Putrescine Upregulates Melanogenesis Through Modulation of MITF Transcription Factor in B16F1 Mouse Melanoma Cells.

Research background: Ageing is a biochemical, metabolic and genetic physiological phenomenon. The suppression of melanin biosynthesis, evident in the greying of the hair, is a hallmark of ageing resulting from translation failure, reduced enzyme activity and cellular senescence. Putrescine, the smallest member of the polyamine family and an organic chemical, is present in living mammalian cells and plays a crucial role in regulating skin melanogenesis. Therefore, the purpose of this study is to explore the effect of putrescine on the signalling pathways of melanogenesis in melanoma cells. Experimental approach: Melanin production capacity of putrescine was analysed using a tyrosinase activity assay. To assess the cell viability of B16F1 cells exposed to putrescine, a tetrazolium dye MTT assay was performed. The effect of putrescine on melanin synthesis in the presence of H2O2 was evaluated using various in vitro assays in B16F1 cells. The effect of putrescine on melanin production in B16F1 cells was determined using a specific melanin production assay. Gene expression was analysed using real-time polymerase chain reaction (RT-PCR). Furthermore, the effect of putrescine on the expression of proteins related to melanin production in the cells treated with H2O2 was analysed by immunofluorescence and Western blot analysis. Results and conclusions: Putrescine increased tyrosinase activity and showed no cytotoxicity in B16F1 cells. In addition, putrescine effectively scavenged H2O2, as shown by the reduction of intracellular H2O2 amounts in 2',7'-dichlorofluorescin diacetate analysis, and promoted melanin production in living cells. The stimulation of melanogenesis by putrescine was attributed to the increased expression of Mitf, Tyr, Trp-1 and Trp-2 genes. Immunofluorescence assays revealed that putrescine enhanced the expression of proteins associated with melanogenesis and upregulated TYR, TRP-1 and TRP-2 via the microphthalmia-associated transcription factor (MITF) and increased the expression of methionine sulfoxide reductases A (MSRA) and B (MSRB) in the cells treated with H2O2, effectively promoting melanogenesis. These results suggest that putrescine can be used to stimulate melanin synthesis. Novelty and scientific contribution: This is the first study to investigate the effect of putrescine on the signalling pathways of melanogenesis in B16F1 melanoma cells. The results confirm that putrescine can promote melanogenesis through the expression of TYR, TRP-1 and TRP-2 via the MITF in cells treated with H2O2. Putrescine can be used exclusively as a cosmetic product to prevent premature greying of hair.

Development of gold nanocluster complex for the detection of tumor necrosis factor-alpha based on immunoassay.

Tumor necrosis factor-alpha, TNF-α, a cytokine recognized as a key regulator of inflammatory responses, is primarily produced by activated monocytes and macrophages. Measuring TNF-α levels serves as a valuable indicator for tracking several diseases and pathological states. Gold nanotechnology has been identified as a highly effective catalyst with unique properties for measuring inflammatory cytokines. This study aimed to synthesize gold nanoclusters (AuNCs) and the AuNCs-streptavidin system, along with their characterizations and spherical morphology. The detection of TNF-α antigen with AuNCs was determined, and a new immunoassay-based AuNCs analytical platform was studied. In this study, it was demonstrated that the synthesized AuNCs and AuNCs-streptavidin showed a bright-yellow appearance with absorption peaks at A600 and A610 nm, respectively. The approximately spherical shape was observed by TEM analysis. The AuNCs demonstrated a sensitivity limit for the detection of the TNF-α antigen, with a linear dose-dependent detection range of less than 1.25 ng/mL. The products of the band sizes and band intensities were proportional to the amount of TNF-α in the range of ∼80 kDa, ∼55 kDa, and âˆ¼ 25 kDa in western blot analysis. The TNF-α in cell lysate was successfully detected using an immunoassay after the activation of RAW264.7 cells with lipopolysaccharide (LPS). This assay may serve as a viable alternative for TNF-α detection with high speed, sensitivity, and qualities, ensuring its broad applications.

Structural properties and immune-enhancing activities of galactan isolated from red seaweed Grateloupia filicina.

A water-soluble polysaccharide (GFP) was isolated from Grateloupia filicina and fractionated using a DEAE Sepharose Fast Flow column to evaluate immunostimulatory activity. Carbohydrates (62.0%-68.4%) and sulfates (29.3%-34.3%) were the major components of GFP and its fractions (GFP-1 and GFP-2), with relatively lower levels of proteins (4.5%-15.4%) and uronic acid (1.4%-3.9%). The average molecular weight (Mw ) for GFP and its fractions was calculated between 98.2%-243.7 kDa. The polysaccharides were composed of galactose (62.1%-87.2%), glucose (4.5%-33.2%), xylose (3.1%-5.3%), mannose (1.4%-2.2%), rhamnose (1.2%-2.0%), and arabinose (0.9%-1.7%) units connected through →3)-Galp-(1→, →4)-Galp-(1→, →2)-Galp-(1→, →6)-Galp-(1→, →3,4)-Galp -(1→, →3,6)-Galp-(1→, →4,6)-Galp-(1→, →3,4,6)-Galp-(1→, →2,3)-Galp-(1→, →2,4)-Galp-(1→, →4)-Glcp-(1→, →6)-Glcp-(1→ and →4,6)-Glcp-(1→residues. The isolated polysaccharides effectively induced RAW264.7 murine macrophages by releasing nitric oxide (NO) and various cytokines via nuclear factor kappa light chain enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. Further, the expression of toll-like receptor-2 (TLR-2) and TLR-4 in RAW264.7 cells indicated their activation through TLR-2 and TLR-4 binding receptors. Among the polysaccharides, GFP-1 highly stimulated the activation of RAW264.7 cells, which was mainly constituted of (→1) terminal-D-galactopyranosyl, (1→3)-linked-ᴅ-galactopyranosyl, (1→4)-linked-ᴅ-galactopyranosyl and (1→3,4) -linked-ᴅ-galactopyranosyl residues. These findings demonstrate that GFP-1 from G. filicina are effective at stimulating the immune system and this warrants further investigation to determine potential biomedical applications.

Structural Elucidation and Immunostimulatory Activities of Quinoa Non-starch Polysaccharide Before and After Deproteinization.

Non-starch polysaccharides derived from natural resources play a significant role in the field of food science and human health due to their extensive distribution in nature and less toxicity. In this order, the immunostimulatory activity of a non-starch polysaccharide (CQNP) from Chenopodium quinoa was examined before and after deproteination in murine macrophage RAW 264.7 cells. The chemical composition of CQNP and deproteinated-CQNP (D-CQNP) were spectrometrically analysed that revealed the presence of carbohydrate (22.7 ± 0.8% and 39.5 ± 0.8%), protein (41.4 ± 0.5% and 20.8 ± 0.5%) and uronic acid (8.7 ± 0.3% and 6.7 ± 0.2%). The monosaccharide composition results exposed that CQNP possesses a high amount of arabinose (34.5 ± 0.3) followed by galactose (26.5 ± 0.2), glucose (21.9 ± 0.3), rhamnose (7.0 ± 0.1), mannose (6.0 ± 0.1) and xylose (4.2 ± 0.2). However, after deproteination, a difference was found in the order of the monosaccharide components, with galactose (41.1 ± 0.5) as a major unit followed by arabinose (34.7 ± 0.5), rhamnose (10.9 ± 0.2), glucose (6.6 ± 0.2), mannose (3.4 ± 0.2) and xylose (3.2 ± 0.2). Further, D-CQNP potentially stimulate the RAW 264.7 cells through the production of nitric oxide (NO), upregulating inducible nitric oxide synthase (iNOS) and various pro-inflammatory cytokines including interleukin (IL)-1ß, IL-6, IL-10, and tumor necrosis factor-alpha (TNF-α). Moreover, stimulation of RAW 264.7 cells by D-CQNP takes place along the NF-κB and the MAPKs signaling pathways through the expression of cluster of differentiation 40 (CD40). This results demonstrate that RAW 264.7 cells are effectively stimulated after removal of the protein content in C. quinoa non-starch polysaccharides, which could be useful for develop a new immunostimulant agent.

Preparation and characterization of folic acid conjugated sulfated polysaccharides on NK cell activation and cellular uptake in HeLa cells.

In this study, the sulfated polysaccharide (SP) of Codium fragile was conjugated to folic acid (SP-FA). FT-IR and 1H NMR techniques revealed the occurrence of esterification reaction between the hydroxyl group of SP and the γ-carboxyl group of FA that confirming the SP-FA conjugation. SP and SP-FA did not show any direct toxicity on NK cells and HeLa cells. However, the treatment of SP and SP-FA enhance the NK cells cytotoxicity against HeLa cells by the upregulation of IFN-γ, TNF-α, perforin, and Granzyme-B. Moreover, NK cells activation was stimulated through NF-кB and MAPK pathways. The binding capacity studies exposed the targeting ability of HeLa cells by folate receptor (FR) which was assessed by a confocal quantitative image cytometer analysis. These results indicate that SP-FA could be used as selective drug delivery systems for targeting FR-overexpressed cancer cells with less toxicity.

Polysaccharide extracted from Taraxacum platycarpum root exerts immunomodulatory activity via MAPK and NF-κB pathways in RAW264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Taraxacum platycarpum Dahlst. (Korean dandelion) is a medicinal herb used in traditional medicine in Korea to treat various disease such as furuncles, mammitis, hepatitis, jaundice. Moreover, a decoction prepared from T. platycarpum leaves and stems is an effective treatment for cancer, glycosuria, liver disease, pleurodynia, and stomach problems. AIM OF THE STUDY: The main objective of this work was to study the composition and structural properties of polysaccharides (TPP) from Taraxacum platycarpum Dahlst. root and investigate the immunostimulatory activity on RAW264.7 cells. MATERIALS AND METHODS: TPP was extracted from T. platycarpum using hot water extraction, ethanol precipitation method and its fractionated using DEAE-Sepharose fast flow column. The composition, molecular weight, and structural characterization of TPP and its fractions were evaluated by various techniques. Further, the immunostimulatory activity of polysaccharides was tested on murine macrophage cell line RAW264.7 by various in vitro assays. The structure effect of TPP on RAW264.7 cells was studied by the removal of sulfate (desulfation) and protein (deproteinization) contents from TPP. RESULTS: We obtained three fractions namely TPP-1, TPP-2, and TPP-3 which mainly consisted of carbohydrates (75.55, 52.71, and 48.41%), sulfate (8.42, 15.19, and 27.67%), uronic acid (1.27, 6.56, and 4.39%), and protein (8.15, 24.85, and 9.73%). The average molecular weight of the fractions was 56.7, 108.2, and 132.3 × 103 g/mol, respectively. The polysaccharides activate the RAW264.7 cell to produce a significant amount of NO and upregulate the various mRNA expression by the activation of MAPK and NF-κB pathways via TLR4, TLR2, and CR3 receptors. The structurally modified deproteinated derivative (DP-TPP-2) more effectively decreases the NO production which means the protein content of TPP-2 mainly contributes to the RAW264.7 cells activation. The structure of DP-TPP-2 primarily consists of 1 â†’ 2)-Galp, 1 â†’ 6)-Glup, 1 â†’ 2) - Rhap, and 1 â†’ 5) - Arap glycosidic linkages. CONCLUSIONS: The present study demonstrated that the polysaccharide isolated from T. platycarpum shows admirable immunostimulatory by the activation of MAPK and NF-κB pathways through TLR4, TLR2, and CR3 receptors. The protein content of polysaccharides mainly contributes to the RAW264.7 cells activation. Our study results could be useful for developing a new immunostimulant agent.

Extraction, structural elucidation and immunostimulating properties of water-soluble polysaccharides from wheat bran.

A water-soluble polysaccharide was extracted from wheat bran (WBP) and investigate their structural characteristics and immunostimulatory activities. The chemical composition of WBP and purified fraction (WBP-F) mainly consists of neutral sugars (91.2 ± 1.2 and 98.7 ± 1.2%), proteins (8.6 ± 0.3 and 0.2 ± 0.1%) and uronic acids (0.7 ± 0.1 and 0.6 ± 0.1%). The molecular weight (Mw ) of WBP and WBP-F was calculated as 911.7 and 510.2 × 103  g/mol, respectively. The WBP-F stimulates the RAW264.7 cells through the production of nitric oxide and various cytokines. The treatment of WBP-F facilitated the phosphorylation of P38, JNK, ERK, and NF-ƘB in RAW264.7 cells suggesting that they might stimulate RAW264.7 cells through the activation of NF-ƙB and MAPKs pathways. Furthermore, the structural details of WBP-F were studied by GC-MS and NMR spectrum, which confirms that the main backbone consists of 4-α-D-linked glucopyranosyl residues with branching points at C-6. PRACTICAL APPLICATIONS: Wheat bran is a potential source of health-promoting compounds. It has been reported that polysaccharides of wheat bran containing numerous beneficial activities. In this study, the wheat bran polysaccharide was extracted, fractionated and investigated their immunostimulatory activities. The results found in this study revealed that the purified polysaccharide from wheat bran potentially enhanced the RAW264.7 cells activation. Hence, these polysaccharides could be utilized as a potent immunity-enhancing agent in food and pharmaceutical industries.

The activation of RAW264.7 murine macrophage and natural killer cells by glucomannogalactan polysaccharides from Tornabea scutellifera.

A water-soluble polysaccharide was isolated from Tornabea scutellifera and fractionated using a DAEA Sepharose FF column to evaluate its capacity to stimulate natural killer (NK) cells and macrophages. Neutral sugars (71.8-93.5%) constituted the major part of crude polysaccharides and fractions (TSF1 and TSF2) with relatively lower levels of proteins (0.4-20.3%) and uronic acids (0.8-4.9%). The weight average molecular weights (Mw) of 152.7-537.3 × 103 g/mol were measured for isolated polysaccharides. The polysaccharides were composed of glucose (14.4-44.0%), galactose (23.2-43.2%), mannose (28.5-34.2%) and rhamnose (2.6-13.9%) units connected through (1→2)-Galp, (1→2,6)-Galp, (1→4)-Glcp, (1→6)-Glcp, (1→3)-Rhap, (1→2)-Rhap and (1→4)-Manp residues. TSF2 polysaccharide effectively induced RAW264.7 murine macrophages to release nitric oxide, TNF-α, IL-1ß and IL-6, and activated NK cells to produce TNF-α, INF-γ, granzyme-B, perforin, NKG2D and FasL through NF-κB and MAPKs signaling pathways. Overall results suggested that polysaccharides from T. scutellifera could be potent immunostimulatory compounds inducing both macrophages and NK cells.