Hydroxysafflor Yellow A Promotes HaCaT Cell Proliferation and Migration by Regulating HBEGF/EGFR and PI3K/AKT Pathways and Circ_0084443.

OBJECTIVE: To investigate the effect and possible mechanism of hydroxysafflor yellow A (HSYA) on human immortalized keratinocyte cell proliferation and migration. METHODS: HaCaT cells were treated with HSYA. Cell proliferation was detected by the cell counting kit-8 assay, and cell migration was measured using wound healing assay and Transwell migration assay. The mRNA and protein expression levels of heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF), EGF receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), mammalian target of rapamycin (mTOR), and hypoxia-inducible factor-1α (HIF-1α) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. Circ_0084443-overexpressing HaCaT cells and empty plasmid HaCaT cells were constructed using the lentiviral stable transfection and treated with HSYA. The expression of circ_0084443 was detected by qRT-PCR. RESULTS: HSYA (800 µmol/L) significantly promoted HaCaT cell proliferation and migration (P<0.05 or P<0.01). It also increased the mRNA and protein expression levels of HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α, and increased the phosphorylation levels of PI3K and AKT (P<0.05 or P<0.01). Furthermore, HSYA promoted HaCaT cell proliferation and migration via the HBEGF/EGFR and PI3K/AKT/mTOR signaling pathways (P<0.01). Circ_0084443 attenuated the mRNA expression levels of HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α (P<0.05). HSYA inhibited the circ_0084443 expression, further antagonized the inhibition of circ_0084443 on HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α, and promoted the proliferation of circ_0084443-overexpressing HaCaT cells (P<0.05 or P<0.01). However, HSYA could not influence the inhibitory effect of circ_0084443 on HaCaT cell migration (P>0.05). CONCLUSION: HSYA played an accelerative role in HaCaT cell proliferation and migration, which may be attributable to activating HBEGF/EGFR and PI3K/AKT signaling pathways, and had a particular inhibitory effect on the keratinocyte negative regulator circ_0084443.

Morphological and anatomical characteristics of exserted stigma sterility and the location and function of SlLst (Solanum lycopersicum Long styles) gene in tomato.

KEY MESSAGE: Anatomical changes in and hormone roles of the exserted stigma were investigated, and localization and functional analysis of SlLst for the exserted stigma were performed using SLAF-BSA-seq, parental resequencing and overexpression of SlLst in tomato. Tomato accession T431 produces stigmas under relatively high temperatures (> 27 °C, the average temperature in Harbin, China, in June-August), so pollen can rarely reach the stigma properly. This allows the percentage of male sterility exceed 95%, making the use of this accession practical for hybrid seed production. To investigate the mechanism underlying the exserted stigma male sterility, the morphological changes of, anatomical changes of, and comparative endogenous hormone (IAA, ABA, GA3, ZT, SA) changes in flowers during flower development of tomato accessions DL5 and T431 were measured. The location and function of genes controlling exserted stigma sterility were analyzed using super SLAF-BSA-seq, parental resequencing, comparative genomics and the overexpression of SlLst in tomato. The results showed that an increase in cell number mainly caused stigma exsertion. IAA played a major role, while ABA had an opposite effect on stigma exertion. Moreover, 26 candidate genes related to the exserted stigma were found, located on chromosome 12. The Solyc12g027610.1 (SlLst) gene was identified as the key candidate gene by functional analysis. A subcellular localization assay revealed that SlLst is targeted to the nucleus and cell membrane. Phenotypic analysis of SlLst-overexpressing tomato showed that SlLst plays a crucial role during stigma exsertion.

The α-Subunit of the Chloroplast ATP Synthase of Tomato Reinforces Resistance to Gray Mold and Broad-Spectrum Resistance in Transgenic Tobacco.

Chloroplast ATP synthase (cpATPase) is responsible for ATP production during photosynthesis. Our previous studies showed that the cpATPase CF1 α subunit (AtpA) is a key protein involved in Clonostachys rosea-induced resistance to the fungus Botrytis cinerea in tomato. Here, we show that expression of the tomato atpA gene was upregulated by B. cinerea and Clonostachys rosea. The tomato atpA gene was then isolated, and transgenic tobacco lines were obtained. Compared with untransformed plants, atpA-overexpressing tobacco showed increased resistance to B. cinerea, characterized by reduced disease incidence, defense-associated hypersensitive response-like reactions, balanced reactive oxygen species, alleviated damage to the chloroplast ultrastructure of leaf cells, elevated levels of ATP content and cpATPase activity, and enhanced expression of genes related to carbon metabolism, photosynthesis, and defense. Incremental Ca2+ efflux and steady H+ efflux were observed in transgenic tobacco after inoculation with B. cinerea. In addition, overexpression of atpA conferred enhanced tolerance to salinity and resistance to the fungus Cladosporium fulvum. Thus, AtpA is a key regulator that links signaling to cellular redox homeostasis, ATP biosynthesis, and gene expression of resistance traits to modulate immunity to pathogen infection and provides broad-spectrum resistance in plants in the process.

[In vitro metabolism of daphnetin in rat liver S9 fractions].

Daphnetin is quickly eliminated in rats after dosing, but the mechanism remains unclear. This study was aimed to investigate the in vitro metabolism of daphnetin using rat liver S9 fractions (RLS9). The metabolites formed in RLS9 were identified and the kinetic parameters for different metabolic pathways were determined. HPLC-DAD-MS analysis showed that daphnetin was biotransformed to six metabolites, which were identified as 7 or 8 mono-glucuronide and mono-sulfate, 8-methylate, and 7-suflo-8-methylate. Methylation and glucuronidation of daphnetin exhibited the Michaelis-Menten kinetic characteristics, whereas the substrate inhibition kinetic and the two-site kinetic were observed for 8-sulfate and 7-sulfate formations. Of the 3 conjugation pathways, the intrinsic clearance rate for sulfation was highest, followed by methylation and glucuronidation. By in vitro-in vivo extrapolation of the kinetic data measured in RLS9, the hepatic clearance were estimated to be 54.9 mL·min−1·kg−1 which is comparable to the system clearance (58.5 mL·min−1·kg−1) observed in rats. In conclusions, the liver might be the main site for daphnetin metabolism in rats. Sulfation, methylation and glucuronidation are important pathways of the hepatic metabolism of daphnetin in rats.

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

Regulatory 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. To investigate the expression of various surface receptors on CD4(+)CD25(high) regulatory T cells and to investigate their capacity for inhibiting the proliferation of CD4(+) CD25(-) effector T cells (Teffs). Peripheral 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 of Teffs were assessed in the absence or presence of autologous Tregs and the TGF-ß1 and IL-10 levels in the culture supernatant and sera were detected by enzyme-linked immunosorbent assay (ELISA). The 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-ß in the culture supernatants of Tregs were lower in the AD group than in the control. The attenuated ability of Tregs to suppress Teff proliferation may be responsible for the autoimmune reaction of severe AD.

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.

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.

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.

[Current status and industrialization of transgenic tomatoes].

In this review, the progress in transgenic tomato research, including disease and insect resistance, herbicide resistance, stress tolerance, long-term storage, quality improvement, and male sterility, were described. The recent researches on producing heterologous proteins using transgenic tomatoes were also reviewed. Furthermore, the industrialization status and problems of transgenic tomatoes were analyzed and the prospects of both research and industrialization in transgenic tomatoes were discussed.

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.

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 CL(int) (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.

Alkaline-stress response in Glycine soja leaf identifies specific transcription factors and ABA-mediated signaling factors.

Transcriptome of Glycine soja leaf tissue during a detailed time course formed a foundation for examining transcriptional processes during NaHCO(3) stress treatment. Of a total of 2,310 detected differentially expressed genes, 1,664 genes were upregulated and 1,704 genes were downregulated at various time points. The number of stress-regulated genes increased dramatically after a 6-h stress treatment. GO category gene enrichment analysis revealed that most of the differentially expressed genes were involved in cell structure, protein synthesis, energy, and secondary metabolism. Another enrichment test revealed that the response of G. soja to NaHCO(3) highlights specific transcription factors, such as the C2C2-CO-like, MYB-related, WRKY, GARP-G2-like, and ZIM families. Co-expressed genes were clustered into ten classes (P < 0.001). Intriguingly, one cluster of 188 genes displayed a unique expression pattern that increases at an early stage (0.5 and 3 h), followed by a decrease from 6 to 12 h. This group was enriched in regulation of transcription components, including AP2-EREBP, bHLH, MYB/MYB-related, C2C2-CO-like, C2C2-DOF, C2C2, C3H, and GARP-G2-like transcription factors. Analysis of the 1-kb upstream regions of transcripts displaying similar changes in abundance identified 19 conserved motifs, potential binding sites for transcription factors. The appearance of ABA-responsive elements in the upstream of co-expression genes reveals that ABA-mediated signaling participates in the signal transduction in alkaline response.

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.