Resveratrol Regulates Activated Hepatic Stellate Cells by Modulating NF‐κB and the PI3K/Akt Signaling Pathway

In the present study, we investigated whether resveratrol could suppress the hepatic fibrogenesis in activated hepatic stellate cells. The immortalized rat hepatic stellate cells, t‐HSC/Cl‐6, were treated with resveratrol 1 h prior to lipopolysaccharide (LPS, 1 μg/mL). Resveratrol decreased t‐HSC/Cl‐6 cell viability at much lower concentrations within 24 h. Resveratrol pretreatment also decreased the LPS‐induced protein expression of α‐SMA and collagen I. In addition, resveratrol significantly reduced the protein expression of Toll‐like receptor 4 (TLR4) and myeloid differentiation primary response gene 88 (MyD88), and the expression of phosphorylated phosphatidylinositol 3‐kinase (PI3K) and phosphorylated serine/threonine kinase B (Akt). Moreover, resveratrol markedly blocked the translocation of nuclear factor (NF)‐κB in LPS‐activated HSCs. Furthermore, resveratrol inhibited HSCs activation through stimulating LXRβ, but did not influence LXRα. Overall, we conclude that the antifibrotic effect of resveratrol is the result of blocking NF‐κB activation and PI3K/Akt phosphorylation, which inhibits HSC activation to obstruct liver fibrosis. Thus, resveratrol may be a natural agent for preventing hepatic fibrosis.     

The protective role of natural phytoalexin resveratrol on inflammation, fibrosis and regeneration in cholestatic liver injury

Liver injuries can trigger a cascade of inflammatory responses and as a result, initiate the process of hepatic regeneration and fibrogenesis. Resveratrol (RSV) has multiple health‐promoting benefits. This study evaluated the potential protective effects and mechanism of RSV as related to cholestatic liver injury. RSV was given (4 mg/kg/day, i.p.) for either 3 days or 7 days after bile duct ligation (BDL) injury. RSV significantly reduced serum ALT, AST but not T‐bil on Day 3. At this early stage of injury, RSV significantly reduced TNF‐α and IL‐6 mRNA and decreased the number of Kupffer cells (CD68⁺) recruited in the injured liver. RSV decreased hepatic fibrosis and reduced collagen Iα1 and TIMP‐1 mRNA on Day 7. At the later stages of injury, RSV increased the number of Ki67⁺ hepatocytes indicating that RSV promoted hepatocyte proliferation. Additionally, it resulted in decreased expression of 4‐hydroxynonenal and increased expression of the hepatocyte growth factor protein and mRNA in the RSV‐treated BDL group. Meanwhile, RSV reduced the mortality rate of BDL mice. In conclusion, RSV attenuated inflammation and reduced Kupffer cells activation. RSV decreased fibrosis and promoted hepatocyte regeneration, which increased the survival of BDL mice. RSV was beneficial for the treatment of cholestatic liver injury.     

Camel and bovine milk lactoferrins activate insulin receptor and its related AKT and ERK1/2 pathways

Lactoferrin (LF) is a milk protein that may be an interesting candidate for the antidiabetic properties of milk due to its well-documented bioactivity and implication in diabetes. Here, we investigated the functional action of LF purified from camel and bovine milk (cLF, bLF) on insulin receptors (IR) and their pharmacology and signaling in hepatocarcinoma (HepG2) and human embryonic kidney (HEK293) cells. For this, we examined IR activation by bioluminescence resonance energy transfer (BRET) technology and the phosphorylation of its key downstream signaling kinases by western blot. The purified cLF and bLF induced phosphorylation of IR, AKT, and ERK1/2 in HepG2 and HEK293 cells. The BRET assays in HEK293 cells confirm the pharmacological action of cLF and bLF on IR, with a possible allosteric mode of action. This reveals for the first time the bioactivity of LF toward IR function, indicating it as a potential bioactive protein behind the antidiabetic properties of camel milk.     

Synthesis of Quercetin‐3‐O‐Glucoside from Rutin by Penicillium decumbens Naringinase

Enzymatic bioconversion of rutin to quercetin‐3‐O‐glucoside (Q‐3‐G) by Penicillium decumbens naringinase was increased with reaction pH increased approximately to pH 6.0. It resulted in greater than 92% production of Q‐3‐G due to the removal of the terminal rhamnose at the controlled pH 6.0. The enzymatic bioconversion of rutin to Q‐3‐G was repetitively performed, yielding 84% after 5 batches with little quercetin formation. Interestingly, the water solubility of Q‐3‐G was enhanced 69‐ and 328‐fold over those of rutin and quercetin, which may make Q‐3‐G more bioavailable in food. Q‐3‐G was approximately 6‐ and 1.4‐fold more potent than rutin as an inhibitor of human intestinal maltase and human DL‐3‐hydroxy‐3‐methylglutalyl coenzyme A reductase. Q‐3‐G was less potent (16‐ and 1.3‐fold, respectively) than quercetin as an inhibitor of these enzymes. However, the results suggest that Q‐3‐G may be confirmed more effective and bioavailable food component than rutin and even quercetin because of its enhanced solubility and inhibitory properties. Practical Application : Bioconverted intermediate, quercetin‐3‐O‐glucoside (Q‐3‐G), was found and confirmed to be largely more soluble than rutin and quercetin in water solution, which might make it more bioavailable as food ingredient. In addition, Q‐3‐G inhibited mildly the intestinal maltase, which might act as antidiabetic substance by modulating the adsorption of glucose in the intestine.     

Chronic dietary intake of quercetin alleviates hepatic fat accumulation associated with consumption of a Western-style diet in C57/BL6J mice

Scope: To determine the effect of consumption of a quercetin‐rich diet on obesity and dysregulated hepatic gene expression. Methods and results: C56BL/6J mice were fed for 20 wk on AIN93G (control) or a Western diet high in fat, cholesterol and sucrose, both with or without 0.05% quercetin. Triglyceride levels in plasma, thiobarbituric acid‐reactive substances (oxidative stress marker) and glutathione levels and peroxisome proliferator‐activated receptor α expression in livers of mice fed with the Western diet were all improved after 8 wk feeding with quercetin. After 20 wk, further reductions of visceral and liver fat accumulation and improved hyperglycemia, hyperinsulinemia, dyslipidemia and plasma adiponectin and TNFα levels in these mice fed with quercetin were observed. The expression of hepatic genes related to steatosis, such as peroxisome proliferator‐activated receptor γ and sterol regulatory element‐binding protein‐1c was also normalized by quercetin. In mice fed with the control diet, quercetin did not affect body weight but reduces the plasma TNFα and hepatic thiobarbituric acid‐reactive substance levels. Conclusion: In mice fed with a Western diet, chronic dietary intake of quercetin reduces liver fat accumulation and improves systemic parameters related to metabolic syndrome, probably mainly through decreasing oxidative stress and reducing PPARα expression, and the subsequent reduced expression in the liver of genes related to steatosis.     

Dietary quercetin alleviates diabetic symptoms and reduces streptozotocin‐induced disturbance of hepatic gene expression in mice

Quercetin is a food component that may ameliorate the diabetic symptoms. We examined hepatic gene expression of BALB/c mice with streptozotocin (STZ)‐induced diabetes to elucidate the mechanism of the protective effect of dietary quercetin on diabetes‐associated liver injury. We fed normal and STZ‐induced diabetic mice with diets containing quercetin for 2 wk and compared the patterns of hepatic gene expression in these groups of mice using a DNA microarray. Diets containing 0.1 or 0.5% quercetin lowered the STZ‐induced increase in blood glucose levels and improved plasma insulin levels. A cluster analysis of the hepatic gene expressions showed that 0.5% quercetin diet suppressed STZ‐induced alteration of gene expression. Gene set enrichment analysis (GSEA) and quantitative RT‐PCR analysis showed that the quercetin diets had greatest suppressive effect on the STZ‐induced elevation of expression of cyclin‐dependent kinase inhibitor p21(WAF1/Cip1) (Cdkn1a). Quercetin also suppressed STZ‐induced expression of Cdkn1a in the pancreas. Dietary quercetin might improve liver and pancreas functions by enabling the recovery of cell proliferation through the inhibition of Cdkn1a expression. Unexpectedly, in healthy control mice the 0.5 and 1% quercetin diets reduced the expression of ubiquitin C (Ubc), which has heat‐shock element (HSE) in the promoter region, in the liver.     

Quercetin properties as a functional ingredient in omega‐3 enriched fish gels fed to rats

Wistar rats were fed with surimi gels containing either sunflower oil, fish oil (ω3), and the same formulation additionally supplemented with 1.05 g kg⁻¹ quercetin (ω3‐Q). Antioxidant capacity was highest in gels with added quercetin when measured by the ferric‐reducing/antioxidant power (FRAP) method, but not by the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) free radical assay. Lipid stability was not enhanced by quercetin since commercial fish oil already contains stabilizers. Quercetin modified neither rheological properties nor water‐holding capacity of the gels; however, it produced a large increase in yellowness (b*). Serum lipid profile of rats was not significantly different. Total serum antioxidant capacity by FRAP was significantly increased only in the ω3‐Q group. Plasma malondialdehyde was similar in the ω3 and ω3‐Q groups, indicating no prooxidative effect of quercetin in vivo. These results suggest that quercetin might be used as a food ingredient in fish gel to improve some nutritional properties of the gel. Copyright © 2005 Society of Chemical Industry     

Salicornia Extract Ameliorates Salt‐Induced Aggravation of Nonalcoholic Fatty Liver Disease in Obese Mice Fed a High‐Fat Diet

High‐fat and high‐salt intakes are among the major risks of chronic diseases including obesity, nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH). Salicornia is a halophytic plant known to exert antioxidant, antidiabetic, and hypolipidemic effects, and Salicornia‐extracted salt (SS) has been used as a salt substitute. In this study, the effects of SS and purified salt (PS) on the aggravation of NAFLD/NASH were compared. C57BL/6J male mice (8‐wk‐old) were fed a high‐fat diet (HFD) for 6 mo and divided into 3 dietary groups, which were additionally fed HFD, HFD + SS, and HFD + PS for 13 wk. PS induced aggravation of NAFLD/NASH in HFD‐fed mice. Although the actual salt intake was same between the PS and SS groups as 1% of the diet (extrapolated from the World Health Organization [WHO] guideline), SS induced less liver injury and hepatic steatosis compared to PS. The hepatic mRNA expressions of inflammatory cytokines and fibrosis marker were significantly lower in the SS group than the PS group. Oxidative stress is one of the major causes of inflammation in NAFLD/NASH. Results of the component analysis showed that the major polyphenols that exhibited antioxidant activity in the Salicornia water extract were ferulic acid, caffeic acid, and isorhamnetin. These results suggest that even the level of salt intake recommended by WHO can accelerate the progression of liver disease in obese individuals consuming HFD. It is proposed that SS can be a salt substitute for obese individuals who consume HFD.     

Ursolic Acid,a Natural Nutraceutical Agent,Targets Caspase3 and Alleviates Inflammation‐Associated Downstream Signal Transduction

1 Scope

Ursolic acid (UA) is a pentacyclicterpenoid carboxylic acid that is present in a wide variety of plant foods. There are many beneficial health effects that are attributed to the properties of UA. However, the specific cellular targets of UA and the mechanism underlying downstream signal transduction processes linked to the anti‐inflammation pathway have not been thoroughly elucidated to date.

2 Methods and results

Chemical biology strategies such as target fishing, click reaction synthesis of a UA probe and molecular imaging were used to identify potential target proteins of UA. Cysteinyl aspartate specific proteinase 3 (CASP3) and its downstream signaling pathway were verified as potential targets by molecular docking, intracellular enzyme activity evaluation and accurate pathway analysis. The results indicated that UA acted on CASP3, ERK1 and JNK2 targets, alleviated inflammation‐associated downstream multiple signal transduction factors, including ERK1, NF‐κB and STAT3, and exhibited anti‐inflammation activities.

3 Conclusion

As a natural dietary supplement, UA demonstrated anti‐inflammation activity via inhibition of CASP3 and shows the potential to improve the therapy effect of several inflammation‐associated diseases.     

Quercetin‐induced apoptotic cascade in cancer cells: Antioxidant versus estrogen receptor α‐dependent mechanisms

The flavonol quercetin, especially abundant in apple, wine, and onions, is reported to have anti‐proliferative effects in many cancer cell lines. Antioxidant or pro‐oxidant activities and kinase inhibition have been proposed as molecular mechanisms for these effects. In addition, an estrogenic activity has been observed but, at the present, it is poorly understood whether this latter activity plays a role in the quercetin‐induced anti‐proliferative effects. Here, we studied the molecular mechanisms of quercetin committed to the generation of an apoptotic cascade in cancer cells devoid or containing transfected estrogen receptor α (ERα; i.e., human cervix epitheloid carcinoma HeLa cells). Although none of tested quercetin concentrations increase reactive oxygen species (ROS) generation in HeLa cells, quercetin stimulation prevents the H₂O₂‐induced ROS production both in the presence and in the absence of ERα. However, this flavonoid induces the activation of p38/MAPK, leading to the pro‐apoptotic caspase‐3 activation and to the poly(ADP‐ribose) polymerase cleavage only in the presence of ERα. Notably, no down‐regulation of survival kinases (i.e., AKT and ERK) was reported. Taken together, these findings suggest that quercetin results in HeLa cell death through an ERα‐dependent mechanism involving caspase‐ and p38 kinase activation. These findings indicate new potential chemopreventive actions of flavonoids on cancer growth.     

Antidiabetic Potential of Green Seaweed Enteromorpha prolifera Flavonoids Regulating Insulin Signaling Pathway and Gut Microbiota in Type 2 Diabetic Mice

This study aimed to investigate the antidiabetic activity of water–ethanol extract of green macroalgae Enteromorpha prolifera (EPW) and its flavonoid‐rich fraction less than 3 kDa (EPW3) in type 2 diabetic mice induced by streptozotocin and a high‐sucrose/high‐fat diet. The major active compounds were identified using ultra‐performance liquid chromatography coupled with quadrupole‐time‐of‐flight‐tandem mass spectrometry. Quantitative gene expression analysis of the insulin signaling pathway was performed. The effects of EPW3 on gut microflora in diabetic mice were analyzed by high‐throughput 16S rRNA gene sequencing. The results showed EPW3 treatment decreased the fasting blood glucose, improved oral glucose tolerance, and protected against liver and kidney injury with reduced inflammation in diabetic mice. The active principle of EPW3 revealed hypoglycemic effect as indicated by activation of the IRS1/PI3K/AKT and inhibition of the JNK1/2 insulin pathway in liver. Furthermore, the treatment significantly enriched the abundance of Lachnospiraceae and Alisties, which were positive correlation of metabolic phenotypes. These findings indicated that EPW3 possessed great therapeutic potential as adjuvant therapy for type 2 diabetes.     

The importance of the long-chain polyunsaturated fatty acid n-6/n-3 ratio in development of non-alcoholic fatty liver associated with obesity

Non-alcoholic fatty liver disease (NAFLD) is the most important cause of chronic liver disease that is characterized by hepatocyte triacylglycerol accumulation (steatosis), which can progress to inflammation, fibrosis, and cirrhosis (steatohepatitis). Overnutrition triggers the onset of oxidative stress in the liver due to higher availability and oxidation of fatty acids (FA), with development of hyperinsulinemia and insulin resistance (IR), and n-3 long-chain polyunsaturated FA (n-3 LCPUFA) depletion, with enhancement in the n-6/n-3 LCPUFA ratio favouring a pro-inflammatory state. These changes may lead to hepatic steatosis by different mechanisms, namely, (i) IR-dependent higher peripheral lipolysis and FA flux to the liver, (ii) n-3 LCPUFA depletion-induced changes in DNA binding activity of sterol regulatory element-binding protein 1c (SREBP-1c) and peroxisome proliferator-activated receptor α (PPAR-α) favouring lipogenesis over FA oxidation, and (iii) hyperinsulinemia-induced activation of lipogenic factor PPAR-γ. Supplementation with n-3 LCPUFA appears to reduce nutritional hepatic steatosis in adults, however, other histopathologic features of NAFLD remain to be studied.     

铁皮石斛对缺血再灌注诱导的心律失常的保护作用

为研究甘氨酸（Glycine,Gly）对肝脏IGF-1表达和分泌的影响,本文一方面采用不同浓度Gly离体处理HepG2肝细胞,另一方面采用小鼠尾静脉注射1.0 g/kg Gly和等摩尔Ala,0.5 h和1 h后采集血液、肝脏和肌肉样品。研究采用定量PCR检测了IGF-1以及IGFBPs基因表达水平的影响,同时运用Western blot法分析了肝细胞和肝脏生长激素受体通路（JAK2/STAT5）与肌肉IGF-I受体通路（ERK/Akt/mTOR）的变化。研究结果发现,不同浓度的Gly能剂量依赖性提高HepG2细胞IGF-1的蛋白和mRNA表达水平;1.0 g/kg Gly能显著提高小鼠血清中的IGF-1和白蛋白含量,且极显著提高肝脏IGF-1和IGFBP-3 mRNA水平,下调IGFBP-1 mRNA水平。细胞和活体试验均表明,Gly能够有效激活肝细胞和肝脏JAK2/STAT5信号通路,同时提高腓肠肌IGF-I受体信号通路磷酸化水平。这些研究说明甘氨酸可能直接提高肝脏生长激素受体信号通路敏感性,从而促进IGF-1的表达和分泌。研究结果为深入揭示甘氨酸的分子营养学功能和作用机制提供了实验依据。     

Change in Flavonoid Composition and Antioxidative Activity during Fermentation of Onion (Allium cepa L.) by Leuconostoc mesenteroides with Different Salt Concentrations

The aim of this study is to investigate the change in flavonoid composition and antioxidative activity during fermentation of onion (Allium cepa L.) by Leuconostoc mesenteroides with different NaCl concentrations. In order to qualify and quantify the flavonoids during fermentation of onion, 7 flavonoids, [quercetin 3,7‐O‐β‐d ‐diglucopyranoside (Q3,7G), quercetin 3,4′‐O‐β‐d ‐diglucopyranoside (Q3,4′G), quercetin 3‐O‐β‐d ‐glucopyranoside (Q3G), quercetin 4′‐O‐β‐d ‐glucopyranoside (Q4′G), isorhamnetin 3‐O‐β‐d ‐glucopyranoside (IR3G), quercetin (Q), and isorhamnetin (IR)], were isolated and identified from onion. During fermentation, the contents of flavonoid glucosides (Q3,7G, Q3,4′G, Q3G, Q4′G, and IR3G) gradually decreased, whereas the contents of flavonoid aglycones (Q, IR) gradually increased. Decline rates of the flavonoid glucosides increased with the addition of L. mesenteroides. Furthermore, the activity of β‐glucosidase, which is produced by L. mesenteroides, is dose‐dependently inhibited with different NaCl concentrations during fermentation. The presence of L. mesenteroides enhanced the antioxidative activity of onion as demonstrated using the 1,1‐diphenyl‐2‐picrylhydrazyl, 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid), and reducing power assays. The enhancement of antioxidative activity was considered because the content of flavonoid aglycones increased during fermentation. However, the addition of NaCl may decrease the antioxidative activity; we surmise that this phenomenon occurs because of the inhibition of β‐glucosidase by NaCl. Therefore, we conclude that the addition of NaCl may be useful for the regulation of antioxidative activity via the control of β‐glucosidase action, during the fermentation of flavonoid glucoside‐rich foods.     

Phenethyl isothiocyanate inhibits STAT3 activation in prostate cancer cells

This study was undertaken to investigate the mechanism by which phenethyl isothiocyanate (PEITC), a natural compound from cruciferous vegetables, exhibits antitumor effect on prostate cancer cells. Cell proliferation, cell cycle, Western blot, gene transfer, and reporter assays were used to test the effects of PEITC on the growth and IL6/JAK/STAT3 pathway in prostate cancer. The result showed that PEITC significantly inhibited DU145 cell proliferation in a dose‐dependent manner and induced the cell arrest at G2‐M phase. PEITC inhibited both constitutive and IL‐6‐induced STAT3 activity in DU145 cells. IL‐6‐stimulated phosphorylation of JAK2, an STAT3 upstream kinase, was also attenuated by PEITC. Moreover, an antioxidant reagent, N‐acetyl‐L ‐cysteine (NAC) which suppresses reactive oxygen species (ROS) generation, reversed the early inhibitory effects of PEITC on cell proliferation, constitutive or IL‐6‐mediated JAK‐STAT3 phosphorylation in PCa cells. Taken together, our data demonstrated that PEITC can inhibit the activation of the JAK‐STAT3 signal‐cascade in prostate cancer cells and the underlying mechanism may be partially involved with blocking cellular ROS production during the early stage of the signaling activation by IL‐6.     

Syringetin,a flavonoid derivative in grape and wine,induces human osteoblast differentiation through bone morphogenetic protein‐2/extracellular signal‐regulated kinase 1/2 pathway

Syringetin (3,5,7,4′‐tetrahydroxy‐3′,5′dimethoxyflavone), a flavonoid derivative, is present in grape and wine. By means of alkaline phosphatase (ALP) activity, osteocalcin, and type I collagen ELISA, we have shown that syringetin exhibits a significant induction of differentiation in MC3T3‐E1 mouse calvaria osteoblasts and human fetal osteoblastic 1.19 cell line human osteoblasts. ALP and osteocalcin are phenotypic markers for early‐stage differentiated osteoblasts and terminally differentiated osteoblasts, respectively. Our results indicate that syringetin stimulates osteoblast differentiation at various stages, from maturation to terminally differentiated osteoblasts. Induction of differentiation by syringetin is associated with increased bone morphogenetic protein‐2 (BMP‐2) production. The BMP‐2 antagonist noggin blocked syringetin‐mediated ALP activity and osteocalcin secretion enhancement, indicating that BMP‐2 production is required in syringetin‐mediated osteoblast maturation and differentiation. Induction of differentiation by syringetin is associated with increased activation of SMAD1/5/8 and extracellular signal‐regulated kinase 1/2 (ERK1/2). Cotreatment of ERK1/2 inhibitor 2′‐amino‐3′‐methoxyflavone inhibited syringetin‐mediated ALP upregulation and osteocalcin production. In conclusion, syringetin increased BMP‐2 synthesis, and subsequently activated SMAD1/5/8 and ERK1/2, and this effect may contribute to its action on the induction of osteoblast maturation and differentiation, followed by an increase of bone mass.