Ethanol Extract of Pinus koraiensis Leaf Ameliorates Alcoholic Fatty Liver via the Activation of LKB1–AMPK Signaling In Vitro and In Vivo

Although Pinus koraiensis leaf (PKL) was reported for its anti‐diabetes, anti‐obesity and anticancer effects as a folk remedy, the inhibitory effect of PKL on alcoholic fatty liver has never been elucidated yet. This study investigated the molecular mechanisms of PKL on alcoholic fatty liver in HepG2 cells, Sprague Dawley (SD) rats and Imprinting Control Region (ICR) mice. Pinus koraiensis leaf increased phosphorylation of liver kinase B1 (LKB1)/AMP‐activated protein kinase signaling, low‐density lipoprotein receptor and decreased fatty acid biosynthesis‐related proteins such as sterol regulatory element‐binding protein 1c, fatty acid synthase, 3‐hydroxy‐3‐methylglutaryl‐CoA reductase in HepG2 cells. In SD rats with 25% alcohol‐induced fatty liver, PKL suppressed the levels of aspartate aminotransferase and triglyceride and also enhanced the activities of antioxidant enzymes including superoxide dismutase, glutathione peroxidase and glutathione s‐transferase compared with untreated control. Furthermore, PKL increased serum alcohol dehydrogenase and serum aldehyde dehydrogenase, but decreased serum alcohol concentration in ICR mice after alcohol administration. Consistently, histochemical analysis revealed that PKL attenuated alcohol‐induced fatty liver in SD rats. Overall, these findings suggest that PKL ameliorates alcohol‐induced fatty liver via activation of LKB1–AMP‐activated protein kinase and modulation of proteins related to lipogenesis synthesis, cholesterol synthesis and fatty acid oxidation. Copyright © 2017 John Wiley & Sons, Ltd.     

Effects of Sinensetin on Lipid Metabolism in Mature 3T3‐L1 Adipocytes

Sinensetin is a rare polymethoxylated flavone found in certain citrus fruits. In this study, we investigated the effects of sinensetin on lipid metabolism in mature 3T3‐L1 adipocytes. Sinensetin decreased the expression of sterol regulatory element‐binding protein 1c (SREBP1c), suggesting its antiadipogeneic property via downreguation of SREBP1c. Also, sinensetin increased the phosphorylation of protein kinase A and hormone‐sensitive lipase, indicating its lipolytic property via a cAMP‐mediated signaling pathway. Moreover, sinensetin inhibited insulin‐stimulated glucose uptake by decreasing the phosphorylation of insulin receptor substrate and Akt. Furthermore, sinensetin increased the phosphorylation of AMP‐activated protein kinase (AMPK) and acetyl‐CoA carboxylase. It also upregulated mRNA expression of carnitine palmitoyltransferase‐1a, suggesting that sinensetin enhances fatty acid β‐oxidation through the AMPK pathway. Taken together, these results suggest that sinensetin may have potential as a natural agent for prevention/improvement of obesity. Copyright © 2012 John Wiley & Sons, Ltd.     

Hypolipidemic Activity of Okra is Mediated Through Inhibition of Lipogenesis and Upregulation of Cholesterol Degradation

Little is known about the hypolipidemic activity of okra; therefore, we investigated the hypolipidemic activity of okra and its interaction with gene expression of several key components involved in lipid homeostasis. Male C57BL/6 mice were randomly divided into three groups and fed with hyperlipidemic diet or two hyperlipidemic diets supplemented with 1% or 2% okra powder for eight weeks. Results demonstrated that okra dose‐dependently decreased serum and hepatic total cholesterol and triglyceride, and enhanced fecal excretion of bile acids. Gene expression analysis revealed that okra upregulated cholesterol 7α‐hydroxylase (CYP7A1) expression, downregulated expression of sterol regulatory element‐binding protein 1c (SREBP1c) and fatty acid synthase (FAS), with no effect on sterol regulatory element‐binding protein 2 (SREBP2), 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMGR), low‐density lipoprotein receptor (LDLR) and carnitine palmitoyltransferase‐1A (CPT1A). It was suggested that hypolipidemic activity of okra was mediated most likely by upregulation of cholesterol degradation through CYP7A1 and by inhibition of lipogenesis through SREBP1c and FAS. Okra raw and fractionated polysaccharide showed strong bile acid binding capacity in vitro, which may contribute to the hypolipidemic activity observed. In conclusion, okra has potential application in the management of hyperlipidemia and its associated metabolic disorders. Copyright © 2013 John Wiley & Sons, Ltd.     

Reactive Oxygen Species‐Mediated Activation of AMP‐Activated Protein Kinase and c‐Jun N‐terminal Kinase Plays a Critical Role in Beta‐Sitosterol‐Induced Apoptosis in Multiple Myeloma U266 cells

Although beta‐sitosterol has been well known to have anti‐tumor activity in liver, lung, colon, stomach, breast and prostate cancers via cell cycle arrest and apoptosis induction, the underlying mechanism of anti‐cancer effect of beta‐sitosterol in multiple myeloma cells was never elucidated until now. Thus, in the present study, the role of reactive oxygen species (ROS) in association with AMP‐activated protein kinase (AMPK) and c‐Jun N‐terminal kinase (JNK) pathways was demonstrated in beta‐sitosterol‐treated multiple myeloma U266 cells. Beta‐sitosterol exerted cytotoxicity, increased sub‐G₁ apoptotic population and activated caspase‐9 and ‐3, cleaved poly (ADP‐ribose) polymerase (PARP) followed by decrease in mitochondrial potential in U266 cells. Beta‐sitosterol promoted ROS production, activated AMPK, acetyl‐CoA carboxylase (ACC) and JNK in U266 cells. Also, beta‐sitosterol attenuated the phosphorylation of AKT, mammalian target of rapamycin and S6K, and the expression of cyclooxygenase‐2 and VEGF in U266 cells. Conversely, AMPK inhibitor compound C and JNK inhibitor SP600125 suppressed apoptosis induced by beta‐sitosterol in U266 cells. Furthermore, ROS scavenger N‐acetyl L‐cysteine attenuated beta‐sitosterol‐mediated sub‐G₁ accumulation, PARP cleavage, JNK and AMPK activation in U266 cells. Overall, these findings for the first time suggest that ROS‐mediated activation of cancer metabolism‐related genes such as AMPK and JNK plays an important role in beta‐sitosterol‐induced apoptosis in U266 multiple myeloma cells. Copyright © 2013 John Wiley & Sons, Ltd.     

Apoptotic effect of lambertianic acid through AMPK/FOXM1 signaling in MDA‐MB231 breast cancer cells

Though lambertianic acid (LA) was known to exert antitumor effect in liver and prostate cancers, its underlying anticancer mechanism is never reported in breast cancers so far. Thus, in this study, apoptotic mechanism of LA was elucidated in MDA‐MB‐231 breast cancer cells. Here, LA increased cytotoxicity in MCF‐7 and MDA‐MB‐231 cells; enhanced sub‐G1 population, G2/M arrest, and cleaved poly(ADP‐ribose) polymerase; activated phosphorylation of AMP‐activated protein kinase (AMPK)/acetyl‐CoA carboxylase pathway; and also suppressed phosphorylation of AKT and the expression of forkhead box M1 (FOXM1), X‐linked inhibitor of apoptosis protein, B‐cell lymphoma 2, and CyclinB1 in MDA‐MB‐231 cells. Furthermore, AMPK inhibitor compound C reversed the effect of LA on FOXM1, Cyclin B1, and cleaved poly(ADP‐ribose) polymerase in MDA‐MB‐231 cells. Notably, immunoprecipitation revealed that LA disturbed the direct binding of AKT and FOXM1 in MDA‐MB‐231 cells. Overall, these findings suggest that LA‐induced apoptosis is mediated via activation of AMPK and inhibition of AKT/FOXM1 signaling pathway.     

Bromophenol (5‐bromo‐3,4‐dihydroxybenzaldehyde) isolated from red alga Polysiphonia morrowii inhibits adipogenesis by regulating expression of adipogenic transcription factors and AMP‐activated protein kinase activation in 3T3‐L1 adipocytes

The aim of the present study was to investigate the effect of 5‐bromo‐3,4‐dihydroxybenzaldehyde (BD) isolated from Polysiphonia morrowii on adipogenesis and differentiation of 3T3‐L1 preadipocytes into mature adipocytes and its possible mechanism of action. Levels of lipid accumulation and triglyceride were significantly lower in BD treated cells than those in untreated cells. In addition, BD treatment reduced protein expression levels of peroxisome proliferator‐activated receptor‐γ, CCAAT/enhancer‐binding proteins α, and sterol regulatory element‐binding protein 1 compared with control (no treatment). It also reduced expression levels of adiponectin, leptin, fatty acid synthase, and fatty acid binding protein 4. AMP‐activated protein kinase activation was found to be one specific mechanism involved in the effect of BD. These results demonstrate that BD possesses inhibitory effect on adipogenesis through activating AMP‐activated protein kinase signal pathway.     

Hypolipidemic activity and mechanisms of the total phenylpropanoid glycosides from Ligustrum robustum (Roxb.) Blume by AMPK‐SREBP‐1c pathway in hamsters fed a high‐fat diet

The aim of this study was to evaluate the hypolipidemic effect and mechanisms of total phenylpropanoid glycosides extracted from Ligustrum robustum (Roxb.) Blume (LRTPG) in hamsters fed a high‐fat diet and to discover bioactive components in HepG2 cell model induced by oleic acid. LRTPG of high (1.2 g/kg), medium (0.6 g/kg), and low (0.3 g/kg) doses was administrated daily for 21 consecutive days in hamsters. We found that in hamsters fed a high‐fat diet, LRTPG effectively reduced the concentrations of plasma triglycerides (TG), free fatty acid, total cholesterol, low‐density lipoprotein cholesterol, and hepatic TG and total cholesterol. And the compounds acteoside, ligupurpuroside A, ligupurpuroside C, and ligupurpuroside D significantly inhibited lipid accumulation in HepG2 cell at the concentration of 50 μmol/L. Mechanism research demonstrated that LRTPG increased the levels of phospho–AMP‐activated protein kinase and phospho‐sterol regulatory element binding protein‐1c in liver, further to suppress the downstream lipogenic genes as stearoyl‐CoA desaturase 1, glycerol‐3‐phosphate acyltransferase, 1‐acylglycerol‐3‐phosphate O‐acyltransferase 2, and diacylglycerol acyltransferase 2. In addition, LRTPG increased the hydrolysis of circulating TG by up‐regulating lipoprotein lipase activities. These results indicate that LRTPG prevents hyperlipidemia via activation of hepatic AMP‐activated protein kinase‐sterol regulatory element binding protein‐1c pathway.     

An In Vitro and Molecular Informatics Study to Evaluate the Antioxidative and β‐hydroxy‐β‐methylglutaryl‐CoA Reductase Inhibitory Property of Ficus virens Ait

The present study is initially intended to evaluate antioxidant and β‐hydroxy‐β‐methylglutaryl‐CoA reductase (HMGR) inhibitory property of Ficus virens Ait., first by in vitro analyses followed by a corroboratory molecular informatics study. Our results show that of all the sequentially extracted fraction of F. virens bark and leaves extract, F. virens bark methanol extract exhibits strong radical scavenging, antioxidant and oxidative DNA damage protective activity, which is well correlated with its total phenolic content. In addition, F. virens bark methanol extract, which is non‐cytotoxic, significantly and non‐covalently inhibit the HMGR activity (IC₅₀ = 3.45 ± 0.45 µg/ml) in comparison with other extracts. The mechanistic aspect of this inhibition activity is authenticated by molecular docking study of bioactive compounds as revealed from gas chromatography–mass spectrometry data, with HMGR. The analysis for the first time indicates that quinic acid (ΔG: ?8.11 kcal/mol) and paravastatin (ΔG: ?8.22 kcal/mol) exhibit almost same binding energy, while other compounds also showed good binding energy, suggesting that quinic acid alone or in combination with other major bioactive compound is probably responsible for HMGR inhibitory property of the extract and plausibly can be used in in vivo system for the management, prevention, and alleviation of hypercholesterolemia as well as hypercholesterolemia‐induced oxidative stress. Copyright © 2013 John Wiley & Sons, Ltd.     

The Novel p53‐Dependent Metastatic and Apoptotic Pathway Induced by Vitexin in Human Oral Cancer OC2 Cells

Vitexin, identified as apigenin‐8‐C‐D‐glucopyranoside, a natural flavonoid compound found in certain herbs such as hawthorn herb, has been reported to exhibit anti‐oxidative, anti‐inflammatory, anti‐metastatic and antitumor properties. The aim of this study was to investigate the possible existence of p53‐dependent pathway underlying vitexin‐induced metastasis and apoptosis in human oral cancer cells, OC2 cells. Vitexin decreased cell viability significantly. Meanwhile, the expression of tumor suppressor p53 and a small group of its downstream genes, p21 ^WAF1 and Bax, were upregulated. The p53 inhibitor pifithrin‐α (PFT‐α) knockdown of the signaling of p53 led vitexin to lose its antitumor effect and inhibited the expression of p53 downstream genes, p21^WAF1 and Bax. Vitexin had anti‐metastatic potential accompanied with increasing plasminogen activator inhibitor 1 (PAI‐1) accumulation and decreasing matrix metalloproteinase‐2 expression. Our present study evidenced, by using p53 inhibitor PFT‐α, PAI‐1 and peroxisome proliferator‐activated receptor γ are downstream genes of p53 in vitexin‐induced signaling. MAPK inhibitor PD98059 decreased the OC2 cells viability significantly. The expression of p53 and its downstream genes p21 ^WAF1 and Bax were enhanced by blocking the activation of p42/p44 MAPK in response to treatment with vitexin. Moreover, p42/p44 MAPK played a negative role in p53‐dependent metastasis and apoptosis. We give evidence for the first time that the novel p53‐dependent metastatic and apoptotic pathway induced by vitexin in human oral cancer OC2 cells. Copyright © 2012 John Wiley & Sons, Ltd.     

A polymethoxy flavonoids‐rich Citrus aurantium extract ameliorates ethanol‐induced liver injury through modulation of AMPK and Nrf2‐related signals in a binge drinking mouse model

Nobiletin and tangeretin are polymethoxy flavonoids (PMFs), found in rich quantities in the peel of citrus fruits. In the present study, we assessed the biological effect of the PMFs on liver damage using a mouse model of binge drinking. First, we extracted PMFs from the peels of Citrus aurantium to make Citrus aurantium extract (CAE). Male C57BL/6 mice were orally treated with silymarin and CAE (50, 100, and 200 mg/kg) for 3 days prior to ethanol (5 g/kg, total of 3 doses) oral gavage. Liver injury was observed in the ethanol alone group, as evidenced by increases in serum hepatic enzymes and histopathologic alteration, as well as by hepatic oxidative status disruption. CAE improved serum marker and hepatic structure and restored oxidative status by enhancing antioxidant enzyme levels and by reducing lipid peroxidation levels. In addition, CAE evidently suppressed inflammation and apoptosis in the livers of mice administered with ethanol, by 85% (tumor necrosis factor‐α) and 44% compared to the control group, respectively. Furthermore, CAE activated lipid metabolism related signals and enhanced phosphorylation of AMP‐activated protein kinase (AMPK) and nuclear factor E2‐related factor 2 (Nrf2) with several cytoprotective proteins including heme oxygenase‐1, NAD(P)H quinone oxidoreductase 1, and γ‐glutamylcysteine synthetase. Taken together, the present study demonstrated that, CAE possesses antioxidant, anti‐inflammatory, and antiapoptotic activity against ethanol‐induced liver injury. Copyright © 2015 John Wiley & Sons, Ltd.     

Retracted: Bilobalide alleviates tumor necrosis factor‐alpha‐induced pancreatic beta‐cell MIN6 apoptosis and dysfunction through upregulation of miR‐153

Type 1 diabetes mellitus (T1DM) is a systemic disease and one classical type of total DM. Bilobalide (BB) is constituted of EGb 761. Our purpose was identifying the role of BB in TIDM in the current study. MIN6 cells were treated by TNF‐α; then, viability, apoptosis, and insulin secretion were assessed by performing Cell Counting Kit‐8 assay, flow cytometry, glucose‐stimulated insulin secretion assay, and western blot. The effects of BB were assessed to identify its function. Further, the above mentioned parameters were reassessed when silencing miR‐153. TNF‐α declined viability and insulin secretion as well as raised apoptosis and inducible nitric oxide synthase (iNOS) expression in MIN6 cells. BB alleviated the apoptosis and dysfunction induced by TNF‐α. MiR‐153 expression was elevated by BB when induced by TNF‐α. Increase of viability and insulin secretion as well as decline of apoptosis and iNOS induced by BB treatment was alleviated by silencing miR‐153. The rates of p/t‐p70S6K, p/t‐mammalian target of rapamycin (mTOR) and p/t‐adenosine monophosphate‐activated protein kinase (AMPK) were raised by BB and suppressed by silencing miR‐153 under TNF‐α induced condition. BB raised viability and insulin secretion, declined apoptosis and iNOS expression by up‐regulating miR‐153. Furthermore, BB activated AMPK/mTOR pathway by up‐regulating miR‐153.     

Regulation of obesity and lipid disorders by extracts from Angelica acutiloba root in high-fat diet-induced obese rats

The aim of this study was to investigate the antiobesity and antihyperlipidemic effects of Angelica acutiloba root (Japanese Dong Quai). High‐fat diet (HFD)‐induced obese rats were treated orally with the polyphenolic‐rich extract of Angelica acutiloba root (AARE) once daily for 8 weeks. The AARE (300 mg/kg per day) supplementation significantly lowered body weight gain, visceral fat‐pad weights and plasma lipid levels, as well as the coronary artery risk index and the atherogenic index of HFD‐fed rats. The AARE caused dose related reductions in the hepatic triglyceride and cholesterol contents, as well as lowered hepatic lipid droplet accumulation and epididymal adipocyte size in the HFD‐fed rats. The AARE reversed the HFD‐induced down‐regulation of the hepatic peroxisome proliferator activated receptor‐α (PPARα). The HFD‐induced decreases of the hepatic protein level of acyl‐CoA oxidase (ACO), and the cytochrome P450 isoform 4A1 (CYP4A1) was up‐regulated by AARE. The elevated expressions of hepatic sterol regulatory element binding proteins (SREBPs) of HFD‐fed rats were lowered by AARE. These results suggest that AARE attenuated visceral fat accumulation and improved hyperlipidemia in HFD‐induced obesity by increasing lipid metabolism through the down‐regulation of SREBPs and enhanced the expression of ACO and CYP4A1 in the liver, which was likely mediated by up‐regulation of the expression of hepatic PPARα. Copyright © 2011 John Wiley & Sons, Ltd.     

Inhibition of LPS‐Induced TNF‐α and NO Production in Mouse Macrophage and Inflammatory Response in Rat Animal Models by a Novel Ayurvedic Formulation,BV‐9238

Rheumatoid arthritis is a chronic crippling disease, where protein‐based tumor necrosis factor‐alpha (TNF‐α) inhibitors show significant relief, but with potentially fatal side effects. A need for a safe, oral, cost‐effective small molecule or phyto‐pharmaceutical is warranted. BV‐9238 is an Ayurvedic poly‐herbal formulation containing specialized standardized extracts of Withania somnifera, Boswellia serrata, Zingiber officinale and Curcuma longa. The anti‐inflammatory and anti‐arthritic effects of BV‐9238 were evaluated for inhibition of TNF‐α and nitric oxide (NO) production, in lipopolysaccharide‐stimulated, RAW 264.7, mouse macrophage cell line. BV‐9238 reduced TNF‐α and NO production, without any cytotoxic effects. Subsequently, the formulation was tested in adjuvant‐induced arthritis (AIA) and carrageenan‐induced paw edema (CPE) rat animal models. AIA was induced in rats by injecting Freund's complete adjuvant intra‐dermally in the paw, and BV‐9238 and controls were administered orally for 21 days. Arthritic scores in AIA study and inflamed paw volume in CPE study were significantly reduced upon treatment with BV‐9238. These results suggest that the anti‐inflammatory and anti‐arthritic effects of BV‐9238 are due to its inhibition of TNF‐α, and NO, and this formulation shows promise as an alternate therapy for inflammatory disorders where TNF‐α and NO play important roles. Copyright © 2014 John Wiley & Sons, Ltd.     

Effects of Lonicera japonica Thunb. on Type 2 Diabetes via PPAR‐γ Activation in Rats

Lonicera japonica Thunb. (Caprifoliaceae) is a traditional herbal medicine and has been used to treat diabetic symptoms. Notwithstanding its use, the scientific basis on anti‐diabetic properties of L. japonica is not yet established. This study is designed to investigate anti‐diabetic effects of L. japonica in type 2 diabetic rats. L. japonica was orally administered at the dose of 100 mg/kg in high‐fat diet‐fed and low‐dose streptozotocin‐induced rats. After the treatment of 4 weeks, L. japonica reduced high blood glucose level and homeostatic model assessment of insulin resistance in diabetic rats. In addition, body weight and food intake were restored by the L. japonica treatment. In the histopathologic examination, the amelioration of damaged β‐islet in pancreas was observed in L. japonica‐treated diabetic rats. The administration of L. japonica elevated peroxisome proliferator‐activated receptor gamma and insulin receptor subunit‐1 protein expressions. The results demonstrated that L. japonica had anti‐diabetic effects in type 2 diabetic rats via the peroxisome proliferator‐activated receptor gamma regulatory action of L. japonica as a potential mechanism. Copyright © 2015 John Wiley & Sons, Ltd.