Targeting histone deacetylase 9 represses fibrogenic phenotypes in buccal mucosal fibroblasts with arecoline stimulation.

Background/purpose: Oral submucosal fibrosis (OSF) is a premalignant disorder positively associated with betel nut chewing. Recent studies supported the promising benefits of histone deacetylase (HDAC) inhibitors for fibrosis treatment. Here we aim to clarify the pro-fibrogenic role of HDAC9 in regulating OSF. Materials and methods: Healthy and OSF specimens were collected to investigate the clinical significance of HDAC9. Chronic arecoline treatment process was used to induce arecoline-mediated myofibroblasts-related activation of primary buccal mucosa fibroblasts (BMFs). Functional analysis of collagen gel contraction, transwell migration, and wound-healing assays were performed to assess the change in pro-fibrogenic properties of BMFs and fibrotic BMFs (fBMFs). Lentiviral-mediated HDAC9 knockdown was used to verify the role of HDAC9 in the pro-fibrogenic process. Results: We found that arecoline significantly increased the mRNA and protein expression of HDAC9 of BMFs in a dose-dependent manner. Knockdown of HDAC9 in BMFs reversed the strengthened effects of arecoline on collagen gel contraction, cell migration, and wound-healing ability. We further demonstrated that knockdown of HDAC9 in fBMFs significantly attenuated its inherent pro-fibrogenic properties. Furthermore, we confirmed a significantly increased expression of HDAC9 mRNA in OSF compared to normal tissues, which suggested a positive correlation between the up-regulation of HDAC9 and OSF. Conclusion: We demonstrated that silencing of HDAC9 inhibited arecoline-induced activation and inherent pro-fibrogenic properties, suggesting potential therapeutics by targeting HDAC9 in the OSF treatment.

Anti-cancer activity and cellular uptake of 7,3',4'- and 7,8,4'-trihydroxyisoflavone in HepG2 cells under hypoxic conditions.

Transarterial chemoembolisation (TACE) is used for unresectable hepatocellular carcinoma (HCC) treatment, but TACE-induced hypoxia leads to poor prognosis. The anti-cancer effects of soybean isoflavones daidzein derivatives 7,3',4'-trihydroxyisoflavone (734THIF) and 7,8,4'-trihydroxyisoflavone (784THIF) were evaluated under hypoxic microenvironments. Molecular docking of these isomers with cyclooxygenase-2 (COX-2) and vascular endothelial growth factor receptor 2 (VEGFR2) was assessed. About 40 µM of 734THIF and 784THIF have the best effect on inhibiting the proliferation of HepG2 cells under hypoxic conditions. At a concentration of 40 µM, 784THIF significantly inhibits COX-2 expression in pre-hypoxia conditions compared to 734THIF, with an inhibition rate of 67.73%. Additionally, 40 µM 784THIF downregulates the expression of hypoxic, inflammatory, and metastatic-related proteins, regulates oxidative stress, and inhibits the expression of anti-apoptotic proteins. The uptake by HepG2 confirmed higher 784THIF level and slower degradation characteristics under post- or pre-hypoxic conditions. In conclusion, our results showed that 784THIF had better anti-cancer effects and cellular uptake than 734THIF.

Pterostilbene Attenuates Particulate Matter-Induced Oxidative Stress, Inflammation and Aging in Keratinocytes.

Particulate matter (PM) is the main indicator of air pollutants, and it may increase the level of reactive oxygen species (ROS) in keratinocytes, leading to skin inflammation, aging, and decreased moisturizing ability. Pterostilbene (PTS) is a dimethylated analog of resveratrol that has antioxidant effects. However, the molecular mechanisms of PTS in preventing PM-induced keratinocyte inflammation and aging have not been investigated yet. Therefore, we used PM-induced human keratinocytes to investigate the protective mechanisms of PTS. The results showed that 20 µM PTS had no toxicity to HaCaT keratinocytes and significantly reduced PM-induced intracellular ROS production. In addition, nuclear translocation of the aryl hydrocarbon receptor (AHR) was inhibited by PTS, leading to reduced expression of its downstream CYP1A1. PTS further inhibited PM-induced MAPKs, inflammation (COX-2), and aging (MMP-9) protein cascades, and rescued moisturizing (AQP-3) protein expression. We analyzed the PTS content in cells at different time points and compared the concentration required for PTS to inhibit the target proteins. Finally, we used the skin penetration assay to show that the PTS essence mainly exists in the epidermal layer and did not enter the system circulation. In conclusion, PTS could protect HaCaT keratinocytes from PM-induced damage and has the potential to become a cosmetic ingredient.

Topical Artocarpus communis Nanoparticles Improved the Water Solubility and Skin Permeation of Raw A. communis Extract, Improving Its Photoprotective Effect.

Antioxidants from plant extracts are often used as additives in skincare products to prevent skin problems induced by environmental pollutants. Artocarpus communis methanol extract (ACM) has many biological effects, such as antioxidant, anti-inflammatory, wound healing, and photoprotective effects; however, the poor water solubility of raw ACM has limited its applications in medicine and cosmetics. Topical antioxidant nanoparticles are one of the drug-delivery systems for overcoming the poor water solubility of antioxidants for increasing their skin penetration. The present study demonstrated that ACM-loaded hydroxypropyl-ß-cyclodextrin and polyvinylpyrrolidone K30 nanoparticles (AHP) were successfully prepared and could effectively increase the skin penetration of ACM through changing the physicochemical characteristics of raw ACM, including reducing the particle size, increasing the surface area, and inducing amorphous transformation. Our results also revealed that AHP had significantly better antioxidant activity than raw ACM for preventing photocytotoxicity because the AHP formulation increased the cellular uptake of the ACM in UVB-irradiated HaCaT keratinocytes. In conclusion, our results suggest that AHP may be used as a good topical antioxidant nanoparticle for delivering ACM into deep layers of the skin for preventing UVB-induced skin problems.

Oleanolic Acid Nanofibers Attenuated Particulate Matter-Induced Oxidative Stress in Keratinocytes.

Airborne particulate matter (PM) is one of the indicators of air pollution, and it is also the main factor causing oxidative stress in the skin. Oleanolic acid (OA), a natural terpenoid compound, effectively inhibited PM-induced skin aging; however, OA has poor water solubility and skin absorption, which limit its application in medicines and cosmetics. The aim of this study was to prepare oleanolic acid nanofibers (OAnf) and evaluate the effects of OA and OAnf in PM-treated keratinocytes. The results showed that OA dissolved in dissolved in dimethyl sulfoxide (DMSO) attenuated PM-induced reactive oxygen species overproduction, stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK) activation, and the expressions of inflammatory and skin-aging-related proteins. In addition, the nanofiber process of OA effectively improved the water solubility of OA more than 99,000-fold through changing its physicochemical properties, including a surface area increase, particle size reduction, amorphous transformation, and hydrogen bonding formation with excipients. The skin penetration ability of OAnf was consistently over 10-fold higher than that of OA. Moreover, when dissolved in PBS, OAnf displayed superior antioxidant, anti-inflammatory, and anti-skin aging activities in PM-treated keratinocytes than OA. In conclusion, our findings suggest that OAnf could be a topical antioxidant formulation to attenuate skin problems caused by PM.

Pterostilbene Nanoparticles Downregulate Hypoxia-Inducible Factors in Hepatoma Cells Under Hypoxic Conditions.

PURPOSE: Transcatheter arterial chemoembolization (TACE) is a common clinical treatment for hepatocellular carcinoma (HCC). However, hypoxia induction after treatment might trigger tumor invasiveness and metastasis. Although pterostilbene (PTS) has antitumor effects, its chemoprevention in HepG2 cells under hypoxia has not been investigated yet. In addition, the poor water solubility of raw PTS limits its clinical application. Here, we prepared nanoparticles of PTS (PSN) and compared their antihepatoma activities with those of raw PTS in HepG2 under hypoxic conditions. MATERIALS AND METHODS: The PTS nanoparticle formulation was prepared by nanoprecipitation, using Eudragit® e100 (EE) and polyvinyl alcohol (PVA) as carriers. We analyzed the physicochemical properties of raw PTS and PSN, including yield, encapsulation efficiency, water-solubility, particle size, morphology, crystalline-to-amorphous transformation, and molecular interaction between PTS and carriers. We also evaluated their antihepatoma activities under hypoxia treatment in HepG2 cells, including cell viability, hypoxia, and apoptosis. RESULTS: The yield and encapsulation efficiency of PSN were 86.33% and >99%, respectively. The water solubility and drug release of PTS were effectively improved after nanoprecipitation corresponding to the reduction in particle size, amorphous transformation, and formation of hydrogen bonding with carriers. PSN had a better cytotoxic effect than raw PTS in HepG2 under pre- and post-hypoxia conditions. In addition, hypoxia- and apoptosis-related proteins in HepG2 cells under two different hypoxic conditions were significantly inhibited by PSN compared with the control group with hypoxia only, except for HIF-1α in the post-hypoxia group. PSN was also significantly better in inhibiting these proteins, except for Bcl2, under pre-hypoxic conditions. CONCLUSION: Our results suggested that PSN could improve the water solubility and drug release of PTS and enhance the efficacy of HCC treatment under hypoxic conditions.

Electrospun Resveratrol-Loaded Polyvinylpyrrolidone/Cyclodextrin Nanofibers and Their Biomedical Applications.

Resveratrol is a naturally occurring polyphenol compound which has been shown to possess antioxidant and anti-inflammatory properties. However, its pharmaceutical applications are limited by its poor water solubility. In this study, we used electrospinning technology to synthesize nanofibers of polyvinylpyrrolidone (PVP) and hydroxypropyl-ß-cyclodextrin (HPBCD) loaded with resveratrol. We used X-ray diffractometry to analyze crystalline structure, Fourier transform infrared spectroscopy to determine intermolecular hydrogen bonding, antioxidant assays to measure antioxidant activity, and Franz diffusion cells to evaluate skin penetration. Our results showed that the aqueous solubility of resveratrol nanofibers was greatly improved (by more than 20,000-fold) compared to the pure compound. Analysis of physicochemical properties demonstrated that following nanofiber formation, resveratrol was converted from a crystalline to amorphous structure, and resveratrol formed new intermolecular bonds with PVP and HPBCD. Moreover, resveratrol nanofibers showed good antioxidant activity. In addition, the skin penetration ability of resveratrol in the nanofiber formulation was greater than that of pure resveratrol. Furthermore, resveratrol nanofibers suppressed particulate matter (PM)-induced expression of inflammatory proteins (COX-2 and MMP-9) in HaCaT keratinocytes. Therefore, resveratrol-loaded nanofibers can effectively improve the solubility and physicochemical properties of resveratrol, and may have potential applications as an antioxidant and anti-inflammatory formulation for topical skin application.

Improvement of Skin Penetration, Antipollutant Activity and Skin Hydration of 7,3',4'-Trihydroxyisoflavone Cyclodextrin Inclusion Complex.

As is known, many antioxidants from plant extracts have been used as additives in skincare products to prevent skin damage following overexposure to environmental pollutants. 7,3',4'-trihydroxyisoflavone (734THIF), an isoflavone compound, possesses various biological activities, including antioxidant, antityrosinase, photodamage protection, and anticancer effects. Unfortunately, 734THIF has poor water solubility, which limits its skin penetration and absorption, and subsequently influences its biological activity. The aim of the present study was to investigate the mechanisms for the improvement in water solubility and skin penetration of 2-hydroxypropyl-ß-cyclodextrin (HPBCD) inclusion complex with 734THIF (5-7HP). We also determined its photostability, antipollutant activity in HaCaT keratinocytes, and moisturizing effect in human subjects. Our results showed that 734THIF was embedded into the lipophilic inner cavity of HPBCD and its water solubility and skin penetration were thereby improved through amorphous transformation, surface area enhancement, and hydrogen bonding formation between 734THIF and HPBCD. In addition, 5-7HP inhibited PM-induced ROS generation and then downregulated ROS-mediated COX-2 and MMP9 production and AQP-3 consumption by inhibiting the phosphorylation of MAPKs. Consequently, we suggest that 5-7HP is a safe and photostable topical ingredient to enhance the skin penetration of 734THIF and skin hydration, and therefore 5-7HP may be used as an antipollutant additive in skin care products.

Preparation, characterizations and anti-pollutant activity of 7,3',4'-trihydroxyisoflavone nanoparticles in particulate matter-induced HaCaT keratinocytes.

BACKGROUND: 7,3',4'-Trihydroxyisoflavone (734THI), a secondary metabolite derived from daidzein in soybean, possesses several biological activities, including antioxidant, skin whitening and anti-atopic dermatitis properties, but the poor aqueous solubility of 734THI has limited its application in medicine and cosmetic industry. METHODS: The aim of the present study was to improve the physicochemical properties of 734THI using planetary ball mill preparation under a solvent-free process to improve its solubility and anti-pollutant activity. RESULTS: 734THI nanoparticle powder (734THIN) was successfully prepared by the planetary ball mill technique using polyvinylpyrrolidone K30 as the excipient. 734THIN effectively increased the aqueous solubility and cellular uptake of 734THI by improving its physicochemical properties, including particle size reduction, crystalline-amorphous transformation and intermolecular hydrogen bonding with polyvinylpyrrolidone K30. In addition, 734THIN inhibited the overexpression of COX-2 and MMP-9 by downregulating MAPK pathway signaling in particulate matter-exposed HaCaT keratinocytes, while raw 734THI in PBS with low aqueous solubility did not show any anti-inflammatory or antiaging activity. CONCLUSION: 734THIN may be used as an additive in anti-pollutant skin care products for preventing particulate matter-induced inflammation and aging in skin.

Design of acid-responsive polymeric nanoparticles for 7,3',4'-trihydroxyisoflavone topical administration.

7,3',4'-Trihydroxyisoflavone (734THIF) is a secondary metabolite of daidzein and has been recently found to possess antioxidant, melanin inhibition, and skin cancer chemopreventive activities. However, the poor water solubility of 734THIF impedes its absorption and skin penetration and, therefore, limits its pharmacological effects when applied topically to the skin. We seek to use the nanoprecipitation method to prepare optimal eudragit E100 (EE)-polyvinyl alcohol (PVA)-loaded 734THIF nanoparticles (734N) to improve its physicochemical properties and thereby increase its water solubility, skin penetration, and biological activities. EE-PVA-loaded 734THIF nanoparticles (734N) were prepared, and their morphology and particle size were evaluated using a particle size analyzer and by electron microscopy. The drug loading and encapsulation efficiencies and in vitro solubility were determined using high-performance liquid chromatography. Hydrogen-bond formation was evaluated by (1)H-nuclear magnetic resonance and Fourier transform infrared spectroscopy, and crystalline-to-amorphous transformation was determined by differential scanning calorimetry and X-ray diffractometry. In vitro skin penetration was analyzed using fresh pig skin mounted on Franz diffusion cells, and cytotoxicity against human keratinocyte HaCaT cells was evaluated using the MTT assay. Antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl-free radical scavenging ability. EE-PVA-loaded 734THIF nanoparticles showed good drug loading and encapsulation efficiencies and were characterized by improved physicochemical properties, including reduction in particle size, amorphous transformation, and intermolecular hydrogen-bond formation. This is associated with increased water solubility and enhanced in vitro skin penetration, with no cytotoxicity toward HaCaT cells. In addition, 734THIF nanoparticles retained their antioxidant activity. In conclusion, 734THIF nanoparticles are characterized by improved physicochemical properties, increased water solubility, and enhanced skin penetration, and these may have potential use in the future as a topical delivery formulation for the treatment of skin diseases.