4-Acetylantroquinonol B Inhibits Osteoclastogenesis by Inhibiting the Autophagy Pathway in a Simulated Microgravity Model.

Astronauts suffer from 1-2% bone loss per month during space missions. Targeting osteoclast differentiation has been regarded as a promising strategy to prevent osteoporosis in microgravity (µXg). 4-acetylantroquinonol B (4-AAQB), a ubiquinone from Antrodia cinnamomea, has shown anti-inflammatory and anti-hepatoma activities. However, the effect of 4-AAQB on µXg-induced osteoclastogenesis remains unclear. In this study, we aimed to explore the mechanistic impact of 4-AAQB on osteoclast formation under µXg conditions. The monocyte/macrophage-like cell line RAW264.7 was exposed to simulated µXg (Rotary Cell Culture System; Synthecon, Houston, TX, USA) for 24 h and then treated with 4-AAQB or alendronate (ALN) and osteoclast differentiation factor receptor activator of nuclear factor kappa-B ligand (RANKL). Osteoclastogenesis, bone resorption activity, and osteoclast differentiation-related signaling pathways were analyzed using tartrate-resistant acid phosphatase (TRAP) staining, actin ring fluorescent staining, bone resorption, and western blotting assays. Based on the results of TRAP staining, actin ring staining, and bone resorption assays, we found that 4-AAQB significantly inhibited µXg-induced osteoclast differentiation. The critical regulators of osteoclast differentiation, including nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), c-Fos, and dendritic cell-specific transmembrane protein (DC-STAMP), were consistently decreased. Meanwhile, osteoclast apoptosis and cell cycle arrest were also observed along with autophagy suppression. Interestingly, the autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) showed similar effects to 4-AAQB. In conclusion, we suggest that 4-AAQB may serve as a potential agent against µXg-induced osteoclast formation.

Protective Effects of Rhodiola Crenulata Extract on Hypoxia-Induced Endothelial Damage via Regulation of AMPK and ERK Pathways.

Rhodiola crenulata root extract (RCE) has been shown to possess protective activities against hypoxia both in vitro and in vivo. However, the effects of RCE on response to hypoxia in the endothelium remain unclear. In this study, we aimed to examine the effects of RCE in endothelial cells challenged with hypoxic exposure and to elucidate the underlying mechanisms. Human umbilical vein endothelial cells were pretreated with or without RCE and then exposed to hypoxia (1% O2) for 24 h. Cell viability, nitric oxide (NO) production, oxidative stress markers, as well as mechanistic readouts were studied. We found that hypoxia-induced cell death, impaired NO production, and oxidative stress. These responses were significantly attenuated by RCE treatment and were associated with the activation of AMP-activated kinase and extracellular signal-regulated kinase 1/2 signaling pathways. In summary, we showed that RCE protected endothelial cells from hypoxic insult and suggested that R. crenulata might be useful for the prevention of hypoxia-associated vascular dysfunction.

In Vitro Anticancer Activity and Structural Characterization of Ubiquinones from Antrodia cinnamomea Mycelium.

Two new ubiquinones, named antrocinnamone and 4-acetylantrocamol LT3, were isolated along with six known ubiquinones from Antrodia cinnamomea (Polyporaceae) mycelium. The developed HPLC analysis methods successfully identified eight different ubiquinones, two benzenoids, and one maleic acid derivative from A. cinnamomea. The ubiquinones 1-8 exhibited potential and selective cytotoxic activity against three human cancer cell lines, with IC50 values ranging from 0.001 to 35.883 µM. We suggest that the different cytotoxicity levels were related to their chemical structures, especially the 4-hydroxycyclohex-2-enone ring and the presence of a free hydroxyl group in the side chain. The suppression by 4-acetylantrocamol LT3 stopped the cell cycle at the beginning of the G2-M phase thus making the cell cycle arrest at the sub-G1 phase as compared with control cells.

Polygonum barbatum extract reduces colorectal cancer cell proliferation, migration, invasion, and epithelial-mesenchymal transition via YAP and ß-catenin pathway regulation.

Colorectal cancer (CRC) is the third most common cancer worldwide with novel therapeutic developmental challenges. Polygonum barbatum has anticancer potential, but its mechanism(s) are unclear. This study investigates the inhibitory effect of P. barbatum on human CRC cells. Polygonum barbatum extract (PBE) and quercetin standard HPLC fingerprints were determined using analytical RP-HPLC and evaluations were completed using the human colon cancer cell line HCT-116 (KRASG13D mutation) and HT-29 (BRAF mutation) cells. Post-PBE treatment, cell viability, colony formation, migration, invasion, and apoptosis, as well as changes in the whole-transcriptome of cells were analyzed. PBE significantly reduced CRC cell growth, migration, and invasion, and the genes responsible for extracellular matrix (ECM) organization, cell motility, and cell growth were suppressed by PBE. The differentially expressed genes revealed that PBE treatment exerted a significant effect on the ECM interaction and focal adhesion pathways. Epithelial-to-mesenchymal transition markers, N-cadherin, vimentin, SLUG, and SNAIL, were shown to be regulated by PBE. These effects were associated with blockade of the Yes-associated protein and the GSK3ß/ß-catenin axis. PBE exerts a significant inhibitory effect on CRC cells and may be applicable in clinical trials.

Uveitis increases the risk of stroke among patients with ankylosing spondylitis: A nationwide population-based longitudinal study.

Ankylosing spondylitis (AS) is known to increase the risk of stroke. Among patients with AS, uveitis is the most common extra-articular manifestation. However, no previous investigations have discussed the association between uveitis and the risk for developing stroke in patients with AS. This retrospective cohort study aimed to explore the relationship between uveitis and the incidence of stroke in patients with AS by obtaining medical records from January 1, 2000, to December 31, 2015, from the National Health Insurance Research Database, according to the International Classification of Diseases, 9th Revision, Clinical Modification diagnosis codes. The primary outcome was the incidence of stroke. Pearson's chi-square test and Fisher's exact test were used to analyze variables. Kaplan-Meier survival curves and univariate and multivariate Cox proportional hazard regression models with and without Fine and Gray's competing risk model were used to analyze data. Total 828 AS patients with uveitis and 3,312 AS patients without uveitis were identified. During the follow-up period, 137 patients in the uveitis group and 344 in the non-uveitis group developed stroke. Uveitis is a significant risk factor for stroke development in patients with AS (adjusted hazard ratio = 1.846, p < 0.001). Age, diabetes mellitus, hyperlipidemia, hypertension, congestive heart failure, chronic obstructive pulmonary disease, asthma, coronary artery disease, and atrial fibrillation were associated with a higher risk of stroke. After subgroup analysis, both anterior uveitis and posterior segment involvement were found to increase the risk of stroke in patients with AS. Uveitis is associated with an increased risk in both ischemic and hemorrhagic strokes in patients with AS. Therefore, when uveitis is identified, clinicians should pay more attention to the cerebrovascular risk in patients with AS, especially in those with underlying comorbidities.

4-Acetylantrocamol LT3 Inhibits Glioblastoma Cell Growth and Downregulates DNA Repair Enzyme O6-Methylguanine-DNA Methyltransferase.

Glioblastoma multiforme (GBM) is a deadly malignant brain tumor that is resistant to most clinical treatments. Novel therapeutic agents that are effective against GBM are required. Antrodia cinnamomea has shown antiproliferative effects in GBM cells. However, the exact mechanisms and bioactive components remain unclear. Thus, the present study aimed to investigate the effect and mechanism of 4-acetylantrocamol LT3 (4AALT3), a new ubiquinone from Antrodia cinnamomeamycelium, in vitro. U87 and U251 cell lines were treated with the indicated concentration of 4AALT3. Cell viability, cell colony-forming ability, migration, and the expression of proteins in well-known signaling pathways involved in the malignant properties of glioblastoma were then analyzed by CCK-8, colony formation, wound healing, and western blotting assays, respectively. We found that 4AALT3 significantly decreased cell viability, colony formation, and cell migration in both in vitro models. The epidermal growth factor receptor (EGFR), phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR), Hippo/yes-associated protein (YAP), and cAMP-response element binding protein (CREB) pathways were suppressed by 4AALT3. Moreover, 4AALT3 decreased the level of DNA repair enzyme O6-methylguanine-DNA methyltransferase and showed a synergistic effect with temozolomide. Our findings provide the basis for exploring the beneficial effect of 4AALT3 on GBM in vivo.

4-Acetylantroquinonol B ameliorates nonalcoholic steatohepatitis by suppression of ER stress and NLRP3 inflammasome activation.

OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD) is an inflammatory lipotoxic disorder with a prevalence of over 25% worldwide. However, safe and effective therapeutic agents for the management of NAFLD are still lacking. We aimed to investigate the hepatoprotective effect and molecular mechanism of 4-acetylantroquinonol B (4-AAQB), a natural ubiquinone derivative obtained from the mycelia of Antrodia cinnamomea. METHODS: RAW264.7 and J774A.1 cells were treated with 4-AAQB and then stimulated with LPS or tunicamycin (TM) for 24 h. Inflammatory responses, markers of endoplasmic reticulum (ER) stress, and NOD-like receptor protein 3 (NLRP3) inflammasome were analyzed in both cell lines. In the applied in vivo model, male C57BL/6J mice were fed with chow or a methionine/choline-deficient (MCD) diet along with vehicle or 4-AAQB (10 mg/kg, i.p. injected, once a day) for 10 consecutive days. Plasma levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured. Liver tissues were analyzed using histological techniques; protein levels involved in ER stress, NLRP3 inflammasome, and inflammatory responses were measured. RESULTS: 4-AAQB significantly ameliorated the plasma levels of ALT and AST as well as the NAFLD activity score (NAS) in mice fed the MCD diet. In addition, 4-AAQB suppressed inflammatory responses, ER stress, and NLRP3 inflammasome activation, but increased the nuclear factor erythroid 2-related factor 2 (Nrf2) and Sirtuin 1 (SIRT1) signaling pathways in both in vitro and in vivo models. CONCLUSIONS: We suggest that 4-AAQB treatment might be a tangible therapeutic strategy in the management of NAFLD/NASH.

Antrodia Cinnamomea Attenuates Non-Alcoholic Steatohepatitis by Suppressing NLRP3 Inflammasome Activation In Vitro and In Vivo.

Blockade of the NOD-like receptor protein 3 (NLRP3) inflammasome has been shown to be effective in halting the progression of non-alcoholic steatohepatitis (NASH). Antrodia cinnamomea is a well-known indigenous medicine used by Taiwanese aboriginal tribes. However, its effect on NASH remains unclear. This study aimed to examine the mechanistic insight of Antrodia cinnamomea extract (ACE) in both in vitro and in vivo models of NASH. Murine RAW264.7 macrophages and human hepatocellular carcinoma HepG2 cells were treated with the indicated concentration of ACE 30 minutes prior to stimulation with lipopolysaccharide (LPS) for 24 h. Levels of inflammatory markers, NLRP3 inflammasome, components, and endoplasmic reticulum (ER) stress markers were analyzed by Western blotting. For the in vivo experiments, male C57BL/6 mice weighing 21-25 g were fed a methionine/choline deficient (MCD) diet along with vehicle or ACE (100 mg/kg) for 10 consecutive days. The serum glutamate pyruvate transaminase (SGPT) levels of the mice were measured. The liver tissues from the mice underwent histological analysis (hematoxylin and eosin staining), and the levels of inflammatory markers, NLRP3 inflammasome components, and autophagy-related proteins were evaluated. ACE significantly inhibited NLRP3 inflammasome activation in vitro and in vivo. In addition, ACE attenuated the severity of MCD-induced steatohepatitis, reduced the levels of oxidative stress markers, and ameliorated inflammatory responses, but restored autophagic flux. Based on these findings, Antrodia cinnamomea could be developed into an anti-non-alcoholic fatty liver disease/NASH agent.

Rhodiola crenulata Suppresses High Glucose-Induced Matrix Metalloproteinase Expression and Inflammatory Responses by Inhibiting ROS-Related HMGB1-TLR4 Signaling in Endothelial Cells.

Rhodiola crenulata, a popular folk medicine for anti-altitude sickness in Tibet, has been shown to have protective effects against high glucose (HG)-induced endothelial cell dysfunction in human umbilical vein endothelial cells (HUVECs). However, its mechanisms of action are unclear. Here, we aimed to examine the effects and the mechanisms of action of Rhodiola crenulata extract (RCE) on matrix metalloproteinases (MMPs) and inflammatory responses under HG conditions. HUVECs were pretreated with RCE or untreated and then exposed to 33mM glucose medium for 24h. The levels of oxidative stress markers, MMPs, endogenous tissue inhibitors of MMPs (TIMPs), and adhesion molecules were determined. Zymography assays were also carried out. We found that RCE significantly decreased HG-induced increases in reactive oxygen species (ROS) and activation of MAPK and NF-κB pathways. In addition, RCE not only significantly reduced the expression and activities of MMPs but also upregulated TIMP protein levels. Consistently, HG-induced activation of the toll-like receptor 4 (TLR4)/myeloid differentiation primary response protein (MyD88) signaling pathway, intracellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and high mobility group box 1 (HMGB1) as well as endothelial cell apoptosis was inhibited by RCE treatment. RCE exerts protective effects on endothelial cells against HG insult, partially by suppressing the HMGB1/TLR4 axis. These findings indicate that Rhodiola crenulata may be a potential therapeutic agent for diabetes-associated vascular diseases.

4-Acetylantrocamol LT3, a New Ubiquinone from Antrodia cinnamomea, Inhibits Hepatocellular Carcinoma HepG2 Cell Growth by Targeting YAP/TAZ, mTOR, and WNT/ß-Catenin Signaling.

4-acetylantrocamol LT3 (4AALT3), a new ubiquinone from the mycelium of Antrodia cinnamomea (Polyporaceae), has been recently shown to possess anticancer activity. However, the detailed mechanisms of such action remain unclear. In this study, the molecular mechanisms of 4AALT3 on hepatocellular carcinoma cells (HCC) were investigated. Human hepatocellular carcinoma cell line HepG2 cells were treated with concentrations of 4AALT3. Cell viability, colony formation, and the underlying mechanisms were then analyzed by CCK-8, colony formation, qPCR, and Western blotting assays. We found that 4AALT3 significantly decreased cell viability and colony formation in a dose-dependent manner. Accordingly, 4AALT3 significantly decreased protein levels of cyclin B, E1, D1, and D3, thereby facilitating cell cycle arrest. In addition, 4AALT3 significantly suppressed the nuclear localization of Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ), mammalian target of rapamycin (mTOR), and WNT/[Formula: see text]-catenin signaling pathways, all of which are well-known signaling pathways that contribute to the malignant properties of HCC. These effects are associated with activation of 5' AMP-activated protein kinase (AMPK) and autophagy. Our findings indicate that 4AALT3 exerts inhibitory effects on HepG2 cell growth via multiple signaling pathways and may be a potential agent for HCC therapy.

Antrolone, a Novel Benzoid Derived from Antrodia cinnamomea, Inhibits the LPS-Induced Inflammatory Response in RAW264.7 Macrophage Cells by Balancing the NF-[Formula: see text]B and Nrf2 Pathways.

Antrodia cinnamomea, a medicinal mushroom, has previously demonstrated anti-inflammatory activity, although the specific compound responsible for the effect remains unclear. The present study was designed to investigate the anti-inflammatory property of antrolone, a novel benzoid derived from A. cinnamomea mycelium, and to clarify the underlying mechanisms of action. To this end, murine macrophage RAW264.7 cells were treated with antrolone (0.1-30[Formula: see text][Formula: see text]M) 30[Formula: see text]min prior to stimulation with lipopolysaccharides (LPS, 0.1[Formula: see text][Formula: see text]g/ml) for 24[Formula: see text]h. Cell viability, nitric oxide (NO) and prostaglandin E2 (PGE2) production, levels of pro-inflammatory cytokines and chemokines, and the signaling pathways involved in the inflammatory cascades were then investigated. Our results show that antrolone significantly decreased LPS-induced NO, PGE2, pro-inflammatory cytokine, and keratinocyte chemoattractant CXCL1 (KC) production and reduced levels of the proteins inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). These effects were independent of the effect of antrolone on macrophage cytotoxicity. Moreover, antrolone significantly inhibited the activation of the NF[Formula: see text]B, MAPK, and AKT pathways, while it increased nuclear factor erythroid-2-related factor (Nrf2) and heme oxygenase-1 (HO-1) levels. Our findings suggest that antrolone exhibits potent anti-inflammatory activity and may, therefore, be a lead compound for the development of an anti-inflammatory drug.

Rhodiola crenulata Attenuates High Glucose Induced Endothelial Dysfunction in Human Umbilical Vein Endothelial Cells.

Rhodiola crenulata root extract (RCE), a traditional Chinese medicine, has been shown to regulate glucose and lipid metabolism via the AMPK pathway in high glucose (HG) conditions. However, the effect of RCE on HG-induced endothelial dysfunction remains unclear. The present study was designed to examine the effects and mechanisms of RCE against hyperglycemic insult in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were pretreated with or without RCE and then exposed to 33[Formula: see text]mM HG medium. The cell viability, nitrite production, oxidative stress markers, and vasoactive factors, as well as the mechanisms underlying RCE action, were then investigated. We found that RCE significantly improved cell death, nitric oxide (NO) defects, and oxidative stress in HG conditions. In addition, RCE significantly decreased the HG-induced vasoactive markers, including endothelin-1 (ET-1), fibronectin, and vascular endothelial growth factor (VEGF). However, the RCE-restored AMPK-Akt-eNOS-NO axis and cell viability were abolished by the presence of an AMPK inhibitor. These findings suggested that the protective effects of RCE were associated with the AMPK-Akt-eNOS-NO signaling pathway. In conclusion, we showed that RCE protected endothelial cells from hyperglycemic insult and demonstrated its potential for use as a treatment for endothelial dysfunction in diabetes mellitus.

Rhodiola crenulata extract suppresses hepatic gluconeogenesis via activation of the AMPK pathway.

BACKGROUND: Rhodiola, a popular herb, has been used for treating high altitude sicknesses, depression, fatigue, and diabetes. However, the detailed mechanisms by which Rhodiola crenulata functions in the liver need further clarification. PURPOSE: The current study was designed to examine the effects of Rhodiola crenulata root extract (RCE) on hepatic glucose production. METHODS: Human hepatoma HepG2 cells were treated with RCE for 6 h. Glucose production, the expression level of p-AMPK, and the expression of key gluconeogenic genes were measured. The effects of RCE were also studied in Sprague-Dawley (SD) rats. The efficacy and underlying mechanism of RCE in the liver were examined. RESULTS: RCE significantly suppressed glucose production and gluconeogenic gene expression in HepG2 cells while activating the AMPK signaling pathway. Interestingly, RCE-suppressed hepatic gluconeogenesis was eliminated by an AMPK-specific inhibitor, but not by the PI3K/AKT-specific inhibitor. In addition, oral administration of RCE significantly increased phosphorylated AMPK levels and inhibited gluconeogenic gene expression in the rat liver. Furthermore, RCE treatment also decreased plasma glucose concentration in rats. CONCLUSION: We present in vitro and in vivo evidence that RCE might exert the glucose-lowering effect partly by inhibiting hepatic gluconeogenesis through activating the AMPK signaling pathway. These findings provide evidence that Rhodiola crenulata may be helpful for the management of type II diabetes.

Anti-cancer agents derived from solid-state fermented Antrodia camphorata mycelium.

Three new ubiquinone derivatives, antrocamol LT1, antrocamol LT2, and antrocamol LT3, along with two known compounds, were isolated from Antrodia camphorata (Polyporaceae) mycelium. The structures of these compounds were established on the basis of extensive 1D and 2D NMR spectroscopic analyses. These ubiquinones exhibited selective cytotoxicities against five human cancer cell lines (CT26, A549, HepG2, PC3 and DU-145) with IC50 values ranging from 0.01 to 1.79µΜ.