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At least 1,063 records · Page 59

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DOE OSTI2016

Molybdenum induces pancreatic β-cell dysfunction and apoptosis via interdependent of JNK and AMPK activation-regulated mitochondria-dependent and ER stress-triggered pathways

Molybdenum (Mo), a well-known toxic environmental and industrial pollutant, causes adverse health effects and diseases in humans and has received attention as a potential risk factor for DM. However, the roles of Mo in the mechanisms of the toxicological effects in pancreatic β-cells are mostly unclear. In this study, the results revealed dysfunction of insulin secretion and apoptosis in the pancreatic β-cell-derived RIN-m5F cells and the isolated mouse islets in response to Mo. These effects were accompanied by a mitochondria-dependent apoptotic signals including a decreased in the MMP, an increase in cytochrome c release, and the activation of caspase cascades and PARP. In addition, ER stress was triggered as indicated by several key molecules of the UPR. Furthermore, exposure to Mo induced the activation of ERK1/2, JNK, AMPKα, and GSK3-α/β. Pretreatment with specific pharmacological inhibitors (in RIN-m5F cells and isolated mouse islets) of JNK (SP600125) and AMPK (Compound C) or transfection with si-RNAs (in RIN-m5F cells) specific to JNK and AMPKα effectively prevented the Mo-induced apoptosis and related signals, but inhibitors of ERK1/2 and GSK3-α/β (PD98059 and LiCl, respectively) did not reverse the Mo-induced effects. Additionally, both the inhibitors and specific si-RNAs could suppress the Mo-induced phosphorylation of JNK and AMPKα each other. Taken together, these results suggest that Mo exerts its cytotoxicity on pancreatic β-cells by inducing dysfunction and apoptosis via interdependent JNK and AMPK activation downstream-regulated mitochondrial-dependent and ER stress-triggered apoptosis pathways. - Highlights: • Molybdenum (Mo) induces pancreatic β-cell dysfunction and apoptosis. • Mo causes β-cell death via mitochondria-dependent caspase cascades signals. • ER stress-triggered apoptotic pathway also regulates Mo-induced β-cell death. • Interdependent of JNK and AMPK activation involves in Mo-induced β-cell apoptosis.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2016

Role of miR-155 in fluorooctane sulfonate-induced oxidative hepatic damage via the Nrf2-dependent pathway

Studies demonstrated that perfluorooctane sulfonate (PFOS) tends to accumulate in the liver and is capable to cause hepatomegaly. In the present study, we investigated the roles of miR-155 in PFOS-induced hepatotoxicity in SD rats and HepG2 cells. Male SD rats were orally administrated with PFOS at 1 or 10 mg/kg/day for 28 days while HepG2 cells were treated with 0–50 μM of PFOS for 24 h or 50 μM of PFOS for 1, 3, 6, 12 or 24 h, respectively. We found that PFOS significantly increased the liver weight and serum alanine transaminase (ALT) and aspartate amino transferase (AST) levels in rats. Morphologically, PFOS caused actin filament remodeling and endothelial permeability changes in the liver. Moreover, PFOS triggered reactive oxygen species (ROS) generation and induced apoptosis in both in vivo and in vitro assays. Immunoblotting data showed that NF-E2-related factor-2 (Nrf2) expression and activation and its target genes were all suppressed by PFOS in the liver and HepG2 cells. However, PFOS significantly increased miR-155 expression. Further studies showed that pretreatment of HepG2 cells with catalase significantly decreased miR-155 expression and substantially increased Nrf2 expression and activation, resulting in reduction of PFOS-induced cytotoxicity and oxidative stress. Taken together, these results indicated that miR-155 plays an important role in the PFOS-induced hepatotoxicity by disrupting Nrf2/ARE signaling pathway. - Highlights: • PFOS is capable to cause hepatotoxicity. • PFOS triggers ROS generation and induces apoptosis both in vivo and in vitro assays. • PFOS-induced ROS inhibits Nrf2 expression and its transactivation function. • PFOS promotes miR155 expression in liver and HepG2 cells. • miR-155 is involved in PFOS-induced hepatotoxicity by disrupting Nrf2/ARE pathway.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2016

Discovery of unique and ENM— specific pathophysiologic pathways: Comparison of the translocation of inhaled iridium nanoparticles from nasal epithelium versus alveolar epithelium towards the brain of rats

The biokinetics of inhaled nanoparticles (NP) is more complex than that of larger particles since NP may NP deposited on the nasal mucosa of the upper respiratory tract (URT) may translocate to the olfactory bulb of the brain and also via the trigeminus (URT neuronal route); and (b) NP deposited in the lower respiratory tract (LRT) may cross the ABB into blood and enter the brain across the blood-brain-barrier (BBB) or take a neuronal route from enervated tracheo-bronchial epithelia via the vagus nerve. Translocation from both - the URT and the LRT - are quantified during the first 24 h after a 1-hour aerosol inhalation of 20 nm-sized, {sup 192}Ir radiolabeled iridium NP by healthy adult rats using differential exposures: (I) nose-only exposure of the entire respiratory tract or (II) intratracheal (IT) inhalation of intubated and ventilated rats, thereby bypassing the URT and extrathoracic nasal passages. After nose-only exposure brain accumulation (BrAcc) is significantly nine-fold higher than after IT inhalation since the former results from both pathways (a + b) while the latter exposure comes only from pathway (b). Interestingly, there are significantly more circulating NP in blood 24 h after nose-only inhalation than after IT inhalation. Distinguishing translocation from URT versus LRT estimated from the differential inhalation exposures, the former is significantly higher (8-fold) than from the LRT. Although the BrAcc fraction is rather low compared to total NP deposition after this short-term exposure, this study proofs that inhaled insoluble NP can accumulate in the brain from both – URT and LRT which may trigger and/or modulate adverse health effects in the central nervous system (CNS) during chronic exposure. - Highlights: • Nanoparticle (NP) translocation from nose versus lungs to brain is differentiated. • Differential exposure of 20 nm radio-NP:nose-only versus intratracheal inhalation • The nose-brain path precedes via nerves, the lungs-brain path via circulation. • NP uptake in the rat brain is nine-fold higher from nose than from lungs. • Relative to deposited NP in both regions it is 45-fold higher than from the nose.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2016

6-Shogaol, an active compound of ginger, alleviates allergic dermatitis-like skin lesions via cytokine inhibition by activating the Nrf2 pathway

Allergic dermatitis (AD) clinically presents with skin erythematous plaques, eruption, and elevated serum IgE, and T helper cell type 2 and 1 (Th2 and Th1) cytokine levels. 6-Shogaol [1-(4-hydroxy-methoxyphenyl)-4-decen-one], a pungent compound isolated from ginger, has shown anti-inflammatory effects, but its inhibitory effects on AD are unknown. The aim of this study was to examine whether 6-shogaol inhibits AD-like skin lesions and their underlying mechanism in vivo and in vitro. An AD-like response was induced by tumor necrosis factor-α (TNF-α) + IFN-γ in human keratinocytes or by 2,4-dinitrochlorobenzene (DNCB) in mice. In vivo, 6-shogaol inhibited the development of DNCB-induced AD-like skin lesions and scratching behavior, and showed significant reduction in Th2/1-mediated inflammatory cytokines, IgE, TNF-α, IFN-γ, thymus and activation-regulated chemokine, IL-1, 4, 12, and 13, cyclooxygenase-2, and nitric oxide synthase levels. In vitro, 6-shogaol inhibited reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) signaling, and increased the levels of total glutathione, heme oxygenase-1, and quinone 1 via nuclear factor erythroid 2 related factor 2 (Nrf2) activation. 6-Shogaol can alleviate AD-like skin lesions by inhibiting immune mediators via regulating the ROS/MAPKs/Nrf2 signaling pathway, and may be an effective alternative therapy for AD. - Highlights: • 6-Shogaol inhibited Th2/1-mediated inflammatory mediators in vitro and in vivo. • 6-Shogaol regulated ROS/MAPKs/Nrf2 signaling pathway. • 6-Shogaol can protect against the development of AD-like skin lesions.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2016

Restoration of enterohepatic bile acid pathways in pregnant mice following short term activation of Fxr by GW4064

The farnesoid X receptor (Fxr) controls bile acid homeostasis by coordinately regulating the expression of synthesizing enzymes (Cyp7a1, Cyp8b1), conjugating enzymes (Bal, Baat) and transporters in the ileum (Asbt, Ostα/β) and liver (Ntcp, Bsep, Ostβ). Transcriptional regulation by Fxr can be direct, or through the ileal Fgf15/FGF19 and hepatic Shp pathways. Circulating bile acids are increased during pregnancy due to hormone-mediated disruption of Fxr signaling. While this adaptation enhances lipid absorption, elevated bile acids may predispose women to develop maternal cholestasis. The objective of this study was to determine whether short-term treatment of pregnant mice with GW4064 (a potent FXR agonist) restores Fxr signaling to the level observed in virgin mice. Plasma, liver and ilea were collected from virgin and pregnant mice administered vehicle or GW4064 by oral gavage. Treatment of pregnant mice with GW4064 induced ileal Fgf15, Shp and Ostα/β mRNAs, and restored hepatic Shp, Bal, Ntcp, and Bsep back to vehicle-treated virgin levels. Pregnant mice exhibited 2.5-fold increase in Cyp7a1 mRNA compared to virgin controls, which was reduced by GW4064. Similarly treatment of mouse primary hepatocytes with plasma isolated from pregnant mice induced Cyp7a1 mRNA by nearly 3-fold as compared to virgin plasma, which could be attenuated by co-treatment with either GW4064 or recombinant FGF19 protein. Collectively, these data reveal that repressed activity of intestinal and hepatic Fxr in pregnancy, as previously demonstrated, may be restored by pharmacological activation. This study provides the basis for a novel approach to restore bile acid homeostasis in patients with maternal cholestasis. - Highlights: • Ileal bile acid pathways are altered in pregnancy in an Fxr-dependent manner. • Ileal Fxr/Fgf contributes to changes in hepatic bile acid synthesis and transport. • Treatment of pregnant mice with an Fxr agonist restores bile acid homeostasis.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2016

Acute respiratory changes and pulmonary inflammation involving a pathway of TGF-β1 induction in a rat model of chlorine-induced lung injury

We investigated acute and delayed respiratory changes after inhalation exposure to chlorine (Cl{sub 2}) with the aim to understand the pathogenesis of the long-term sequelae of Cl{sub 2}-induced lung-injury. In a rat model of nose-only exposure we analyzed changes in airway hyperresponsiveness (AHR), inflammatory responses in airways, expression of pro-inflammatory markers and development of lung fibrosis during a time-course from 5 h up to 90 days after a single inhalation of Cl{sub 2}. A single dose of dexamethasone (10 mg/kg) was administered 1 h following Cl{sub 2}-exposure. A 15-min inhalation of 200 ppm Cl{sub 2} was non-lethal in Sprague-Dawley rats. At 24 h post exposure, Cl{sub 2}-exposed rats displayed elevated numbers of leukocytes with an increase of neutrophils and eosinophils in bronchoalveolar lavage (BAL) and edema was shown both in lung tissue and the heart. At 24 h, the inflammasome-associated cytokines IL-1β and IL-18 were detected in BAL. Concomitant with the acute inflammation a significant AHR was detected. At the later time-points, a delayed inflammatory response was observed together with signs of lung fibrosis as indicated by increased pulmonary macrophages, elevated TGF-β expression in BAL and collagen deposition around airways. Dexamethasone reduced the numbers of neutrophils in BAL at 24 h but did not influence the AHR. Inhalation of Cl{sub 2} in rats leads to acute respiratory and cardiac changes as well as pulmonary inflammation involving induction of TGF-β1. The acute inflammatory response was followed by sustained macrophage response and lack of tissue repair. It was also found that pathways apart from the acute inflammatory response contribute to the Cl{sub 2}-induced respiratory dysfunction. - Highlights: • Inhalation of Cl{sub 2} leads to acute lung inflammation and airway hyperreactivity. • Cl{sub 2} activates an inflammasome pathway of TGF-β induction. • Cl{sub 2} leads to a fibrotic respiratory disease. • Treatment with corticosteroids alone is insufficient to counteract acute lung injury.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2016

A mouse model of alcoholic liver fibrosis-associated acute kidney injury identifies key molecular pathways

Clinical data strongly indicate that acute kidney injury (AKI) is a critical complication in alcoholic hepatitis, an acute-on-chronic form of liver failure in patients with advanced alcoholic fibrosis. Development of targeted therapies for AKI in this setting is hampered by the lack of an animal model. To enable research into molecular drivers and novel therapies for fibrosis- and alcohol-associated AKI, we aimed to combine carbon tetrachloride (CCl{sub 4})-induced fibrosis with chronic intra-gastric alcohol feeding. Male C57BL/6J mice were administered a low dose of CCl{sub 4} (0.2 ml/kg 2 × week/6 weeks) followed by alcohol intragastrically (up to 25 g/kg/day for 3 weeks) and with continued CCl{sub 4}. We observed that combined treatment with CCl{sub 4} and alcohol resulted in severe liver injury, more pronounced than using each treatment alone. Importantly, severe kidney injury was evident only in the combined treatment group. This mouse model reproduced distinct pathological features consistent with AKI in human alcoholic hepatitis. Transcriptomic analysis of kidneys revealed profound effects in the combined treatment group, with enrichment for damage-associated pathways, such as apoptosis, inflammation, immune-response and hypoxia. Interestingly, Havcr1 and Lcn2, biomarkers of AKI, were markedly up-regulated. Overall, this study established a novel mouse model of fibrosis- and alcohol-associated AKI and identified key mechanistic pathways. - Highlights: • Acute kidney injury (AKI) is a critical complication in alcoholic hepatitis • We developed a novel mouse model of fibrosis- and alcohol-associated AKI • This model reproduces key molecular and pathological features of human AKI • This animal model can help identify new targeted therapies for alcoholic hepatitis.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2016

UDCA and CDCA alleviate 17α-ethinylestradiol-induced cholestasis through PKA-AMPK pathways in rats

Estrogen-induced cholestasis, known as intrahepatic cholestasis of pregnancy (ICP), is an estrogen-related liver disease that is widely recognized as female or pregnancy-specific. Our previous findings showed that the synthetic estrogen, 17α-ethinylestradiol (EE), induced cholestatic injury through ERK1/2-LKB1-AMP-activated protein kinase (AMPK) signaling pathway and its mediated suppression of farnesoid X receptor (FXR). To investigate the role played by bile acids in EE-induced cholestasis, we evaluated the effects of chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA) and deoxycholic acid (DCA) on sandwich cultured rat primary hepatocytes (SCRHs) and an in vivo rat model. Our results showed that, both CDCA and UDCA significantly induced time- and concentration-dependent reduction in AMPK phosphorylation in SCRHs. Despite having different effects on FXR activation, CDCA and UDCA both inhibited EE-induced AMPK activation, accompanied with the up-regulation of FXR and its downstream bile acid transporters. However, although DCA activates FXR and induces SHP, it was unable to alleviate EE-induced FXR suppression and further aggravated EE-induced cholestasis. We further demonstrated that both CDCA and UDCA, but not DCA, activated cyclic AMP dependent protein kinase (PKA) in SCRHs and the livers of male rats (8 weeks old) liver. Furthermore, PKA antagonist, H89, blocked the AMPK inhibition by CDCA and UDCA, and pharmacological and genetic activation of PKA suppressed EE-induced AMPK activation and its downstream effects. Collectively, these results suggest that CDCA and UDCA protect against estrogen-induced cholestatic injury via PKA signaling pathway and up-regulation of EE-suppressed FXR, which suggests a potential therapeutic target for ICP. - Highlights: • AMPK is involved in cholestatic liver injury with bile acid dysregulation. • CDCA and UDCA inhibit the phosphorylation of AMPK and alleviate estrogen-induced cholestasis. • PKA activation contributes to the CDCA- and UDCA-induced protective effects. • FXR up-regulation may be critical for improvement of cholestasis.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2016

Identification of the key pathway of oxazolinoanthracyclines mechanism of action in cells derived from human solid tumors

Oxazolinodoxorubicin (O-DOX) and oxazolinodaunorubicin (O-DAU) are novel anthracycline derivatives with a modified daunosamine moiety. In the present study, we evaluated the cytotoxicities, genotoxicities and abilities of O-DOX and O-DAU to induce apoptosis in cancer cell lines (SKOV-3; A549; HepG2), and compared the results with their parent drugs. We assessed antiproliferative activity by MTT assay. We evaluated apoptosis-inducing ability by double-staining with fluorescent probes (Hoechst 33258/propidium iodide), and by determining expression levels of genes involved in programmed cell death by reverse transcription-polymerase chain reaction. Genotoxicities of the compounds were tested by comet assays. Oxazolinoanthracyclines demonstrated high anti-tumor activity. O-DOX had significantly higher cytotoxicity, apoptosis-inducing ability, and genotoxicity compared with parental doxorubicin (DOX) in all tested conditions, while O-DAU activity differed among cell lines. The mechanism of oxazoline analog action appeared to involve the mitochondrial pathway of programmed cell death. These results provide further information about oxazoline derivatives of commonly used anthracycline chemotherapy agents. O-DOX and O-DAU have the ability to induce apoptosis in tumor cells. - Highlights: • Substituted amino group increased the anticancer activity of anthracyclines. • Mitochondrial apoptotic pathway seems to be involved in the mechanism of action. • Favorable biological properties of oxazoline derivatives were confirmed.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

Amentoflavone protects dopaminergic neurons in MPTP-induced Parkinson's disease model mice through PI3K/Akt and ERK signaling pathways

Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic neurons in substantia nigra pars compacta (SNpc). Mitochondrial dysfunction and cell apoptosis are suggested to be actively involved in the pathogenesis of PD. In the present study, the neuroprotective effect of amentoflavone (AF), a naturally occurring biflavonoid from Selaginella tamariscina, was examined in PD models both in vitro and in vivo. On SH-SY5Y cells, AF treatment dose-dependently reduced 1-methyl-4-phenylpyridinium (MPP{sup +})-induced nuclear condensation and loss of cell viability without obvious cytotoxicity. It inhibited the activation of caspase-3 and p21 but increased the Bcl-2/Bax ratio. Further study disclosed that AF enhanced the phosphorylation of PI3K, Akt and ERK1/2 down-regulated by MPP{sup +} in SH-SY5Y cells, the effect of which could be blocked by LY294002, the inhibitor of PI3K. Consistently, AF alleviated the behavioral deterioration in pole and traction tests and rescued the loss of dopaminergic neurons in SNpc and fibers in striatum in methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced mice. It also could enhance the activation of PI3K and Akt as well as Bcl-2/Bax ratio in SN. Moreover, AF alleviated gliosis as well as the gene expression levels of IL-1β and iNOS in SN. Collectively, these results suggested that AF protected dopaminergic neurons against MPTP/MPP{sup +}-induced neurotoxicity, which might be mediated through activation of PI3K/Akt and ERK signaling pathways in dopaminergic neurons and attenuation of neuroinflammation. - Highlights: • AF protected dopaminergic neurons against MPTP/MPP{sup +}-induced neurotoxicity. • AF modulated PI3K/Akt and ERK signaling pathways. • AF could alleviate neuroinflammation in SN.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

Mono-2-ethylhexyl phthalate inhibits human extravillous trophoblast invasion via the PPARγ pathway

Concerns over the adverse reproductive outcomes in human have been raised, more evidence including the underlying mechanism are required. Since extravillous trophoblast (EVT) invasion is an important physiological step during early development, the effects of mono-2-ethylhexyl phthalate (MEHP), the bioactive metabolite of DEHP, on EVT invasion were investigated using Matrigel-coated transwell chambers and cell line HTR-8/SVneo. In the transwell-based invasive assay, MEHP exposure inhibited EVT invasion as judged by decreased invasion index. Further analysis showed that MEHP exposure significantly inhibited the activity of matrix metalloproteinase-9 (MMP-9), which is an important positive regulator of EVT invasion. Meanwhile, the protein levels of tissue inhibitor matrix metalloproteinase-1 (TIMP-1), one key negative regulator of EVT invasion, were upregulated by MEHP treatment. Finally, inactivation of PPARγ pathway by either PPARγ inhibitors or PPARγ shRNA knockdown rescued the MEHP-induced inhibited invasion of HTR-8/SVneo cells, which is accompanied by the recovery of inhibited MMP-9 expression. The present study provides the evidence that MEHP exposure inhibits trophoblast invasion via PPARγ at concentrations comparable to those found in humans, which provides an insight in understanding the mechanisms of DEHP-associated early pregnancy loss. - Highlights: • MEHP inhibits HTR-8/SVneo cell invasion. • MEHP exposure imbalanced the expression of MMP-9/TIMP-1 in HTR-8/SVneo cells. • This effect of MEHP is mediated via the PPARγ pathway.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

Hsp90 inhibitor geldanamycin attenuates the cytotoxicity of sunitinib in cardiomyocytes via inhibition of the autophagy pathway

Sunitinib malate (sunitinib) is an orally available, multitargeted tyrosine kinase inhibitor with antitumor and antiangiogenic activities. Although sunitinib is effective for the treatment of patients with gastrointestinal stromal tumor, advanced renal cell carcinoma, or pancreatic neuroendocrine tumor, adverse cardiac events associated with sunitinib administration have been reported. Here, we examined the effect of geldanamycin, an inhibitor of heat shock protein (Hsp) 90, on sunitinib-induced cytotoxicity in cardiomyocytes. First, we found that treatment with geldanamycin or other Hsp90 inhibitors (tanespimycin, ganetespib, or BIIB021) significantly attenuated sunitinib-induced cytotoxicity in rat H9c2 cardiomyocytes, suggesting a drug-class effect of Hsp90 inhibitors. We then examined the mechanisms underlying sunitinib-induced cytotoxicity and found that sunitinib induced autophagy in H9c2 cells and that pretreatment with geldanamycin inhibited the induction of autophagy by promoting degradation of the autophagy-related proteins Atg7, Beclin-1, and ULK1. Pharmacological assessment with autophagy inhibitors confirmed that geldanamycin attenuated the cytotoxicity of sunitinib by interfering with autophagy. In addition, we found that the molecular chaperone Hsp70, which is induced by geldanamycin, was not involved in the attenuation of sunitinib-induced cytotoxicity. Finally, to provide more clinically relevant data, we confirmed that geldanamycin attenuated sunitinib-induced cytotoxicity in human induced pluripotent stem cell-derived cardiomyocytes. Together, these data suggest that geldanamycin attenuates sunitinib-induced cytotoxicity in cardiomyocytes by inhibiting the autophagy pathway. Thus, the further investigation of combination or sequential treatment with an Hsp90 inhibitor and sunitinib is warranted as a potential strategy of attenuating the cardiotoxicity associated with sunitinib administration in the clinical setting. - Highlights: • Hsp90 inhibitors attenuated sunitinib-induced cytotoxicity in cardiomyocytes. • Sunitinib induced autophagic flux in cardiomyocytes. • Geldanamycin degraded autophagy regulator proteins in cardiomyocytes. • Inhibition of the autophagy pathway attenuated sunitinib-induced cardiotoxicity. • Hsp70 induction was not involved in attenuating sunitinib-induced cardiotoxicity.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

High doses of baicalin induces kidney injury and fibrosis through regulating TGF-β/Smad signaling pathway

Baicalin is a major flavonoid compound purified from Scutellariae radix, which has been described as an herb in the Chinese Pharmacopoeia. Previous studies have suggested baicalin possessed extensive anti-inflammatory, anti-cancer, anti-viral properties. However, up to known, there have been no reports of safety and toxicity in the rats following oral administration of baicalin. In this present study, we showed the first evidence that treatment of baicalin (400, 800 and 1600 mg/kg/day) induced significantly kidney injury and fibrosis. The collagen synthesis and fibrosis-related protein expression were increased in the kidney of Sprague-Dawley (SD) rats after treatment with high doses of baicalin. We further investigated the potential molecular mechanism of baicalin-mediated renal fibrosis and revealed that baicalin activated the transforming growth factor-β (TGF-β)/Smad signaling pathway in a dose-dependent manner. Moreover, we also observed that baicalin induced Smad3 interaction with transcriptional coactivator p300 accompanying with increment of Smad3 acetylation. Our results may contribute to better understanding of the future pharmacological and toxicological studies of Scutellaria baicalensis Georgi and its active compounds on the human disease. - Highlights: • High doses of baicalin induced kidney injury and fibrosis. • High doses of baicalin activated TGF-β/Smad signaling pathway. • High doses of baicalin increased the interaction of p300 and Smad3, and subsequent acetylation of Smad3.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2013

Failure in activation of the canonical NF-κB pathway by human T-cell leukemia virus type 1 Tax in non-hematopoietic cell lines

Human T-cell leukemia virus type 1 (HTLV-1) Tax (Tax1) plays crucial roles in leukemogenesis in part through activation of NF-κB. In this study, we demonstrated that Tax1 activated an NF-κB binding (gpκB) site of the gp34/OX40 ligand gene in a cell type-dependent manner. Our examination showed that the gpκΒ site and authentic NF-κB (IgκB) site were activated by Tax1 in hematopoietic cell lines. Non-hematopoietic cell lines including hepatoma and fibroblast cell lines were not permissive to Tax1-mediated activation of the gpκB site, while the IgκB site was activated in those cells in association with binding of RelB. However RelA binding was not observed in the gpκB and IgκB sites. Our results suggest that HTLV-1 Tax1 fails to activate the canonical pathway of NF-κB in non-hematopoietic cell lines. Cell type-dependent activation of NF-κB by Tax1 could be associated with pathogenesis by HTLV-1 infection. - Highlights: • HTLV-1 Tax1 does not activate RelA of NF-κB in non-hematopoietic cell lines. • Tax1 activates the NF-κB non-canonical pathway in non-hematopoietic cell lines. • Tax1 does not induce RelA nuclear translocation in those cell lines, unlike TNFα. • The OX40L promoter κB site is activated by ectopic, but not endogenous, RelA.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

Kobuvirus VP3 protein restricts the IFN-β-triggered signaling pathway by inhibiting STAT2-IRF9 and STAT2-STAT2 complex formation

Emerged porcine kobuvirus (PKV) has adversely affected the global swine industry since 2008, but the etiological biology of PKV is unclear. Screening PKV-encoded structural and non-structural proteins with a type I IFN-responsive luciferase reporter showed that PKV VP3 protein inhibited the IFN-β-triggered signaling pathway, resulting in the decrease of VSV-GFP replication. QPCR data showed that IFN-β downstream cytokine genes were suppressed without cell-type specificity as well. The results from biochemical experiments indicated that PKV VP3 associated with STAT2 and IRF9, and interfered with the formation of the STAT2-IRF9 and STAT2-STAT2 complex, impairing nuclear translocation of STAT2 and IRF9. Taken together, these data reveal a new mechanism for immune evasion of PKV. - Highlights: •PKV VP3 inhibits the IFN-β-triggered signaling pathway. •VP3 associates with STAT2 and IRF9. •VP3 blocks the STAT2-IRF9 nuclear translocation. •VP3 utilizes a novel strategy for innate immune evasion.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2015

Adrenomedullin attenuates interleukin-1β-induced inflammation and apoptosis in rat Leydig cells via inhibition of NF-κB signaling pathway

The aim of this paper is to investigate the protective effects of adrenomedullin (ADM) on interleukin-1β (IL-1β)-induced inflammation and apoptosis in rat Leydig cells and its underlying molecular mechanisms. Leydig cells were isolated from adult Sprague–Dawley rats. The cell culture was established by adding ADM 2 h prior to 24 h treatment with IL-1β-induced cytotoxicity. We detected cell viability and concentrations of testosterone, reactive oxygen species (ROS), malondialdehyde (MDA), and reduced glutathione (GSH). Gene expression levels were measured for inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2). Concentrations were detected for nitric oxide (NO) and prostaglandin E2 (PGE2). Apoptosis was assessed using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Levels of gene expression and protein were detected for Bcl-2, Bax, caspase-3, and poly adenosine diphosphate-ribose polymerase (PARP). Protein levels were measured for nuclear factor kappa B (NF-κB) p65 and IκBα. ADM reduced IL-1β-induced cytotoxicity. ADM pretreatment significantly increased testosterone concentrations and decreased ROS, MDA, and GSH concentrations. ADM pretreatment inhibited IL-1β-induced inflammation in Leydig cells by decreasing the gene expression levels of iNOS and COX-2, as well as the concentrations of NO and PGE2. ADM pretreatment further decreased the number of TUNEL-positive stained Leydig cells, as confirmed by the increase in gene expression and protein levels of Bcl-2 and the decrease of Bax, caspase-3, and PARP levels. Moreover, ADM pretreatment inhibited NF-κB p65 phosphorylation and IκBα phosphorylation and degradation. ADM has potential anti-inflammatory and anti-apoptotic properties in IL-1β-induced rat Leydig cells, which might be related to NF-κB signaling pathway. - Highlights: • ADM attenuates IL-1β-induced oxidative stress in rat Leydig cells. • ADM attenuates IL-1β-induced uncontrolled inflammation in rat Leydig cells. • ADM inhibits IL-1β-induced apoptosis in rat Leydig cells. • ADM effects on rat Leydig cells via inhibition of NF-κB signaling pathway.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2016

Curcumin inhibits metastasis in human papillary thyroid carcinoma BCPAP cells via down-regulation of the TGF-β/Smad2/3 signaling pathway

Thyroid cancers usually possess a good prognosis while the risks of recurrence and metastasis turn out to be a disturbing issue. Curcumin [bis(4-hydroxy-3-methoxy-phenyl)-1,6-heptadiene-3,5-dione] is a natural polyphenolic compound mainly found in turmeric (Curcuma longa). Our previous studies have demonstrated that curcumin showed proliferation-inhibitory and apoptosis-inducing effects on K1 papillary thyroid cancer cells. However, the mechanism underlying the inhibition effects of curcumin on thyroid cancer cells remains unclear. Herein, we demonstrated that curcumin remarkably increased the expression of the epithelial marker E-cadherin and repressed the expression of the mesenchymal marker vimentin in human papillary thyroid carcinoma BCPAP cells. Curcumin also suppressed multiple metastatic steps of BCPAP cells, including cell attachment, spreading as well as migration. In addition, the transcription, secretion and activation of matrix metalloproteinases (MMPs) induced by transforming growth factor-β1 (TGF-β1) in BCPAP cells were mitigated upon curcumin treatment. Further evidence showed that curcumin decreased TGF-β1-mediated phosphorylation of Smad2 and Smad3. These results revealed that curcumin inhibited the TGF-β1-induced epithelial–mesenchymal transition (EMT) via down-regulation of Smad2/3 signaling pathways. Our findings provide new evidence that the anti-metastatic and anti-EMT activities of curcumin may contribute to the development of chemo-preventive agents for thyroid cancer treatment. - Highlights: • Curcumin inhibits the adhesion, spreading and migration of BCPAP cells. • Curcumin inhibits TGF-β1-induced EMT in BCPAP cells. • Curcumin inhibits TGF-β/Smad2/3 signaling pathway in BCPAP cells.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2016

MAT2B promotes adipogenesis by modulating SAMe levels and activating AKT/ERK pathway during porcine intramuscular preadipocyte differentiation

Intramuscular fat (IMF) has been demonstrated as one of the crucial factors of livestock meat quality. The MAT2B protein with MAT2α catalyzes the formation of methyl donor S- adenosylmethionine (SAMe) to mediate cell metabolism including proliferation and apoptosis. However, the regulatory effect of MAT2B on IMF deposition is still unclear. In this study, the effect of MAT2B on adipogenesis and its potential mechanism during porcine intramuscular preadipocyte differentiation was studied. The results showed that overexpression of MAT2B promoted adipogenesis and significantly up-regulated the mRNA and protein levels of adipogenic marker genes including FASN, PPARγ and aP2, consistently, knockdown of MAT2B inhibited lipid accumulation and down-regulated the mRNA and protein levels of the above genes. Furthermore, flow cytometry and EdU-labeling assay indicated that MAT2B regulate adipogenesis was partly due to influence intracellular SAMe levels and further affect cell clonal expansion. Also, increased expression of MAT2B activated the phosphorylations of AKT and ERK1/2, whereas knockdown of MAT2B blocked AKT signaling and repressed the phosphorylation of ERK1/2. Moreover, the inhibitory effect of LY294002 (a specific PI3K inhibitor) on the activities of AKT and ERK1/2 was partially recovered by overexpression of MAT2B in porcine intramuscular adipocytes. Finally, Co-IP experiments showed that MAT2B can directly interact with AKT. Taken together, our findings suggested that MAT2B acted as a positive regulator through modifying SAMe levels as well as activating AKT/ERK signaling pathway to promote porcine intramuscular adipocyte differentiation. - Highlights: • MAT2B up-regulates the expression of adipogenic marker genes and promotes porcine intramuscular preadipocyte differentiation. • MAT2B influences intracellular SAMe levels and further affects cell clonal expansion. • MAT2B interacts with AKT and activates AKT/ERK signaling pathway.

60 APPLIED LIFE SCIENCES↗