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.

Ameliorative Effects of Pueraria lobata Extract on Postmenopausal Symptoms through Promoting Estrogenic Activity and Bone Markers in Ovariectomized Rats.

Pueraria lobata (Willd.) Ohwi, known as kudzu, is one of the most popular traditional medicines in Asian countries. It has been widely used as a natural alternative to hormone replacement therapy for treating postmenopausal symptoms. This study aimed to investigate the estrogenic effect of P. lobata extract (PE) against postmenopausal osteoporosis in ovariectomized (OVX) rats. OVX rats were treated with PE (25-1600 mg/kg) for 8 weeks. Biochemical parameters, estradiol, and bone turnover markers (e.g., osteocalcin, C-terminal telopeptide fragment of type I collagen, deoxypyridinoline, and pyridinoline) were measured in plasma samples. In addition, estrogen receptor-alpha (ER-α) protein expression and morphology of uterine were evaluated. Long-term treatment with PE did not cause liver damage in OVX rats. PE supplementation reduced body weight gain in obese rats with high lipid accumulation induced by ovariectomy. Furthermore, PE exhibited a protective effect against insulin resistance, hyperlipidemia, and hepatic lipid peroxidation. PE treatment increased uterine weight and thickness of the uterine layers in cases of uterus atrophy due to removal of ovaries. The levels of bone turnover markers, which were significantly increased in OVX rats, were decreased by PE treatment. Western blotting analysis showed that ER-α protein expression was upregulated in PE-treated rats compared with OVX rats. These results suggest that PE could be a promising alternative functional food for improving menopausal symptoms.

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.

Platycarya strobilacea leaf extract inhibits tumor necrosis factor-α production and bone loss induced by Porphyromonas gingivalis-derived lipopolysaccharide.

OBJECTIVE: Remodeling of alveolar bone is controlled by osteoclast-mediated bone resorption and osteoblast-induced bone formation. LPS of Porphyromonas gingivalis, a major causative agent of periodontitis, produces proinflammatory cytokines in host immune cells, which thereby triggers osteoclastogenesis and leads to alveolar bone resorption. We investigated the anti-periodontitis potential of Platycarya strobilacea leaf extract (PLE), which is used as a traditional medicine in Asian countries. DESIGN: TNF-α levels in cell culture media were measured using a commercially available enzyme-linked immunosorbent assay kit. Osteoclast differentiation was observed by tartrate-resistant acid phosphatase staining, and the expression levels of osteoclastogenic genes were measured by quantitative real-time PCR. Bone-resorbing activity was confirmed by the resorption pit formation, gelatin zymographic, and the cathepsin K activity assays. Osteogenic differentiation was confirmed with an ALP activity assay and alizarin red S staining. RESULTS: PLE treatment inhibited the production of TNF-α in P. gingivalis LPS-stimulated RAW264.7 macrophages. In bone marrow-derived macrophages serving as osteoclast precursors, PLE treatment blocked RANKL-induced osteoclastogenesis and gene expression levels of the osteoclastogenic transcription factor NFATc1, DC-STAMP for osteoclast fusion, and cathepsin K for osteoclast activity. In addition, PLE treatment reduced the formation of resorption pits and the secretion of MMP 9 and cathepsin K from the differentiated osteoclasts. Furthermore, PLE treatment induced osteogenesis by increasing ALP activity and calcium content in preosteoblastic cells. CONCLUSION: PLE inhibits P. gingivalis LPS-induced TNF-α production and bone resorption and induces bone formation. PLE may be a beneficial agent to promote oral health by inhibiting periodontitis-induced alveolar bone loss.

Anti-Diabetic Effect of Aster sphathulifolius in C57BL/KsJ-db/db Mice.

In this study, we investigated the anti-diabetic effect of Aster sphathulifolius (AS) extract in C57BL/KsJ-db/db mice. The db/db mice were orally administered with AS 50% ethanol extract at concentrations of 50, 100, and 200 mg/kg/day (db/db-AS50, db/db-AS100, and db/db-AS200, respectively) for 10 weeks. Food and water intake, fasting blood glucose concentrations, blood glycosylated hemoglobin levels, and plasma insulin levels were significantly lower in the db/db-AS200 group than in the vehicle-treated db/db group; whereas glucose tolerance was significantly improved in the db/db-AS200 group. Moreover, AS dose dependently increased both insulin receptor substrate 1 and glucose transporter type 4 expression in skeletal muscle, significantly increased glucokinase expression, and decreased glucose 6-phosphatase and phosphoenolpyruvate carboxykinase expressions in the liver. The expressions of transcription factors, such as sterol-regulatory element-binding protein, peroxisome proliferator-activated receptor γ, and adipocyte protein 2, were upregulated in adipose tissue. Furthermore, immunohistochemical analysis showed that AS upregulated insulin production by increasing pancreatic ß-cell mass. In summary, AS extract normalized hyperglycemia by multiple mechanisms: inhibition of glyconeogenesis, acceleration of glucose metabolism and lipid metabolism, and increase of glucose uptake. Using in vivo assays, this study has shown the potential of AS as a medicinal food and suggests the efficacy of AS for the use of prevention of diabetes.

Sage weed (Salvia plebeia) extract antagonizes foam cell formation and promotes cholesterol efflux in murine macrophages.

Lipid-laden peripheral tissue cells release cholesterol to an extracellular acceptor such as high-density lipoprotein (HDL). Foam cells are formed at the first stage of atherosclerosis development. This study investigated whether sage weed (Salvia plebeia) extract (SWE) influences cholesterol handling of J774A1 murine macrophages. A murine macrophage cell line, J774A1, was used in this study. Oxidized low-density lipoproteins (LDL) treatment was used for foam cell formation, which was confirmed using Oil red O staining. The oxidized LDL uptake and cholesterol efflux from lipid-laden foam cell-associated proteins were detected by western blot analysis. Also, transcriptional levels of these associated genes were examined using reverse transcription-PCR. Also, cholesterol efflux was measured using NBD-cholesterol efflux assay. Non-toxic SWE at ≥10 µg/ml attenuated scavenger receptor (SR)-B1 expression of macrophages induced by oxidized LDL for 6 h, which was achieved at its transcriptional levels. Consistently, SWE suppressed oxidized LDL-stimulated cellular lipid accumulation and foam cell formation due to downregulated SR-B1. SWE upregulated the protein expression and mRNA levels of ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) in lipid-laden foam cells, both responsible for cholesterol efflux. In addition, SWE promoted apolipoprotein E (apoE) secretion from oxidized LDL-induced foam cells. Cholesterol efflux was enhanced by ≥10 µg/ml SWE most likely through the induction of ABCA1 and ABCG1 and the secretion of apoE. Although 10 µM homoplantaginin, a compound mainly present in sage weeds, did not influence cellular expression of ABCA1 and ABCG1, it suppressed oxidized LDL-enhanced SR-B1 induction and foam cell formation. These results demonstrate that SWE antagonized oxidized LDL uptake and promoted cholesterol efflux in lipid-laden macrophages. Therefore, SWE may serve as a protective therapeutic agent against the development of atherosclerosis.

Acute effect of high-dose isoflavones from Pueraria lobata (Willd.) Ohwi on lipid and bone metabolism in ovariectomized mice.

We investigated the acute metabolic effects of isoflavones from Pueraria lobata (Willd.) Ohwi (IPL) in ovariectomized (OVX) mice. After 4 weeks of IPL feeding at 500 mg/day/kg body weight (OVX500), plasma 17ß-estradiol concentrations were significantly higher (+25%, p < 0.05), whereas plasma triglyceride levels were significantly lower in OVX mice (-15%, p < 0.05) compared with controls. Abdominal adipose tissue weight was marginally reduced in IPL-fed groups compared with OVX controls and the plasma levels of liver enzymes were unchanged. In addition, IPL significantly inhibited the reduction of bone mineral density in the femurs of OVX mice (OVX200, +22%; OVX500, +26%; p < 0.05) compared with controls after 4 weeks of IPL feeding. In quantitative polymerase chain reaction analysis the expression of aromatase was significantly suppressed and SULT1E1 was increased by IPL feeding, showing that IPL feeding may not alter the risk for breast cancer in mice. Our results suggest that IPL could ameliorate menopausal symptoms in mice. Further studies will confirm the effects of IPL in humans.

Design and evaluation of a potential mutagen for Hepatitis C virus.

Pyrrolopyrimidine nucleoside 1 was designed and synthesized as a potential mutagen for HCV. An in vitro HCV NS5B enzymatic assay indicated that pyrrolopyrimidine triphosphate acts as a CTP analog rather than a UTP analog. The SATE-prodrug of pyrrolopyrimidine monophosphate showed a weak inhibitory activity in an HCV replicon system (EC(50)=60 microM) and did not exhibit cytotoxicity (CC(50)>100 microM). Investigation of phosphorylation events using nucleoside kinases and LC-MS analysis revealed that the second phosphorylation step, from monophosphate ester to diphosphate ester, is unfavorable.