Juglone Suppresses LPS-induced Inflammatory Responses and NLRP3 Activation in Macrophages.

The NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome has been implicated in a variety of diseases, including atherosclerosis, neurodegenerative diseases, and infectious diseases. Thus, inhibitors of NLRP3 inflammasome have emerged as promising approaches to treat inflammation-related diseases. The aim of this study was to explore the effects of juglone (5-hydroxyl-1,4-naphthoquinone) on NLRP3 inflammasome activation. The inhibitory effects of juglone on nitric oxide (NO) production were assessed in lipopolysaccharide (LPS)-stimulated J774.1 cells by Griess assay, while its effects on reactive oxygen species (ROS) and NLRP3 ATPase activity were assessed. The expression levels of NLRP3, caspase-1, and pro-inflammatory cytokines (IL-1ß, IL-18) and cytotoxicity of juglone in J774.1 cells were also determined. Juglone was non-toxic in J774.1 cells when used at 10 µM (p < 0.01). Juglone treatment inhibited the production of ROS and NO. The levels of NLRP3 and cleaved caspase-1, as well as the secretion of IL-1ß and IL-18, were decreased by treatment with juglone in a concentration-dependent manner. Juglone also inhibited the ATPase activities of NLRP3 in LPS/ATP-stimulated J774.1 macrophages. Our results suggested that juglone could inhibit inflammatory cytokine production and NLRP3 inflammasome activation in macrophages, and should be considered as a therapeutic strategy for inflammation-related diseases.

Immunomodulatory Effects of Extracellular ß-Glucan Isolated from the King Oyster Mushroom Pleurotus eryngii (Agaricomycetes) and Its Sulfated Form on Signaling Molecules Involved in Innate Immunity.

The aim of this study was to determine, using murine RAW 264.7 macrophages, the immunomodulatory effect of extracellular ß-glucan isolated from Pleurotus eryngii (PEBG) and its sulfated derivative (PEBG-S) on signaling molecules implicated in host innate immunity. ß-Glucan was extracted and purified from the mycelial culture using optimal medium concentrations. It was then chemically converted to its sulfated form. Monosaccharide composition of ß-glucan was characterized with p-aminobenzoic acid ethyl ester-derivatized sugars through highperformance liquid chromatography analysis. Fourier transform infrared structural analysis showed an S=O bond at 1250 cm-1 and C-S-O binding at 815 cm-1 in PEBG-S. 13C nuclear magnetic resonance analysis showed 1,3-linked α-D-mannopyranosyl and 1,3-ß-D-glucopyranosyl in PEBG-S. A concentration-dependent increase of nitric oxide production was noticed in RAW 264.7 cells treated with PEBG-S or PEBG; those treated with PEBG-S showed less cytotoxicity than those treated with PEBG. Cellular levels of tumor necrosis factor-α, interleukin-1ß, and interleukin-6 were increased by PEBG and PEBG-S treatment, suggesting that they have immunomodulatory activity. Real-time polymerase chain reaction array revealed that the expression levels of nuclear factor-κB and Toll-like receptor signaling genes in cells were upregulated by PEBG and PEBG-S. Moreover, the expression of the ß-glucan receptor dectin-2 was significantly upregulated by PEBG and PEBG-S treatment, reflecting immune activation through the dectin-2-Syk-(CARD9/Bcl-10/MALT1) pathway. Our results suggest that PEBG-S could be used as an effective adjuvant or immune enhancer that can be sustainably produced by recycling the by-product of mycelial culture.

Schizandra chinensis exhibits phytoestrogenic effects by regulating the activation of estrogen receptor-α and -ß.

OBJECTIVE: To investigate the phytoestrogenic effects of Schizandra chinensis (SC) extract by regulating the activation of estrogen receptor. METHODS: Western blotting assay was performed to investigate the effect of SC extract (1, 10, 100 µg/mL) on the expression of estrogen receptor (ER)-α and -ß in MCF-7 human breast cancer cells. Cell viability and the levels of c-fos and c-Jun were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Western blot analysis, respectively, to further confirm the anti-cancer effect of SC extract. RESULTS: SC extract increased the expressions of ER-α and -ß (P<0.001), whereas cell viability and the expressions of growth factors (c-fos and c-Jun) were inhibited (P< and <0.001, respectively) following treatment. CONCLUSIONS: SC extract has phytoestrogenic effects, and its biological action includes ER binding ability with low cancer risk. Therefore, SC might be a potential source for the development of a new alternative to hormone therapy in menopause.

Ameliorative effects of Schizandra chinensis on osteoporosis via activation of estrogen receptor (ER)-α/-ß.

Estrogen deficiency in menopausal women is the main cause of osteoporosis. Phytoestrogen could be a suitable candidate for treatment of post-menopausal osteoporosis. Recent studies showed that S. chinensis contains several lignans, which may be phytoestrogen. In this study, we investigated the ameliorative effects of S. chinensis on post-menopausal osteoporosis. 30% ethanol extract of S. chinensis (SC) was administered orally for 6 weeks after 7 weeks of ovariectomized-induced osteoporosis. Bone mineral density was significantly increased following increased serum osteocalcin levels by SC treatment. Histological analysis showed that SC reduced the increased growth plate of the epiphyseal plate in femur. In addition, pores within bone marrow cells filling the lateral and medial epicondyle were decreased. Serum estradiol concentration was significantly increased in the SC-treated group. The expressions of estrogen receptor-α and -ß were increased in uterus and MCF-7 breast cancer cells by SC treatment. And two transcriptions of proto-oncogenes, c-fos and c-Jun, were suppressed by treatment of SC. From these data, we propose that S. chinensis attenuates post-menopausal osteoporosis with its phytoestrogenic effects. S. chinensis may have the potential to be used as an alternative for treatment of osteoporosis.

Effects of different selenium levels on growth and regulation of laccase and versatile peroxidase in white-rot fungus, Pleurotus eryngii.

This study was conducted to evaluate the physiological effects of different selenium (Se) levels on the growth of white-rot fungus, Pleurotus eryngii, with special reference to the regulation of ligninolytic enzymes such as laccase and versatile peroxidase. The fungus was grown in medium supplemented with 1, 10, 100, 1,000 and 10,000 µM of sodium selenite. Mycelial growth was stronger at lower Se levels, but declined significantly at higher concentrations of 1,000 and 10,000 µM, highlighting its association in mediating toxic responses. Inhibition of fungal growth was accompanied with dense and entangled hyphae taking the shape of irregular short strips. Additionally, hyphal swellings and septation were noticed which lead to a reduction in the advancement of the mycelium. Along with the inhibition of fungal biomass, the reducing sugar and protein concentrations increased to about 30.2 and 3.5 mg/ml respectively in the growth medium. Additionally, the laccase gene expression showed a twofold upregulation at higher levels of Se, although the activity of the enzyme was compromised with an inverse relationship with increased gene transcripts. The versatile peroxidase transcript showed a complete downregulation at 10,000 µM after an upregulation at lower levels of Se. We also confirmed the direct relationship of different Se levels on laccase activity of Rhus vernicifera that showed similar behavior to the fungal laccase. The results of the present study suggest that Se supplementation regulates mRNA levels of laccase and versatile peroxidase depending on exposure and may play a role in the toxicity associated with Se.