Ephedrine-induced mitophagy via oxidative stress in human hepatic stellate cells.

The herb Ephedra sinica (also known as Chinese ephedra or Ma Huang), used in traditional Chinese medicine, contains alkaloids identical to ephedrine and pseudoephedrine as its principal active constituents. Recent studies have reported that ephedrine has various side effects in the cardiovascular and nervous systems. In addition, herbal Ephedra, a plant containing many pharmacologically active alkaloids, principally ephedrine, has been reported to cause acute hepatitis. Many studies reported clinical cases, however, the cellular mechanism of liver toxicity by ephedrine remains unknown. In this study, we investigated hepatotoxicity and key regulation of mitophagy in ephedrine-treated LX-2 cells. Ephedrine triggered mitochondrial oxidative stress and depolarization. Mitochondrial swelling and autolysosome were observed in ephedrine-treated cells. Ephedrine also inhibited mitochondrial biogenesis, and the mitochondrial copy number was decreased. Parkin siRNA recovered the ephedrine-induced mitochondrial damage. Excessive mitophagy lead to cell death through imbalance of autophagic flux. Moreover, antioxidants and reducing Parkin level could serve as therapeutic targets for ephedrine-induced hepatotoxicity.

The Effects of Gymnema sylvestre in High-Fat Diet-Induced Metabolic Disorders.

This study used an integrated approach to investigate the effects of Gymnema sylvestre (GS) extract as a functional dietary supplement with a high-fat diet. This approach examined insulin resistance, the dysfunction of adipose tissue, and liver steatosis. Male C57BL/6J mice were fed a normal chow or high-fat diet (HFD) for the acute and chronic study, in addition to GS in different doses (100, 250 and 500[Formula: see text]mg/kg body weight). Their body composition changes, serum lipid and glucose parameters, adipose and liver tissue histology, and gene expression were measured. It was found that GS significantly suppressed the increase of body weight, serum levels of lipid, insulin and leptin, and adipose tissue, and liver inflammation. GS also demonstrated hypoglycemic effects due to the amylase inhibition activity. Our results support the existence of a relationship between the HFD induced insulin resistance, adipose dysfunction and liver steatosis. In conclusion, GS works as a functional dietary supplement with preventative effects against metabolic disorder.

Biodistribution and toxicity of spherical aluminum oxide nanoparticles.

With the rapid development of the nano-industry, concerns about their potential adverse health effects have been raised. Thus, ranking accurately their toxicity and prioritizing for in vivo testing through in vitro toxicity test is needed. In this study, we used three types of synthesized aluminum oxide nanoparticles (AlONPs): γ-aluminum oxide hydroxide nanoparticles (γ-AlOHNPs), γ- and α-AlONPs. All three AlONPs were spherical, and the surface area was the greatest for γ-AlONPs, followed by the α-AlONPs and γ-AlOHNPs. In mice, γ-AlOHNPs accumulated the most 24 h after a single oral dose. Additionally, the decreased number of white blood cells (WBC), the increased ratio of neutrophils and the enhanced secretion of interleukin (IL)-8 were observed in the blood of mice dosed with γ-AlOHNPs (10 mg kg(-1)). We also compared their toxicity using four different in vitro test methods using six cell lines, which were derived from their potential target organs, BEAS-2B (lung), Chang (liver), HACAT (skin), H9C2 (heart), T98G (brain) and HEK-293 (kidney). The results showed γ-AlOHNPs induced the greatest toxicity. Moreover, separation of particles was observed in a transmission electron microscope (TEM) image of cells treated with γ-AlOHNPs, but not γ-AlONPs or α-AlONPs. In conclusion, our results suggest that the accumulation and toxicity of AlONPs are stronger in γ-AlOHNPs compared with γ-AlONPs and α-AlONPs owing their low stability within biological system, and the presence of hydroxyl group may be an important factor in determining the distribution and toxicity of spherical AlONPs.

Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells.

In this study, we examined the mechanism underlying the effect of Saururus chinensis Baill (saururaceae) on hepatocellular carcinoma HepG2 cells. HepG2 cells and Chang cells were exposed to various concentrations of S. chinensis Baill extract (SC-E) for 24 h. SC-E affected more significantly HepG2 cells than Chang cells in terms of cell viability and ATP production. Therefore, current study examined detailed mechanism how SC-E affected HepG2 cell survival. We found that SC-E (75 and 150 µg/ml) induced apoptosis via oxidative stress. SC-E also caused CCAAT-enhancer-binding protein homologous protein (CHOP) activation by dissociating the binding immunoglobulin protein (BiP) from inositol-requiring 1α (IRE1α) in the endoplasmic reticulum (ER) and induced Bax, cytochrome c release to cytosol, caspase-3 activation, and poly ADP ribose polymerase (PARP) cleavage, resulting in HepG2 cell apoptosis. Furthermore, SC-E caused ER Ca(2+) leakage into the cytosol; ER dilation and mitochondrial membrane damage were observed in transmission electron microscopy (TEM). Taken together, our results demonstrated that SC-E induced cancer cell apoptosis specifically through ER stress.

Target-specific near-IR induced drug release and photothermal therapy with accumulated Au/Ag hollow nanoshells on pulmonary cancer cell membranes.

Au/Ag hollow nanoshells (AuHNSs) were developed as multifunctional therapeutic agents for effective, targeted, photothermally induced drug delivery under near-infrared (NIR) light. AuHNSs were synthesized by galvanic replacement reaction. We further conjugated antibodies against the epidermal growth factor receptor (EGFR) to the PEGylated AuHNS, followed by loading with the antitumor drug doxorubicin (AuHNS-EGFR-DOX) for lung cancer treatment. AuHNSs showed similar photothermal efficiency to gold nanorods under optimized NIR laser power. The targeting of AuHNS-EGFR-DOX was confirmed by light-scattering images of A549 cells, and doxorubicin release from the AuHNSs was evaluated under low pH and NIR-irradiated conditions. Multifunctional AuHNS-EGFR-DOX induced photothermal ablation of the targeted lung cancer cells and rapid doxorubicin release following irradiation with NIR laser. Furthermore, we evaluated the effectiveness of AuHNS-EGFR-DOX drug delivery by comparing two drug delivery methods: receptor-mediated endocytosis and cell-surface targeting. Accumulation of the AuHNS-EGFR-DOX on the cell surfaces by targeting EGFR turned out to be more effective for lung cancer treatments than uptake of AuHNS-EGFR-DOX. Taken together, our data suggest a new and optimal method of NIR-induced drug release via the accumulation of targeted AuHNS-EGFR-DOX on cancer cell membranes.

Comparison of the toxicity of aluminum oxide nanorods with different aspect ratio.

Aluminum oxide nanoparticles are listed among 14 high-priority nanomaterials published by the Organization for Economic Co-operation and Development, but limited information is available on their potential hazards. In this study, we compared the toxicity of two different aluminum oxide nanorods (AlNRs) commercially available in vivo and in vitro. Considering aspect ratio, one was 6.2 ± 0.6 (long-AlNRs) and the other was 2.1 ± 0.4 (short-AlNRs). In mice, long-AlNRs induced longer and stronger inflammatory responses than short-AlNRs, and the degree reached the maximum on day 7 for both types and decreased with time. In addition, in vitro tests were performed on six cell lines derived from potential target organs for AlNPs, HEK-293 (kidney), HACAT (skin), Chang (liver), BEAS-2B (lung), T98G (brain), and H9C2 (heart), using MTT assay, ATP assay, LDH release, and xCELLigence system. Long-AlNRs generally produced stronger toxicity than short-AlNRs, and HEK-293 cells were the most sensitive for both AlNRs, followed by BEAS-2B cells, although results from 4 kinds of toxicity tests conflicted among the cell lines. Based on these results, we suggest that toxicity of AlNRs may be related to aspect ratio (and resultant surface area). Furthermore, novel in vitro toxicity testing methods are needed to resolve questionable results caused by the unique properties of nanoparticles.

A 13-week repeated-dose oral toxicity and bioaccumulation of aluminum oxide nanoparticles in mice.

Because of an increase in the commercial applications of manufactured nanoparticles, the issue of potential adverse health effects of nanoparticles following intended or unintended exposure is rapidly gaining attention. In this study, we evaluated the toxicity of aluminum oxide nanoparticles (AlNPs, rod-type, 1.5, 3, and 6 mg/kg) after oral administration to mice for 13 weeks. Compared with the control group, the consumption of diet and drinking water and body weight gain decreased in the group treated with AlNPs. The group treated with 6 mg/kg AlNPs also showed a marked elevation in the count of white blood cells that associated with a significant decrease and increase to the proportion of eosinophils and lymphocytes, respectively. In addition, the secretion of IL-6 and monocyte chemotactic protein-1 increased in a dose-dependent manner in the treated groups. Furthermore, AlNPs showed the highest accumulation in the liver and kidneys compared with the control group, increased the lactate dehydrogenase level in the blood, and induced the development of a pathological lesion in the liver and kidneys. Taken together, we suggest that the target organs of rod-type AlNPs may be the liver, kidneys and the immune system, and the not-observed adverse effect level may be lower than 6 mg/kg.

Ascidian tunicate extracts attenuate rheumatoid arthritis in a collagen-induced murine model.

Murine rheumatoid arthritis models are often used to investigate the potential therapeutic effects of candidate drugs. The present study has been conducted in order to investigate the therapeutic efficacy of ascidian tunicate extracts in a collagen-induced arthritis DBA1/J mice model. Four types of formulas, ascidian tunicate extracts (ATE), crude ascidian tunicate glycans (ATEC), ascidian tunicate extracts with licorice extracts (ATEL), and crude ascidian tunicate glycans with licorice extracts (ATECL) were orally administered into DBA/1J mice for 3 weeks and paw edema and thickness were evaluated. Changes in inflammatory proteins and cytokines levels were monitored in hind leg tissues by Western blot and quantitative PCR analysis. The oral administration of ascidian tunicate extracts alleviated paw edema and improved the histological hind leg cartilage status. The extracts also reduced the matrix metalloproteinase-9 (MMP-9) protein and prostaglandin E synthase (PGES) levels. In addition, the extracts-treated groups showed increased interleukin-10 (IL-10) levels compared with the non-treated group. These findings suggest that orally administered ascidian tunicate extracts might have potential therapeutic effects for the treatment of rheumatoid arthritis.

Therapeutic effect of Broussonetia papyrifera and Lonicera japonica in ovalbumin-induced murine asthma model.

Broussonetia papyrifera (L.) Vent. and Lonicera japonica Thunb. have been used in recent medicinal research for their antioxidative and anti-inflammatory properties. The present study investigated the therapeutic efficacy of B. papyrifera and L. japonica ethanolic extracts in a murine model of ovalbumin-induced asthma, in which intra-peritoneal (IP) injections and aerosol ovalbumin delivery were used to induce allergic asthma. Bronchioalveolar lavage fluid (BALF), serum samples, lungs and livers were collected from the experimental groups. In the groups treated with B. papyrifera and L. japonica extracts, CD3, CD4, serum IgE and IL-4 levels; activities of matrix metalloproteinase (MMP)-2 and MMP-9; and eotaxin levels in the BALF significantly decreased to near normal levels. Results of a histopathological analysis showed that the level of inflammation and mucous secretions reduced in the treated groups compared to the corresponding levels in the other groups. Moreover, results of a serum enzymatic analysis showed the non-toxic nature of the extracts in the B. papyrifera and L. japonica treated groups. Taken together, these results clearly indicate that the B. papyrifera and L. japonica extracts may be very effective against asthma and inflammation related diseases.

Subchronic inhalation toxicity of silver nanoparticles.

The subchronic inhalation toxicity of silver nanoparticles was studied in Sprague-Dawley rats. Eight-week-old rats, weighing approximately 253.2 g (males) and 162.6 g (females), were divided into four groups (10 rats in each group): fresh-air control, low dose (0.6 x 10(6) particle/cm(3), 49 microg/m(3)), middle dose (1.4 x 10(6) particle/cm(3), 133 microg/m(3)), and high dose (3.0 x 10(6) particle/cm(3), 515 microg/m(3)). The animals were exposed to silver nanoparticles (average diameter 18-19 nm) for 6 h/day, 5 days/week, for 13 weeks in a whole-body inhalation chamber. In addition to mortality and clinical observations, body weight, food consumption, and pulmonary function tests were recorded weekly. At the end of the study, the rats were subjected to a full necropsy, blood samples were collected for hematology and clinical chemistry tests, and the organ weights were measured. Bile-duct hyperplasia in the liver increased dose dependently in both the male and female rats. Histopathological examinations indicated dose-dependent increases in lesions related to silver nanoparticle exposure, including mixed inflammatory cell infiltrate, chronic alveolar inflammation, and small granulomatous lesions. Target organs for silver nanoparticles were considered to be the lungs and liver in the male and female rats. No observable adverse effect level of 100 microg/m(3) is suggested from the experiments.

Recovery from welding-fume-exposure-induced MRI T1 signal intensities after cessation of welding-fume exposure in brains of cynomolgus monkeys.

The shortening of the MRI T1 relaxation time, indicative of a high signal intensity in a T1-weighted MRI, is known as a useful biomarker for Mn exposure after short-term welding-fume exposure. A previous monkey experimental study found that the T1 relaxation times decreased time-dependently after exposure, and a visually detectable high signal intensity appeared after 150 days of exposure. The nadir for the shortening of the T1 relaxation time was also previously found to correspond well with the blood Mn concentration in welders, suggesting a correlation between a prolonged high blood Mn concentration and shortened T1 relaxation time. Accordingly, to clarify the clearance of the brain Mn concentration after the cessation of welding-fume exposure, cynomolgus monkeys were assigned to 3 groups-unexposed, low dose (31 mg/m(3) total suspended particulate (TSP), 0.9 mg Mn/m(3)), and high dose (62 mg/m(3) TSP, 1.95 mg Mn/m(3))-and exposed to manual metal-arc stainless steel (MMA-SS) welding fumes for 2 h per day for 8 mo in an inhalation chamber system equipped with an automatic fume generator. After reaching the peak MRI T1 signal intensity (shortest T1 relaxation time), the monkeys were allowed to recover by ceasing the welding-fume exposure. Within 2 mo, the MRI T1 signal intensities for the exposed monkeys returned to nearly the same level as those for the unexposed monkeys, indicating the potential for recovery from a high MRI T1 signal intensity induced by welding-fume exposure, even after prolonged exposure. Clearance of the Mn tissue concentration was also demonstrated in the globus pallidus, plus other tissues from the brain, liver, spleen, and blood. In contrast, there was no clearance of the lung concentrations of Mn, indicating that a soluble form of Mn was transported to the blood and brain. Therefore, the solubility of Mn in welding fumes would appear to be an important determinant as regards the retention of blood Mn levels and brain tissue Mn concentrations in welders.

Enhanced efficacy of 7-hydroxy-3-methoxycadalene via glycosylation in in vivo xenograft study.

7-Hydroxy-3-methoxycadalene, isolated from Zelkova serrata Makino, was confirmed as a biologically active natural compound. In this study, the efficacy of cadalene as an anticancer agent was tested. In order to address the poor physicochemical properties of cadalene, we designed and synthesized glycosylated cadalene derivatives for improved solubility and efficient drug delivery as a potential prodrug. In vitro cell viability assays confirmed that glycosylated cadalenes were less toxic and more soluble than cadalene. In an in vivo xenograft study in mice, the oral administration of glycosylated cadalenes caused a significant reduction in tumor size.

Antimicrobial effects of silver nanoparticles.

The antimicrobial effects of silver (Ag) ion or salts are well known, but the effects of Ag nanoparticles on microorganisms and antimicrobial mechanism have not been revealed clearly. Stable Ag nanoparticles were prepared and their shape and size distribution characterized by particle characterizer and transmission electron microscopic study. The antimicrobial activity of Ag nanoparticles was investigated against yeast, Escherichia coli, and Staphylococcus aureus. In these tests, Muller Hinton agar plates were used and Ag nanoparticles of various concentrations were supplemented in liquid systems. As results, yeast and E. coli were inhibited at the low concentration of Ag nanoparticles, whereas the growth-inhibitory effects on S. aureus were mild. The free-radical generation effect of Ag nanoparticles on microbial growth inhibition was investigated by electron spin resonance spectroscopy. These results suggest that Ag nanoparticles can be used as effective growth inhibitors in various microorganisms, making them applicable to diverse medical devices and antimicrobial control systems.

Effect of Evodiae fructus extracts on gene expressions related with alcohol metabolism and antioxidation in ethanol-loaded mice.

The effects of Evodiae fructus on alcohol concentration in blood plasma and the relative expression of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), Cu-Zn superoxide dismutase (Cu-Zn SOD), glutathione peroxidase type 5 (GPX5), and catalase (CAT) in mice were assessed 1, 2, 3, and 4h after acute alcohol consumption. Evodiae fructus extracts (2.5, 5, and 10 mL/kg-bw), a commercial hangover removal drug (2.5, 5, and 10 mL/kg-bw), and saline solution, referred to as treatment, positive control, and negative control group, respectively, were orally given at the same time as alcohol administered to mice (20% and 40% concentration). Alcohol concentrations were the lowest in all of the Evodiae fructus treatment groups, as well as one (10 mL/kg-bw) of the positive control groups, 4h after ethanol exposure (0-5mg/dL and 60-110 mg/dL for 20% and 40% ethanol-loaded mice, respectively). Overall, the relative expression of ADH and Zn-Cu SOD were higher in treatment groups than in positive controls; whereas, the relative expression of GPX5 was higher in positive control groups than in treatment groups. The relative expression of ALDH and CAT was unchanged between treatments and positive controls. This study suggests that Evodiae fructus extracts can be used as a possible therapy for alcohol-induced hangover symptoms by stimulating the expression of hepatic alcohol metabolizing and antioxidant enzymes.

Manganese distribution in brains of Sprague-Dawley rats after 60 days of stainless steel welding-fume exposure.

Welders working in a confined space, as in the shipbuilding industry, are at risk of being exposed to high concentrations of welding fumes and developing pneumoconiosis or other welding-fume exposure related diseases. Among such diseases, manganism resulting from welding-fume exposure remains a controversial issue, as the movement of manganese into specific brain regions has not yet been clearly established. Accordingly, to investigate the distribution of manganese in the brain after welding-fume exposure, male Sprague-Dawley rats were exposed to welding fumes generated from manual metal arc-stainless steel (MMA-SS) at concentrations of 63.6 +/- 4.1 mg/m(3) (low dose, containing 1.6 mg/m(3) Mn) and 107.1 +/- 6.3 mg/m(3) (high dose, containing 3.5 mg/m(3) Mn) total suspended particulate (TSP) for 2 h per day in an inhalation chamber over a 60-day period. Blood, brain, lung, and liver samples were collected after 2 h, 15, 30, and 60 days of exposure and the tissues analyzed for their manganese concentrations using an atomic absorption spectrophotometer. Although dose- and time-dependent increases in the manganese concentrations were found in the lungs and livers of the rats exposed for 60 days, only slight manganese increases were observed in the blood during this period. Major statistically significant increases in the brain manganese concentrations were detected in the cerebellum after 15 days of exposure and up until 60 days. Slight increases in the manganese concentrations were also found in the substantia nigra, basal ganglia (caudate nucleus, putamen, and globus pallidus), temporal cortex, and frontal cortex, thereby indicating that the pharmacokinetics and distribution of the manganese inhaled from the welding fumes were different from those resulting from manganese-only exposure.

Inhibitors of 5alpha -reductase type I in LNCaP cells from the roots of Angelica koreana.

A prenylated coumarin, osthenol (1) and a sesquiterpene, bisabolangelone (2) have been isolated as active principles with 5alpha-reductase type I inhibitory effects in LNCaP cells from the roots of Angelica koreana Max. by bioassay-guided chromatographic fractionation. Osthenol exhibited a highly potent inhibitory activity on 5alpha-reductase type I in LNCaP cells with an IC50 value of 0.1 microg/ml, which is about 200 times more potent than the positive control, finasteride (IC50 = 19.8 microg/ml). Bisabolangelone also inhibited the activity of 5alpha-reductase type I in LNCaP cells (IC50 = 11.6 microg/ml), indicating that these compounds are possible candidates for the development of new drugs to treat human endocrine disorders associated with overproduction of DHT by 5 alpha-reductase type I. In addition, four compounds isooxypeucedanin, oxypeucedanin hydrate, oxypeucedanin and isoimperatorin were also isolated and found to be inactive in the 5alpha-reductase assay systems used in the present study.