Uptake and toxicity of cerium dioxide nanoparticles with different aspect ratio.

Cerium dioxide nanoparticles (CeONPs) have been extensively applied in research for future energy development due to two common oxidation states on their surface. Considering that shape (aspect ratio) is a key determinant of NPs-induced toxicity, we compared the toxicity of hexagonal (H)- and rod-shaped (R)-CeONPs in mice. At 24 h after pharyngeal aspiration, both types of CeONPs recruited surrounding immune cells (monocytes and neutrophils) into the lung, and R-CeONPs induced a more severe pulmonary inflammatory response compared with H-CeONPs. To identify an indicator to predict pulmonary inflammatory responses at the cellular level, we also investigated their responses in alveolar macrophage cells. At 24 h after treatment, both types of CeONPs were mainly located within the vacuoles (partially, in the lysosome) in the cytoplasm. Mitochondrial damage, intracellular calcium accumulation, and increased NO production were observed in cells exposed to both types of CeONPs, ultimately resulting in a decrease in cell viability. More interestingly, both types of CeONPs formed multinucleated giant cells. Meanwhile, contrary to when suspended in deionized water, R-CeONPs were strongly aggregated with a negative charge in cell culture media, whereas H-CeONPs were relatively well-dispersed with a positive charge. R-CeONPs-induced lysosomal extension was also recovered by premix with negatively charged DNA, and even NPs suspended in cell culture media without cells were detected under the FACS system, suggesting interference by protein corona. Therefore, we suggest that shape (aspect ratio) is an important factor determining inhaled NPs-induced pathology and that the effect of the surface charge and protein corona should be carefully considered in interpreting results derived from in vitro tests. Furthermore, we propose that the relationship between the formation of multinucleated giant cells and the inflammatory response of inhaled CeONPs should be further studied.

Extremely low-frequency electromagnetic field induces neural differentiation of hBM-MSCs through regulation of (Zn)-metallothionein-3.

Extremely low-frequency electromagnetic field (ELFEMF) can stimulate neural differentiation in human bone marrow-derived mesenchymal cells (hBM-MSCs), and this provides an opportunity for research on neurodegenerative diseases such as Alzheimer's disease (AD). Metallothionein-3 (MT3), an isoform of the metal-binding proteins, metallothioneins, involved in maintaining intracellular zinc (Zn) homeostasis and the deregulation of zinc homeostasis, has separately been implicated in AD. Here, we investigated the effect of ELFEMF-induced neural differentiation of hBM-MSCs on Zn-MT3 homeostatic interaction. Exposure to ELFEMF induced neural differentiation of hBM-MSCs, which was characterized by decreased proliferation and enhanced neural-like morphology. We observed expression of neuronal markers such as ß-tubulin3, pleiotrophin, and neurofilament-M at the mRNA level and MAP2 at the protein level. ELFEMF-induced neural differentiation correlated with decreased expression of metal-response element-transcription factor 1 and MT3, as well as decreased intracellular Zn concentration. In addition, upregulation of dihydropyrimidinase-related protein 2 was observed, but there was no change in γ-enolase expression. These data indicate a possible regulatory mechanism for MT3 during neural differentiation. Our findings provide considerable insight into molecular mechanisms involved in neural differentiation, which is useful for developing new treatments for neurodegenerative diseases. Bioelectromagnetics. 38:364-373, 2017. © 2017 Wiley Periodicals, Inc.

Anti-diabetic effect of Wen-pi-tang-Hab-Wu-ling-san extract in streptozotocin-induced diabetic rats.

OBJECTIVES: Wen-pi-tang-Hab-Wu-ling-san (WHW) is an oriental herbal prescription formulated using 14 herbs and has been used to cure chronic renal failure in Korean oriental medicine. In this study, we investigated the anti-diabetic effect of WHW in the streptozotocin-induced diabetic rats. MATERIALS AND METHODS: Diabetes was induced by streptozotocin (STZ, 60 mg/kg, i.p.) in rats. WHW extract (100 mg/kg) was orally dosed once a day for four weeks. The results were compared with standard antidiabetic drug, glibenclamide (3 mg/kg, p.o). RESULTS: Significant decrease in body weight and insulin levels and increase in blood glucose, triglycerides, urea nitrogen (BUN), and creatinine were detected in STZ-induced diabetic rats with disruption and disappearance of pancreatic and kidney cells and decrease in insulin producing beta cells. However, these diabetic changes were significantly inhibited by treatment with WHW extract. In the oral glucose tolerance test, the extract produced a significant decrease in glycemia 60 minutes after the glucose pulse. CONCLUSIONS: Based on these results, we suggest that WHW extract has favorable effects in protecting the STZ-induced hyperglycemia, renal damage, and beta-cell damage in rats.

Hexane fraction of Zingiberis Rhizoma Crudus extract inhibits the production of nitric oxide and proinflammatory cytokines in LPS-stimulated BV2 microglial cells via the NF-kappaB pathway.

Excessive production of inflammatory mediators such as nitric oxide (NO), prostaglandin E(2) (PGE2), and proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) from activated microglia contributes to uncontrolled inflammation in neurodegenerative diseases. It seems possible that treatment with anti-inflammatory agents, including plants used in Oriental medicine, might delay the progression of neurodegeneration through the inhibition of microglial activation. The present study is focused on the inhibitory effect of the rhizome hexane fraction extract of Zingiber officinale Roscoe (ginger hexan extract; GHE) on the production of inflammatory mediators such as NO, PGE(2), and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated BV-2 cells, a mouse microglial cell line. GHE significantly inhibited the excessive production of NO, PGE(2), TNF-alpha, and IL-1beta in LPS-stimulated BV2 cells. In addition, GHE attenuated the mRNA expressions and protein levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and proinflammatory cytokines. The molecular mechanisms that underlie GHE-mediated attenuation are related to the inhibition of the phosphorylation of three mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase (JNK), and the activation of nuclear factor-kappaB (NF-kappaB). Our results indicate that GHE exhibits anti-inflammatory properties by suppressing the transcription of inflammatory mediator genes through the MAPK and NF-kappaB signaling pathways. The anti-inflammatory properties of GHE may make it useful as a therapeutic candidate for the treatment of human neurodegenerative diseases.

Wen-Pi-Tang-Hab-Wu-Ling-San extract inhibits the release of inflammatory mediators from LPS-stimulated mouse macrophages.

AIM OF THE STUDY: Wen-Pi-Tang is a traditional herbal prescription that has been used traditionally for the treatment of various inflammatory diseases, including chronic renal failure, renal injury, renal tubular cell damage and diabetic nephropathy. In this study, we investigated the pharmacological activity of modified Wen-Pi-Tang, Wen-Pi-Tang-Hab-Wu-Ling-San (WHW) extract. MATERIALS AND METHODS: Production of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6 and NO in supernatant, mRNA expression of TNF-alpha, IL-1beta, IL-6 and iNOS, protein expression of iNOS, phosphorylation of mitogen-activated protein kinases (MAPKs) and activation of nuclear factor-kappa B in the extract were assayed. RESULTS: We found that WHW extract had potent anti-inflammatory effects in LPS-stimulated RAW264.7 cells and primary peritoneal macrophages. WHW extract strongly inhibited the excessive production of inflammatory mediators, nitric oxide (NO), TNF-alpha (TNF-alpha), interleukin 1-beta (IL-1beta) and IL-6 in LPS-stimulated macrophages. The inhibition of inducible nitric oxide synthase (iNOS) and these cytokines resulted from the reduced expressions of mRNAs of iNOS and these cytokines, respectively. WHW extract attenuated the phosphorylation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinase (JNK), as well as the activation of nuclear factor-kappa B (NF-kappaB) in LPS-stimulated RAW264.7 cells. CONCLUSIONS: These data suggest that WHW extract may exhibit anti-inflammatory effects through the modulation of MAPK and the NF-kappaB-dependent pathway involved in inflammation.

Wen-pi-tang-Hab-Wu-ling-san attenuates kidney ischemia/reperfusion injury in mice. A role for antioxidant enzymes and heat-shock proteins.

The purpose of this study was to investigate the effects of Wen-pi-tang-Hab-Wu-ling-san (WHW) extract, which has been used for treatment of renal diseases, on kidney ischemia/reperfusion (I/R) injury. Thirty minutes of bilateral renal ischemia resulted in disruption of kidney tubular epithelial cells and increased plasma creatinine levels in mice, however these effects were significantly attenuated when WHW was administered prior to I/R. WHW-administration also inhibited post-ischemic decreases of catalase, copper-zinc superoxide dismutase (CuZnSOD), and manganese superoxide dismutase (MnSOD) activity in kidney tissue, leading to decreased tissue hydrogen peroxide levels and lipid peroxidation. Post-ischemic increases of heat-shock protein (HSP)-27 and -72 expressions were greater in mouse kidneys that received WHW. In conclusion, WHW-administration reduced kidney susceptibility to I/R injury, and this reduced susceptibility was associated with greater post-ischemic activation of catalase, CuZnSOD and MnSOD, resulting in reduced hydrogen peroxide levels and lipid peroxidation, as well as higher post-ischemic expression of HSP-27 and -72.

Wen-pi-tang-Hab-Wu-ling-san, an oriental herbal prescription, attenuates epithelial-mesenchymal transdifferentiation stimulated by TGF-beta1 in kidney cells.

Wen-pi-tang-Hab-Wu-ling-san (WHW), an oriental herbal prescription, is currently used in oriental clinics for the treatment of chronic renal failure (CRF). While its effectiveness has been supported by a series of modern studies, the underlying mechanism remains poorly understood. CRF progression involves tubulointerstitial fibrosis where transforming growth factor-beta1 (TGF-beta1) plays a critical role by inducing epithelial-mesenchymal transdifferentiation (EMT). This study examined whether WHW extract attenuated the TGF-beta1-induced EMT in Madin-Darby canine kidney cells. When the cells were stimulated by TGF-beta1 (2.5 ng/mL), they exhibited an elongated, spindle-shaped appearance but this morphological change was significantly suppressed by WHW extract (1 mg/mL). The WHW extract did not show notable cytotoxicity and even mitigated the cytotoxic effects of TGF-beta1. It inhibited the expression of alpha-smooth muscle actin (alpha-SMA), a marker of EMT, but not the secretion of matrix metalloproteinases stimulated by TGF-beta1. The WHW extract also inhibited the phosphorylation of Smad2 that mediates TGF-beta1 signaling leading to alpha-SMA expression. The present study suggests that WHW extract may provide renal protective effects through modulation of the TGF-beta1/Smad2/alpha-SMA pathway involved in fibrosis.

Effects of Bambusae concretio Salicea (Chunchukhwang) on amyloid beta-induced cell toxicity and antioxidative enzymes in cultured rat neuronal astrocytes.

Bambusae concretio Salicea (BCS; plant family name: Phyllostachys bambusoides Siebold et Zuccarinii) is a medicinal plant used in Korea for the treatment of various symptoms accompanying hypertension and cerebrovascular disorders. Previously, it was shown that BCS is an effective protectant against oxidative glutamate toxicity in the murine neuroblastoma cells and human neuroblastoma cells. Treatment with BCS increased the secretion of the non-amyloidogenic amyloid precursor protein fragment, and decreased the secretion of amyloid-beta (Abeta) peptides from neuronal cells [Jeong, J.C., Seo, Y.J., Kim, H.M., Lee, Y.C., Kim, C.H., 2003. Inhibitory effects of Bombusae concretio Salicea on neuronal secretion of Alzheimer's beta-amyloid peptides, a neuro-degenerative peptide. Neurochemical Research 28, 1785-1792.]. To further examine the pharmacological activity of BCS, we studied the protective effect of the water extracts on Abeta25-35 peptide-induced neuronal death by microscopic observation and lactate dehydrogenase (LDH) assay, and action on antioxidative enzymes using cultured astrocyte cells. Ten microM Abeta25-35-induced cell death was protected by the application of water extract of BCS in a dose-dependent manner, and concentrations of 1-10 microg/ml had a significant effect compared to exposure to Abeta25-35 only. When antioxidative enzyme activities such as catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) were assayed after Abeta25-35 treatment, the enzymes were decreased in a similar fashion. However, those activities were enhanced by BCS treatment and this may have resulted from the potentiation of antioxidative ability by BCS. The ability of BCS to reduce cellular cytotoxicity induced by 10 microM Abeta25-35 suggests that BCS may be a protective agent for free radical generating compounds such as Abeta25-35, and that Abeta25-35 is not only a potent lipid peroxide inducer, but also causes changes in antioxidative enzymes. From the results, it was concluded that BCS has a protective effect on Abeta-induced neuronal death in cultured astrocyte cells through the inhibition of lipid peroxidation and protection of antioxidative enzymes.

Effects of Drynariae rhizoma on the proliferation of human bone cells and the immunomodulatory activity.

Drynariae Rhizoma (DR), a traditional Korea medicine, which is known for its effect to strengthen myoskeletal systems, frequently appears as the main ingredient in prescriptions for bone injuries. However, it is unclear how it pharmacologically contributes to the reformation of bone. In this study, the effect of DR on bone cells was investigated in vitro for the first time. The human osteoprecursor cells (OPC-1) were incubated in the medium with different concentrations of DR and the cell proliferation was studied. When the concentration of DR was < or = 120 microg ml(-1), the proliferation of OPC-1 was enhanced. However, the proliferation of OPC-1 was inhibited by DR with the concentrations of > 250 microg ml(-1). Under most treatments, the cells presented very pale expression for cyclooxygenase-2 (Cox-2) protein; slightly intensified band showed at the highest DR concentration, 120 microg ml(-1) during the course of culture. On the other hand, we investigated the immunomodulatory activity of DR on cellular and humoral immunity. When different doses of ethanolic and water extracts of DR was administered to mice, it was dose-dependently potentiated the delayed-type hypersensitivity reaction induced by both sheep red blood cells (SRBC) and oxazolone. It significantly enhanced the production of circulating antibody titre in mice in response to SRBC. But, DR did not any effect on macrophage phagocytosis. Prolonged administration of DR significantly ameliorated the total white blood cell count and also restored the immunosuppressive effects induced by cyclophosphamide. The present investigation reveals that DR possesses immunomodulatory activity. From the results, it was concluded that DR directly stimulated the proliferation, alkaline phosphatase activity, protein secretion and particularly type I collagen synthesis of OPC-1 at dose-dependent manner, and stimulated both the cellular and the humoral immunity.

Stimulative effects of Drynariae Rhizoma extracts on the proliferation and differentiation of osteoblastic MC3T3-E1 cells.

Pharmacological factors are needed to prevent bone loss that occurs with increasing age. The chemical compounds that act on bone metabolism in herbal medicines, however, are poorly understood. Effects of traditional Korean medicine, Drynariae Rhizoma [Drynaria fortunei (kunze) J. Sm] extract (DR), on the osteoblastic proliferation and differentiation were investigated. The effect of DR, a natural phyto herb, on the proliferation and osteoblastic differentiation in non-transformed osteoblastic cells (MC3T3-E1) was studied. DR dose-dependently increased DNA synthesis (significant at 50-150 microg/ml). DR increased alkaline phosphatase (ALP) activity and prolyl hydroxylase activity of MC3T3-E1 cells (50-150 microg/ml). Antiestrogen tamoxifen eleminated the stimulation of proliferation and ALP activity of MC3T3-E1, which were induced by DR. DR at concentrations ranged from 30-100 microg/ml inhibited prostaglandin E2 production in MC3T3-E1. These results indicate that DR directly stimulates cell proliferation and differentiation of osteoblasts. These results also suggest and DR is effective for bone anti-resorptive action in bone cells.

Inhibitory activity of Drynariae rhizoma extracts on cathepsin having bone resorption activity.

Effects of traditional Korean (Hanbang) medicine, Drynariae rhizoma (DR), on the protease activity of bone loss-initiation in rats and mice were investigated. Ethanol extracts-DR (EE-DR) and water extracts-DR (WE-DR) were identified as potent inhibitor of cathepsins K and L. The original WE-DR inhibits cathepsins K and L with IC50 values of 3.7 microg/ml and 4.5 microg/ml, respectively. EE-DR was more potent than that of WE-DR, because the inhibitions of cathepsin K and L increased to 0.5 microg/ml and 0.8 microg/ml, respectively. The EE-DR was proved to be the most potent. EE-DR was found to be a potent inhibitor of cathepsins K with a Ki value of 5.0 microg/ml for cathepsin K. The activity was increased by 10-fold when the assay is performed in the presence of glutathione at pH 7.0, which favors the formation of a GSH thiolate anion. Thus, it is suggested that this increase in potency is probably due to an enhanced chemical reactivity of the extract mixtures toward the thiolate of the active site of the enzyme. WE-DR exhibited time-dependet inhibition which allowed us to determine the association and dissociation rate constants with cathepsin K. Finally, EE-DR inhibits bone resorption in an in vitro assay involving mouse osteoclasts and bovine bone with an IC50 value of 70 microg/ml. WE-DR represents a new herbal formulation inhibiting cathepsin K and L activity and proteolysis of bone collagen. These results strongly suggest that DR is effective for preventing the development of bone loss induced by cathepsin K. This result also suggested that the DR is effective for bone resorptive action in bone cells.

Drynariae Rhizoma promotes osteoblast differentiation and mineralization in MC3T3-E1 cells through regulation of bone morphogenetic protein-2, alkaline phosphatase, type I collagen and collagenase-1.

In a previous study (Jeong et al., 2003, Inhibition of Drynariae Rhizoma extracts on bone resorption mediated by processing of cathepsin K in cultured mouse osteoclasts. International Immunopharmacology 3, 1685-1697), treatment of osteoclasts-containing long bone cells with Drynariae Rhizoma (DR) extract prevented the intracellular maturation of cathepsin K and thus, it was considered that DR is a pro-drug of a potent bone resorption inhibitor. To further clarify the role of DR in ossification, we investigated the effects of DR on the proliferation and differentiation of osteoblastic cell lines in vitro. In this study, the bone effect of DR is studied. We assessed the effects of DR on osteoblastic differentiation in nontransformed osteoblastic cells (MC3T3-E1) and rat bone marrow cells. DR enhanced alkaline phosphatase (ALP) activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the DR was observed at relatively low doses (significant at 50-150 microg/ml and maximal at 150 microg/ml). Northern blot analysis showed that the DR (100 microg/ml) increased in bone morphogenetic protein-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. DR (60 microg/ml) slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 15 and 20 of culture. These results indicate that DR has anabolic effects on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic bone diseases such as osteoporosis.

Inhibitory effects of Bombusae concretio Salicea on neuronal secretion of Alzheimer's beta-amyloid peptides, a neurodegenerative peptide.

Alzheimer's disease (AD) is characterized by the age-related deposition of beta-amyloid (A beta) 40/42 peptide aggregates in vulnerable brain regions. Multiple levels of evidence implicate a central role for A beta in the pathophysiology of AD. A beta is generated by the regulated cleavage of a = 700 amino acid A beta precursor protein (betaAPP). Full-length betaAPP can undergo proteolytic cleavage either within the A beta domain to generate secreted sbetaAPP alpha or at the N-terminal and C-terminal domain(s) of A beta to generate amyloidogenic A beta peptides. Several epidemiological studies have reported that estrogen replacement therapy protects against the development of AD in postmenopausal women. The aim of this study was to elucidate the antioxidant neuroprotective mechanism of Bombusae concretio Salicea (BC). BC was effective protectants against oxidative glutamate toxicity in the murine neuroblastoma cells (N2a) and human neuroblastoma cells (SK-N-MC). BC exhibited similar protective properties against oxidative glutamate toxicity and H2O2 toxicity. BC exhibited an antioxidant activity at approximately 20 microg/ml. BC of 5 microg/ml was ineffective in preventing the oxidative modification of LDL. The half-maximal effective concentration for BC was 16 microg/ml. These results suggested that BC supplementation in elderly men may be protective in the treatment of Alzheimer's disease (AD). We report here that treatment with BC increases the secretion of the nonamyloidogenic APP fragment, sbetaAPP alpha and decreases the secretion of A beta peptides from N2a cells and rat primary cerebrocortical neurons. These results raise the possibility that BC supplementation in elderly men may be protective in the treatment of AD.