Asaronic acid inhibits ER stress sensors and boosts functionality of ubiquitin-proteasomal degradation in 7ß-hydroxycholesterol-loaded macrophages.

BACKGROUND: Misfolded proteins are formed in the endoplasmic reticulum (ER) due to diverse stimuli including oxidant production, calcium disturbance, and inflammatory factors. Accumulation of these non-native proteins in the ER evokes cellular stress involving the activation of unfolded protein response (UPR) and the execution of ER-associated degradation (ERAD). Naturally-occurring plant compounds are known to interfere with UPR due to their antioxidant and anti-inflammatory activities, leading to inhibition of ER stress. However, there are few studies dealing with the protective effects of natural compounds on the functionality of ERAD. PURPOSE: The current study examined whether asaronic acid enhanced ubiquitin-proteasomal degradation in J774A.1 murine macrophages exposed to 7ß-hydroxycholesterol, a risk factor for atherosclerosis. Asaronic acid (2,4,5-trimethoxybenzoic acid), identified as one of purple perilla constituents, has anti-diabetic and anti-inflammatory effects. Little is known regarding the effects of asaronic acid on the ERAD process and the ubiquitin-proteasomal degradation. METHODS AND RESULTS: Murine macrophages were incubated with 28 µM 7ß-hydroxycholesterol in absence and presence of 1-20 µΜ asaronic acid for up to 24 h. Nontoxic asaronic acid in macrophage diminished the activation of the ER stress sensors of ATF6, IRE1 and PERK stimulated by 7ß-hydroxycholesterol. This methoxybenzoic acid down-regulated the oxysterol-induced expression of EDEM1, OS9, Sel1L-Hrd1 and p97/VCP1, all required for the recognition, recruitment and dislocation of misfolded proteins. On the other hand, asaronic acid enhanced the ubiquitin-proteasomal degradation of non-native proteins dislocated to the cytosol by 7ß-hydroxycholesterol, which entailed the induction of the chaperones of Hsp70 and CHIP and the increased colocalization of ubiquitin and proteasomes. Taken together, asaronic acid attenuated the induction of the UPR-associated sensors and the dislocation-linked transmembrane components in the ER. Conversely, this compound enhanced the proteasomal degradation of dislocated non-native proteins in concert with the chaperones of Hsp70 and CHIP through ubiquitination. CONCLUSION: These observations demonstrate that asaronic acid may be a potent atheroprotective agent as a natural chaperone targeting ER stress-associated macrophage injury.

Hydroxycoumarin Scopoletin Inhibits Bone Loss through Enhancing Induction of Bone Turnover Markers in a Mouse Model of Type 2 Diabetes.

Diabetes induces bone deterioration, which leads to increased risk of fracture, osteopenia, and osteoporosis. Thus, diabetes-associated bone fragility has been recognized as a diabetic complication. However, the pathophysiological effects of hyperglycemia on bone turnover remain unclear. Literature evidence demonstrates that anti-diabetic medications increase the risk of fractures in individuals with type 2 diabetes. Scopoletin is a naturally occurring hydroxycoumarin potentially exhibiting anti-inflammatory and antioxidant activities and ameliorating insulin resistance as an anti-diabetic agent. However, little is known regarding the effects of scopoletin on the impairment of bone remodeling that is caused by diabetes. The aim of this study was to identify that scopoletin was capable of inhibiting the impairment of bone remodeling and turnover in a mouse model of type 2 diabetes. Submicromolar scopoletin accelerated the formation TRAP-positive multinucleated osteoclasts (40.0 vs. 105.1%) and actin ring structures impaired by 33 mM glucose. Further, 1-20 µM scopoletin enhanced bone resorption and the induction of matrix-degrading enzymes in diabetic osteoclasts. The oral administration of 10 mg/kg scopoletin elevated serum RANKL/OPG ratio and osteocalcin level reduced in db/db mice along with an increase in BMD by ~6-14%; however, it was not effective in lowering blood glucose and hemoglobin glycation. In addition, the supplementation of scopoletin elevated the formation of trabecular bones and collagen fibers in femoral epiphysis and metaphysis with a thicker epiphyseal plate and cortical bones. Furthermore, 1-20 µM scopoletin enhanced ALP activity (4.39 vs. 7.02 nmol p-nitrophenyl phosphate/min/mg protein) and deposits of mineralized bone nodules in cultured osteoblasts reduced by 33 mM glucose. The treatment of diabetic osteoblasts with scopoletin stimulated the cellular induction of BMP-2 and osteopontin and Runx2 transcription. Accordingly, the administration of scopoletin protected mice from type 2 diabetes-associated bone loss through boosting bone remodeling via the robust induction of bone turnover markers of both osteoclasts and osteoblasts. These findings suggest that scopoletin could be a potential osteoprotective agent for the treatment of diabetes-associated bone loss and fractures.

Dietary Collagen Hydrolysates Ameliorate Furrowed and Parched Skin Caused by Photoaging in Hairless Mice.

Collagen hydrolysates have been suggested as a favorable antiaging modality in skin photoaged by persistent exposure to ultraviolet radiation (UV). The current study evaluated the beneficial effect of collagen hydrolysates (fsCH) extracted from Pangasius hypophthalmus fish skin on wrinkle formation and moisture preservation in dorsal skin of hairless mice challenged with UV-B. Inter-comparative experiments were conducted for anti-photoaging among fsCH, retinoic acid (RA), N-acetyl-D-glucosamine (NAG), and glycine-proline-hydroxyproline (GPH). Treating human HaCaT keratinocytes with 100-200 µg/mL fsCH reciprocally ameliorated the expression of aquaporin 3 (AQP3) and CD44 deranged by UV-B. The UV-B-induced deep furrows and skin thickening were improved in parched dorsal skin of mice supplemented with 206-412 mg/kg fsCH as well as RA and GPH. The UV-B irradiation enhanced collagen fiber loss in the dorsal dermis, which was attenuated by fsCH through enhancing procollagen conversion to collagen. The matrix metalloproteinase expression by UV-B in dorsal skin was diminished by fsCH, similar to RA and GPH, via blockade of collagen degradation. Supplementing fsCH to UV-B-irradiated mice decreased transepidermal water loss in dorsal skin with reduced AQP3 level and restored keratinocyte expression of filaggrin. The expression of hyaluronic acid synthase 2 and hyaluronidase 1 by UV-B was remarkably ameliorated with increased production of hyaluronic acid by treating fsCH to photoaged mice. Taken together, fsCH attenuated photoaging typical of deep wrinkles, epidermal thickening, and skin water loss, like NAG, RA, or GPH, through inhibiting collagen destruction and epidermal barrier impairment.

Astragalin Inhibits Cigarette Smoke-Induced Pulmonary Thrombosis and Alveolar Inflammation and Disrupts PAR Activation and Oxidative Stress-Responsive MAPK-Signaling.

Epidemiological evidence shows that smoking causes a thrombophilic milieu that may play a role in the pathophysiology of chronic obstructive pulmonary disease (COPD) as well as pulmonary thromboembolism. The increased nicotine level induces a prothrombotic status and abnormal blood coagulation in smokers. Since several anticoagulants increase bleeding risk, alternative therapies need to be identified to protect against thrombosis without affecting hemostasis. Astragalin is a flavonoid present in persimmon leaves and green tea seeds and exhibits diverse activities of antioxidant and anti-inflammation. The current study investigated that astragalin attenuated smoking-induced pulmonary thrombosis and alveolar inflammation. In addition, it was explored that molecular links between thrombosis and inflammation entailed protease-activated receptor (PAR) activation and oxidative stress-responsive mitogen-activated protein kinase (MAPK)-signaling. BALB/c mice were orally administrated with 10-20 mg/kg astragalin and exposed to cigarette smoke for 8 weeks. For the in vitro study, 10 U/mL thrombin was added to alveolar epithelial A549 cells in the presence of 1-20 µM astragalin. The cigarette smoking-induced the expression of PAR-1 and PAR-2 in lung tissues, which was attenuated by the administration of ≥10 mg/kg astragalin. The oral supplementation of ≥10 mg/kg astragalin to cigarette smoke-challenged mice attenuated the protein induction of urokinase plasminogen activator, plasminogen activator inhibitor-1and tissue factor, and instead enhanced the induction of tissue plasminogen activator in lung tissues. The astragalin treatment alleviated cigarette smoke-induced lung emphysema and pulmonary thrombosis. Astragalin caused lymphocytosis and neutrophilia in bronchoalveolar lavage fluid due to cigarette smoke but curtailed infiltration of neutrophils and macrophages in airways. Furthermore, this compound retarded thrombin-induced activation of PAR proteins and expression of inflammatory mediators in alveolar cells. Treating astragalin interrupted PAR proteins-activated reactive oxygen species production and MAPK signaling leading to alveolar inflammation. Accordingly, astragalin may interrupt the smoking-induced oxidative stress-MAPK signaling-inflammation axis via disconnection between alveolar PAR activation and pulmonary thromboembolism.

Linarin and its aglycone acacetin abrogate actin ring formation and focal contact to bone matrix of bone-resorbing osteoclasts through inhibition of αvß3 integrin and core-linked CD44.

BACKGROUND: Since enhanced bone resorption due to osteoclast differentiation and activation cause skeletal diseases, there is a growing need in therapeutics for combating bone-resorbing osteoclasts. Botanical antioxidants are being increasingly investigated for their health-promoting effects on bone. Edible Cirsium setidens contains various polyphenols of linarin, pectolinarin, and apigenin with antioxidant and hepatoprotective effects. PURPOSE: This study aimed to determine whether linarin present in Cirsium setidens water extracts (CSE) and its aglycone acacetin inhibited osteoclastogenesis of RANKL-exposed RAW 264.7 murine macrophages for 5 days. METHODS: This study assessed the osteoprotective effects of CSE, linarin and acacetin on RANKL-induced differentiation and activation of osteoclasts by using MTT assay, TRAP staining, Western blot analysis, bone resorption assay actin ring staining, adhesion assay and immunocytochemical assay. This study explored the underlying mechanisms of their osteoprotection, and identified major components present in CSE by HPLC analysis. RESULTS: Linarin and pectolinarin were identified as major components of CSE. Nontoxic linarin and acacetin as well as CSE, but not pectolinarin attenuated the RANKL-induced macrophage differentiation into multinucleated osteoclasts, and curtailed osteoclastic bone resorption through reducing lacunar acidification and bone matrix degradation in the osteoclast-bone interface. Linarin and acacetin in CSE reduced the transmigration and focal contact of osteoclasts to bone matrix-mimicking RGD peptide. Such reduction was accomplished by inhibiting the induction of integrins, integrin-associated proteins of paxillin and gelsolin, cdc42 and CD44 involved in the formation of actin rings. The inhibition of integrin-mediated actin ring formation by linarin and acacetin entailed the disruption of TRAF6-c-Src-PI3K signaling of bone-resorbing osteoclasts. The functional inhibition of c-Src was involved in the loss of F-actin-enriched podosome core protein cortactin-mediated actin assembly due to linarin and acacetin. CONCLUSION: These observations demonstrate that CSE, linarin and acacetin were effective in retarding osteoclast function of focal adhesion to bone matrix and active bone resorption via inhibition of diffuse cloud-associated αvß3 integrin and core-linked CD44.

Anti-inflammatory effects of Dendrobium nobile derived phenanthrenes in LPS-stimulated murine macrophages.

Dendrobium nobile belongs to the Orchidaceae family and is one of the medicinal herbs used in traditional Chinese medicine as a therapeutic agent for gastrointestinal and cardiovascular diseases. In this study, we separated three phenanthrenes (ephemeranthol A (EA), 1,5,7-trimethoxyphenanthren-2-ol (TP), dehydroorchinol (DO)) from D. nobile, and compared their anti-inflammatory activities. TP is a new phenanthrene compound and its structure was determined from (1)H, (13)C NMR and HR-ESI-MS data. To analyze the anti-inflammatory activities of the phenanthrenes, Raw 264.7 cells were used, since they are immature-macrophages and easily matured by LPS stimulation. EA and DO showed anti-inflammatory activities in the activated Raw 264.7 cells. That is, we showed that EA is a potent inhibitor of the production of nitric oxide and pro-inflammatory cytokines. The inhibitory activities of phenanthrenes were found to be caused by blockage of NF-κB activation and the phosphorylation of MAP kinases in the macrophages. These results are expected to serve as a guide for future studies on the ability of phenanthrenes to inhibit acute and chronic inflammatory diseases.

Anti-inflammatory effects of gomisin N, gomisin J, and schisandrin C isolated from the fruit of Schisandra chinensis.

Schiandra chinensis is a well-known Chinese traditional medicine for the treatment of hepatic disease. In this study, we investigated whether the nine major compounds of Schiandra chinensis could be applied to suppress lipopolysaccharide (LPS)-induced inflammatory responses in murine macrophages (Raw 264.7 cells). Among the nine lignans, three, gomisin J, gomisin N, and schisandrin C, were found to reduce nitric oxide (NO) production from LPS-stimulated Raw 264.7 cells. These three lignans showed low cytotoxic effects in Raw 264.7 cells. Pre-treatment of Raw 264.7 cells with gomisin J, gomisin N, or schisandrin C reduced the expression of mRNA and the secretion of pro-inflammatory cytokines. These inhibitory effects were found to be caused by blockage of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases 1 and 2 (ERK 1/2), and c-Jun N-terminal kinase (JNK) phosphorylation.