Suppression of Inflammation in Adipocyte-Macrophage Coculture by Passion Fruit Seed Extract: Insights into the p38 and NF-Ò¡B Pathway.

Obesity, which is characterized by chronic low-grade inflammation, involves the infiltration of immune cells into adipose tissue, leading to the secretion of inflammatory cytokines and subsequent inflammation. Therefore, the aim of this study was to examine the potential of passion fruit seed extract (PSEE) in mitigating lipopolysaccharide (LPS)-induced inflammation in a coculture system comprising macrophages and adipocytes. PSEE demonstrated significant reductions in reactive oxygen species (ROS) and nitric oxide (NO) levels, primarily achieved through the downregulation of inducible nitric oxide synthase (iNOS) protein expression in LPS-induced adipocyte-macrophage cocultures. Furthermore, PSEE effectively suppressed the secretion of TNF-α and IL-1ß by attenuating the gene expression of these cytokines, as well as other inflammation-related genes such as MMP-2, IL-6, and MCP-1. Notably, PSEE exhibited potent inhibitory effects on the p38 and NF-κB signaling pathways, thus alleviating inflammation in the LPS-induced adipocyte-macrophage cocultures. Additionally, PSEE led to a decrease in the expression of ACC, HSL, and FaSN, while aP2 and ATGL showed increased expression in LPS-induced cocultured macrophages and adipocytes. These findings suggest that passion fruit seed extract effectively combats inflammation by suppressing the p38 and NF-κB signaling pathways, resulting in reduced levels of proinflammatory cytokines, NO, and ROS production.

Impact of Time and Enzyme Concentration on Sangyod Rice Bran Hydrolysate: Phytochemicals, Antioxidants, Amino Acids, and Cytotoxicity.

This study investigated the production of Sangyod rice bran hydrolysate (SYRB) from Sangyod rice, focusing on incubation times (1, 3, and 5 h) and alcalase enzyme concentrations (0, 0.7, and 1% v/v). The results demonstrated a concentration-dependent relationship: higher alcalase concentrations increased hydrolysate yield. Prolonged incubation, especially with alcalase, enhanced substrate breakdown, further increasing hydrolysate production. The degree of hydrolysis, reflecting peptide bond cleavage, depended on both incubation time and enzyme concentration, emphasizing the role of enzyme activity in efficiency. Moreover, color analysis (L*, a*, b*) and color difference (∆E) revealed intricate changes from enzymatic hydrolysis. Proximate composition analysis showed higher protein and lipid content with increased enzyme concentration and longer incubation times, whereas ash content varied with both factors. Hydrolysate powders exhibited higher moisture content than raw rice bran, indicating the impact of the hydrolysis process. The study also explored SYRB's antioxidant properties and cytotoxicity, which were sensitive to incubation time and alcalase concentration. Longer incubation increased DPPH scavenging activity, with the highest efficacy at 3 h. Meanwhile, ABTS scavenging displayed a delicate balance with alcalase concentration. The cytotoxicity study of SYRB revealed that all concentrations of SYRB were non-toxic to C2C12 cells, with cell viability values exceeding 70%.

Antimetastatic effect of fucoidan against non-small cell lung cancer by suppressing non-receptor tyrosine kinase and extracellular signal-related kinase pathway.

BACKGROUND/OBJECTIVES: Fucoidan, a polysaccharide content in brown algae, has been reported to inhibit the growth of cancer cells. The present study aimed to investigate the suppression effects of fucoidan on A549 non-small cell lung cancer cells migration. MATERIALS/METHODS: The anti-migratory activity of fucoidan in A549 cells was examined by wound healing assay and phalloidin-rhodamine staining in response to fucoidan (0-100 µg/mL) treatment for 48 h. Western blot analysis was performed to clarify the protein expressions relevant to migratory activity. RESULTS: Fucoidan (25-100 µg/mL) significantly suppressed A549 cells migration together with reduced the intensity of phalloidin-rhodamine which detect filopodia and lamellipodia protrusions at 48 h of treatment. The protein expression indicated that fucoidan significantly suppressed the phosphorylation of focal adhesion kinase (FAK), Src, and extracellular signal-related kinase (ERK). In addition, the phosphorylation of p38 in A549 cells was found to be increased. CONCLUSIONS: Our data conclude that fucoidan exhibits anti-migratory activities against lung cancer A549 cells mediated by inhibiting ERK1/2 and FAK-Src pathway.

Upregulation of TRPA1 and reduction of NF-κB translocation could be part of the immunomodulatory process during primary tooth inflammation.

Transient receptor potential ankyrin 1 (TRPA1) is expressed on neurons and immune and endothelial cells, and acts as a chemosensor that is activated by bacterial components and endotoxins. This study aimed to better understand how TRPA1 responds to inflammation induced by bacterial-related stimuli during dental caries. The pulp of 40 primary teeth and 46 permanent teeth were categorized into three stages of carious progression: intact dentin, exposed dentin and exposed pulp. We measured the percentages of cells with NF-κB nuclear translocation to verify the severity of inflammation, and then assessed TRPA1 expression by immunofluorescence technique in the pulpal horn, subodontoblastic and mid-coronal regions of the dental pulp samples at various stages of caries. We found NF-κB nuclear translocation gradually reduced during the progression of caries in all three areas of the dental pulp in both primary and permanent teeth. TRPA1 was gradually upregulated in the pulp of primary teeth as caries progressed, but did not significantly vary during caries in the pulp of permanent teeth. This study of TRPA1 expression demonstrates primary and permanent teeth exhibit distinct responses when undergoing carious infection.

Self-assembled polydopamine nanoparticles improve treatment in Parkinson's disease model mice and suppress dopamine-induced dyskinesia.

Although Levodopa (l-DOPA), a dopamine precursor, exhibits a high risk of dyskinesia, it remains the primary treatment in Parkinson's disease (PD), a progressive neurodegenerative disorder. In this study, we designed poly(l-DOPA)-based self-assembled nanodrug (NanoDOPA) from amphiphilic block copolymer possessing poly(l-DOPA(OAc)2), which is a precursor of l-DOPA as a hydrophobic segment, for treatment in a PD model mouse. Under physiological enzyme treatment, the poly(l-DOPA(OAc)2) in the block copolymer was hydrolyzed to liberate l-DOPA gradually. Using the MPTP-induced PD mouse model, we observed that mice treated with NanoDOPA demonstrated a significant improvement of PD symptoms compared to the l-DOPA treatment. Interestingly, the NanoDOPA treatment did not cause the dyskinesia symptoms, which was clearly observed in the l-DOPA-treated mice. Furthermore, NanoDOPA exhibited remarkably lower toxicity in vitro compared to l-DOPA, in addition with no noticeable NanoDOPA toxicity observed in the treated mice. These results suggested that self-assembled NanoDOPA is a promising therapeutic in the treatment of PD. STATEMENT OF SIGNIFICANCE: In this study, we proposed a therapeutic approach for the effective treatment of Parkinson's disease (PD) using newly designed poly(l-DOPA)-based self-assembled nanodrug (NanoDOPA) prepared from amphiphilic block copolymers possessing poly(l-DOPA(OAc)2), which is a precursor of l-DOPA as a hydrophobic segment, for treatment in a PD model mouse. Under physiological enzyme treatments, NanoDOPA was hydrolyzed to liberate l-DOPA gradually, improving the pharmacokinetic value of l-DOPA. The mice treated with NanoDOPA significantly improved PD symptoms compared to the l-DOPA treatment in a neurotoxin-induced PD mouse model. Interestingly, NanoDOPA treatment did not cause dyskinesia symptoms, which was observed in the l-DOPA-treated mice. The obtained results in this study suggested that self-assembled NanoDOPA is a promising therapeutic in the treatment of PD.

Luteolin attenuates migration and invasion of lung cancer cells via suppressing focal adhesion kinase and non-receptor tyrosine kinase signaling pathway.

BACKGROUND/OBJECTIVES: Non-small cell lung cancer is mostly recognized among other types of lung cancer with a poor prognosis by cause of chemotherapeutic resistance and increased metastasis. Luteolin has been found to decrease cell metastasis. However, its underlying mechanisms remain unresolved. The objective of this study was to examine the effect (and its mechanism) of luteolin on the migration and invasion of human non-small cell lung cancer A549 cells. MATERIALS/METHODS: Cell viability was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Wound healing and transwell assays were evaluated to assess migration and invasion, respectively. Western blot analysis and immunofluorescence were further performed to investigate the role of luteolin and its mechanisms of action. RESULTS: Administration with up to 40 µM luteolin showed no cytotoxic activity on lung cancer A549 cells or non-cancer MRC-5 cells. Additionally, luteolin at 20-40 µM significantly suppressed A549 cells' migration, invasion, and the formation of filopodia in a concentration-dependent manner at 24 h. This is similar with western blot analysis, which revealed diminished the phosphorylated focal adhesion kinase (pFAK), phosphorylated non-receptor tyrosine kinase (pSrc), Ras-related C3 botulinum toxin substrate 1 (Rac1), cell division control protein 42 (Cdc42), and Ras homolog gene family member A (RhoA) expression levels. CONCLUSIONS: Overall, our data indicate that luteolin plays a role in controlling lung cancer cells' migration and invasion via Src/FAK and its downstream Rac1, Cdc42, and RhoA pathways. Luteolin might be considered a promising candidate for suppressing invasion and metastasis of lung cancer cells.

Standardized extract of Centella asiatica ECa 233 inhibits lipopolysaccharide-induced cytokine release in skin keratinocytes by suppressing ERK1/2 pathways

Objective: To evaluate the effect of standardized extract of Centella asiatica ECa 233 on inflammatory mediator production through cyclooxygenase-2 (COX-2), extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor-κB (NF-κB) pathway in keratinocyte HaCaT cells.Methods: HaCaT cells were treated with 0.1, 1, 10 and 100 μg/mL ECa 233 in the presence of lipopolysaccharide (LPS). Proinflammatory cytokines and prostaglandin E2 were assessed with ELISA. Western blotting was used to determine the inhibition of COX-2, ERK1/2 and NF-κB protein expression. Results: ECa 233 suppressed LPS-induced release of interleukin-1β, tumor necrosis factor-α, and prostaglandin E2. ECa 233 also inhibited COX-2, phosphorylation of ERK1/2 and the activation of NF-κB. Moreover, the formation of reactive oxygen species (ROS) was decreased in response to LPS-inflamed keratinocytes. Conclusions: ECa 233 inhibits LPS-stimulated production of inflammatory mediators in keratinocytes via suppressing ERK1/2 and NF-κB pathways. The suppressive effect of ECa 233 may be related to an inhibition of ROS production.

Metformin Inhibit Cervical Cancer Migration by Suppressing the FAK/Akt Signaling Pathway.

BACKGROUND: Metformin, an antidiabetic drug, has been previously reported to have anti-cancer activities. However, its role in the control of cancer cell migration remains elusive. METHODS: To examine the possible effect of metformin on migration of cervical cancer cells. The related mechanisms were further determined by immunocytochemistry and Western's blotting assay. RESULTS: The results showed that metformin treatment substantially inhibited the migration ability of cervical cancer cells. Consistently, the filopodia and lamellipodia formation were depleted after exposure to metformin. The suppression of migration mediated through the regulatory proteins such as focal adhesion kinase (FAK), ATP-dependent tyrosine kinase (Akt), Rac1 and RhoA after metformin treatment. CONCLUSION: Metformin displays antimigration effects in cervical cancer cells by inhibiting filopodia and lamellipodia formation through the suppression of FAK, Akt and its downstream Rac1 and RhoA protein. We propose that metformin could be a novel potential candidate as an antimetastatic cancer drug in the cervical cancer management.

ECa 233 Suppresses LPS-Induced Proinflammatory Responses in Macrophages via Suppressing ERK1/2, p38 MAPK and Akt Pathways.

A current anti-inflammatory agent often targets the prevention of inflammatory disorder development. The standardized Centella asiatica ECa 233 extract has been previously reported for anti-inflammatory effect. This study aimed to investigate its anti-inflammatory effect and mechanisms of ECa 233 in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages, through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, nitric oxide (NO) assay, reactive oxygen species (ROS) production assay, enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Our results found that ECa 233 significantly inhibited LPS-stimulated pro-inflammatory mediators production including ROS, NO and prostaglandin E2 (PGE2), and pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α and interleukin (IL)-1ß without cytotoxicity. In addition, ECa 233 downregulated not only the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), but also the activation of nuclear factor-kappa B (NF-κB), activated protein kinase B (Akt), extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinases (MAPK) induced by LPS. The inhibition of LPS-induced inflammation due to ECa 233 offered an opportunity as a tentatively potential candidate for the prevention and treatment of inflammatory diseases.

Metformin Promotes Neuronal Differentiation via Crosstalk between Cdk5 and Sox6 in Neuroblastoma Cells.

Metformin has recently emerged as a key player in promotion of neuroblastoma differentiation and neurite outgrowth. However, molecular mechanisms of how metformin promotes cellular differentiation have not yet been fully elucidated. In this study, we investigated how metformin promotes cell differentiation, via an inhibition of cell proliferation, by culturing SH-SY5Y neuroblastoma cells with or without metformin. Pretreatment with reactive oxygen species (ROS) scavenger, NAC, revealed that ROS plays a crucial role in induction of cell differentiation. Cell differentiation was observed under various morphological criteria: extension of neuritic processes and neuronal differentiation markers. Treatment with metformin significantly increased neurite length, number of cells with neurite, and expression of neuronal differentiation markers, ß-tubulin III and tyrosine hydroxylase (TH) compared with untreated control. Further investigation found that metformin significantly decreased Cdk5 but increased Sox6 during cell differentiation. Analysis of the mechanism underlying these changes using Cdk5 inhibitor, roscovitine, indicated that expressions of Cdk5 and Sox6 corresponded to metformin treatment. These results suggested that metformin produces neuronal differentiation via Cdk5 and Sox6. In addition, phosphorylated Erk1/2 was decreased while phosphorylated Akt was increased in metformin treatment. Taken together, these findings suggest that metformin promotes neuronal differentiation via ROS activation through Cdk5/Sox6 crosstalk, relating to Erk1/2 and Akt signaling.

Missing and overexpressing proteins in domestic cat oocytes following vitrification and in vitro maturation as revealed by proteomic analysis.

BACKGROUND: The domestic cat serves as an animal model for assisted reproductive studies of endangered felid species. To date, there are no data on the protein alterations following cryopreservation of oocytes in felid family. METHODS: Immature (germinal vesicle) domestic cat oocytes were vitrified in the vitrification solution containing 35% ethylene glycol, 20% DMSO and 0.5 mM sucrose. The vitrified-warmed oocytes were matured (metaphase II) in vitro and subjected to proteomic analysis using 1DE SDS-PAGE prefractionation combined with LC-MS/MS. RESULTS: A total of 1712 proteins were identified in in vitro matured oocytes. Of the 1712 proteins, 1454 proteins were found in both groups, whereas, 258 proteins were differentially expressed between control and vitrified-warmed groups. In vitrified-warmed oocytes, the missing proteins were membrane and nuclear proteins; whereas, apoptosis and DNA repair proteins were overrepresented. CONCLUSIONS: The identified missing and overexpressed proteins in vitrified-warmed oocytes represent potential markers of cryoinjuries and the developmental pathways of oocytes. The findings of differential expressed proteins may contribute to effective ways of proteome analysis of oocyte/embryo quality in order to assess safety of cryopreservation in felid species.

Induction of keratinocyte migration by ECa 233 is mediated through FAK/Akt, ERK, and p38 MAPK signaling.

Centella asiatica is widely considered the most important medicinal plant for treating and relieving skin diseases. Recently developed standardized extract of Centella asiatica ECa 233 has demonstrated positive effects on wound healing of incision and burn wound in rats. However, knowledge associated with wound healing mechanism of ECa 233 was scare. Therefore, this study aimed to investigate the effect and underlying molecular mechanisms of ECa 233 on the migration of a human keratinocyte cell line (HaCaT) using scratch wound healing assay. Formation of filopodia, a key protein in cell migration as well as signaling pathways possibly involved were subsequently assessed. It was found that HaCaT cell migration was significantly enhanced by ECa 233 in a concentration- and time-dependent manner. The filopodia formations were accordingly increased in exposure to ECa 233 at concentrations of 0.1-100 µg/ml. Furthermore, ECa 233 was found to significantly upregulate the expression of Rac1 and RhoA and to induce phosphorylation of FAK and Akt as well as ERK and p38 MAPK. Taken all together, it is suggestive that ECa 233 induces cell migration and subsequently promotes wound healing activity, through the activation of FAK, Akt, and MAPK signaling pathways thereby supporting the role of ECa 233 to be further developed for the clinical treatment of wound.

Missing and overexpressing proteins in domestic cat oocytes following vitrification and in vitro maturation as revealed by proteomic analysis

BACKGROUND: The domestic cat serves as an animal model for assisted reproductive studies of endangered felid species. To date, there are no data on the protein alterations following cryopreservation of oocytes in felid family. METHODS: Immature (germinal vesicle) domestic cat oocytes were vitrified in the vitrification solution containing 35% ethylene glycol, 20% DMSO and 0.5 mM sucrose. The vitrified-warmed oocytes were matured (metaphase II) in vitro and subjected to proteomic analysis using 1DE SDS-PAGE prefractionation combined with LC-MS/MS. RESULTS: A total of 1712 proteins were identified in in vitro matured oocytes. Of the 1712 proteins, 1454 proteins were found in both groups, whereas, 258 proteins were differentially expressed between control and vitrified-warmed groups. In vitrified-warmed oocytes, the missing proteins were membrane and nuclear proteins; whereas, apoptosis and DNA repair proteins were overrepresented. CONCLUSIONS: The identified missing and overexpressed proteins in vitrified-warmed oocytes represent potential markers of cryoinjuries and the developmental pathways of oocytes. The findings of differential expressed proteins may contribute to effective ways of proteome analysis of oocyte/embryo quality in order to assess safety of cryopreservation in felid species.

Astaxanthin induces migration in human skin keratinocytes via Rac1 activation and RhoA inhibition.

BACKGROUND/OBJECTIVES: Re-epithelialization has an important role in skin wound healing. Astaxanthin (ASX), a carotenoid found in crustaceans including shrimp, crab, and salmon, has been widely used for skin protection. Therefore, we investigated the effects of ASX on proliferation and migration of human skin keratinocyte cells and explored the mechanism associated with that migration. MATERIAL/METHOD: HaCaT keratinocyte cells were exposed to 0.25-1 µg/mL of ASX. Proliferation of keratinocytes was analyzed by using MTT assays and flow cytometry. Keratinocyte migration was determined by using a scratch wound-healing assay. A mechanism for regulation of migration was explored via immunocytochemistry and western blot analysis. RESULTS: Our results suggest that ASX produces no significant toxicity in human keratinocyte cells. Cell-cycle analysis on ASX-treated keratinocytes demonstrated a significant increase in keratinocyte cell proliferation at the S phase. In addition, ASX increased keratinocyte motility across the wound space in a time-dependent manner. The mechanism by which ASX increased keratinocyte migration was associated with induction of filopodia and formation of lamellipodia, as well as with increased Cdc42 and Rac1 activation and decreased RhoA activation. CONCLUSIONS: ASX stimulates the migration of keratinocytes through Cdc42, Rac1 activation and RhoA inhibition. ASX has a positive role in the re-epithelialization of wounds. Our results may encourage further in vivo and clinical study into the development of ASX as a potential agent for wound repair.

Apium graveolens extract influences mood and cognition in healthy mice.

Apium graveolens is a food flavoring which possesses various health promoting effects. This study investigates the effect of a sub-acute administration of A. graveolens on cognition and anti-depression behaviors via antioxidant and related neurotransmitter systems in mice brains. Cognition and depression was assessed by various models of behavior. The antioxidant system of glutathione peroxidase (GPx), % inhibition of superoxide anion (O2-), and lipid peroxidation were studied. In addition, neurochemical parameters including acetylcholinesterase (AChE) and monoamine oxidase-type A (MAO-A) were also evaluated. Nine groups of male mice were fed for 30 days with different substances-a control, vehicle, A. graveolens extract (65-500 mg/kg), and reference drugs (donepezil and fluoxetine). The results indicated that the effect of the intake of A. graveolens extract (125-500 mg/kg) was similar to the reference drugs, as it improved both spatial and non-spatial memories. Moreover, there was a decrease in immobility time in both the forced swimming and tail suspension tests. In addition, the A. graveolens extract reduced lipid peroxidation of the brain and increased GPx activity and the % inhibition of O2-, whereas the activities of AChE and MAO-A were decreased. Thus, our data have shown that the consumption of A. graveolens extract improved cognitive function and anti-depression activities as well as modulating the endogenous antioxidant and neurotransmitter systems in the brain, resulting in increased neuronal density. This result indicated an important role for A. graveolens extract in preventing age-associated decline in cognitive function associated with depression.

Anxiolytic and free radical scavenging potential of Chinese celery (Apium graveolens) extract in mice

Objective: To elucidate the anxiolytic and free radical scavenging effect of methanolic extract of Apium graveolens (A. graveolens) in adult C57BL/6 mice. Methods: Sixty male mice were divided into 6 groups:control, vehicle, positive control and A. graveolens (125, 250 and 500 mg/kg). Different behavioral models of elevated plus maze, open field, light/dark, hole-board and pentobarbital-induced sleep were used to assess anxiety-like behavior. Biochemical parameters including monoamine oxidase-A (MAO-A) activity, lipid peroxidation,%inhibition of superoxide anion and glutathione peroxidase activity were measured. Histologic studies were also examined. Results: Mice receiving various doses of A. graveolens (125, 250 and 500 mg/kg) showed an alleviation of anxiety-like behavior as evidenced by the battery of behavioral tests. Likewise, A. graveolens treatment was found to significantly decrease MAO-A activity, lipid peroxidation as well as cause a significant increase of%inhibition of su-peroxide anion and glutathione peroxidase activity in both cortex and striatum. The total number of survival neurons found in the frontal cortex and striatum was significantly higher than that of the vehicle-treated group. Conclusions: Taken together, we showed that A. graveolens improve the behavioral changes which might be related to the inhibition of free radicals and modulation of MAO-A activity resulting in an increased number of survival neurons. Our findings suggest the therapeutic potential of A. graveolens in the treatment of anxiety.

Effects of Apium graveolens Extract on the Oxidative Stress in the Liver of Adjuvant-Induced Arthritic Rats.

Apium graveolens Linn. (Apiaceae) is an indigenous plant of the North and South Americas, Southern Europe, and Asia and has been widely used as a food or a traditional medicine for treatment of inflammation and arthritis. The purpose of this study was to investigate the antioxidant effects of a methanolic extract of A. graveolens (AGE) against liver oxidative stress in an adjuvant-induced arthritic rat model. The AGE (250, 500, and 1,000 mg/kg) was given orally for 24 consecutive days after induction by injecting complete Freund's adjuvant. Liver and spleen weights were recorded. The superoxide anion level, total peroxide (TP), glutathione peroxidase (GPx) activity, superoxide dismutase (SOD) activity, total antioxidant status, and oxidative stress index (OSI) were also measured. AGE treatment significantly decreased the levels of the superoxide anion, TP, and OSI whereas the GPx and SOD activities significantly increased in the liver of the arthritic rats. These results indicated that AGE showed an ameliorative effect against liver oxidative stress in adjuvant-induced arthritic rats by reducing the generation of liver free radicals and increasing the liver antioxidant enzyme activity.