Anti-Photoaging Effects of Upcycled Citrus junos Seed Anionic Peptides on Ultraviolet-Radiation-Induced Skin Aging in a Reconstructed Skin Model.

Side streams and byproducts of food are established sources of natural ingredients in cosmetics. In the present study, we obtained upcycled low-molecular-weight anionic peptides (LMAPs) using byproducts of the post-yuzu-juicing process by employing an enzyme derived from Bacillus sp. For the first time, we isolated anionic peptides less than 500 Da in molecular weight from Citrus junos TANAKA seeds via hydrolysis using this enzyme. The protective effect of LMAPs against UVR-induced photoaging was evaluated using a reconstructed skin tissue (RST) model and keratinocytes. The LMAPs protected the keratinocytes by scavenging intracellular reactive oxygen species and by reducing the levels of paracrine cytokines (IL-6 and TNF-α) in UVR (UVA 2 J/cm2 and UVB 15 mJ/cm2)-irradiated keratinocytes. Additionally, the increase in melanin synthesis and TRP-2 expression in RST caused by UVR was significantly inhibited by LMAP treatment. This treatment strongly induced the expression of filaggrin and laminin-5 in UVR-irradiated RST. It also increased type I collagen expression in the dermal region and in fibroblasts in vitro. These results suggest that a hydrolytic system using the enzyme derived from Bacillus sp. can be used for the commercial production of LMAPs from food byproducts and that these LMAPs can be effective ingredients for improving photoaging-induced skin diseases.

Activation of Hair Cell Growth Factors by Linoleic Acid in Malva verticillata Seed.

Hair loss by excessive stress from work and lifestyle changes has become a growing concern, particularly among young individuals. However, most drugs for alopecia impose a plethora of side effects. We have found the powerful impact of Malva verticillata seed extracts on alleviating hair loss. This study further isolated effective chemicals in M. verticillata seed extracts by liquid silica gel column chromatography. Under the screening for the growth rate (%) of human follicles dermal papilla cells (HFDPCs), we identified linoleic acid (LA) and oleic acid in n-hexane of M. verticillate (MH)2 fraction. LA treatment activated Wnt/ß-catenin signaling and induced HFDPCs growth by increasing the expression of cell cycle proteins such as cyclin D1 and cyclin-dependent kinase 2. LA treatment also increased several growth factors, such as vascular endothelial growth factor, insulin-like growth factor-1, hepatocyte growth factor, and keratinocyte growth factor, in a dose-dependent manner. Besides, LA significantly inhibited Dickkopf-related protein expression (DKK-1), a primary alopecia signaling by dihydrotestosterone. Our findings suggest that LA treatment may alleviate a testosterone-induced signaling molecule and induces HFDPCs growth by activating Wnt/ß-catenin signaling.

Anti-melanogenic effect of gomisin N from Schisandra chinensis (Turcz.) Baillon (Schisandraceae) in melanoma cells.

Overproduction and accumulation of melanin in the skin can lead to pigmentation disorders, such as freckles and melasma. Many researchers are studying the regulation of melanogenesis in the skin with the aim of developing whitening cosmetics. In this study, bioactivity-guided separation of the hexane fraction of Schisandra chinensis (Turcz.) Baillon extract yielded five major compounds, ß-chamigrenal, α-ylangenol, gomisin N, gomisin A, and schisandrin. The structures were identified by using 1H-NMR and 13C-NMR spectroscopy and comparing the spectral data with published data. Melanogenesis inhibitory activity assay in alpha-melanocyte-stimulating hormone (α-MSH)-induced B16F10 mouse melanoma cells revealed that gomisin N was the active component and significantly inhibited melanin synthesis in a concentration-dependent manner. Thus, we evaluated the mechanism underlying its anti-melanogenic effect. Gomisin N inhibited the expression of tyrosinase and microphthalmia-associated transcription factor (MITF) in B16F10 cells, while it did not affect cAMP response element binding protein (CREB) phosphorylation. Additionally, gomisin N activated AKT phosphorylation, which inhibits MITF expression. Thus, our results strongly suggest that the active compound, gomisin N, has potential for use in cosmetics to reduce hyperpigmentation.

Saucerneol D inhibits dendritic cell activation by inducing heme oxygenase-1, but not by directly inhibiting toll-like receptor 4 signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Saururus chinensis is a medicinal plant used to treat jaundice, pneumonia, edema, fever, and several inflammatory diseases. Saucerneol D (SD), a lignan constituent of this plant, has antioxidant, anti-asthmatic, and anti-inflammatory activities. SD has been previously reported to inhibit the pro-inflammatory responses of RAW264.7 cells and primary mast cells. In this study, we investigated the effect of SD on the functions of dendritic cells (DCs). MATERIALS AND METHODS: SD was isolated from methanol extract of the roots of S. chinensis. Bone marrow-derived DCs were used as target cells. The effects of SD on the following DC functions were examined: surface molecule expression, cytokine expression, migration, allogenic T cell activation, heme oxygenase-1 expression, and Toll-like receptor 4 signaling. RESULTS: In lipopolysaccharide (LPS)-treated DCs, SD inhibited the expression of cell surface molecules (MHC I/II, CD40, CD80, and CD86), the production of inflammatory mediators (nitric oxide, IL-12, IL-1ß, and TNF-α), and allogenic T cell activation capacity. SD also inhibited DC migration toward MIP-3ß by down-regulating CCR7 expression. SD attenuated LPS-induced activation of NF-κB and MAPK signaling in DCs, but did not directly inhibit kinase activities of IRAK1, IRAK4, TAK1, or IKKß in enzymatic assays. SD did not inhibit LPS binding to myeloid differentiation protein-2, co-receptor of TLR4. SD increased the production of reactive oxygen species, Nrf-2, and heme oxygenase (HO)-1, which degrades the heme to immunosuppressive carbon monoxide and biliverdin, which may underlie the anti-inflammatory effects in SD-treated DCs. CONCLUSIONS: Taken together, these data suggest that SD suppresses LPS-induced activation of DCs through the induction of HO-1, but not by directly affecting Toll-like receptor 4 signaling.

Validation of cyclooxygenase-2 as a direct anti-inflammatory target of 4-O-methylhonokiol in zymosan-induced animal models.

4-O-methylhonokiol (MH) is known to inhibit inflammation by partially understood mechanisms. Here, the anti-inflammatory mechanisms of MH were examined using enzymatic, cellular, and animal assays. In enzymatic assays, MH inhibited COX-2 activity with an IC50 of 0.062 µM, and also COX-1 with an IC50 of 2.4 µM. In cellular assays, MH was immunotoxic above 10 µM. At non-toxic concentrations (below 3 µM), MH strongly inhibited COX-2-mediated prostaglandin production with an IC50 of 0.1 µM, whereas did not or slightly affect other functions of B cells, T cells, dendritic cells, and macrophages. In an animal model, MH inhibited the increase in footpad thickness and popliteal lymph node weight in zymosan-injected mice. When analyzed the draining pLNs of zymosan-injected mice on day 5, MH inhibited the overall inflammatory responses. However, MH inhibited cyclooxygenase (COX)-2-mediated prostaglandin production without affecting tumor necrosis factor-α production in inflamed tissues within 6 h after zymosan injection. In summary, our data suggest that COX-2 may be a direct anti-inflammatory target of MH in vitro and in vivo.

Tussilagone inhibits dendritic cell functions via induction of heme oxygenase-1.

Sesquiterpenoid tussilagone (TUS) has a variety of pharmacological activities, such as anti-oxidant, anti-cancer, and anti-inflammatory activities. In this study, we investigated the effects of TUS on dendritic cell (DC) functions and the underlying mechanisms. TUS inhibited lipopolysaccharide (LPS)-induced activation of DCs, as shown by decrease in surface molecule expression, cytokine production, cell migration, and allo-T cell activation. In addition, TUS inhibited LPS-induced activation of NF-κB, MAPKs, and IRF-3 signalings in DCs, although it did not directly affect kinase activities of IRAK1/4, TAK1, and IKK, which suggests that TUS might indirectly inhibit TLR signaling in DCs. As a critical mechanism, we showed that TUS activated heme oxygenase-1 (HO-1), which degrades heme to immunosuppressive products, such as carbon monoxide and bilirubin. HO-1 inhibitor reversed the inhibitory activity of TUS in DCs. In conclusion, this study suggests that TUS inhibits DC function through the induction of HO-1.

Platycodon grandiflorum polysaccharide induces dendritic cell maturation via TLR4 signaling.

Dendritic cell (DC) maturation is critical for initiation of the adoptive immune response. DC maturation is often attenuated in several pathological conditions including cancer. In this study, we report the effect of Platycodon grandiflorum polysaccharide (PG) on DC maturation. PG induced phenotypic maturation of DCs, as proved by the increase in the expression of CD40, CD80, CD86, and major histocompatibility complex (MHC)-I/II on the cell surface. PG also induced functional maturation of DCs, as proved by elevated production of interleukin (IL)-12, tumor necrosis factor-α, IL-1ß, IL-6, IL-10, and interferon-ß, and by enhanced allogeneic T cell stimulation ability of PG-treated DCs. PG efficiently induced maturation of DCs from C3H/HeN mice, which have normal Toll-like receptor-4 (TLR4), but not that of DCs from C3H/HeJ mice, which have mutated TLR4, suggesting that TLR4 might be one of the PG receptors in DCs. In line with TLR4 activation, PG increased the phosphorylation of ERK, p38, and JNK, and the nuclear translocation of p-c-Jun, p-CREB, and c-Fos. PG also activated NF-κB signaling, as evidenced by degradation of IκBα/ß and nuclear translocation of p65 and p50. In summary, our data suggest that PG induces DC maturation by activating MAPK and NF-κB signaling downstream of TLR4.

Activation of macrophages by polysaccharide isolated from Paecilomyces cicadae through toll-like receptor 4.

Paecilomyces cicadae have been reported to have immunomodulatory properties. In this study, we investigated the effect of polysaccharide (PCP) isolated from P. cicadae on the macrophages. PCP increased the production of nitric oxide (NO) and the gene expression of IL-1ß, IL-6, and TNF-α in RAW 264.7 cells. To investigate the membrane receptor, we examined the effect of PCP on primary macrophages isolated from wild type C3H/HeN and C3H/HeJ mice having mutant-TLR4. PCP induced NO production and cytokine gene expression in macrophages from C3H/HeN, but not from tlr4-mutated C3H/HeJ mice, which suggests that TLR4 is the membrane receptor for PCP. PCP induced the phosphorylation of ERK, JNK, and p38, and the nuclear translocation of NF-κB p50/p65, which are the main signaling molecules downstream from TLR4. Among them, p38 and NF-κB signaling played a crucial role in PCP-induced NO production by macrophages. These results indicate that PCP activates macrophages through the TLR4 signaling pathway.

Adjuvant effect of a natural TLR4 ligand on dendritic cell-based cancer immunotherapy.

The clinical efficacy of dendritic cell (DC) vaccine in cancer patients has been unsatisfactory due, at least in part, to the deficiency of maturation and impaired migration of ex vivo generated DCs to the draining lymph nodes. To solve this problem, we used angelan, a natural TLR4 ligand, to enhance the maturation and migration of DCs. Angelan increased the expression of MHC-I/II, CD80, and CD86, DC maturation markers, through the NF-κB pathway. This compound also increased CCR7 expression in DCs through NF-κB and p38 pathway and enhanced their migration against CCL19, which is a key chemokine that guides DCs into lymph nodes. We also showed that angelan enhanced in vivo DC homing from tissues to draining lymph nodes. When treated to DCs in vitro and vivo, angelan increased antitumor activity of DCs in B16F10 syngeneic tumor model. Taken together, the present data suggest that a natural TLR4 ligand might be helpful for overcoming the disadvantages of DC-based cancer therapy, such as impaired maturation and poor migration in cancer patients.

Phenotypic and functional maturation of dendritic cells induced by polysaccharide isolated from Paecilomyces cicadae.

Paecilomyces cicadae Miquel Samson is the anamorph of Cordyceps cicadae Shing and is used in functional foods for the prevention and treatment of various diseases. In the present study, we examined the effects of P. cicadae polysaccharide (PCP) on dendritic cell (DC) maturation. Phenotypic maturation of DCs by PCP was confirmed by the elevated expressions of CD80, CD86, major histocompatibility complex (MHC)-I, and MHC-II molecules and functional maturation by increased expression of interleukin-12, interleukin-1ß, and tumor necrosis factor-α, enhanced allogenic T cell stimulation, and decreased endocytosis. PCP induced the maturation of DCs from C3H/HeN and C57BL/6 mice but not from Toll-like receptor (tlr) 4⁻/⁻ knockout mice and TLR4-mutated C3H/HeJ mice, which suggests that TLR4 is the membrane receptor for PCP. PCP increased the degradation of inhibitor of nuclear factor-κB (NF-κB) α/ß, which enhanced the nuclear translocation of NF-κB p50/p65 and induced the phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases, which are signaling molecules downstream of TLR4. These results indicate that PCP induces DC maturation through TLR4 signaling.

Adoptive immunotherapy of human gastric cancer with ex vivo expanded T cells.

Surgical resection of gastric cancer has made significant progress, but majority of patients with advanced gastric cancer face relapse and die within five years. In this study, the antitumor activity of ex vivo expanded T cells against the human gastric cancer was evaluated in vitro and in vivo. Human peripheral blood mononuclear cells were cultured with IL-2-containing medium in anti-CD3 antibody-coated flasks for 5 days, followed by incubation in IL-2-containing medium for 9 days. The resulting populations were mostly CD3(+) T cells (97%) and comprised 1% CD3⁻CD56(+), 36% CD3(+)CD56(+), 11% CD4(+), and 80% CD8(+). This heterogeneous cell population was also called cytokine-induced killer (CIK) cells. CIK cells strongly produced IFN-γ, moderately TNF-α, but not IL-2 and IL-4. At an effector-target cell ratio of 30:1, CIK cells destroyed 58% of MKN74 human gastric cancer cells, as measured by the 5¹Cr-release assay. In addition, CIK cells at doses of 3 and 10 million cells per mouse inhibited 58% and 78% of MKN74 tumor growth in nude mouse xenograft assays, respectively. This study suggests that CIK cells may be used as an adoptive immunotherapy for gastric cancer patients.

Induction of dendritic cell maturation by ß-glucan isolated from Sparassis crispa.

Sparassis crispa is a medicinal mushroom containing high 6-branched 1,3-ß-D-glucan (sparan) content, which exhibits immune-mediated antitumor activity. In the present study, we investigated the stimulating effect of sparan on phenotypic and functional maturation of dendritic cells (DCs). Phenotypic maturation was confirmed by the elevated expressions of CD40, CD80, CD86, and MHC-I/II molecules. Functional activation was proved by increased cytokine production of IL-12, IL-1ß, TNF-α, and IFN-α/ß, enhanced IL-2 production and proliferation of allogenic T cells, and decreased endocytosis. The role of toll-like receptor 4 (TLR4) as a membrane receptor of sparan was proved by the impaired maturation of DCs generated from bone marrow cells of tlr4⁻/⁻ knock-out mice and TLR4-mutated C3H/HeJ mice, and by using anti-MD-2/TLR4 neutralizing antibody. Sparan increased phosphorylation of ERK, p38, and JNK, and enhanced nuclear translocation of NF-κB p50/p65 in DCs. These results indicate that sparan activates DCs via MAPK and NF-κB signaling pathways, which are signaling molecules downstream of TLR4.

Inhibition of bone marrow-derived dendritic cell maturation by glabridin.

Glabridin has multiple pharmacological activities including anti-microbial, anti-atherosclerotic, anti-nephritic, anti-inflammatory and cardiovascular protective activities. In this study, we investigated the effect of glabridin on dendritic cells, which play an essential role in innate and adaptive immune responses. Glabridin inhibited lipopolysaccharide-, poly (I:C)-, or zymosan-induced phenotypic maturation of dendritic cells (DCs), as proven by the decreased expression of CD40, CD80, CD86, MHC-I, and MHC-II. Glabridin decreased the functional maturation of DCs, in that glabridin attenuated pro-inflammatory cytokine production of IL-12, IL-1ß, TNF-α, and IFN-α/ß, enhanced antigen capture capacity, inhibited migration to SDF-1α and MIP-3ß, and impaired induction of allogenic T cell activation. We also showed that glabridin inhibited zymosan-induced inflammation in mice. As a mode of action, we showed that glabridin inhibited degradation of IκΒα/ß, nuclear translocation of NF-κB p65/p50, and phosphorylation of ERK, JNK, and p38 MAPKs. Taken together, the present results show that glabridin inhibits dendritic cell maturation by blocking NF-κB and MAPK signalings.