Luteolin, apigenin, and chrysin inhibit lipotoxicity-induced NLRP3 inflammasome activation and autophagy damage in macrophages by suppressing endoplasmic reticulum stress.

Lipotoxicity leads to numerous metabolic disorders such as nonalcoholic steatohepatitis. Luteolin, apigenin, and chrysin are three flavones with known antioxidant and anti-inflammatory properties, but whether they inhibit lipotoxicity-mediated NLRP3 inflammasome activation was unclear. To address this question, we used J774A.1 macrophages and Kupffer cells stimulated with 100 µM palmitate (PA) in the presence or absence of 20 µM of each flavone. PA increased p-PERK, p-IRE1α, p-JNK1/2, CHOP, and TXNIP as well as p62 and LC3-II expression and induced autophagic flux damage. Caspase-1 activation and IL-1ß release were also noted after 24 h of exposure to PA. In the presence of the PERK inhibitor GSK2656157, PA-induced CHOP and TXNIP expression and caspase-1 activation were mitigated. Compared with PA treatment alone, Bcl-2 coupled to beclin-1 was elevated and autophagy was reversed by the JNK inhibitor SP600125. With luteolin, apigenin, and chrysin treatment, PA-induced ROS production, ER stress, TXNIP expression, autophagic flux damage, and apoptosis were ameliorated. Moreover, TXNIP binding to NLRP3 and IL-1ß release in response to LPS/PA challenge were reduced. These results suggest that luteolin, apigenin, and chrysin protect hepatic macrophages against PA-induced NLRP3 inflammasome activation and autophagy damage by attenuating endoplasmic reticulum stress.

Benzyl isothiocyanate inhibits TNFα-driven lipolysis via suppression of the ERK/PKA/HSL signaling pathway in 3T3-L1 adipocytes.

Tumor necrosis factor α (TNFα), an inflammatory cytokine, induces lipolysis and increases circulating concentrations of free fatty acids. In addition, TNFα is the first adipokine produced by adipose tissue in obesity, contributing to obesity-associated metabolic disease. Given that benzyl isothiocyanate (BITC) is a well-known anti-inflammatory agent, we hypothesized that BITC can ameliorate TNFα-induced lipolysis and investigated the working mechanisms involved. We first challenged 3T3-L1 adipocytes with TNFα to induce lipolysis, which was confirmed by increased glycerol release, decreased protein expression of peroxisome proliferator-activated receptor γ (PPARγ) and perilipin 1 (PLIN1), and increased phosphorylation of ERK, protein kinase A (PKA), and hormone-sensitive lipase (HSL). However, inhibition of ERK or PKA significantly attenuated the lipolytic activity of TNFα. Meanwhile, pretreatment with BITC significantly ameliorated the lipolytic activity of TNFα; the TNFα-induced phosphorylation of ERK, PKA, and HSL; the TNFα-induced ubiquitination of PPARγ; the TNFα-induced decrease in PPARγ nuclear protein binding to PPAR response element; and the TNFα-induced decrease in PLIN1 protein expression. Our results indicate that BITC ameliorates TNFα-induced lipolysis by inhibiting the ERK/PKA/HSL signaling pathway, preventing PPARγ proteasomal degradation, and maintaining PLIN1 protein expression.

Protective Effect of Alpha-Linolenic Acid on Human Oral Squamous Cell Carcinoma Metastasis and Apoptotic Cell Death.

Oral cancer ranks sixth among Taiwan's top 10 cancers and most patients with poor prognosis acquire metastases. The essential fatty acid alpha-linolenic acid (ALA) has been found to diminish many cancer properties. However, the anti-cancer activity of ALA in oral cancer has yet to be determined. We examined the mechanisms underlying ALA inhibition of metastasis and induction of apoptotic cell death in oral squamous cell carcinoma (OSCC). Migration and invasion assays confirmed the cancer cells' EMT capabilities, whereas flow cytometry and Western blotting identified molecular pathways in OSCC. ALA dramatically reduced cell growth in a concentration-dependent manner according to the findings. Low concentrations of ALA (100 or 200 µM) inhibit colony formation, the expression of Twist and EMT-related proteins, the expression of MMP2/-9 proteins, and enzyme activity, as well as cell migration and invasion. Treatment with high concentrations of ALA (200 or 400 µM) greatly increases JNK phosphorylation and c-jun nuclear accumulation and then upregulates the FasL/caspase8/caspase3 and Bid/cytochrome c/caspase9/caspase3 pathways, leading to cell death. Low concentrations of ALA inhibit SAS and GNM cell migration and invasion by suppressing Twist and downregulating EMT-related proteins or by decreasing the protein expression and enzyme activity of MMP-2/-9, whereas high concentrations of ALA promote apoptosis by activating the JNK/FasL/caspase 8/caspase 3-extrinsic pathway and the Bid/cytochrome c/caspase 9 pathway. ALA demonstrates potential as a treatment for OSCC patients.

Andrographolide Attenuates Oxidized LDL-Induced Activation of the NLRP3 Inflammasome in Bone Marrow-Derived Macrophages and Mitigates HFCCD-Induced Atherosclerosis in Mice.

Andrographolide (AND) is a bioactive component of the herb Andrographis paniculata and a well-known anti-inflammatory agent. Atherosclerosis is a chronic inflammatory disease of the vasculature, and oxidized LDL (oxLDL) is thought to contribute heavily to atherosclerosis-associated inflammation. The aim of this study was to investigate whether AND mitigates oxLDL-mediated foam cell formation and diet-induced atherosclerosis (in mice fed a high-fat, high-cholesterol, high-cholic acid [HFCCD] diet) and the underlying mechanisms involved. AND attenuated LPS/oxLDL-mediated foam cell formation, IL-1[Formula: see text] mRNA and protein (p37) expression, NLR family pyrin domain containing 3 (NLRP3) mRNA and protein expression, caspase-1 (p20) protein expression, and IL-1[Formula: see text] release in BMDMs. Treatment with oxLDL significantly induced protein and mRNA expression of CD36, lectin-like oxLDL receptor-1 (LOX-1), and scavenger receptor type A (SR-A), whereas pretreatment with AND significantly inhibited protein and mRNA expression of SR-A only. Treatment with oxLDL significantly induced ROS generation and Dil-oxLDL uptake; however, pretreatment with AND alleviated oxLDL-induced ROS generation and Dil-oxLDL uptake. HFCCD feeding significantly increased aortic lipid accumulation, ICAM-1 expression, and IL-1[Formula: see text] mRNA expression, as well as blood levels of glutamic pyruvic transaminase (GPT), total cholesterol, and LDL-C. AND co-administration mitigated aortic lipid accumulation, the protein expression of ICAM-1, mRNA expression of IL-1[Formula: see text] and ICAM-1, and blood levels of GPT. These results suggest that the working mechanisms by which AND mitigates atherosclerosis involve the inhibition of foam cell formation and NLRP3 inflammasome-dependent vascular inflammation as evidenced by decreased SR-A expression and IL-1[Formula: see text] release, respectively.

Andrographolide Inhibits Lipotoxicity-Induced Activation of the NLRP3 Inflammasome in Bone Marrow-Derived Macrophages.

Andrographolide is the major bioactive component of the herb Andrographis paniculata and is a potent anti-inflammatory agent. Obesity leads to an excess of free fatty acids, particularly palmitic acid (PA), in the circulation. Obesity also causes the deposition of ectopic fat in nonadipose tissues, which leads to lipotoxicity, a condition closely associated with inflammation. Here, we investigated whether andrographolide could inhibit PA-induced inflammation by activating autophagy, activating the antioxidant defense system, and blocking the activation of the NLRP3 inflammasome. Bone marrow-derived macrophages (BMDMs) were primed with lipopolysaccharide (LPS) and then activated with PA. LPS/PA treatment increased both the mRNA expression of NLRP3 and IL-1[Formula: see text] and the release of IL-1[Formula: see text] in BMDMs. Andrographolide inhibited the LPS/PA-induced protein expression of caspase-1 and the release of IL-1[Formula: see text]. Furthermore, andrographolide attenuated LPS/PA-induced mtROS generation by first promoting autophagic flux and catalase activity, and ultimately inhibiting activation of the NLRP3 inflammasome. Our results suggest that the mechanisms by which andrographolide downregulates LPS/PA-induced IL-1[Formula: see text] release in BMDMs involve promoting autophagic flux and catalase activity. Andrographolide may thus be a candidate to prevent obesity- and lipotoxicity-driven chronic inflammatory disease.

Luteolin ameliorates palmitate-induced lipotoxicity in hepatocytes by mediating endoplasmic reticulum stress and autophagy.

Abnormal accumulation of lipids in liver leads to uncontrolled endoplasmic reticulum (ER) stress and autophagy. Luteolin is known to have antioxidant, anti-inflammatory, and anti-cancer properties, but whether it protects against lipotoxicity in liver remains unclear. In this study, we challenged AML12 liver cells and mouse primary hepatocytes with palmitic acid (PA) with or without luteolin pretreatment. In the presence of PA, reactive oxygen species (ROS) production was increased at 3 h, followed by enhancement of expression of p-PERK, ATF4, p-eIF2α, CHOP, and TXNIP (ER stress markers) and p-p62 and LC3II/LC3I ratio (autophagy markers), in both primary hepatocytes and AML12 cells. When PA treatment was extended up to 24 h, apoptosis was induced as evidenced by an increase in caspase-3 activation. RFP-GFP-LC3B transfection further revealed that the fusion of autophagosomes with lysosomes was damaged by PA. With luteolin treatment, the expression of antioxidant enzymes, i.e., heme oxygenase-1 and glutathione peroxidase, was upregulated, and PA-induced ROS production, ER stress, and cell death were dose-dependently ameliorated. Luteolin could also reverse the damage caused to autophagic flux. These results indicate that luteolin protects hepatocytes against PA assault by enhancing antioxidant defense, which can attenuate ER stress and autophagy as well as promote autophagic flux.

By-Products of the Black Soybean Sauce Manufacturing Process as Potential Antioxidant and Anti-Inflammatory Materials for Use as Functional Foods.

To assess the potential of by-products of the black bean fermented soybean sauce manufacturing process as new functional food materials, we prepared black bean steamed liquid lyophilized product (BBSLP) and analysed its antioxidant effects in vitro. RAW264.7 macrophages were cultured and treated with BBSLP for 24 h, and 1 µg/mL lipopolysaccharide (LPS) was then used for another 24 h to induce inflammation. The cellular antioxidant capacity and inflammatory response were then analysed. Activation of nuclear factor kappa B (NF-κB) signaling in RAW264.7 macrophages was also analysed. Results showed BBSLP had 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium (ABTS+) radical-scavenging abilities and reducing power in vitro. The levels of both reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) were reduced after RAW264.7 macrophages were treated with BBSLP after LPS induction. After RAW264.7 macrophage treatment with BBSLP and induction by LPS, the levels of inflammatory molecules, including nitric oxide (NO), prostaglandin E2 (PGE2), IL-1α, IL-6 and TNF-α, decreased. NF-κB signaling activity was inhibited by reductions in IκB phosphorylation and NF-κB DNA-binding activity after RAW264.7 macrophages were treated with BBSLP after LPS induction. In conclusion, BBSLP has antioxidant and anti-inflammatory capabilities and can be a supplement material for functional food.

Docosahexaenoic acid promotes the formation of autophagosomes in MCF-7 breast cancer cells through oxidative stress-induced growth inhibitor 1 mediated activation of AMPK/mTOR pathway.

Docosahexaenoic acid (DHA) is known to regulate autophagy in cancer cells. We explored whether oxidative stress-induced growth inhibitor 1 (OSGIN1) is involved in the regulation of autophagy by DHA in breast cancer cells and the possible mechanisms involved. DHA upregulated the levels of OSGIN1, LC3-II and SQSTM1/p62. By contrast, DHA dose-dependently decreased the levels of mTOR and p-mTORS2448 expression. Using GFP/RFP-LC3 fluorescence staining, we showed that cells treated with DHA showed a dose-dependent response in autophagic signals. OSGIN1 Overexpression mimicked DHA treatment in that LC3-II and GFP/RFP-LC3 signals as well as the expression of p-AMPKαT172 and p-RaptorS792 were significantly increased, whereas mTOR, p-mTORS2448, and p-ULK1S757 expression were decreased. With knockdown of OSGIN1 expression, these outcomes were reversed. Moreover, OSGIN1 overexpression transiently elevated the accumulation of OSGIN1 and reactive oxygen species (ROS) in the mitochondrial fraction and subsequently increased p-AMPKαT172 and p-RaptorS792 expression. Upon pretreatment with Mito-TEMPO, a scavenger of mitochondrial ROS, these outcomes were reversed. Taken together, these results suggest that DHA can transiently elevate the generation of ROS in mitochondria and promote autophagosome formation through activation of the p-AMPKαT172/p-Raptor S792 and inactivation of the p-mTORS2448/p-ULK1Ser757 signaling pathways, and these effects depend on OSGIN1 protein in MCF-7 cells.

α-Linolenic acid inhibits the migration of human triple-negative breast cancer cells by attenuating Twist1 expression and suppressing Twist1-mediated epithelial-mesenchymal transition.

α-Linolenic acid (ALA), an essential fatty acid, has anticancer activity in breast cancer, but the mechanism of its effects in triple-negative breast cancer (TNBC) remains unclear. We investigated the effect of ALA on Twist1, which is required to initiate epithelial-mesenchymal transition (EMT) and promotes tumor metastasis, and Twist1-mediated migration in MDA-MB231, MDA-MB468 and Hs578T cells. Twist1 protein was constitutively expressed in these TNBC cells, particularly MDA-MB-231 cells. Treatment with 100 µM ALA and Twist1 siRNA markedly decreased the Twist1 protein level and cell migration. Moreover, ALA transiently attenuated the nuclear accumulation of STAT3α as well as Twist1 mRNA expression. Treatment with ALA significantly attenuated the phosphorylation of JNK, ERK and Akt and decreased the phosphorylation of Twist1 at serine 68 in MDA-MB-231 cells. ALA accelerated Twist1 degradation in the presence of cycloheximide, whereas the ubiquitination and degradation of Twist1 by ALA was suppressed by MG-132. Pretreatment with ALA mimicked Twist1 siRNA, increased the protein expression of epithelial markers such as E-cadherin, and decreased the protein expression of mesenchymal markers including Twist1, Snail2, N-cadherin, vimentin, and fibronectin. Our findings suggest that ALA can be used not only to abolish EMT but also to suppress Twist1-mediated migration in TNBC cells.

1,25(OH)2 D3 attenuates indoxyl sulfate-induced epithelial-to-mesenchymal cell transition via inactivation of PI3K/Akt/ß-catenin signaling in renal tubular epithelial cells.

OBJECTIVES: Indoxyl sulfate (IS), a uremic toxin, has been shown to promote the epithelial-to-mesenchymal transition (EMT) of human proximal tubular cells and to accelerate the progression of chronic kidney disease (CKD). Despite the well-known protective role of 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3] in EMT, the effect of 1,25(OH)2 D3 on IS-induced EMT in human proximal tubular epithelial cells and the underlying mechanism remain unclear. The aim of this study was to determine whether IS (0-1 mM) dose-dependently inhibited the protein expression of E-cadherin and increased the protein expression of alpha-smooth muscle actin, N-cadherin, and fibronectin. METHODS: This study investigated the molecular mechanism by which 1,25(OH)2 D3 attenuates IS-induced EMT. HK-2 human renal tubular epithelial cells was used as the study model, and the MTT assay, Western Blotting, siRNA knockdown technique were used to explore the effects of 1,25(OH)2 D3 on EMT in the presence of IS. RESULTS: Pretreatment with 1,25(OH)2 D3 inhibited the IS-induced EMT-associated protein expression in HK-2 cells. IS induced the phosphorylation of Akt (S473) and ß-catenin (S552) and subsequently increased the nuclear accumulation of ß-catenin. Pretreatment with 1,25(OH)2 D3 and LY294002 (phosphoinositide 3-kinase [PIK3] inhibitor) significantly inhibited the IS-induced phosphorylation of Akt and ß-catenin, nuclear ß-catenin accumulation, and EMT-associated protein expression. CONCLUSIONS: Results from the present study revealed that the anti-EMT effect of 1,25(OH)2 D3 is likely through inhibition of the PI3K/Akt/ß-catenin pathway, which leads to down-regulation of IS-driven EMT-associated protein expression in HK-2 human renal tubular epithelial cells.

Docosahexaenoic acid inhibits TNFα-induced ICAM-1 expression by activating PPARα and autophagy in human endothelial cells.

Inflammation plays a key role in the development of cardiovascular disease (CVD), and docosahexaenoic acid (DHA) is recognized to fight against CVD. PPARα belongs to the nuclear hormone receptor superfamily and can interfere with inflammatory processes. Autophagy can degrade inflammasome proteins and counteract inflammation. Overexpression of intercellular adhesion molecule (ICAM) 1 in endothelial cells contributes to monocyte migration into the vascular intima. Here we investigated the mechanisms by which DHA inhibits TNFα-induced ICAM-1 expression in EA. hy926 endothelial cells. DHA markedly activated PPARα and suppressed TNFα-induced ICAM-1 expression, ICAM-1 promoter activity, p65 nuclear translocation, NFκB and DNA binding activity, and THP-1 cell adhesion. PPARα knockdown abolished the ability of DHA to inhibit TNFα-induced ICAM-1 expression and THP-1 cell adhesion. The PPARα antagonist GW6471 reversed the inhibitory effect of DHA on TNFα-induced ICAM-1 expression, p65 nuclear translocation, NFκB and DNA binding activity, and THP-1 cell adhesion. DHA significantly activated autophagy as evidenced by the formation of autophagosomes and increased LC3II protein expression. By contrast, wortmannin, which inhibits autophagy, abrogated DHA-induced autophagy and the inhibition of TNFα-induced ICAM-1 protein expression by DHA. Our results suggest that DHA likely inhibits TNFα-induced ICAM-1 expression by activating PPARα and autophagy.

Epigallocatechin-3-Gallate Reduces Hepatic Oxidative Stress and Lowers CYP-Mediated Bioactivation and Toxicity of Acetaminophen in Rats.

Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol in green tea. To investigate the effects of dietary EGCG on oxidative stress and the metabolism and toxicity of acetaminophen in the liver, rats were fed diets with (0.54%) or without EGCG supplementation for four weeks and were then injected intraperitoneally with acetaminophen (1 g/kg). The results showed that EGCG lowered hepatic oxidative stress and cytochrome P450 (CYP) 1A2, 2E1, and 3A, and UDP-glucurosyltransferase activities prior to acetaminophen injection. After acetaminophen challenge, the elevations in plasma alanine aminotransferase activity and histological changes in the liver were ameliorated by EGCG treatment. EGCG reduced acetaminophen-induced apoptosis by lowering the Bax/Bcl2 ratio in the liver. EGCG mildly increased autophagy by increasing the LC3B II/I ratio. Lower hepatic acetaminophen-glutathione and acetaminophen-protein adducts contents were observed after EGCG treatment. EGCG increased glutathione peroxidase and NAD(P)H quinone 1 oxidoreductase activities and reduced organic anion-transporting polypeptides 1a1 expression in the liver after acetaminophen treatment. Our results indicate that EGCG may reduce oxidative stress and lower the metabolism and toxicity of acetaminophen. The reductions in CYP-mediated acetaminophen bioactivation and uptake transporter, as well as enhanced antioxidant enzyme activity, may limit the accumulation of toxic products in the liver and thus lower hepatotoxicity.

Carnosine Suppresses Human Colorectal Cell Migration and Intravasation by Regulating EMT and MMP Expression.

Carnosine is an endogenous dipeptide found in the vertebrate skeletal muscles that is usually obtained through the diet. To investigate the mechanism by which carnosine regulates the migration and intravasation of human colorectal cancer (CRC) cells, we used cultured HCT-116 cells as an experimental model in this study. We examined HCT-116 cell migratory and intravasive abilities and expression of epithelial-mesenchymal transition (EMT)-associated molecules and matrix metalloproteinases (MMPs) after carnosine treatment. The results showed that both migration and invasion were inhibited in cells treated with carnosine. We found significant decreases in Twist-1 protein levels and increases in E-cadherin protein levels in HCT-116 cells after carnosine exposure. Although plasminogen activator (uPA) and MMP-9 mRNA and protein levels were decreased, TIMP-1 mRNA and protein levels were increased. Furthermore, the cytosolic levels of phosphorylated I κ B (p-I κ B) and NF- κ B DNA-binding activity were reduced after carnosine treatment. These results indicate that carnosine inhibits the migration and intravasation of human CRC cells. The regulatory mechanism may occur by suppressing NF- κ B activity and modulating MMP and EMT-related gene expression in HCT-116 cells.

Andrographis paniculata diterpenoids and ethanolic extract inhibit TNFα-induced ICAM-1 expression in EA.hy926 cells.

BACKGROUND: Andrographis paniculata (A. paniculata), a traditional herb in Southeastern Asia, is used to treat inflammation-mediated diseases. PURPOSE: The two major bioactive diterpenoids in A. paniculata are andrographolide (AND) and 14-deoxy-11,12-didehydroandrographolide (deAND). Because of the anti-inflammatory evidence for AND, we hypothesized that deAND might possess similar potency for inhibiting monocyte adhesion to the vascular endothelium, which is a critical event for atherosclerotic lesion formation. MATERIAL: In the present study, we used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine cell viability. We evaluated the production of intracellular reactive oxygen species (ROS) by using DCFDA assay. We assayed the protein expression by using Western blot analysis, the mRNA expression by using RT-PCR, and the nuclear protein-DNA binding activity by using EMSA. RESULTS: We showed that pretreatment of EA.hy926 cells with A. paniculata ethanolic extract (APE), deAND, and AND significantly inhibited TNFα-induced ICAM-1 protein and mRNA expression, ICAM-1 promoter activity, and monocyte adhesion. TNFα-stimulated IKKß phosphorylation, IκBα phosphorylation and degradation, p65 nuclear translocation, and NFκB nuclear protein-DNA binding activity were attenuated by pretreatment with APE, deAND, and AND. APE, deAND, and AND attenuated TNFα-induced Src phosphorylation and membrane translocation of the NOX subunits p47phox and p67phox. Both APE and AND induced protein expression of heme oxygenase 1 and the glutamate cysteine ligase modifier subunit and enhanced glutathione content. Pretreatment with AND and deAND inhibited TNFα-induced ROS generation. CONCLUSION: These results suggest that the mechanism by which APE, deAND, and AND down-regulates TNFα-induced ICAM-1 expression in EA.hy926 cells is via attenuation of activation of the IKK/IκB/NFκB pathway.

14-Deoxy-11,12-didehydroandrographolide suppresses adipogenesis of 3 T3-L1 preadipocytes by inhibiting CCAAT/enhancer-binding protein ß activation and AMPK-mediated mitotic clonal expansion.

Obesity is highly correlated with several metabolic disorders. Adipocyte differentiation is a key process in determining obesogenesis. 14-Deoxy-11,12-didehydroandrographolide (deAND) is a diterpenoid rich in Andrographis paniculata (Burm.f.) Nees., a herbal medicine commonly used to treat colds, infections, and liver diseases. We investigated whether deAND inhibits the adipogenesis of 3T3-L1 cells and the underlying mechanisms. We found that deAND (0-15 µM) dose-dependently inhibits the mRNA and protein expression of peroxisome proliferator-activated receptor γ, sterol regulatory element-binding protein 1c, fatty acid synthase, and stearoyl-CoA desaturase-1. Cellular lipid accumulation was decreased by deAND, and the early phase of adipocyte differentiation was critical for this inhibition. Immunoblotting revealed that deAND attenuated differentiation medium-induced protein kinase A (PKA) and cAMP response element-binding protein (CREB) activation, which leads to down-regulating C/EBPß transcription. Moreover, deAND inhibited ERK- and GSK3ß-mediated C/EBPß transcriptional activity. Flow cytometry analysis showed that deAND impaired the progression of mitotic clonal expansion (MCE) by arresting the cell cycle at the G0/G1 phase, while the expression of cyclin D1, cyclin E, CDK6, and CDK2 was attenuated. deAND increased the phosphorylation of AMPK and raptor, an mTOR-interacting partner, which inhibited the mTOR-driven phosphorylation of P70S6K and eukaryotic translation initiation factor 4E binding protein. In the presence of compound C, deAND modulation of AMPK-mTOR signaling and inhibition of cell cycle regulator expression were reversed. Our results reveal that the anti-adipogenic effect of deAND is likely through inhibition of the PKA-CREB-C/EBPß and AMPK/mTOR pathways, which leads to down-regulating C/EBPß-driven lipogenic protein expression and halting MCE progression.

CYP450-mediated mitochondrial ROS production involved in arecoline N-oxide-induced oxidative damage in liver cell lines.

BACKGROUND: IARC has classified the betel nut as a human environmental carcinogen. Previous studies have found that arecoline (AR) is the major alkaloid present in the saliva of betel quid chewers. Saliva contains a large content of AR which has been further shown to cause mutation of oral mucosa cells, resulting in oral cancer. Whereas, to date, there are only few studies reported the hepatotoxicity associated with arecoline and betel nut chewing. Therefore, the main purpose of this study was to determine the toxic effects of AR and its oxidative metabolite, arecoline N-oxide (ARNO), in normal liver cell lines. METHODS: The cytotoxic, genotoxic, and mutagenic effects were detected by crystal violet staining, alkaline comet assay, and Salmonella mutagenicity test, respectively. Measurement of intracellular reactive oxygen species (ROS) generation was determined using the H2-DCFDA assay. RESULTS: Our results demonstrated that ARNO exerted higher cytotoxicity, DNA damage, and mutagenicity than its parent compound arecoline in liver cells. Antioxidants, such as N-acetylcysteine, Trolox, and penicillamine, strongly protected liver cells from ARNO-induced DNA damage and ROS production. Furthermore, co-treatment with Mito-TEMPO also effectively blocked ARNO-induced ROS production in liver cells. Besides antioxidants, co-treatment with 1-aminobenzotriazole and methimazole nearly completely suppressed ARNO-induced ROS production in liver cells. CONCLUSIONS: Our data suggest that arecoline ingested from the habit of chewing betel quid can be primarily oxidized to ARNO, thereby enhancing its toxicity through increased ROS production. Considering the excellent protective effects of both mitochondria-targeted antioxidant and CYP450 inhibitor on ARNO-induced ROS production in liver cells, mitochondria CYP450-mediated metabolism of ARNO may be a key mechanism. Collectively, our results provide novel cellular evidence for the positive connection between habitual betel quid chewing and the risk for liver damage.

Anti-inflammatory effect of cinnamaldehyde and linalool from the leaf essential oil of Cinnamomum osmophloeum Kanehira in endotoxin-induced mice.

Cinnamomum osmophloeum Kanehira is a Taiwan native plant that belongs to genus Cinnamomum and is also known as pseudocinnamomum or indigenous cinnamon. Its leaf is traditionally used by local people in cooking and as folk therapy. We previously demonstrated the chemical composition and anti-inflammatory effect of leaf essential oil of Cinnamomum osmophloeum Kanehira of linalool chemotype in streptozotocin-induced diabetic rats and on endotoxin-injected mice. The aim of the present study is to evaluate whether cinnamaldehyde and linalool the active anti-inflammatory compounds in leaf essential oil of Cinnamomum osmophloeum Kanehira. Before the injection of endotoxin, C57BL/6 mice of the experimental groups were administered cinnamaldehyde (0.45 or 0.9 mg/kg body weight) or linalool (2.6 or 5.2 mg/kg body weight), mice of the positive control group were administered the leaf essential oil (13 mg/kg body weight), and mice of the negative group were administered vehicle (corn oil, 4 mL/kg body weight) by gavage every other day for two weeks. All mice received endotoxin (i.p. 10 mg/mL/kg body weight) the next day after the final administration and were killed 12 h after the injection. Normal control mice were pretreated with vehicle followed by the injection with saline. None of the treatment found to affect body weight or food or water intake of mice before the injection of endotoxin. Cinnamaldehyde and linalool were found significantly reversed endotoxin-induced body weight loss and lymphoid organ enlargement compared with vehicle (P < 0.05). Both compounds also significantly lowered endotoxin-induced levels of peripheral nitrate/nitrite, interleukin (IL)-1ß, IL-18, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and High-mobility group box 1 protein (HMGB-1), and levels of nitrate/nitrite, IL-1ß, TNF-α, and IFN-γ in spleen and mesenteric lymph nodes (MLNs) (P < 0.05). Endotoxin-induced expression of toll-like receptor 4 (TLR4), Myeloid differentiation primary response gene 88 (MyD88), myeloid differentiation protein 2 (MD2), Nod-like receptor family, pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), and caspase-1 in spleen and mesenteric lymph nodes (MLNs) were inhibited by all tested doses of cinnamaldehyde and linalool (P < 0.05). Subsequently, the activation of nuclear factor (NF)-κB and the activity of caspase-1 in spleen and MLNs were also suppressed by these two compounds (P < 0.05). In addition, cinnamaldehyde and linalool at the dose equivalent to their corresponding content in the tested dose of the leaf essential oil, which was 0.9 mg/kg and 5.2 mg/kg, respectively, showed similar or slightly less inhibitory activity for most of these inflammatory parameters compared with that of the leaf essential oil. Our data confirmed the potential use of leaf essential oil of Cinnamomum osmophloeum Kanehira as an anti-inflammatory natural product and provide evidence for cinnamaldehyde and linalool as two potent agents for prophylactic use in health problems associated with inflammations that being attributed to over-activated TLR4 and/or NLRP3 signaling pathways.

Effects of lemongrass oil and citral on hepatic drug-metabolizing enzymes, oxidative stress, and acetaminophen toxicity in rats.

The essential oil from a lemongrass variety of Cymbopogon flexuosus [lemongrass oil (LO)] is used in various food and aroma industry products and exhibits biological activities, such as anticancer and antimicrobial activities. To investigate the effects of 200 LO (200 mg/kg) and 400 LO (400 mg/kg) and its major component, citral (240 mg/kg), on drug-metabolizing enzymes, oxidative stress, and acetaminophen toxicity in the liver, male Sprague-Dawley rats were fed a pelleted diet and administered LO or citral by gavage for 2 weeks. After 2 weeks of feeding, the effects of LO and citral on the metabolism and toxicity of acetaminophen were determined. The results showed that rats treated with 400 LO or citral had significantly reduced hepatic testosterone 6ß-hydroxylation and ethoxyresorufin O-deethylation activities. In addition, NAD(P)H:quinone oxidoreductase 1 activity was significantly increased by citral, and Uridine 5'-diphospho (UDP) glucurosyltransferase activity was significantly increased by 400 LO in the rat liver. Treatment with 400 LO or citral reduced lipid peroxidation and reactive oxygen species levels in the liver. After acetaminophen treatment, however, LO and citral treatment resulted in little or no change in plasma alanine aminotransferase activity and acetaminophen-protein adducts content in the liver. Our results indicate that LO and citral may change the activities of drug-metabolizing enzymes and reduce oxidative stress in the liver. However, LO and citral may not affect the detoxification of acetaminophen.

Caffeic acid and resveratrol ameliorate cellular damage in cell and Drosophila models of spinocerebellar ataxia type 3 through upregulation of Nrf2 pathway.

Polyglutamine (polyQ)-expanded mutant ataxin-3 protein, which is prone to misfolding and aggregation, leads to cerebellar neurotoxicity in spinocerebellar ataxia type 3 (SCA3), an inherited PolyQ neurodegenerative disease. Although the exact mechanism is unknown, the pathogenic effects of mutant ataxin-3 are associated with dysregulation of transcription, protein degradation, mitochondrial function, apoptosis, and antioxidant potency. In the present study we explored the protective role and possible mechanism of caffeic acid (CA) and resveratrol (Res) in cells and Drosophila expressing mutant ataxin-3. Treatment with CA and Res increased the levels of antioxidant and autophagy protein expression with consequently corrected levels of reactive oxygen species, mitochondrial membrane potential, mutant ataxin-3, and the aggregation of mutant ataxin-3 in SK-N-SH-MJD78 cells. Moreover, in SK-N-SH-MJD78 cells, CA and Res enhanced the transcriptional activity of nuclear factor erythroid-derived-2-like 2 (Nrf2), a master transcription factor that upregulates the expression of antioxidant defense genes and the autophagy gene p62. CA and Res improved survival and motor performance in SCA3 Drosophila. Additionally, the above-mentioned protective effects of CA were also observed in CA-supplemented SCA3 Drosophila. Notably, blockade of the Nrf2 pathway by use of small interfering RNA annulled the health effects of CA and Res on SCA3, which affirmed the importance of the increase in Nrf2 activation by CA and Res. Additional studies are need to dissect the protective role of CA and Res in modulating neurodegenerative progression in SCA3 and other polyQ diseases.