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At least 973 records · Page 54
All sourcesNASA NTRSDOE OSTI
DOE OSTI2014

SIRT2 ameliorates lipopolysaccharide-induced inflammation in macrophages

Highlights: • Knockout of SIRT2 attenuates lipopolysaccharide-induced iNOS expression. • Lipopolysaccharide-induced NO production is decreased in SIRT2 KO macrophage. • SIRT2 deficiency suppresses lipopolysaccharide-induced ROS production in macrophage. • M1-macrophage related factors are decreased in SIRT2 deficient cells. • SIRT2 deficiency decreases lipopolysaccharide-induced activation of NFκB. - Abstract: Introduction: SIRT2 is a NAD(+)-dependent deacetylases and associated with numerous processes such as infection, carcinogenesis, DNA damage and cell cycle regulation. However, the role of SIRT2 in inflammatory process in macrophage remains unclear. Materials and methods: In the present study, we have evaluated the regulatory effects of SIRT2 in lipopolysaccharide (LPS)-stimulated macrophages isolated from SIRT2 knockout (KO) and wild type (WT) mice or Raw264.7 macrophage cells. As inflammatory parameters, expression of inducible nitric oxide synthase (iNOS), the productions of nitric oxide, reactive oxygen species (ROS) and M1-macrophage-related factors were evaluated. We also examined the effects of SIRT2 on activation of nuclear factor-kappaB (NFκB) signaling. Results: SIRT2 deficiency inhibits LPS-induced iNOS mRNA and protein expression in bone marrow derived macrophages. SIRT2-siRNA transfection also suppressed LPS-induced iNOS expression in Raw264.7 macrophage cells. Bone marrow derived macrophages isolated from SIRT2 KO mice produced lower nitric oxide and expressed lower levels of M1-macrophage related markers including iNOS and CD86 in response to LPS than WT mice. Decrease of SIRT2 reduced the LPS-induced reactive oxygen species production. Deficiency of SIRT2 resulted in inhibition of NFκB activation through reducing the phosphorylation and degradation of IκBα. The phosphorylation and nuclear translocation of p65 was significantly decreased in SIRT2-deficient macrophages after LPS stimulation. Discussion: Our data suggested that deficiency of SIRT2 ameliorates iNOS, NO expression and reactive oxygen species production with suppressing LPS-induced activation of NFκB in macrophages.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

DNA–PKcs–SIN1 complexation mediates low-dose X-ray irradiation (LDI)-induced Akt activation and osteoblast differentiation

Highlights: • LDI increases ALP activity, promotes type I collagen (Col I)/Runx2 mRNA expression. • LDI induces DNA–PKcs activation, which is required for osteoblast differentiation. • Akt activation mediates LDI-induced ALP activity and Col I/Runx2 mRNA increase. • DNA–PKcs–SIN1 complexation mediates LDI-induced Akt Ser-473 phosphorylation. • DNA–PKcs–SIN1 complexation is important for osteoblast differentiation. - Abstract: Low-dose irradiation (LDI) induces osteoblast differentiation, however the underlying mechanisms are not fully understood. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA–PKcs)–Akt signaling in LDI-induced osteoblast differentiation. We confirmed that LDI promoted mouse calvarial osteoblast differentiation, which was detected by increased alkaline phosphatase (ALP) activity as well as mRNA expression of type I collagen (Col I) and runt-related transcription factor 2 (Runx2). In mouse osteoblasts, LDI (1 Gy) induced phosphorylation of DNA–PKcs and Akt (mainly at Ser-473). The kinase inhibitors against DNA–PKcs (NU-7026 and NU-7441) or Akt (LY294002, perifosine and MK-2206), as well as partial depletion of DNA–PKcs or Akt1 by targeted-shRNA, dramatically inhibited LDI-induced Akt activation and mouse osteoblast differentiation. Further, siRNA-knockdown of SIN1, a key component of mTOR complex 2 (mTORC2), also inhibited LDI-induced Akt Ser-473 phosphorylation as well as ALP activity increase and Col I/Runx2 expression in mouse osteoblasts. Co-immunoprecipitation (Co-IP) assay results demonstrated that LDI-induced DNA–PKcs–SIN1 complexation, which was inhibited by NU-7441 or SIN1 siRNA-knockdown in mouse osteoblasts. In summary, our data suggest that DNA–PKcs–SIN1 complexation-mediated Akt activation (Ser-473 phosphorylation) is required for mouse osteoblast differentiation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

CXC195 suppresses proliferation and inflammatory response in LPS-induced human hepatocellular carcinoma cells via regulating TLR4-MyD88-TAK1-mediated NF-κB and MAPK pathway

Highlights: • CXC195 exhibited significant anti-proliferative effect and induced cell cycle arrest in LPS-induced HepG2 cells. • CXC195 suppressed the release of pro-inflammatory mediators in LPS-induced HepG2 cells. • CXC195 regulated TLR4-MyD88-TAK1-mediated NF-κB and MAPK pathway in LPS-induced HepG2 cells. - Abstract: CXC195 showed strong protective effects in neuronal apoptosis by exerting its antioxidant activity. However, the anti-cancer effects of CXC195 is still with limited acquaintance. Here, we investigated the role of CXC195 in lipopolysaccharide (LPS)-induced human hepatocellular carcinoma (HCC) cells lines (HepG2) and the possible signaling pathways. CXC195 exhibited significant anti-proliferative effect and induced cell cycle arrest in LPS-induced HepG2 cells. In addition, CXC195 suppressed the release of pro-inflammatory mediators in LPS-induced HepG2 cells, including TNF-α, iNOS, IL-1β, IL-6, CC chemokine ligand (CCL)-2, CCL-22 and epidermal growth factor receptor (EGFR). Moreover, CXC195 inhibited the expressions and interactions of TLR4, MyD88 and TAK1, NF-κB translocation to nucleus and its DNA binding activity, phosphorylation of ERK1/2, p38 and JNK. Our results suggested that treatment with CXC195 could attenuate the TLR4-mediated proliferation and inflammatory response in LPS-induced HepG2 cells, thus might be beneficial for the treatment of HCC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Eicosapentaenoic acid (EPA) induced apoptosis in HepG2 cells through ROS–Ca{sup 2+}–JNK mitochondrial pathways

Highlights: • EPA evoked ROS formation, [Ca{sup 2+}]{sub c} accumulation, the opening of MPTP and the phosphorylation of JNK. • EPA-induced [Ca{sup 2+}]{sub c} elevation was depended on production of ROS. • EPA-induced ROS generation, [Ca{sup 2+}]{sub c} increase, and JNK activated caused MPTP opening. • The apoptosis induced by EPA was related to release of cytochrome C through the MPTP. • EPA induced HepG2 cells apoptosis through ROS–Ca{sup 2+}–JNK mitochondrial pathways. - Abstract: Eicosapentaenoic acid (EPA), a well-known dietary n−3 PUFAS, has been considered to inhibit proliferation of tumor cells. However, the molecular mechanism related to EPA-induced liver cancer cells apoptosis has not been reported. In this study, we investigated the effect of EPA on HepG2 cells proliferation and apoptosis mechanism through mitochondrial pathways. EPA inhibited proliferation of HepG2 cells in a dose-dependent manner and had no significant effect on the cell viability of humor normal liver L-02 cells. It was found that EPA initially evoked ROS formation, leading to [Ca{sup 2+}]{sub c} accumulation and the mitochondrial permeability transition pore (MPTP) opening; EPA-induced HepG2 cells apoptosis was inhibited by N-acetylcysteine (NAC, an inhibitor of ROS), 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, a chelator of calcium) and CsA (inhibitor of MPTP). The relationship between ROS production, the increase of cytoplasmic Ca and MPTP opening was detected. It seems that ROS may act as an upstream regulator of EPA-induced [Ca{sup 2+}]{sub c} generation, moreover, generation of ROS, overload of mitochondrial [Ca{sup 2+}]{sub c}, and JNK activated cause the opening of MPTP. Western blotting results showed that EPA elevated the phosphorylation status of JNK, processes associated with the ROS generation. Simultaneously, the apoptosis induced by EPA was related to release of cytochrome C from mitochondria to cytoplasm through the MPTP and activation of caspase-9 and caspase-3. These results suggest that EPA induces apoptosis through ROS–Ca{sup 2+}–JNK mitochondrial pathways.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Amyloid β oligomers induce interleukin-1β production in primary microglia in a cathepsin B- and reactive oxygen species-dependent manner

Amyloid β (Aβ) peptide, a causative agent of Alzheimer's disease, forms two types of aggregates: oligomers and fibrils. These aggregates induce inflammatory responses, such as interleukin-1β (IL-1β) production by microglia, which are macrophage-like cells located in the brain. In this study, we examined the effect of the two forms of Aβ aggregates on IL-1β production in mouse primary microglia. We prepared Aβ oligomer and fibril from Aβ (1–42) peptide in vitro. We analyzed the characteristics of these oligomers and fibrils by electrophoresis and atomic force microscopy. Interestingly, Aβ oligomers but not Aβ monomers or fibrils induced robust IL-1β production in the presence of lipopolysaccharide. Moreover, Aβ oligomers induced endo/phagolysosome rupture, which released cathepsin B into the cytoplasm. Aβ oligomer-induced IL-1β production was inhibited not only by the cathepsin B inhibitor CA-074-Me but also by the reactive oxygen species (ROS) inhibitor N-acetylcysteine. Random chemical crosslinking abolished the ability of the oligomers to induce IL-1β. Thus, multimerization and fibrillization causes Aβ oligomers to lose the ability to induce IL-1β. These results indicate that Aβ oligomers, but not fibrils, induce IL-1β production in primary microglia in a cathepsin B- and ROS-dependent manner. - Highlights: • We prepared amyloid β (Aβ) fibrils with minimum contamination of Aβ oligomers. • Primary microglia (MG) produced IL-1β in response to Aβ oligomers, but not fibrils. • Only Aβ oligomers induced leakage of cathepsin B from endo/phagolysosomes. • IL-1β production in response to Aβ oligomers depended on both cathepsin B and ROS. • Crosslinking reduced the ability of the Aβ oligomers to induce IL-1β from MG.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Fenoterol inhibits LPS-induced AMPK activation and inflammatory cytokine production through β-arrestin-2 in THP-1 cell line

The AMP-activated protein kinase (AMPK) pathway is involved in regulating inflammation in several cell lines. We reported that fenoterol, a β{sub 2}-adrenergic receptor (β{sub 2}-AR) agonist, had anti-inflammatory effects in THP-1 cells, a monocytic cell line. Whether the fenoterol anti-inflammatory effect involves the AMPK pathway is unknown. In this study, we explored the mechanism of β{sub 2}-AR stimulation with fenoterol in a lipopolysaccharide (LPS)-induced inflammatory cytokine secretion in THP-1 cells. We studied whether fenoterol and β-arrestin-2 or AMPKα1 subunit knockdown could affect LPS-induced AMPK activation, nuclear factor-kappa B (NF-κB) activation and inflammatory cytokine secretion. LPS-induced AMPK activation and interleukin 1β (IL-1β) release were reduced with fenoterol pretreatment of THP-1 cells. SiRNA knockdown of β-arrestin-2 abolished the fenoterol inhibition of LPS-induced AMPK activation and interleukin 1β (IL-1β) release, thus β-arrestin-2 mediated the anti-inflammatory effects of fenoterol on LPS-treated THP-1 cells. In addition, siRNA knockdown of AMPKα1 significantly attenuated the LPS-induced NF-κB activation and IL-1β release, so AMPKα1 was a key signaling molecule involved in LPS-induced inflammatory cytokine production. These results suggested the β{sub 2}-AR agonist fenoterol inhibited LPS-induced AMPK activation and IL-1β release via β-arrestin-2 in THP-1 cells. The exploration of these mechanisms may help optimize therapeutic agents targeting these pathways in inflammatory diseases. - Highlights: • β{sub 2}-AR agonist fenoterol exerts its protective effect on LPS-treated THP-1 cells. • Fenoterol inhibits LPS-induced AMPK activation and IL-1β production. • β-arrestin2 mediates fenoterol-inhibited AMPK activation and IL-1β release. • AMPKα1 is involved in LPS-induced NF-κB activation and IL-1β production.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

12-oxo-phytodienoic acid, a plant-derived oxylipin, attenuates lipopolysaccharide-induced inflammation in microglia

Jasmonates are plant lipid–derived oxylipins that act as key signaling compounds in plant immunity, germination, and development. Although some physiological activities of natural jasmonates in mammalian cells have been investigated, their anti-inflammatory actions in mammalian cells remain unclear. Here, we investigated whether jasmonates protect mouse microglial MG5 cells against lipopolysaccharide (LPS)–induced inflammation. Among the jasmonates tested, only 12-oxo-phytodienoic acid (OPDA) suppressed LPS-induced expression of the typical inflammatory cytokines interleukin-6 and tumor necrosis factor α. In addition, only OPDA reduced LPS-induced nitric oxide production through a decrease in the level of inducible nitric oxide synthase. Further mechanistic studies showed that OPDA suppressed neuroinflammation by inhibiting nuclear factor κB and p38 mitogen-activated protein kinase signaling in LPS-activated MG5 cells. In addition, OPDA induced expression of suppressor of cytokine signaling-1 (SOCS-1), a negative regulator of inflammation, in MG5 cells. Finally, we found that the nuclear factor erythroid 2-related factor 2 signaling cascade induced by OPDA is not involved in the anti-inflammatory effects of OPDA. These results demonstrate that OPDA inhibited LPS-induced cell inflammation in mouse microglial cells via multiple pathways, including suppression of nuclear factor κB, inhibition of p38, and activation of SOCS-1 signaling. -- Highlights: •OPDA attenuates LPS-induced inflammatory cytokines such as IL-6 and TNF-α. •OPDA reduces LPS-induced iNOS expression and NO production. •OPDA suppresses NF-κB and p38 pathways and activates SOCS-1 signaling.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Cyclophilin B protects SH-SY5Y human neuroblastoma cells against MPP{sup +}-induced neurotoxicity via JNK pathway

Parkinson's disease (PD) is the second most common neurodegenerative disorder of aging. PD involves a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyidine (MPTP) and its toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) inhibit the complex I of the mitochondrial electron transport chain, and have been widely used to construct PD models. Cyclophilin B (CypB) is an endoplasmic reticulum protein that binds to cyclosporine A as a cyclophilin family member. CypB has peptidyl-prolyl cis-trans isomerase (PPIase) activity. We investigated the protective effects of overexpressed CypB on MPP+-induced neurocytotoxicity in SH-SY5Y human neuroblastoma cells. Overexpressed CypB decreased MPP{sup +}-induced oxidative stress through the modulation of antioxidant enzymes including manganese superoxide dismutase and catalase, and prevented neurocytotoxicity via mitogen-activated protein kinase, especially the c-Jun N-terminal kinase pathway. In addition, CypB inhibited the activation of MPP{sup +}-induced the pro-apoptotic molecules poly (ADP-ribose) polymerase, Bax, and Bcl-2, and attenuated MPP{sup +}-induced mitochondrial dysfunction. The data suggest that overexpressed CypB protects neuronal cells from MPP+-induced dopaminergic neuronal cell death. - Highlights: • CypB protects SH-SY5Y cells against MPP{sup +}-induced cell death. • CypB suppresses MPP{sup +}-induced oxidative stress. • CypB inhibits the activation of MPP{sup +}-induced pro-apoptotic and MAPK proteins. • CypB attenuates MPP{sup +}-induced mitochondrial dysfunction.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

WNT/β-catenin pathway modulates the TNF-α-induced inflammatory response in bronchial epithelial cells

In this study, TNF-α was found to activate the WNT/β-catenin pathway in BEAS-2B human bronchial epithelial cells. Levels of phospho-LRP6, Dvl-2, and phospho-GSK-3β were elevated, while that of Axin was reduced by TNF-α treatment. Nuclear translocation of β-catenin and the reporter activity of a β-catenin-responsive promoter were increased by TNF-α treatment. Under the same experimental conditions, TNF-α activated the NF-κB signaling, which includes the phosphorylation and degradation of IκB and nuclear translocation and target DNA binding of NF-κB, and it was found that an inhibitor of NF-κB activation, JSH-23, inhibited TNF-α-induced Wnt signaling as well as NF-κB signaling. It was also found that recombinant Wnt proteins induced NF-κB nuclear translocations and its target DNA binding, suggesting that Wnt signaling and NF-κB signaling were inter-connected. TNF-α-induced modulations of IκB and NF-κB as well as pro-inflammatory cytokine expression were significantly suppressed by the transfection of β-catenin siRNA compared to that of control siRNA. Transfection of a β-catenin expression plasmid augmented the TNF-α-induced modulations of IκB and NF-κB as well as pro-inflammatory cytokine expression. These results clearly demonstrated that the WNT/β-catenin pathway modulates the inflammatory response induced by TNF-α, suggesting that this pathway may be a useful target for the effective treatment of bronchial inflammation. - Highlights: • TNF-α activated the WNT/β-catenin pathway in bronchial epithelial cells. • TNF-α-induced WNT/β-catenin pathway was inhibited by NF-κB inhibitor. • Wnt proteins induced nuclear translocation and target DNA binding of NF-κB. • TNF-α-induced NF-κB signaling was suppressed by β-catenin knock-down. • TNF-α-induced NF-κB signaling was augmented by ectopic β-catenin expression.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Pyruvic acid prevents Cu2+/Zn2+-induced neurotoxicity by suppressing mitochondrial injury

Highlights: • Pyruvic acid clearly suppressed Cu{sup 2+}/Zn{sup 2+}-induced neurotoxicity in mouse hypothalamic neuronal cells (GT1-7 cells). • Pyruvic acid significantly suppressed cytochrome c release into cytoplasm, an index of mitochondrial injury. • Pyruvic acid may be therapeutically beneficial for neurological diseases involving neuronal cell death. Zinc (Zn) is known as a co-factor for over 300 metalloproteins or enzymes, and has essential roles in many physiological functions. However, excessively high Zn concentrations are induced in pathological conditions such as interruption of blood flow in stroke or transient global ischemia-induced neuronal cell death. Furthermore, we recently found that copper (Cu{sup 2+}) significantly exacerbates Zn{sup 2+} neurotoxicity in mouse hypothalamic neuronal cells, suggesting that Zn{sup 2+} interaction with Cu{sup 2+} is important for the development of neurological disease. Meanwhile, organic acids such as pyruvic acid and citric acid are reported to prevent neuronal cell death induced by various stresses. Thus, in this study, we focused on organic acids and searched for compounds that inhibit Cu{sup 2+}/Zn{sup 2+}-induced neurotoxicity. Initially, we examined the protective effect of various organic acids on Cu{sup 2+}/Zn{sup 2+}-induced neurotoxicity, and found that pyruvic acid clearly suppresses Cu{sup 2+}/Zn{sup 2+}-induced neurotoxicity in GT1-7 cells. Next, we examined the protective mechanisms of pyruvic acid against Cu{sup 2+}/Zn{sup 2+}-induced neurotoxicity. Specifically, we examined the possibilities that pyruvic acid chelates Cu{sup 2+} and Zn{sup 2+} or suppresses the ER stress response, but found that neither was suppressed by pyruvic acid treatment. In contrast, pyruvic acid significantly suppressed cytochrome c release into cytoplasm, an index of mitochondrial injury, in a dose-dependent manner. These results suggest that pyruvic acid prevents Cu{sup 2+}/Zn{sup 2+}-induced neuronal cell death by suppressing mitochondrial injury. Based on our results, we assume that pyruvic acid may be therapeutically beneficial for neurological diseases involving neuronal cell death such as vascular dementia.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Role of LRRK2 in manganese-induced neuroinflammation and microglial autophagy

Highlights: • Manganese exposure up-regulated LRRK2 expression in substantia nigra. • LRRK2 expression was increased in BV2 microglia after Mn treatment. • Inhibiting LRRK2 expression inhibited Mn-induced microglia neuroinflammation. • LRRK2 regulated Mn-induced microglia autophagic disorders. Overexposure to manganese (Mn) leads to manganism and neurotoxicity induced by Mn is the focus of recent research. Microglia play a vital role in Mn-induced neurotoxicity, and our previous studies firstly showed that Mn could stimulate activation of microglia, leading to the neuroinflammation, and inhibition of microglial inflammation effectively attenuated Mn-induced death of dopamine neurons. However, the detailed mechanism of manganese-induced neuroinflammation is still unclear. Leucine rich repeat kinase 2 (LRRK2) is a key molecule in the pathogenesis of many neurodegenerative disorders. Recent studies have indicated that LRRK2, which is highly expressed in microglia, plays a specific role in microglia and autophagy process. In this paper, we try to find the effect of LRRK2 on Mn-triggered neuroinflammation and its possible mechanism in vivo and in vitro. By establishing a Mn exposure animal model, our studies found that Mn exposure could induce dopaminergic neurons damage and activate microglia. Activated microglia triggered neuroinflammation by releasing multiple inflammatory cytokines, and the expression of LRRK2 was upregulated in vivo and in vitro. We also found that Mn exposure induced autophagy dysfunction in vivo and in vitro. Next, we used LRRK2 siRNA and LRRK2-IN-1 to inhibit the expression of LRRK2, and found that inhibition of LRRK2 could not only decrease the expression of inflammatory cytokines, but also recover autophagic function of microglia. Our investigation not only reveals the role of LRRK2 in Mn-induced neuroinflammation but also sheds light on the prevention and protection of manganism.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Yoda1-induced phosphorylation of Akt and ERK1/2 does not require Piezo1 activation

Highlights: • Yoda1 induces activation of both Akt and ERK1/2 in endothelial cells (ECs). • Gadolinium and ruthenium red both block Yoda1-induced Akt phosphorylation. • GsMTx4 does not block Yoda1-induced Akt or ERK1/2 phosphorylation. • Yoda1-induced activation of Akt and ERK1/2 in ECs are Piezo1-independent. Piezo1 is a mechanosensitive cation channel that is activated by shear stress in endothelial cells (ECs). It has been shown to mediate shear-induced EC responses, including increased calcium influx, and vascular functions, such as vascular tone and blood pressure. Yoda1, a selective Piezo1 activator, has been shown to mimic shear-induced responses in ECs. Since shear-induced calcium influx causes Akt and ERK1/2 activation in ECs, we examined the effects of Yoda1 and the role of Piezo1 on their activation. Here, we show that Yoda1 robustly activates Akt and ERK1/2 in ECs. Additionally, the Piezo1 antagonists, gadolinium and ruthenium red, but not GsMTx4, effectively blocks Yoda1-induced Akt activation. Our results suggest that Yoda1-induced Akt and ERK1/2 activation is not dependent on Piezo1.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Sennoside A protects mitochondrial structure and function to improve high-fat diet-induced hepatic steatosis by targeting VDAC1

Highlights: • SA inhibited VDAC1 expression in livers of HFD-induced hepatic steatosis mice. • SA suppressed the ATP surplus by inhibiting mitochondrial complex I in livers of HFD-induced hepatic steatosis mice. • SA protected mitochondrial function and structure in livers of HFD-induced hepatic steatosis mice. • SA improved HFD-induced hepatic steatosis and hepatomegaly. Mitochondrial dysfunction is mainly associated with high-fat-diet (HFD)-induced hepatic steatosis. Sennoside A (SA), a commonly used clinical stimulant laxative, is reported to improve energy metabolism and insulin resistance. However, the effect and mechanism of SA on HFD-induced hepatic steatosis remain largely unknown. The aim of this study was to determine the effect and mechanism of SA on HFD-induced hepatic steatosis in mice. We examined the liver and body weight of mice to evaluate the physical changes in the liver. Hematoxylin and eosin (H&E) and oil red O staining were used to detect the lipid accumulation. The mitochondrial structure and function were tested by transmission electron microscopy and the Seahorse XF24 Analyzer. Furthermore, mitochondrial complexes I, II, and IV and voltage-dependent anion channel 1 (VDAC1) protein activity were detected to understand the mechanism of the protective effect on mitochondria. As a result, damage to the structure and function in the hepatic mitochondria of HFD-induced hepatic steatosis was observed in mice. The structural damage was in the form of loss of cristae, mitochondrial swelling, vacuolization and even rupturing of the outer mitochondrial membrane (OMM). Functional alterations were found by activation of complex I and deficiency in complexes II and IV. The VDAC1 activity and the total ATP in the liver tissue was increased under hepatic steatosis conditions. The above effects were reversed by SA. These data suggest that inhibition of VDAC1 may be an underlying mechanism of SA for protecting mitochondria in HFD-induced hepatic steatosis in mice. Thus, VDAC1 may be a promising target for treating fatty liver disease.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Metformin ameliorates TGF-β1–induced osteoblastic differentiation of human aortic valve interstitial cells by inhibiting β-catenin signaling

Highlights: • β-catenin mediates TGF-β–induced osteoblastic differentiation of human AVICs. • Metformin ameliorates osteoblastic differentiation of AVICs by activating AMPKα. • Smad2/3 and JNK signaling mediates TGF-β1–induced activation of β-catenin in AVICs. • Metformin mitigates TGF-β1–induced β-catenin signaling by inhibiting Smad2/3 and JNK. Osteoblastic differentiation of aortic valve interstitial cells (AVICs) is the central process in the development of calcific aortic valve disease (CAVD). Metformin is a widely used first-line antidiabetic drug, and recently, pleiotropic benefits of metformin beyond hypoglycemia have been reported in the cardiovascular system. Here, we examined the effect of metformin on the osteoblastic differentiation of human AVICs. Our results showed that metformin ameliorated TGF-β1–induced production of osteogenic proteins Runx2 and osteopontin as well as calcium deposition in the cultured human AVICs. Experiments using AICAR, Compound C and AMPKα siRNA showed that the beneficial effect of metformin on TGF-β1–induced osteoblastic differentiation of human AVICs was mediated by AMPKα. Moreover, metformin inhibited the TGF-β1–induced activation of β-catenin, and β-catenin siRNA blocked TGF-β1–induced osteoblastic differentiation of AVICs. Smad2/3 and JNK were phosphorylated to promote the TGF-β1–induced activation of β-catenin and osteoblastic differentiation of AVICs, and metformin also alleviated TGF-β1–induced activation of Smad2/3 and JNK. In conclusion, our results suggest a beneficial effect of metformin based on the prevention of osteoblastic differentiation of human AVICs via inhibition of β-catenin, which indicates the therapeutic potential of metformin for CAVD.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2011

5-Methoxyflavanone induces cell cycle arrest at the G2/M phase, apoptosis and autophagy in HCT116 human colon cancer cells

Natural flavonoids have diverse pharmacological activities, including anti-oxidative, anti-inflammatory, and anti-cancer activities. In this study, we investigated the molecular mechanism underlying the action of 5-methoxyflavanone (5-MF) which has a strong bioavailability and metabolic stability. Our results show that 5-MF inhibited the growth and clonogenicity of HCT116 human colon cancer cells, and that it activated DNA damage responses, as revealed by the accumulation of p53 and the phosphorylation of DNA damage-sensitive proteins, including ataxia-telangiectasia mutated (ATM) at Ser1981, checkpoint kinase 2 (Chk2) at Thr68, and histone H2AX at Ser139. 5-MF-induced DNA damage was confirmed in a comet tail assay. We also found that 5-MF increased the cleavage of caspase-2 and -7, leading to the induction of apoptosis. Pretreatment with the ATM inhibitor KU55933 enhanced 5-MF-induced {gamma}-H2AX formation and caspase-7 cleavage. HCT116 cells lacking p53 (p53{sup -/-}) or p21 (p21{sup -/-}) exhibited increased sensitivity to 5-MF compared to wild-type cells. 5-MF further induced autophagy via an ERK signaling pathway. Blockage of autophagy with the MEK inhibitor U0126 potentiated 5-MF-induced {gamma}-H2AX formation and caspase-2 activation. These results suggest that a caspase-2 cascade mediates 5-MF-induced anti-tumor activity, while an ATM/Chk2/p53/p21 checkpoint pathway and ERK-mediated autophagy act as a survival program to block caspase-2-mediated apoptosis induced by 5-MF. - Graphical abstract: Display Omitted Highlights: > 5-MF inhibits the proliferation of HCT116 colon cancer cells. > 5-MF inhibits cell cycle progression and induces apoptosis. > Inhibition of autophagy triggers 5-MF-induced apoptosis. > Inhibition of ERK signaling blocks 5-MF-induced autophagy but activates apoptosis. > Treatment with 5-MF in combination with an ERK inhibitor may be a potential therapeutic strategy in human colon cancer.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2012

Cadmium-induced teratogenicity: Association with ROS-mediated endoplasmic reticulum stress in placenta

The placenta is essential for sustaining the growth of the fetus. An increased endoplasmic reticulum (ER) stress has been associated with the impaired placental and fetal development. Cadmium (Cd) is a potent teratogen that caused fetal malformation and growth restriction. The present study investigated the effects of maternal Cd exposure on placental and fetal development. The pregnant mice were intraperitoneally injected with CdCl{sub 2} (4.5 mg/kg) on gestational day 9. As expected, maternal Cd exposure during early limb development significantly increased the incidences of forelimb ectrodactyly in fetuses. An obvious impairment in the labyrinth, a highly developed tissue of blood vessels, was observed in placenta of mice treated with CdCl{sub 2}. In addition, maternal Cd exposure markedly repressed cell proliferation and increased apoptosis in placenta. An additional experiment showed that maternal Cd exposure significantly upregulated the expression of GRP78, an ER chaperone. Moreover, maternal Cd exposure induced the phosphorylation of placental eIF2α, a downstream molecule of PERK signaling. In addition, maternal Cd exposure significantly increased the level of placental CHOP, another target of PERK signaling, indicating that the unfolded protein response (UPR) signaling was activated in placenta of mice treated with CdCl{sub 2}. Interestingly, alpha-phenyl-N-t-butylnitrone, a free radical spin-trapping agent, significantly alleviated Cd-induced placental ER stress and UPR. Taken together, these results suggest that reactive oxygen species (ROS)-mediated ER stress might be involved in Cd-induced impairment on placental and fetal development. Antioxidants may be used as pharmacological agents to protect against Cd-induced fetal malformation and growth restriction. -- Highlights: ► Cd induces fetal malformation and growth restriction. ► Cd induced placental ER stress and UPR. ► PBN alleviates Cd-induced ER stress and UPR in placenta. ► ROS-mediated ER stress might be involved in Cd-induced placental impairments. ► ROS-mediated ER stress might be involved in Cd-induced fetal malformations.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2012

Sodium fluoride induces apoptosis in mouse embryonic stem cells through ROS-dependent and caspase- and JNK-mediated pathways

Sodium fluoride (NaF) is used as a source of fluoride ions in diverse applications. Fluoride salt is an effective prophylactic for dental caries and is an essential element required for bone health. However, fluoride is known to cause cytotoxicity in a concentration-dependent manner. Further, no information is available on the effects of NaF on mouse embryonic stem cells (mESCs). We investigated the mode of cell death induced by NaF and the mechanisms involved. NaF treatment greater than 1 mM reduced viability and DNA synthesis in mESCs and induced cell cycle arrest in the G{sub 2}/M phase. The addition of NaF induced cell death mainly by apoptosis rather than necrosis. Catalase (CAT) treatment significantly inhibited the NaF-mediated cell death and also suppressed the NaF-mediated increase in phospho-c-Jun N-terminal kinase (p-JNK) levels. Pre-treatment with SP600125 or z-VAD-fmk significantly attenuated the NaF-mediated reduction in cell viability. In contrast, intracellular free calcium chelator, but not of sodium or calcium ion channel blockers, facilitated NaF-induced toxicity in the cells. A JNK specific inhibitor (SP600125) prevented the NaF-induced increase in growth arrest and the DNA damage-inducible protein 45α. Further, NaF-mediated loss of mitochondrial membrane potential was apparently inhibited by pifithrin-α or CAT inhibitor. These findings suggest that NaF affects viability of mESCs in a concentration-dependent manner, where more than 1 mM NaF causes apoptosis through hydroxyl radical-dependent and caspase- and JNK-mediated pathways. -- Highlights: ► The mode of NaF-induced cell death and the mechanisms involved were examined. ► NaF induced mainly apoptotic death of mouse embryonic stem cells (mESCs). ► NaF induced mitochondrial-mediated and caspase-dependent apoptosis. ► JNK- and p53-mediated pathways are involved in NaF-mediated apoptosis in the cells. ► ROS are the up-stream effector in NaF-mediated activation of JNK and p53 in mESCs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2012

H1-antihistamines induce vacuolation in astrocytes through macroautophagy

H1-antihistamines induce vacuolation in vascular smooth muscle cells, which may contribute to their cardiovascular toxicity. The CNS toxicity of H1-antihistamines may also be related to their non-receptor-mediated activity. The aim of this study was to investigate whether H1-antihistamines induce vacuolation in astrocytes and the mechanism involved. The H1-antihistamines induced large numbers of giant vacuoles in astrocytes. Such vacuoles were marked with both the lysosome marker Lysotracker Red and the alkalescent fluorescence dye monodansylcadaverine, which indicated that these vacuoles were lysosome-like acidic vesicles. Quantitative analysis of monodansylcadaverine fluorescence showed that the effect of H1-antihistamines on vacuolation in astrocytes was dose-dependent, and was alleviated by extracellular acidification, but aggravated by extracellular alkalization. The order of potency to induce vacuolation at high concentrations of H1-antihistamines (diphenhydramine > pyrilamine > astemizole > triprolidine) corresponded to their pKa ranking. Co-treatment with histamine and the histamine receptor-1 agonist trifluoromethyl toluidide did not inhibit the vacuolation. Bafilomycin A1, a vacuolar (V)-ATPase inhibitor, which inhibits intracellular vacuole or vesicle acidification, clearly reversed the vacuolation and intracellular accumulation of diphenhydramine. The macroautophagy inhibitor 3-methyladenine largely reversed the percentage of LC3-positive astrocytes induced by diphenhydramine, while only partly reversing the number of monodansylcadaverine-labeled vesicles. In Atg5{sup −/−} mouse embryonic fibroblasts, which cannot form autophagosomes, the number of vacuoles induced by diphenhydramine was less than that in wild-type cells. These results indicated that H1-antihistamines induce V-ATPase-dependent acidic vacuole formation in astrocytes, and this is partly mediated by macroautophagy. The pKa and alkalescent characteristic of H1-antihistamines may be the major determinants of vacuolation, which may contribute to their CNS toxicity. -- Highlights: ► H1-antihistamines induce vacuolar-ATPase-dependent acidic vacuolation in astrocytes. ► Vacuolation may be due to the sequestration of H1-antihistamines in astrocytes. ► The vacuolation induced by H1-antihistamines is partly mediated by macroautophagy. ► The pKa and alkalescent characteristic are the major determinants of vacuolation. ► MDC is not a specific marker for macroautophagy, but suitable for vacuolation.

60 APPLIED LIFE SCIENCES↗