Chrysin Inhibits High Glucose-Induced Migration on Chorioretinal Endothelial Cells via VEGF and VEGFR Down-Regulation.

BACKGROUND: Diabetes mellitus (DM) is a chronic inflammatory disease, which causes multiple complications. Diabetic retinopathy (DR) is among these complications and is a dominant cause of vision loss for diabetic patients. Numerous studies have shown that chrysin, a flavonoid, has many biological activities such as anti-oxidation and anti-inflammation. However, it is rarely used in ocular diseases. In this study, we examined the inhibitory effects of flavonoid on high glucose induced migration of chorioretinal endothelial cells (RF/6A cells) and its mechanism. MATERIALS AND METHODS: The viability of RF/6A cells treated with chrysin was examined with a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The migration of RF/6A cells was assessed by the transwell migration and scratch wound assays. The expression of AKT, ERK, vascular endothelial growth factor (VEGF), HIF-1α and MMP-2 were determined by western blotting. To observe the mRNA expression of VEGF receptor (VEGFR), qRT-PCR, was utilized. RESULTS: The results showed that chrysin can dose-dependently inhibit the RF/6A cell migration in vitro transwell and the scratch wound assays which are induced by high glucose. After pretreatment of RF/6A cells with different concentrations of chrysin, they did not produce any cytotoxicity in MTT assay. Moreover, chrysin down-regulated both phosphorylated AKT and ERK, as well as attenuated the expression levels of MMP-2. It also decreased the expression of the VEGF transcription factor and VEGF. Furthermore, it was shown that chrysin could suppress the protein and mRNA expression levels of VEGFR. CONCLUSION: The results indicate that chrysin could down-regulate the phosphorylation of AKT, ERK and MMP-2 and reduce the effects of VEGF and VEGFR in a high glucose environment. It further inhibits the high glucose-induced migration of RE/6A cells. Therefore, chrysin may have the potential for visual protection.

The Inhibitory Effects of Gold Nanoparticles on VEGF-A-Induced Cell Migration in Choroid-Retina Endothelial Cells.

BACKGROUND: Vascular endothelial growth factor (VEGF) is upregulated by hypoxia and is a crucial stimulator for choroidal neovascularization (CNV) in age-related macular degeneration and pathologic myopia, as well as retinal neovascularization in proliferative diabetic retinopathy. Retinal and choroidal endothelial cells play key roles in the development of retinal and CNV, and subsequent fibrosis. At present, the effects of gold nanoparticles (AuNPs) on the VEGF-induced choroid-retina endothelial (RF/6A) cells are still unknown. In our study, we investigated the effects of AuNPs on RF/6A cell viabilities and cell adhesion to fibronectin, a major ECM protein of fibrovascular membrane. Furthermore, the inhibitory effects of AuNPs on RF/6A cell migration induced by VEGF and its signaling were studied. METHODS: The cell viability assay was used to determine the viability of cells treated with AuNPs. The migration of RF/6A cells was assessed by the Transwell migration assay. The cell adhesion to fibronectin was examined by an adhesion assay. The VEGF-induced signaling pathways were determined by western blotting. RESULTS: The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay revealed no cytotoxicity of AuNPs on RF/6A cells. AuNPs inhibited VEGF-induced RF/6A cell migration in a concentration-dependent manner but showed no significant effects on RF/6A cell adhesion to fibronectin. Inhibitory effects of AuNPs on VEGF-induced Akt/eNOS were found. CONCLUSIONS: These results suggest that AuNPs are an effective inhibitor of VEGF-induced RF/6A cell migration through the Akt/eNOS pathways, but they have no effects on their cell viabilities and cell adhesion to fibronectin.

An Efficient Approach for Lipase-Catalyzed Synthesis of Retinyl Laurate Nutraceutical by Combining Ultrasound Assistance and Artificial Neural Network Optimization.

Although retinol is an important nutrient, retinol is highly sensitive to oxidation. At present, some ester forms of retinol are generally used in nutritional supplements because of its stability and bioavailability. However, such esters are commonly synthesized by chemical procedures which are harmful to the environment. Thus, this study utilized a green method using lipase as a catalyst with sonication assistance to produce a retinol derivative named retinyl laurate. Moreover, the process was optimized by an artificial neural network (ANN). First, a three-level-four-factor central composite design (CCD) was employed to design 27 experiments, which the highest relative conversion was 82.64%. Further, the optimal architecture of the CCD-employing ANN was developed, including the learning Levenberg-Marquardt algorithm, the transfer function (hyperbolic tangent), iterations (10,000), and the nodes of the hidden layer (6). The best performance of the ANN was evaluated by the root mean squared error (RMSE) and the coefficient of determination (R²) from predicting and observed data, which displayed a good data-fitting property. Finally, the process performed with optimal parameters actually obtained a relative conversion of 88.31% without long-term reactions, and the lipase showed great reusability for biosynthesis. Thus, this study utilizes green technology to efficiently produce retinyl laurate, and the bioprocess is well established by ANN-mediated modeling and optimization.

Green fluorescent protein chromophore derivative suppresses ultraviolet A-induced JNK-signalling and apoptosis in keratinocytes and adverse effects in zebrafish embryos.

Solar ultraviolet (UV) light has been recognized as the important environmental hazard and contributes to diverse skin damage such as cell death, photoageing and even carcinogenesis. Revelation of harmful responses attributed to UVA radiation has promoted the development of photoprotective agents against UVA-induced skin damage. In the present study, we tried to evaluate the potential protective effects of a synthetic green fluorescent protein (GFP) chromophore derivative, 4-chlorobenzyldene-1, 2-dimethylimidazolinone (Cl-BDI, called TC-22) on UVA- and UVB- induced stress responses in skin. The HaCaT keratinocytes were used to evaluate the cellular effects. Zebrafish (Danio rerio), which is regarded as a useful and cost-effective alternative to some mammalian models, was applied as the in vivo animal model. In HaCaT keratinocytes, TC-22 was able to obviously decrease UVA-induced cell death. Dissection of the UVA-induced signalling pathways revealed that TC-22 could suppress the activation of JNK and caspase 3, but not of ERK and p38. Reduction of UVA-induced cleavage of caspase 3 and sub-G1 phase accumulation by pretreatment of TC-22 was also observed. In zebrafish, we showed that UVA irradiation could decrease the survival and hatching rate, suppress heart beats of embryos and enhance the pigmentation of larvae. Pretreatment of TC-22 could significantly reverse UVA-induced the suppression in hatching of eggs and heart beating of embryos and also lowered the UVA-induced pigmentation in zebrafish. Collectively, we demonstrate that TC-22, a GFP chromophore derivative, can ameliorate the UVA-induced stress responses in both epidermal keratinocytes and zebrafish, suggesting the potential use of TC-22 in photoprotection in the future.

Protective effects of resveratrol against UVA-induced damage in ARPE19 cells.

Ultraviolet radiation, especially UVA, can penetrate the lens, reach the retina, and induce oxidative stress to retinal pigment epithelial (RPE) cells. Even though it is weakly absorbed by protein and DNA, it may trigger the production of reactive oxygen species (ROS) and generate oxidative injury; oxidative injury to the retinal pigment epithelium has been implicated to play a contributory role in age-related macular degeneration (AMD). Studies showed that resveratrol, an abundant and active component of red grapes, can protect several cell types from oxidative stress. In this study, adult RPE cells being treated with different concentrations of resveratrol were used to evaluate the protective effect of resveratrol on RPE cells against UVA-induced damage. Cell viability assay showed that resveratrol reduced the UVA-induced decrease in RPE cell viability. Through flow cytometry analysis, we found that the generation of intracellular H2O2 induced by UVA irradiation in RPE cells could be suppressed by resveratrol in a concentration-dependent manner. Results of Western blot analysis demonstrated that resveratrol lowered the activation of UVA-induced extracellular signal-regulated kinase, c-jun-NH2 terminal kinase and p38 kinase in RPE cells. In addition, there was also a reduction in UVA-induced cyclooxygenase-2 (COX-2) expression in RPE cells pretreated with resveratrol. Our observations suggest that resveratrol is effective in preventing RPE cells from being damaged by UVA radiation, and is worth considering for further development as a chemoprotective agent for the prevention of early AMD.

Quercetin inhibits histamine-induced calcium influx in human keratinocyte via histamine H4 receptors.

Histamine is released from mast cells when tissues are inflamed or stimulated by allergens. Activation of histamine receptors and calcium influx via TRPV1 could be related to histamine-induced itch and skin inflammation. Quercetin is known to have anti-inflammatory and anti-itching effects. This study aims to understand whether quercetin can directly affect histamine-induced calcium influx in human keratinocyte. In it, we investigated quercetin, which acts on histamine-induced intracellular free calcium ([Ca2+]i) elevation in human keratinocyte. Changes in [Ca2+]i were measured using spectrofluorometry and confocal Imaging. We detected the expression of IL-8 after treatment of quercetin using qRT-PCR and evaluated its anti-itching effect in BALB/c mice. We also performed a docking study to estimate the binding affinity of quercetin to H4 receptors. We found that quercetin pretreatment decreased histamine-induced [Ca2+]i elevation in a concentration-dependent manner. The inhibitory effect of quercetin on histamine-induced [Ca2+]i elevation was blocked by JNJ7777120, a selective H4 antagonist, as well as by U73122, a PLC inhibitor, and by GF109203X, a PKC inhibitor. We also found that H4 agonist (4-methylhistamine)-induced [Ca2+]i elevation could be inhibited by quercetin. Moreover, the selective TRPV1 blocker capsazepine significantly suppressed the quercetin-mediated inhibition of histamine-induced [Ca2+]i elevation, whereas the TRPV4 blocker GSK2193874 had no effect. Last, quercetin decreased histamine and H4 agonist-induced IL-8 expression in keratinocyte and inhibited the scratching behavior-induced compound 48/80 in BALB/c mice. The molecular docking study also showed that quercetin exhibited high binding affinities with H4 receptors (autodock scores for H4 = -8.7 kcal/mol). These data suggest that quercetin could decrease histamine 4 receptor-induced calcium influx through the TRPV1 channel and could provide a molecular mechanism of quercetin in anti-itching, anti-inflammatory, and unpleasant sensations.

Anti-Proliferative and Anti-Migratory Activities of Hispidulin on Human Melanoma A2058 Cells.

Melanoma represents less than 5% of skin cancers, but is the most lethal, mainly because of its high-metastatic potential and resistance to various therapies. Therefore, it is important to develop effective treatments, especially chemotherapeutic drugs with cytotoxicity, anti-metastaticity, and few side effects. One such natural product is hispidulin, a flavone distributed in plants of the Asteraceae. Previous studies have demonstrated that hispidulin has various pharmacological benefits, such as anti-tumor, anti-inflammation, and anti-allergic effects. This study aims to explore the effects of hispidulin against melanoma in vitro and in vivo. Results revealed that hispidulin selectively decreased the cell viability of A2058 cells in a dose- and time-dependent manner. Hispidulin induced cells accumulated in the sub-G1 phase via activating caspase 8 and 9, increased cleaved caspase 3, and cleaved PARP expression. Hispidulin was able to decrease AKT and ERK phosphorylation, which facilitated cell growth and survival. Moreover, hispidulin promoted reactive oxygen species generation in cells and suppressed cell migration through downregulated matrix metalloproteinase-2 expression. Hispidulin significantly inhibited tumor growth in a xenograft model. Based on these results, hispidulin produces its anti-melanoma effects by inducing cancer cell apoptosis and reducing its migration. Therefore, we suggest hispidulin as a potent therapeutic candidate for melanoma treatment.

An isoflavone extract from soybean cake suppresses 2,4-dinitrochlorobenzene-induced contact dermatitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Numerous epidemiological and clinical studies have demonstrated the protective role of dietary isoflavones against development of several chronic diseases. ISO-1, one fraction of isoflavone powders derived from soybean cake, is reported to attenuate inflammation and photodamage. AIM OF THE STUDY: Contact dermatitis is a common inflammatory skin disease, which accounts for most occupational skin disorders. Instead of oral administration, we aimed to explore the effects of topical ISO-1 application on contact dermatitis by using 2,4-dinitrochlorobenzene (DNCB)-stimulated HaCaT keratinocytes and DNCB-induced mouse dermatitis as models. MATERIALS AND METHODS: In the in vitro study, we first evaluated the biologic effects of DNCB on HaCaT keratinocytes. HaCaT keratinocytes were treated with 2,4-dinitrochlorobenzene (DNCB), and cell viability was measured by MTT assay. Then, we detect the prominent induction of IL-8 mRNA expression after DNCB and ISO-1 treatment by reverse transcription polymerase chain reaction (RT-PCR), and release of IL-8 from HaCaT keratinocytes was measured by ELISA assay. HaCaT keratinocytes were pretreated with ISO-1 and then treated with DNCB, phosphorylation of JNK, p38, ERK and IκBα was analyzed by western blot. In the in vivo study, the hairless mice were used for an induced contact dermatitis model. The surface changes in the dorsal skin after DNCB and ISO-1 treatment were recorded using photography, and TEWL, erythema were measured using an MPA-580 cutometer. Blood was also collected from mice for measurement of white blood cell counts. RESULTS: Results showed ISO-1 inhibited DNCB-induced IL-8 production and also suppressed DNCB-induced phosphorylation of JNK and p38, and IκBα in HaCaT. In the animal model of DNCB-induced contact dermatitis, topical ISO-1 treatment significantly decreased DNCB-induced erythema and transepidermal water loss (TEWL) in mouse skin. ISO-1 also reduced DNCB-induced skin thickening and increase of white blood cell count. CONCLUSIONS: ISO-1 is promising for improvement of DNCB-induced inflammation and skin barrier impairment, suggesting the potential application of topical ISO-1 for inflammatory dermatoses.

Chrysin alleviates imiquimod-induced psoriasis-like skin inflammation and reduces the release of CCL20 and antimicrobial peptides.

Psoriasis is a common non-contagious chronic inflammatory skin lesion, with frequent recurrence. It mainly occurs due to aberrant regulation of the immune system leading to abnormal proliferation of skin cells. However, the pathogenic mechanisms of psoriasis are not fully understood. Although most of the current therapies are mostly efficient, the side effects can result in therapy stop, which makes the effectiveness of treatment strategies limited. Therefore, it is urgent and necessary to develop novel therapeutics. Here, we investigated the efficacy of chrysin, a plant flavonoid, which we previously reported to possess strong antioxidant and anti-inflammatory effects, against psoriasis-like inflammation. Our results revealed that chrysin significantly attenuated imiquimod-induced psoriasis-like skin lesions in mice, and improved imiquimod-induced disruption of skin barrier. Moreover, the TNF-α, IL-17A, and IL-22-induced phosphorylation of MAPK and JAK-STAT pathways, and activation of the NF-κB pathway were also attenuated by chrysin pretreatment of epidermal keratinocytes. Most importantly, chrysin reduced TNF-α-, IL-17A-, and IL-22-induced CCL20 and antimicrobial peptide release from epidermal keratinocytes. Thus, our findings indicate that chrysin may have therapeutic potential against inflammatory skin diseases. Our study provides a basis for further investigating chrysin as a novel pharmacologic agent and contributes to the academic advancement in the field of Chinese herbal medicine.

The Therapeutic Potential and Molecular Mechanism of Isoflavone Extract against Psoriasis.

Psoriasis is a common inflammatory disease. It affects 1-3% of the population worldwide and is associated with increasing medical costs every year. Typical psoriatic skin lesions are reddish, thick, scaly plaques that can occur on multiple skin sites all over the body. Topical application of imiquimod (IMQ), a toll-like receptor (TLR)7 agonist and potent immune system activator, can induce and exacerbate psoriasis. Previous studies have demonstrated that isoflavone extract has an antioxidant effect which may help decrease inflammation and inflammatory pain. Through in vivo studies in mice, we found that the topical application to the shaved back and right ear of mice of isoflavone extract prior to IMQ treatment significantly decreased trans-epidermal water loss (TEWL), erythema, blood flow speed, and ear thickness, while it increased surface skin hydration, and attenuated epidermal hyperplasia and inflammatory cell infiltration. Through in vitro experiments, we found that isoflavone extract can reduce IL-22, IL-17A, and TNF-α-induced MAPK, NF-κB, and JAK-STAT activation in normal human epidermal keratinocytes. At the mRNA level, we determined that isoflavone extract attenuated the increased response of the TNF-α-, IL-17A-, and IL-22- related pathways. These results indicate that isoflavone extract has great potential as an anti-psoriatic agent and in the treatment of other inflammatory skin diseases.

18ß-glycyrrhetinic acid derivative promotes proliferation, migration and aquaporin-3 expression in human dermal fibroblasts.

Licorice (Glycyrrhiza) species have been widely used as a traditional medicine and a natural sweetener in foods. The 18ß-glycyrrhetinic acid (18ß-GA) is a bioactive compound in licorice that exhibits potential anti-cancer, anti-inflammatory, and anti-microbial activities. Many synthesized derivatives of 18ß-GA have been reported to be cytotoxic and suggested for the treatment of malignant diseases. In this study, we explored the possible pharmacological roles of an 18ß-GA derivative in skin biology using primary human dermal fibroblasts and HaCaT keratinocytes as cell models. We found that this 18ß-GA derivative did not cause cell death, but significantly enhanced the proliferation of dermal fibroblasts and HaCaT keratinocytes. A scratch wound healing assay revealed that the 18ß-GA derivative promoted the migration of fibroblasts. Due to the important role of aquaporin-3 in cell migration and proliferation, we also investigated the expression of aquaporin-3 and found this compound up-regulated the expression of aquaporin-3 in dermal fibroblasts and HaCaT keratinocytes. In dermal fibroblasts, the 18ß-GA derivative induced the phosphorylation of Akt, ERK, and p38. The inhibitor of Akt predominantly suppressed the 18ß-GA derivative-induced expression of aquaporin-3. Collectively, this compound had a positive effect on the proliferation, migration, and aquaporin-3 expression of skin cells, implying its potential role in the treatment of skin diseases characterized by impaired wound healing or dermal defects.

Lutein Induces Autophagy via Beclin-1 Upregulation in IEC-6 Rat Intestinal Epithelial Cells.

Lutein is a carotenoid with anti-oxidant properties. Autophagy, an evolutionarily conserved catabolic cellular pathway for coping with stress conditions, is responsive to reactive oxygen species (ROS) and degrades damaged organelles. We previously demonstrated that lutein can induce anti-oxidant enzymes to relieve methotrexate-induced ROS stress. We therefore hypothesized that lutein, which activates ROS-scavenging enzymes, can also induce autophagy for cell survival. In this study, we demonstrated that lutein treatment attenuated the reduction in cell viability caused by H2O2. Lutein dose-dependently induced the processing of microtubule-associated protein light chain 3 (LC3)-II, an autophagy marker protein, and accumulation of LC3-positive puncta in rat intestinal IEC-6 cells. Furthermore, (a) direct observation of autophagosome formation through transmission electron microscopy, (b) upregulation of autophagy-related genes including ATG4A, ATG5, ATG7, ATG12, and beclin-1 (BENC1), and (c) increased BECN1/Bcl-2 ratio confirmed the induction of autophagy by lutein. The results revealed that bafilomycin-A1-induced inhibition of autophagy reduced cell viability and increased apoptosis in lutein-treated cells, indicating a protective role of lutein-induced autophagy. Lutein treatment also activated adenosine monophosphate-activated protein kinase (AMPK), c-Jun N-terminal kinase (JNK), and p-38, but had no effects on the induction of extracellular signal-related kinase or inhibition of mTOR; however, the inhibition of activated AMPK, JNK, or p-38 did not attenuate lutein-induced autophagy. Finally, increased BECN1 expression levels were detected in lutein-treated cells, and BECN1 knockdown abolished autophagy induction. These results suggest that lutein-induced autophagy was mediated by the upregulation of BECN1 in IEC-6 cells. We are the first to demonstrate that lutein induces autophagy. Elevated autophagy in lutein-treated IEC-6 cells may have a protective role against various stresses, and this warrants further investigation.

Photoprotective Effects of Cycloheterophyllin against UVA-Induced Damage and Oxidative Stress in Human Dermal Fibroblasts.

Ultraviolet (UV) radiation, particularly ultraviolet A (UVA), is known to play a major role in photoaging of the human skin. Many studies have demonstrated that UV exposure causes the skin cells to generate reactive oxygen species and activates the mitogen-activated protein kinase (MAPK) pathway. Previous studies have also demonstrated that cycloheterophyllin has an antioxidant effect and can effectively scavenge free radicals. Extending the aforementioned investigations, in this study, human dermal fibroblasts were used to investigate the protective effect of cycloheterophyllin against UV-induced damage. We found that cycloheterophyllin not only significantly increased cell viability, but also attenuated the phosphorylation of MAPK after UVA exposure. Furthermore, cycloheterophyllin could reduce hydrogen peroxide (H2O2) generation and down-regulate H2O2-induced MAPK phosphorylation. In the in vivo studies, the topical application of cycloheterophyllin before UVA irradiation significantly decreased trans-epidermal water loss (TEWL), erythema, and blood flow rate. These results indicate that cycloheterophyllin is a photoprotective agent that inhibits UVA-induced oxidative stress and damage, and could be used in the research on and prevention of skin photoaging.

The differential effects of a selective kappa-opioid receptor agonist, U50488, in guinea pig heart tissues.

The differential effects of a selective kappa- (κ-) opioid receptor agonist, U50488, were elucidated by monitoring the contraction of isolated guinea pig atrial and ventricular muscles. In electrically driven left atria, U50488 in nanomolar concentration range decreased the contractile force. Norbinaltorphimine (norBNI), a selective κ-receptor antagonist, and pertussis toxin (PTX) abolished the negative inotropic effect of U50488. In contrast, the inhibitory effect was not affected by the pretreatment of atropine or propranolol. Even though U50488 exerted a negative inotropic effect in the left atrium, it did not affect the contractile force of the right atrium and ventricles paced at 2 Hz. Similarly, the beating rate of the spontaneously beating right atrium was also unaffected by U50488. These results indicate that the activation of κ-opioid receptors can only produce negative inotropic effect in left atria via activation of PTX-sensitive G protein in guinea pigs. The absence of negative inotropic effects in right atria and ventricles suggests that there may be a greater distribution of functional κ-opioid receptors in guinea pig left atria than in right atria and ventricles, and the distribution of the receptors may be species-specific.