Penazaphilones J-L, Three New Hydrophilic Azaphilone Pigments from Penicillium sclerotiorum cib-411 and Their Anti-Inflammatory Activity.

Penazaphilones J-L (1-3), three new hydrophilic azaphilone pigments, as well as six known compounds, were discovered from the filamentous fungus Penicillium sclerotiorum cib-411. Compounds 1-3 were structurally elucidated by the detailed interpretation of their 1D and 2D NMR spectroscopic data. Compound 1 is an unprecedented hybrid of an azaphilone and a glycerophosphate choline. Compounds 2 and 3 each contain an intact amino acid moiety. The bioassay showed that compound 3 exhibited significant anti-inflammatory activity. Concretely, compound 3 significantly suppressed the NO production, the expression levels of COX-2, IL-6, IL-1ß, and iNOS mRNA in LPS-stimulated RAW264.7 cells. Moreover, treatment of compound 3 prevented the translocation of NF-κB through inhibiting the phosphorylation of PI3K, PDK1, Akt, and GSK-3ß. Thus, the inhibition of compound 3 against LPS-induced inflammation should rely on its inactivation on NF-κB.

Systematical Ingredient Investigations of Ficus tikoua Bur. Fruit and Immunoregulatory and Antioxidant Effects of Different Fractions.

Although the fruit of Ficus tikoua Bur. has been consumed by montanic people in China for centuries, its chemical and biological composition was still unclear. A series of comprehensive investigations on its chemical constituents and bioactivities were carried out for the first time. As a result, six compounds were isolated and identified as the main components in this fruit. GC-MS analysis of the lipid components demonstrated that Ficus tikoua Bur. fruit contains some wholesome constituents such as fatty acids, vitamins, triterpenoids, and phytosterols. The fatty acids are mainly composed of linolenic acid (61.27%) and linoleic acid (22.79%). Furthermore, this fruit contains a relative high content of crude protein (9.41 ± 0.03%), total amino acids (9.28%), and total polyphenols (0.86 ± 0.01 g/100 g). The analysis of monosaccharide composition showed that the total polysaccharide mainly consists of glucose, glucuronic acid, xylose, arabinose, mannose, galactose, galacturonic acid, and rhamnose. The polysaccharide, polyphenol, water, ethanol, and flavonoid extracts exhibited prominent antioxidant activity determined by ABTS, DPPH, and FRAPS methods. Meanwhile, the total polysaccharide exhibited significant immunomodulatory effect by enhancing the release of cytokines and expression of iNOS and COX-2 in RAW264.7 cells, significantly decreasing the expression of c-Jun and p65 proteins in the cytoplasm; increasing the translocation of c-Jun and p65 to the nucleus; and regulating the phosphorylation level of Akt, PI3K, and PDK1 in the PI3K/AKT signaling pathway. This study proved that the fruit of F. tikoua is a reliable source of functional food.

Evaluation of a Novel Phosphorylated Red Dragon Fruit Peel Pectin for Enhancement of Thermal Stability and Functional Activity.

Red dragon fruit peel, as a fruit waste, is rich in plant-based nutritional pectins that can be applied as food additives. The present study aims to characterize a novel phosphorylated red dragon fruit peel pectin (PRDFP-P) and to explore its functional activities. The thermal analysis, morphology analysis, antibacterial, antioxidant and antitumor activities of PRDFP-P were evaluated. The results showed that the phosphorylated derivative PRDFP-P had typical phosphate groups. Compared with the native red dragon fruit peel pectin (PRDFP), PRDFP-P possessed superior thermal stability and exhibited significant inhibition effects on Escherichia coli and Staphylococcus aureus. Moreover, the phosphate groups on the derivative PRDFP-P chains remarkably enhanced the scavenging ability of hydroxyl radicals and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. In addition, PRDFP-P showed a significant inhibition effect on growth of human hepatic carcinoma cells (HepG2) and the IC50 value was determined to be 248.69 µg/mL (P < 0.05). Our results suggested that the phosphorylated derivative PRDFP-P might be potentially applied as stabilizing, thickening and gelling agents with functional activities in the food industry.

Stress Resistance and Pathogenicity of Nonthermal-Plasma-Induced Viable-but-Nonculturable Staphylococcus aureus through Energy Suppression, Oxidative Stress Defense, and Immune-Escape Mechanisms.

The occurrence of viable-but-nonculturable (VBNC) bacteria poses a potential risk to food safety due to failure in conventional colony detection. In this study, induction of VBNC Staphylococcus aureus was conducted by exposure to an atmospheric-pressure air dielectric barrier discharge-nonthermal-plasma (DBD-NTP) treatment with an applied energy of 8.1 kJ. The stress resistance profiles and pathogenicity of VBNC S. aureus were further evaluated. We found that VBNC S. aureus showed levels of tolerance of heat, acid, and osmosis challenges comparable to those shown by culturable S. aureus, while VBNC S. aureus exhibited enhanced resistance to oxidative and antibiotic stress, relating to the mechanisms of cellular energy depletion, antioxidant response initiation, and multidrug efflux pump upregulation. Regarding pathogenicity, NTP-induced VBNC S. aureus retained the capacity to infect the HeLa host cells. Compared with the culturable counterparts, VBNC S. aureus caused reduced immune responses (Toll-like receptor [TLR], nucleotide-binding oligomerization domain [NOD]) in HeLa cells, which was attributed to suppression of biosynthesis of the recognized surface ligands (e.g., peptidoglycan). Additionally, the proteomic analysis revealed that upregulation of several virulence factors (ClfB, SdrD, SCIN, SasH, etc.) could ensure that VBNC S. aureus would adhere to and internalize into host cells and avoid the host attack. The camouflaged mechanisms described above led to VBNC S. aureus causing less damage to the host cells, and their activity might result in longer intracellular persistence, posing potential risks during NTP processing.IMPORTANCE The consumer demand for freshness and nutrition has accelerated the development of mild decontamination technologies. The incomplete killing of nonthermal (NT) treatments might induce pathogens to enter into a viable-but-nonculturable (VBNC) status as a survival strategy. The use of nonthermal plasma (NTP) as a novel food decontamination technology received increased attention in food industry during recent decades. Our previous work confirmed that the foodborne pathogen S. aureus was induced into VBNC status in response to NTP exposure. This work further revealed the development of stress resistance and virulence retention of NTP-induced VBNC S. aureus through the mechanisms of energy suppression, oxidative stress defense, and immune escape. The data provide fundamental knowledge of the potential risks posed by NTP-induced VBNC S. aureus, which require further parameter optimization of the NTP process or combination with other techniques to avoid the occurrence of VBNC bacteria.

Appraising the Genetic Architecture of Kernel Traits in Hexaploid Wheat Using GWAS.

Kernel morphology is one of the major yield traits of wheat, the genetic architecture of which is always important in crop breeding. In this study, we performed a genome-wide association study (GWAS) to appraise the genetic architecture of the kernel traits of 319 wheat accessions using 22,905 single nucleotide polymorphism (SNP) markers from a wheat 90K SNP array. As a result, 111 and 104 significant SNPs for Kernel traits were detected using four multi-locus GWAS models (mrMLM, FASTmrMLM, FASTmrEMMA, and pLARmEB) and three single-locus models (FarmCPU, MLM, and MLMM), respectively. Among the 111 SNPs detected by the multi-locus models, 24 SNPs were simultaneously detected across multiple models, including seven for kernel length, six for kernel width, six for kernels per spike, and five for thousand kernel weight. Interestingly, the five most stable SNPs (RAC875_29540_391, Kukri_07961_503, tplb0034e07_1581, BS00074341_51, and BobWhite_049_3064) were simultaneously detected by at least three multi-locus models. Integrating these newly developed multi-locus GWAS models to unravel the genetic architecture of kernel traits, the mrMLM approach detected the maximum number of SNPs. Furthermore, a total of 41 putative candidate genes were predicted to likely be involved in the genetic architecture underlining kernel traits. These findings can facilitate a better understanding of the complex genetic mechanisms of kernel traits and may lead to the genetic improvement of grain yield in wheat.

Two New Cucurbitane Glycosides from the Fruits of Siraitia grosvenori.

Two novel cucurbitane glycosides, named as 11-oxomogroside III A1 and 7ß-methoxy-mogroside V, along with sixteen known ones were isolated from the fruits of Siraitia grosvenori SWINGLE. The structures of the new compounds were characterized by chemical and extensive spectral methods.

Target-Guided Isolation of Three Main Antioxidants from Mahonia bealei (Fort.) Carr. Leaves Using HSCCC.

Mahonia bealei (Fort.) Carr. is an economically important plant that is widely cultivated in Southwest China. Its leaves are commonly used for tea and contain an abundance of antioxidant compounds. However, methods of the systematic purification of antioxidants from M. bealei are lacking. In this study, antioxidants from this plant were effectively and rapidly enriched. First, antioxidants were screened using online 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH)-high performance liquid chromatography (HPLC), followed by separation using high-speed countercurrent chromatography with an optical solvent system composed of n-hexane/ethyl acetate/methanol/water (1:5:1:5, v/v/v/v). Three phenolics-chlorogenic acid (1, 8.3 mg), quercetin-3-O-ß-d-glucopyranoside (2, 20.5 mg), and isorhamnetin-3-O-ß-d-glucopyranoside (3, 28.4 mg)-were obtained from the ethyl acetate-soluble fraction (240 mg) by one-step separation. The chemical structures of the phenolics were characterized by MS and NMR techniques, and the purity of each compound was >92.0% as determined by HPLC. The isolated compounds were assessed by scavenging activities on DPPH and superoxide radicals as well as cytoprotective assays, all of which showed similar trends regarding the antioxidant capacities of the compounds. Moreover, compounds 1-3 significantly attenuated the lipid peroxidation and antioxidant enzyme activities in H2O2-treated RAW264.7 cells. Our study demonstrated the efficiency of a newly developed integrative system for the comprehensive characterization of pure compounds from M. bealei, which will allow their use as reference substances.

20-Hydroxy-3-Oxolupan-28-Oic Acid Attenuates Inflammatory Responses by Regulating PI3K⁻Akt and MAPKs Signaling Pathways in LPS-Stimulated RAW264.7 Macrophages.

20-Hydroxy-3-oxolupan-28-oic acid (HOA), a lupane-type triterpene, was obtained from the leaves of Mahonia bealei, which is described in the Chinese Pharmacopeia as a remedy for inflammation and related diseases. The anti-inflammatory mechanisms of HOA, however, have not yet been fully elucidated. Therefore, the objective of this study was to characterize the molecular mechanisms of HOA in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. HOA suppressed the release of nitric oxide (NO), pro-inflammatory cytokine tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) in LPS-stimulated RAW264.7 macrophages without affecting cell viability. Quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) analysis indicated that HOA also suppressed the gene expression of inducible NO synthase (iNOS), TNF-α, and IL-6. Further analyses demonstrated that HOA inhibited the phosphorylation of upstream signaling molecules, including p85, PDK1, Akt, IκBα, ERK, and JNK, as well as the nuclear translocation of nuclear factor κB (NF-κB) p65. Interestingly, HOA had no effect on the LPS-induced nuclear translocation of activator protein 1 (AP-1). Taken together, these results suggest that HOA inhibits the production of cytokine by downregulating iNOS, TNF-α, and IL-6 gene expression via the downregulation of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinases (MAPKs), and the inhibition of NF-κB activation. Our findings indicate that HOA could potentially be used as an anti-inflammatory agent for medical use.

Preparation and in vitro and in vivo evaluation of quercetin-loaded mixed micelles for oral delivery.

Quercetin (QT) is a plant polyphenol with various pharmacological properties. However, the low water solubility limits its therapeutic efficacy. In the present study, QT-loaded sodium taurocholate-Pluronic P123 (QT-loaded ST/P123) mixed micelles were developed and characterized, and the effect of the formulation on improving the water solubility of QT was investigated. QT-loaded ST/P123 mixed micelles were prepared by thin film hydration-direct dissolution and optimized by uniform design. The optimal formulation possessed high drug loading (12.6%) and entrapment efficiency (95.9%) in small (16.20 nm) spherically-shaped micelles. A low critical micelle concentration indicated that the micelles were stable, and they showed a sustained release pattern, as determined in vitro in simulated gastric fluid and intestinal fluid. Pharmacokinetic evaluation showed the Cmax and AUC0-24 were 1.8-fold and 1.6-fold higher than the QT suspension. The present results indicate that QT-loaded ST/P123 micelles are potential candidates to improve the solubility and oral bioavailability of QT.

A new steroidal saponin from Polygonatum sibiricum.

A new furostan type steroidal saponin, kingianoside Z (1), along with four known compounds (2-5), was isolated from the ethanolic extract of Polygonatum sibiricum Delar. ex Redoute. Their structures were determined by spectroscopical method and by comparison with previously reported spectroscopic data. Compounds 3-5 showed significant cytotoxicity against HepG2 cell lines with IC50 values of 14.2, 12.1 and 8.5 µM, respectively.

Methylation of neurofilament light polypeptide promoter is associated with cell invasion and metastasis in NSCLC.

The role of NEFL in NSCLC remains largely unknown. Immunohistochemistry was performed to investigate the expression of NEFL in 108 lung cancer specimens. NEFL expression was associated with decreased lymph node metastases and favorable prognosis. Furthermore, real-time PCR and Western blot were used to investigate the expression of the NEFL gene in NSCLC cell lines. Subsequently, lentivirus-mediated RNA interference and overexpression were used to demonstrate that knocked-down of NEFL enhanced the invasion and migration of A549 and H460 NSCLC cells, whereas NEFL overexpression resulted in a suppression of the invasion and migration of GLC-82 and L78 cells in vitro. In addition, bisulfite sequence PCR assay demonstrated that NEFL downregulation was associated with promoter methylation, and NEFL expression was restored after treatment with 5-Aza-dC. Finally, we demonstrated that NEFL inhibited the NF-κB pathway, thereby suppressing the expression of uPA and decreasing NSCLC invasiveness and migration. Our studies suggest that NEFL methylation is a novel mechanism for NSCLC invasion and metastasis and that NEFL may represent a candidate biomarker for recurrence and survival in patients with NSCLC.

Early adventitial activation characterized by NADPH oxidase expression and neovascularization in an aortic transplantation model.

Increasing evidence has suggested that arterial adventitia may contribute to pathological vessel remodeling by producing reactive oxygen species and promoting neovascularization. However, these processes have not been studied yet in transplantation-induced vascular pathologies. We characterized the dynamic changes in NADPH oxidase expression and adventitial angiogenic response in a model of allograft aortic transplantation. The thoracic aorta from Fischer 344 rats were transplanted into the abdominal aorta of Lewis rats. Graft specimens were collected on days 0.5, 3, 7, and 14 for morphometry, immunohistochemistry, immunofluorescence staining, and quantitative PCR tests. Following transplantation, adventitial thickening was found as early as day 3, while neointima was observed from day 7. As compared to normal adventitial tissue, the expression levels of NADPH oxidase subunits gp91phox and p47phox in graft adventitia were elevated from day 3 and further increased up to day 14. Immunohistochemistry staining showed that infiltrating macrophages appeared to be a major source of NADPH oxidase expression. Increases in NADPH oxidase expression were also detected in fibroblasts isolated from the graft adventitia. Gene silencing of p47phox significantly suppressed proliferation and migration of the graft fibroblast cells. We also showed that adventitial thickening was accompanied by increased adventitial neovascularization; at day 14, there was a positive correlation between the density of adventitial microvessels and the neointimal thickness. Transplantation injury induces NADPH oxidase expression and neovascularization in the adventitia, raising the possibility that the activated adventitia may represent a target site for prevention of transplantation-induced transplant vasculopathy.

A new phenylethanoid glycoside from Incarvillea compacta.

A new phenylethanoid glycoside, 3'''-O-methylcampneoside I (1), was isolated from the 90% ethanolic extract of the roots of Incarvillea compacta, together with three known compounds, campneoside I (2), ilicifolioside A (3), and campneoside II (4). Their structures were determined spectroscopically and compared with previously reported spectral data. Compound 1 existed as epimers and displayed better 1,1-diphenyl-2-picrylhydrazyl (DPPH)-free radical scavenging activity using di-tert-butyl-4-methylphenol (BHT) as the positive control. In addition, pretreatment of human HepG2 cells with compound 1 significantly increased the viability on CCl4-induced cell death.

Comparative Transcriptional Analysis of Loquat Fruit Identifies Major Signal Networks Involved in Fruit Development and Ripening Process.

Loquat (Eriobotrya japonica Lindl.) is an important non-climacteric fruit and rich in essential nutrients such as minerals and carotenoids. During fruit development and ripening, thousands of the differentially expressed genes (DEGs) from various metabolic pathways cause a series of physiological and biochemical changes. To better understand the underlying mechanism of fruit development, the Solexa/Illumina RNA-seq high-throughput sequencing was used to evaluate the global changes of gene transcription levels. More than 51,610,234 high quality reads from ten runs of fruit development were sequenced and assembled into 48,838 unigenes. Among 3256 DEGs, 2304 unigenes could be annotated to the Gene Ontology database. These DEGs were distributed into 119 pathways described in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. A large number of DEGs were involved in carbohydrate metabolism, hormone signaling, and cell-wall degradation. The real-time reverse transcription (qRT)-PCR analyses revealed that several genes related to cell expansion, auxin signaling and ethylene response were differentially expressed during fruit development. Other members of transcription factor families were also identified. There were 952 DEGs considered as novel genes with no annotation in any databases. These unigenes will serve as an invaluable genetic resource for loquat molecular breeding and postharvest storage.

5-Methoxyl Aesculetin Abrogates Lipopolysaccharide-Induced Inflammation by Suppressing MAPK and AP-1 Pathways in RAW 264.7 Cells.

For the first time, a pale amorphous coumarin derivative, 5-methoxyl aesculetin (MOA), was isolated from the dried bark of Fraxinus rhynchophylla Hance (Oleaceae). MOA modulates cytokine expression in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages, but the precise mechanisms are still not fully understood. We determined the effects of MOA on the production of inflammatory mediators and pro-inflammatory cytokines in the LPS-induced inflammatory responses of RAW 264.7 macrophages. MOA significantly inhibited the LPS-induced production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), interleukin-6, and interleukin-1ß. It also effectively attenuated inducible nitric oxide (NO) synthase, cyclooxygenase-2, and TNF-α mRNA expression and significantly decreased the levels of intracellular reactive oxygen species. It inhibited phosphorylation of the extracellular signal-regulated kinase (ERK1/2), thus blocking nuclear translocation of activation protein (AP)-1. In a molecular docking study, MOA was shown to target the binding site of ERK via the formation of three hydrogen bonds with two residues of the kinase, which is sufficient for the inhibition of ERK. These results suggest that the anti-inflammatory effects of MOA in RAW 264.7 macrophages derive from its ability to block both the activation of mitogen-activated protein kinases (MAPKs) and one of their downstream transcription factors, activator protein-1 (AP-1). Our observations support the need for further research into MOA as a promising therapeutic agent in inflammatory diseases.

Anti-Inflammatory Effects of Chloranthalactone B in LPS-Stimulated RAW264.7 Cells.

Chloranthalactone B (CTB), a lindenane-type sesquiterpenoid, was obtained from the Chinese medicinal herb Sarcandra glabra, which is frequently used as a remedy for inflammatory diseases. However, the anti-inflammatory mechanisms of CTB have not been fully elucidated. In this study, we investigated the molecular mechanisms underlying these effects in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. CTB strongly inhibited the production of nitric oxide and pro-inflammatory mediators such as prostaglandin E2, tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), and IL-6 in RAW264.7 cells stimulated with LPS. A reverse-transcription polymerase chain reaction assay and Western blot further confirmed that CTB inhibited the expression of inducible nitric oxide synthase, cyclooxygenase-2, TNF-α, and IL-1ß at the transcriptional level, and decreased the luciferase activities of activator protein (AP)-1 reporter promoters. These data suggest that inhibition occurred at the transcriptional level. In addition, CTB blocked the activation of p38 mitogen-activated protein kinase (MAPK) but not c-Jun N-terminal kinase or extracellular signal-regulated kinase 1/2. Furthermore, CTB suppressed the phosphorylation of MKK3/6 by targeting the binding sites via formation of hydrogen bonds. Our findings clearly show that CTB inhibits the production of inflammatory mediators by inhibiting the AP-1 and p38 MAPK pathways. Therefore, CTB could potentially be used as an anti-inflammatory agent.

Synergistic Induction of Erlotinib-Mediated Apoptosis by Resveratrol in Human Non-Small-Cell Lung Cancer Cells by Down-Regulating Survivin and Up-Regulating PUMA.

BACKGROUND/AIM: Treatment of human non-small-cell lung cancer (NSCLC) often involves uses of multiple therapeutic strategies with different mechanisms of action. Here we found that resveratrol (RV) enhanced the anti-tumor effects of epidermal growth factor receptor (EGFR) inhibitor erlotinib in NSCLC cells. METHODS: Cell viability was measured by MTT assay and clonogenicity assay. Western blot was applied to assess the protein expression levels of target genes. Cell apoptosis was monitored by AnnexinV-FITC assay and sub-G1 population assay. Intracellular ROS were measured by flow cytometric analysis. Cell caspase activities were carried out by fluorometric assays. RESULTS: Exposure of H460, A549, PC-9 and H1975 cells to minimal or non-toxic concentrations of RV and erlotinib synergistically reduced cell viability, colony formation and induced cell apoptosis. Furthermore, RV synergistically enhanced erlotinib-induced apoptosis was involved in ROS production. Additionally, co-treatment with RV and erlotinib repressed the expressions of anti-apoptosis proteins, such as survivin and Mcl-1, whereas promoted p53 and PUMA expression and caspase 3 activity. Moreover, the combination was also more effective at inhibiting the AKT/mTOR/S6 kinase pathway. Subsequently, small interfering RNA (siRNA) depletion of PUMA and overexpression of survivin significantly attenuated NSCLC cells apoptosis induced by the combination of the two drugs. CONCLUSION: Our findings suggested that RV synergistically enhanced the anti-tumor effects of erlotinib in NSCLC cells were involved in decrease of survivin expression and induction of PUMA expression. In conclusion, based on the observations from our study, we indicated that the combined administration of these two drugs might be considered as a novel therapeutic regimen for treating NSCLC.

Protopanaxatriol activates EGFR and HER2 to strengthen the molecules of skin protection in human keratinocytes.

BACKGROUND: Protopanaxatriol (PPT) is an important ginsenoside produced by ginseng, a tonic plant used in many areas. PPT has beneficial effects against many disease states including inflammation, diabetes, and cancer. However, PPT's protective effects on skin integrity have been rarely studied. Previously, we reported that PPT can maintain skin moisture through activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) pathways. However, the cellular targets for enhancing skin moisturizing effects via PPT are still unknown. PURPOSE: We wanted to identify the upstream targets of PPT on upregulating moisturizing factor (HAS-2) expression. STUDY DESIGN: We investigated which upstream proteins can be directly stimulated by PPT to modulate NF-κB, MAPKs and other signaling cascades. Then, the targeted proteins were overexpressed to check the relationship with HAS-2. Next, the cellular thermal shift assay (CETSA) was conducted to check the relationship between targeted proteins and PPT. METHODS: A human keratinocyte HaCaT were employed to measure the levels of moisturizing factors and the signaling proteins activated by PPT. Transfection conditions were established with DNA constructs expressing epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) and their mutants prepared by site-directed mutagenesis. Further investigation on molecular mechanisms was conducted by RT-PCR, luciferase reporter gene assay, CETSA, or Western blot. RESULTS: We found that PPT can activate the phosphorylation of EGFR and HER2. These stimulations caused Src phosphorylation, which resulted in the activation of phosphoinositide 3-kinases (PI3K)/pyruvate dehydrogenase kinase 1 (PDK1)/protein kinase B (AKT)/NF-κB and MAPKs signaling cascades. Additionally, EGFR and HER2 activation resulted in phosphorylation of signal transducer and activator of transcription 3 (STAT3) and calcium/calmodulin-dependent protein kinase II (CaMKII). This induced the AMP-activated protein kinase alpha (AMPKα) signaling pathway. Additionally, PPT blocked peroxisome proliferator activated receptor gamma (PPARγ), which also contributed to the phosphorylation of Src. CONCLUSION: Overall, we first found that PPT offers excellent protection of the skin barrier and hydrogen supply in keratinocytes. Moreover, growth factor receptors such as EGFR and HER2 were revealed to be central enzymes to be directly targeted by PPT. These results suggest a potentially valuable role as a cosmetic ingredient.

Insight into the allergenicity and structure changes of parvalbumin from Trachinotus ovatus induced by dense-phase carbon dioxide.

Parvalbumin (PV) is a major allergen in fish, and traditional treatments cannot reduce its sensitization. The effects of dense-phase carbon dioxide (DPCD) treatment on the sensitization and spatial structure of PV in Trachinotus ovatus were evaluated in this study. Western blotting and indirect ELISA were used to determine the allergenicity changes and spatial conformations of PV treated by DPCD. Tris-tricine-SDS-PAGE, circular dichroism, surface hydrophobicity, endogenous fluorescence, UV spectrophotometry, free amino group, total sulfhydryl group and SEM analyses were applied to characterize PV structure. The results showed that DPCD treatment (15 MPa, 30 min, 50 °C) could reduce PV-induced allergic reactions by 39-41 %, which destroyed the normal conformational epitopes and reduced the risk of PV-induced allergy. The secondary structure changed from ordered to disordered with a decreased content of α-helical groups, while the internal hydrophobic groups were exposed. The total sulfhydryl group content decreased significantly (P < 0.05). The surface hydrophobicity and ultraviolet absorption spectrum were enhanced, and the endogenous fluorescence peak shifted to a long wavelength. Meanwhile, the content of free amino groups increased significantly (P < 0.05). This study could provide a theoretical basis and a promising technical approach for reduction of PV allergenicities.

Asiaticoside exerts neuroprotection through targeting NLRP3 inflammasome activation.

BACKGROUND: Parkinson's disease (PD), a neurodegenerative disorder, is characterized by motor symptoms due to the progressive loss of dopaminergic neurons in the substantia nigra (SN) and striatum (STR), alongside neuroinflammation. Asiaticoside (AS), a primary active component with anti-inflammatory and neuroprotective properties, is derived from Centella asiatica. However, the precise mechanisms through which AS influences PD associated with inflammation are not yet fully understood. PURPOSE: This study aimed to explore the protective mechanism of AS in PD. METHODS: Targets associated with AS and PD were identified from the Swiss Target Prediction, Similarity Ensemble Approach, PharmMapper, and GeneCards database. A protein-protein interaction (PPI) network was constructed to identify potential therapeutic targets. Concurrently, GO and KEGG analyses were performed to predict potential signaling pathways. To validate these mechanisms, the effects of AS on 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD in mice were investigated. Furthermore, neuroinflammation and the activation of the NLRP3 inflammasome were assessed to confirm the anti-inflammatory properties of AS. In vitro experiments in BV2 cells were then performed to investigate the mechanisms of AS in PD. Moreover, CETSA, molecular docking, and molecular dynamics simulations (MDs) were performed for further validation. RESULTS: Network pharmacology analysis identified 17 potential targets affected by AS in PD. GO and KEGG analyses suggested the biological roles of these targets, demonstrating that AS interacts with 149 pathways in PD. Notably, the NOD-like receptor signaling pathway was identified as a key pathway mediating AS's effect on PD. In vivo studies demonstrated that AS alleviated motor dysfunction and reduced the loss of dopaminergic neurons in MPTP-induced PD mice. In vitro experiments demonstrated that AS substantially decreased IL-1ß release in BV2 cells, attributing this to the modulation of the NLRP3 signaling pathway. CETSA and molecular docking studies indicated that AS forms a stable complex with NLRP3. MDs suggested that ARG578 played an important role in the formation of the complex. CONCLUSION: In this study, we first predicted that the potential target and pathway of AS's effect on PD could be NLRP3 protein and NOD-like receptor signaling pathway by network pharmacology analysis. Further, we demonstrated that AS could alleviate symptoms of PD induced by MPTP through its interaction with the NLRP3 protein for the first time by in vivo and in vitro experiments. By binding to NLRP3, AS effectively inhibits the assembly and activation of the inflammasome. These findings suggest that AS is a promising inhibitor for PD driven by NLRP3 overactivation.