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.

Benzyl isothiocyanate attenuates activation of the NLRP3 inflammasome in Kupffer cells and improves diet-induced steatohepatitis.

The NLRP3 inflammasome plays an important role in the pathogenesis of numerous inflammation-related diseases. Benzyl isothiocyanate (BITC) is rich in cruciferous vegetables and possesses potent antioxidant, anti-inflammatory, anti-cancer, and anti-obesogenic properties. In this study, we investigated the role of the NLRP3 inflammasome in the protection by BITC against steatohepatitis and insulin resistance. A mouse model of high-fat/cholesterol/cholic acid diet (HFCCD)-induced steatohepatitis, LPS/nigericin-stimulated primary Kupffer cells, and IL-1ß treated primary hepatocytes were used. BITC attenuated LPS/nigericin-induced activation of the NLRP3 inflammasome by enhancing protein kinase A-dependent NLRP3 ubiquitination, which increased the degradation of NLRP3 and reduced IL-1ß secretion in Kupffer cells. In hepatocytes, BITC pretreatment reversed the IL-1ß-induced decrease in the phosphorylation of IR, AKT, and GSK3ß in response to insulin. After 12 weeks of HFCCD feeding, increases in blood alanine aminotransferase (ALT) and glucose levels were ameliorated by BITC. Hepatic IL-1ß production, macrophage infiltration, and collagen expression induced by HFCCD were also mitigated by BITC. BITC suppresses activation of the NLRP3 inflammasome in Kupffer cells by enhancing the PKA-dependent ubiquitination of NLRP3, which leads to suppression of IL-1ß production and subsequently ameliorates hepatic inflammation and insulin resistance.

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.

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.

14-Deoxy-11,12-Didehydroandrographolide Ameliorates Glucose Intolerance Enhancing the LKB1/AMPK[Formula: see text]/TBC1D1/GLUT4 Signaling Pathway and Inducing GLUT4 Expression in Myotubes and Skeletal Muscle of Obese Mice.

14-Deoxy-11,12-didehydroandrographolide (deAND), a bioactive component of Andrographis paniculata, has antidiabetic activity. AMP-activated protein kinase (AMPK) regulates glucose transport and ameliorates insulin resistance. The aim of the present study was to investigate whether activation of AMPK is involved in the mechanism by which deAND ameliorates insulin resistance in muscles. deAND amounts up to 40 [Formula: see text]M dose-dependently activated phosphorylation of AMPK[Formula: see text] and TBC1D1 in C2C12 myotubes. In addition, deAND significantly activated phosphorylation of LKB1 at 6 h after treatment, and this activation was maintained up to 48 h. deAND increased glucose uptake at 18 h after treatment, and this increase was time dependent up to 72 h. Compound C, an inhibitor of AMPK, suppressed deAND-induced phosphorylation of AMPK[Formula: see text] and TBC1D1 and reversed the effect on glucose uptake. In addition, the expression of GLUT4 mRNA and protein in C2C12 myotubes was up-regulated by deAND in a time-dependent manner. Promotion of GLUT4 gene transcription was verified by a pGL3-GLUT4 (837 bp) reporter assay. deAND also increased the nuclear translocation of MEF-2A and PPAR[Formula: see text]. After 16 weeks of feeding, the high-fat diet (HFD) inhibited phosphorylation of AMPK[Formula: see text] and TBC1D1 in skeletal muscle of obese C57BL/6JNarl mice, and deactivation of AMPK[Formula: see text] and TBC1D1 by the HFD was abolished by deAND supplementation. Supplementation with deAND significantly promoted membrane translocation of GLUT4 compared with the HFD group. Supplementation also significantly increased GLUT4 mRNA and protein expression in skeletal muscle compared with the HFD group. The hypoglycemic effects of deAND are likely associated with activation of the LKB1/AMPK[Formula: see text]/TBC1D1/GLUT4 signaling pathway and stimulation of MEF-2A- and PPAR[Formula: see text]-dependent GLUT4 gene expression, which account for the glucose uptake into skeletal muscle and lower blood glucose levels.

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.

Andrographolide inhibits IL-1ß release in bone marrow-derived macrophages and monocyte infiltration in mouse knee joints induced by monosodium urate.

Andrographolide (AND) is the major diterpenoid in A. paniculata with wide clinical application and has been shown to be a potent anti-inflammatory agent. Gout is the leading inflammatory disease of the joints, and the deposition of urate in the articular cavity attracts immune cells that release inflammatory cytokines. Monosodium urate (MSU) is known to be one of the activators of the NLRP3 (NLR family pyrin domain containing 3) inflammasome. After activation, the NLRP3 inflammasome releases interleukin-1ß (IL-1ß), which causes the development of many inflammatory diseases. The aim of the present study was to investigate whether AND attenuates the release of IL-1ß mediated by the NLRP3 inflammasome. The effects of AND were studied in bone marrow-derived macrophages (BMDMs) treated with lipopolysaccharide (LPS) and MSU and in mice with MSU-induced joint inflammation. AND suppressed MSU phagocytosis dose-dependently and markedly inhibited LPS- and MSU-induced IL-1ß release in BMDMs. Moreover, AND pretreatment inhibited the LPS-induced NLRP3 inflammasome priming stage by inhibiting the IKK/NFκB signaling pathway, which resulted in decreased protein expression of NLRP3 and proIL-1ß. AND induced HO-1 protein expression in a dose-dependent manner and attenuated MSU-induced ROS generation. Silencing HO-1 mitigated AND inhibition of LPS/MSU-induced IL-1ß release in J774A.1 cells. In addition, AND decreased MSU-mediated ASC binding to NLRP3. Oral administration of AND attenuated MSU-induced monocyte infiltration in mouse knee joints. These results suggest that the working mechanisms by which AND down-regulates MSU-induced joint inflammation might be via HO-1 induction and attenuation of ROS-mediated NLRP3 inflammasome assembly and subsequent IL-1ß release.

Benzyl Isothiocyanate Ameliorates High-Fat Diet-Induced Hyperglycemia by Enhancing Nrf2-Dependent Antioxidant Defense-Mediated IRS-1/AKT/TBC1D1 Signaling and GLUT4 Expression in Skeletal Muscle.

Obesity caused lipotoxicity, which results in insulin resistance. We studied whether benzyl isothiocyanate (BITC) improved insulin resistance in muscle. BITC was studied in vivo in mice fed a high-fat diet (HFD) and in vitro in C2C12 myotubes treated with palmitic acid (PA). In C2C12 cells, BITC mitigated PA inhibition of glucose uptake and phosphorylation of IRS-1, AKT, and TBC1D1 in response to insulin. BITC upregulated the expression of HO-1, GSTP, and GCLM mRNA and protein as well as GSH contents, which suppressed oxidative damage. Knockdown of Nrf2 abrogated BITC enhancement of antioxidant defense and subsequently reversed BITC protection against PA-induced insulin resistance. Moreover, BITC upregulated the expression of GLUT4, PPARγ, and C/EBPα. In HFD-fed mice, plasma total cholesterol, nonesterified fatty acid, and glucose levels and HOMA-IR were dose-dependently decreased with 0.05 or 0.1% BITC administration. In gastrocnemius muscle, compared with the HFD group, BITC increased the phosphorylation of AKT and TBC1D1, GSH contents, and the expression of antioxidant enzymes as well as GLUT4. These results indicate that BITC ameliorates obesity-induced hyperglycemia by enhancing insulin sensitivity in muscle. This is partly attributed to its inhibition of lipotoxicity-induced oxidative insult and upregulation of GLUT4 expression.

Andrographis paniculata Improves Insulin Resistance in High-Fat Diet-Induced Obese Mice and TNFα-Treated 3T3-L1 Adipocytes.

Pro-inflammatory cytokines interfere with blood glucose homeostasis, which leads to hyperglycemia. Andrographis paniculata (AP) has been shown to possess anti-inflammatory activity and to reduce blood glucose levels in diabetes. The two major bioactive diterpenoids in AP, andrographolide (AND) and 14-deoxy-11,12-didehydroandrographolide (deAND), have potent anti-inflammatory activity. We studied whether APE (an ethanolic extract of AP), AND, and deAND could improve a high-fat diet (HFD)-induced hyperglycemia in vivo and TNF[Formula: see text]-induced impairment of insulin signaling in vitro. Male C57BL/6JNarl mice were fed a normal diet (ND) or the HFD, and the fatty mice were treated with APE, deAND, or AND for 16 weeks. 3T3-L1 cells were used to study the underlying mechanisms by which APE, deAND, or AND attenuated TNF[Formula: see text]-induced insulin resistance. The HFD significantly induced obesity, hyperglycemia, insulin resistance, and inflammation, whereas APE and deAND significantly ameliorated HFD-induced obesity, hyperglycemia, insulin resistance, and TNF[Formula: see text] production. The HFD significantly impaired insulin signaling by decreasing the protein expression of p-IRS1 tyr632 and p-AKT ser473, as well as the membrane translocation of GLUT4 in response to insulin stimulation in epididymal adipose tissue. HFD-impaired the membrane translocation of GLUT4 was significantly reversed by deAND and APE. In addition, deAND and APE markedly reversed the detrimental effect of TNF[Formula: see text] on the insulin signaling pathway and glucose uptake in 3T3-L1 cells. Despite no significant positive effect on p-AS160, a trend for recovery by deAND and APE was observed. These results suggest that deAND and APE protect against HFD-induced insulin resistance by ameliorating inflammation-driven impairment of insulin sensitivity.

Benzyl isothiocyanate ameliorates high-fat/cholesterol/cholic acid diet-induced nonalcoholic steatohepatitis through inhibiting cholesterol crystal-activated NLRP3 inflammasome in Kupffer cells.

Incidence of nonalcoholic fatty liver disease is increasing worldwide. Activation of the NLRP3 inflammasome is central to the development of diet-induced nonalcoholic steatohepatitis (NASH). We investigated whether benzyl isothiocyanate (BITC) ameliorates diet-induced NASH and the mechanisms involved. C57BL/6 J mice fed a high-fat diet containing cholesterol and cholic acid (HFCCD) and Kupffer cells stimulated with LPS and cholesterol crystals (CC) were studied. LPS/CC increased the expression of the active form of caspase 1 (p20) and the secretion of IL-1ß by Kupffer cells, and these changes were reversed by MCC950, an NLRP3 inflammasome inhibitor. LPS/CC-induced NLRP3 inflammasome activation and IL-1ß production were dose-dependently attenuated by BITC. BITC decreased cathepsin B release from lysosomes and binding to NLRP3 induced by LPS/CC. Compared with a normal diet, the HFCCD increased serum levels of ALT, AST, total cholesterol, and IL-1ß and hepatic contents of triglycerides and total cholesterol. BITC administration (0.1% in diet) reversed the increase in AST and hepatic triglycerides in the HFCCD group. Moreover, BITC suppressed lipid accumulation, macrophage infiltration, fibrosis, crown-like structure formation, and p20 caspase 1 and p17 IL-1ß expression in liver in the HFCCD group. These results suggest that BITC ameliorates HFCCD-induced steatohepatitis by inhibiting the activation of NLRP3 inflammasome in Kupffer cells and may protect against diet-induced NASH.

A Diterpenoid, 14-Deoxy-11, 12-Didehydroandrographolide, in Andrographis paniculata Reduces Steatohepatitis and Liver Injury in Mice Fed a High-Fat and High-Cholesterol Diet.

14-Deoxy-11,12-didehydroandrographolide (deAND), a diterpenoid in Andrographis paniculata (Burm. f.) Nees, acts as a bioactive phytonutrient that can treat many diseases. To investigate the protective effects of deAND on reducing fatty liver disease, male mice were fed a high-fat and high-cholesterol (HFHC) diet without or with 0.05% and 0.1% deAND supplementation. Cholesterol accumulation, antioxidant, and anti-inflammatory activities in liver and liver injury were evaluated after deAND treatment. The results show that deAND treatment for seven weeks reduced plasma alanine aminotransferase activity and lowered hepatic cholesterol accumulation, tumor nuclear factor-α, and histological lesions. The 0.1% deAND treatment reduced HFHC diet-induced apoptosis by lowering the caspase 3/pro-caspase 3 ratio. After 11 weeks of deAND treatment, increased NOD-like receptor protein 3 (NLRP3), capase-1, and interleukin-1ß protein levels in liver were suppressed by deAND treatment. In addition, nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA expression, heme oxygenase-1 protein expression, and the activities of glutathione peroxidase and glutathione reductase were increased in mice fed the HFHC diet. However, those activities of antioxidant enzymes or proteins were also upregulated by 0.1% deAND treatment. Furthermore, deAND treatment tended to lower hepatic lipid peroxides. Finally, deAND treatment reversed the depletion of hepatic glutamate level induced by the HFHC diet. These results indicate that deAND may ameliorate HFHC diet-induced steatohepatitis and liver injury by increasing antioxidant and anti-inflammatory activities.

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.

Benzyl Isothiocyanate and Phenethyl Isothiocyanate Inhibit Adipogenesis and Hepatosteatosis in Mice with Obesity Induced by a High-Fat Diet.

Benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) are organosulfur phytochemicals rich in cruciferous vegetables. We investigated the antiobesity and antihepatosteatosis activities of BITC and PEITC and the working mechanisms involved. C57BL/6J mice were fed a low-fat diet (LFD), a high-fat diet (HFD), or a HFD supplemented with 0.5 (L) or 1 g/kg (H) BITC or PEITC for 18 weeks. Compared with the HFD group, BITC or PEITC decreased the final body weight of mice in a dose-dependent manner [39.0 ± 3.1 (HFD), 34.4 ± 3.2 (BITC-L), 32.4 ± 2.8 (BITC-H), 36.2 ± 4.4 (PEITC-L), and 32.8 ± 2.9 (PEITC-H) g, p < 0.05], relative weight of epididymal fat [5.7 ± 0.4 (HFD), 4.7 ± 0.7 (BITC-L), 3.7 ± 0.3 (BITC-H), 4.4 ± 1.0 (PEITC-L), and 3.2 ± 0.6 (PEITC-H) %, p < 0.05], hepatic triglycerides [98.4 ± 6.0 (HFD), 81.0 ± 8.9 (BITC-L), 63.5 ± 5.6 (BITC-H), 69.3 ± 5.6 (PEITC-L), and 49.4 ± 2.9 (PEITC-H) mg/g, p < 0.05], and plasma total cholesterol [140 ± 21.3 (HFD), 109 ± 5.6 (BITC-L), 101 ± 11.3 (BITC-H), 126 ± 8.3 (PEITC-L), and 91.8 ± 12.7 (PEITC-H) mg/dL, p < 0.05]. Q-PCR and immunoblotting assays revealed that BITC and PEITC suppressed the expression of liver X receptor α, sterol regulatory element-binding protein 1c, stearoyl-CoA desaturase 1, fatty acid synthase, and acetyl-CoA carboxylase in both epididymal adipose and liver tissues. After a single oral administration of 85 mg/kg BITC or PEITC, the maximum plasma concentrations ( Cmax) of BITC and PEITC were 5.8 ± 2.0 µg/mL and 4.3 ± 1.9 µg/mL, respectively. In 3T3-L1 adipocytes, BITC and PEITC dose-dependently reduced adipocyte differentiation and cell cycle was arrested in G0/G1 phase. These findings indicate that BITC and PEITC ameliorate HFD-induced obesity and fatty liver by down-regulating adipocyte differentiation and the expression of lipogenic transcription factors and enzymes.

Bioavailability of the diterpenoid 14-deoxy-11,12-didehydroandrographolide in rats and up-regulation of hepatic drug-metabolizing enzyme and drug transporter expression.

BACKGROUND: 14-Deoxy-11,12-didehydroandrographolide (deAND) is the second most abundant diterpenoid in Andrographis paniculata (Burm. f.) Nees, a traditional medicine used in Asia. To date, the biological activity of deAND has not been clearly investigated. PURPOSE: In this study, we intended to examine the modulatory effect of deAND on hepatic drug metabolism as well as its bioavailability. STUDY DESIGN: deAND prepared from A. paniculata was orally given to Sprague-Dawley rats and changes in plasma deNAD were determined by HPLC-MS. Modulation of deAND on drug-metabolizing enzyme and drug transporter expression as well as the possible mechanism involved was examined in primary rat hepatocytes. RESULTS: After a single oral administration of 50 mg/kg deAND to rats, the maximum plasma concentration (Cmax), time to reach the Cmax, area under the curve (AUC0-24h), mean retention time, and half-life (t1/2) of deAND were 2.65 ± 0.68 µg/ml, 0.29 ± 0.15 h, 6.30 ± 1.66 µg/ml•h, 5.55 ± 2.52 h, and 3.56 ± 1.05 h, respectively. The oral bioavailability was 3.42%. In primary rat hepatocytes treated with up to 10 µM deAND, a dose-dependent increase was noted in the expression of cytochrome P450 (CYP) 1A1/2, CYP2C6, and CYP3A1/2; UDP-glucuronosyltransferase (UGT) 1A1, NAD(P)H:quinone oxidoreductase (NQO1), π form of GSH S-transferase (GSTP), multidrug resistance-associated protein 2, p-glycoprotein, and organic anion transporter protein 2B1. Immunoblotting assay and EMSA revealed that deAND increases the nuclear translocation and DNA binding activity of aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), and nuclear factor erythroid-derived 2-related factor 2 (Nrf2). Knockdown of AhR and Nrf2 expression abolished deAND induction of CYP isozymes and UGT1A1, NQO1, and GSTP expression, respectively. CONCLUSION: These results indicate that deAND quickly passes through enterocytes in rats and effectively up-regulates hepatic drug-metabolizing enzyme and drug transporter expression in an AhR-, PXR-, and Nrf2-dependent manner.

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.