB pathway. Conversely, miR-132 increased activity of the STAT3 and ERK pathways, thereby promoting keratinocyte growth. Silencing of the miR-132 target heparin-binding EGF-like growth factor (HB-EGF) phenocopied miR-132 overexpression in keratinocytes. Using mouse and human ex vivo wound models, we found that miR-132 blockade delayed healing, which was accompanied by severe inflammation and deficient keratinocyte proliferation. Together, our results indicate that miR-132 is a critical regulator of skin wound healing that facilitates the transition from the inflammatory to the proliferative phase.

Targeting RAS/RAF/MEK/ERK signaling in metastatic melanoma

The RAS/RAF/MEK/ERK pathway has been reported to be activated in over 80% of all cutaneous melanomas, making it the focus of many scientific studies in the melanoma field. Discoveries of mutations and aberrant expression of components in this cascade, in particular, BRAF and NRAS render a deeper understanding of the mechanisms responsible for oncogenesis and provide new therapeutic strategies for this deadly disease. This review starts with a comprehensive discussion on the role of this pathway in initiation and progress of melanoma. Mechanistically, mutated BRAF and NRAS exert most of the oncogenic effects through the activation of the MAPK pathway, which both drive the uncontrolled growth of melanoma cells and regulate the cell survival. In a subsequent section, clinical efficacy of targeted small‐molecule inhibitors is highlighted. BRAF‐targeted therapies (e.g., vemurafenib, dabrafenib) have showed impressive results in systemic therapy for melanoma harboring activating BRAF V600E mutations. MEK inhibitors show limited activity in phase I trials, and inhibitors directly targeting mutated NRAS, to date, have not been realized. Furthermore, the emerging mechanisms underlying both intrinsic and acquired drug resistance as well as approaches to prevent or abrogate the onset of therapeutic escape are addressed. Finally, the promising vistas and major challenges involving small‐molecule inhibitors targeting this MAPK pathway in melanoma therapy are briefly discussed. It can be envisaged that disseminated melanoma is no longer such a bleak prognosis in future given the research and development of new signal transduction inhibitors based on our evolving understanding of melanoma genetics and intracellular signaling.

Inhibitory effects of sanguinarine on human liver cytochrome P450 enzymes

Sanguinarine (SAG) has been recognized as an anticancer drug candidate. However, the drug-drug interactions (DDI) potential for SAG via the inhibition against human cytochrome P450 (CYP) enzymes remains unclear. In the present study, the inhibitory effects of SAG on seven major human CYP isoforms 1A2, 2A6, 2E1, 2D6, 2C8, 2C9 and 3A4 were investigated with human liver microsomes (HLM). The results showed that SAG was a potent noncompetitive inhibitor of CYP2C8 activity (Ki=8.9 μM), and competitive inhibitor of CYP1A2, CYP2C9 and CYP3A4 activities (Ki=2.7, 3.8 and 2.0 μM, respectively). Furthermore, SAG exhibited time- and NADPH-dependent inhibition towards CYP1A2 and CYP3A4 with KI/kinact values of 13.3/0.087 and 5.58/0.029 min(-1) μM(-1), respectively. Weak inhibition of SAG against CYP2E1, CYP2D6 and CYP2A6 was also observed. In vitro-in vivo extrapolation (IV-IVE) from HLM data showed that more than 35.9% of CYP1A2, CYP2C9, CYP2C8 and CYP3A4 activities in vivo could be inhibited by SAG, suggesting that harmful DDIs could occur when SAG or its medical preparations are co-administered with drugs primarily cleared by these CYP isoforms. Further in vivo studies are needed to evaluate the clinical significance of the data presented herein.

MicroRNA-31 Is Overexpressed in Cutaneous Squamous Cell Carcinoma and Regulates Cell Motility and Colony Formation Ability of Tumor Cells

Cutaneous squamous cell carcinoma (cSCC) is a malignancy of epidermal keratinocytes that is responsible for approximately 20% of skin cancer-related death yearly. We have previously compared the microRNA (miRNA) expression profile of cSCC to healthy skin and found the dysregulation of miRNAs in human cSCC. In this study we show that miR-31 is overexpressed in cSCC (n = 68) compared to healthy skin (n = 34) and precancerous skin lesions (actinic keratosis, n = 12). LNA in situ hybridization revealed that miR-31 was specifically up-regulated in tumor cells. Mechanistic studies of inhibition of endogenous miR-31 in human metastatic cSCC cells revealed suppressed migration, invasion and colony forming ability, whereas overexpression of miR-31 induced these phenotypes. These results indicate that miR-31 regulates cancer-associated phenotypes of cSCC and identify miR-31 as a potential target for cSCC treatment.

New insights into T cells and their signature cytokines in atopic dermatitis

An imbalance of the adaptive immune system mediated by various T cells plays a pivotal role in the pathogenesis of atopic dermatitis (AD). Traditionally, sustained exposure of pathogens tailors immune responses and drives the development of specialized T helper (Th) 2‐bias cytokine environment. The increasing understanding of T cell biology has refreshed the roles of classical Th2 responses and regulatory T cells in the development of AD. In particular, the identification of novel CD4+ T cell subsets such as Th9, Th17, and Th22 cells provide further interpretation of immunological mechanisms underlying AD. In this report, we reviewed the functional roles of CD4+ T cell subsets and their derived signature cytokines in AD. We focused on important discoveries of the contribution of CD4+ T cell cytokines to immunomodulation in AD, and particularly, highlighted the multiple consequences of immune dysregulation on the barrier defect of the skin. We subsequently discussed the flexibility and plasticity of these T cells in vivo in terms of cytokine production. T cells involved in innate immunity were also mentioned. Taking the pivotal and varied roles of T‐cell subpopulations and the functional cytokine milieus into account, T cell targeting therapy may thus open up new opportunities for mechanism‐based therapeutic intervention.

Apigenin attenuates dopamine-induced apoptosis in melanocytes via oxidative stress-related p38, c-Jun NH2-terminal kinase and Akt signaling

BACKGROUND Accumulating evidence suggests that the occurrence of oxidative stress leads to melanocyte degeneration in vitiligo. Elevated level of dopamine (DA), an initiator of oxidative stress, reportedly is found in patients with vitiligo and induces melanocyte death in vitro. DA-treated melanocytes have been used as a model to search for antioxidants for treating vitiligo. OBJECTIVE We investigated the protective effects of apigenin against DA-induced apoptosis in melanocytes and the molecular mechanism underlying those effects. METHODS Melanocytes with or without pretreatment with apigenin were exposed to DA. Then cell viabilities were measured by MTT assay. Cellular reactive oxygen species (ROS) levels and the percentage of apoptotic cells were detected by flow cytometry analysis. Activation of caspase 3, poly(ADP-ribose) polymerase (PARP) and oxidative stress-related signaling, including p38, c-Jun NH2-terminal kinase (JNK) and Akt, were assessed by Western blotting. RESULTS Apigenin attenuated DA-induced apoptotic cell death, relieved ROS accumulation and activated caspase 3 and PARP, suggesting the protective effects of apigenin against DA-induced oxidative stress and apoptosis in melanocytes. Moreover, DA induced phosphorylation of p38, JNK and Akt, while inhibitors of p38, JNK and Akt significantly decreased DA-induced apoptosis. However, pretreatment with apigenin significantly inhibited DA-triggered activation of p38, JNK and Akt, suggesting the involvement of p38, JNK and Akt in the protective effects of apigenin against DA-induced cytotoxicity. CONCLUSION These results suggest that apigenin attenuates dopamine-induced apoptosis in melanocytes via oxidative stress-related p38, JNK and Akt signaling and therefore could be a potential agent in treating vitiligo.

Ginsenosides Rb1 and Rg1 Stimulate Melanogenesis in Human Epidermal Melanocytes via PKA/CREB/MITF Signaling.

Reduced or defective melanin skin pigmentation may cause many hypopigmentation disorders and increase the risk of damage to the skin triggered by UV irradiation. Ginsenosides Rb1 and Rg1 have many molecular targets including the cAMP-response element-binding protein (CREB), which is involved in melanogenesis. This study aimed to investigate the effects of ginsenosides Rb1 and Rg1 on melanogenesis in human melanocytes and their related mechanisms. The effects of Rb1 and Rg1 on cell viability, tyrosinase activity, cellular melanin content and protein levels of tyrosinase, microphthalmia-associated transcription factor (MITF), and activation of CREB in melanocytes were assessed. Results showed that Rb1 or Rg1 significantly increased cellular melanin content and tyrosinase activity in a dose-dependent manner. By contrast, the cell viability of melanocytes remained unchanged. After exposure to Rb1 or Rg1, the protein levels of tyrosinase, MITF, and phosphorylated CREB were significantly increased. Furthermore, pretreatment with the selective PKA inhibitor H-89 significantly blocked the Rb1- or Rg1-induced increase of melanin content. These findings indicated that Rb1 and Rg1 increased melanogenesis and tyrosinase activity in human melanocytes, which was associated with activation of PKA/CREB/MITF signaling. The effects and mechanisms of Rb1 or Rg1 on skin pigmentation deserve further study.

Characteristics of peripheral blood CD4+CD25+ regulatory T cells and related cytokines in severe atopic dermatitis

BackgroundRegulatory T cells (Tregs) have been suggested to play a role in the pathogenesis of atopic dermatitis (AD). However, alterations in the ability of Tregs remain to be determined.ObjectivesTo investigate the expression of various surface receptors on CD4+CD25high regulatory T cells and to investigate their capacity for inhibiting the proliferation of CD4+ CD25- effector T cells (Teffs).Materials and methodsPeripheral blood samples were obtained from 15 patients with severe atopic dermatitis (AD) and 20 control subjects. FACs was then carried out to analyze the expression levels of FoxP3, CD152 (CTLA-4), CD39, CD73, CD223 (LAG-3), CCR4, CCR5, and CCR10 on Tregs. The proliferative responses ofTeffs were assessed in the absence or presence of autologous Tregs and the TGF-β1 and IL-10 levels in the culture supernatant and sera were detected by enzyme-linked immunosorbent assay (ELISA).ResultsThe CD152, CD39, CD73, CCR4, and CCR5 expression levels on Tregs were higher in patients with severe AD than in the controls. Tregs showed an attenuated suppressive function of the proliferation of autologous Teffs in severe AD. The concentrations of IL-10 and TGF-β in the culture supernatants of Tregs were lower in the AD group than in the control.ConclusionThe attenuated ability of Tregs to suppress Teff proliferation may be responsible for the autoimmune reaction of severe AD.

Rapid profiling and target analysis of principal components in Fuling Decoctions by UFLC-DAD-ESI-MS

The traditional Chinese medicine formula Fuling Decoction (FD) has been clinically used for eczema treatment, but the unclear chemical distribution and the lack of quality control have strongly restricted its application. In this study, an analytical method incorporating ultra-fast liquid chromatography (UFLC) with MS and UV detection was developed for rapid profiling of the chemical constitutes from FD. Fourteen constitutes were identified by UFLC-ESI-MS, while four major components including genipingentiobioside, geniposide, paeoniflorin and liquiritin were quantified simultaneously by UFLC-DAD. The UFLC-based method was fully validated and can be applied to screening and determination of principal components in commercially FD prescriptions.

C5-Hydroxylation of liquiritigenin is catalyzed selectively by CYP1A2

Liquiritigenin (7,4′-dihydroxyflavone), the primary active component of a traditional Chinese medicine Glycyrrhizae radix, has a wide range of pharmacological activities. Six oxidative metabolites of liquiritigenin (7,3′,4′-trihydroxyflavone, a hydroxyl quinine metabolite, two A-ring dihydroxymetabolites, 7,4′-dihydroxyflavone, and 7-hydroxychromone) have been detected in rat liver microsomes (RLMs), and one CYP3A4-catalyzed metabolite (7,4′-dihydroxyflavone) has been identified in human liver microsomes (HLMs) recently. In this study, a novel mono-hydroxylated metabolite was detected in reaction catalyzed by HLMs, and was identified as 4′,5,7-trihydroxyflavanone by comparing the tandem mass spectra and the chromatographic retention time with that of the standard compound. Significant difference in CLint (9-fold) was found between these two oxidative pathways of liquiritigenin, and C5-hydroxylation pathway was identified as the major oxidative metabolism of liquiritigenin. The study with chemical selective inhibitor, cDNA-expressed human CYPs, correlation assay, and kinetic study demonstrated that CYP1A2 was the specific isozyme responsible for the C5-hydroxylation metabolism of liquiritigenin in HLMs.