Mulberry Anthocyanin Extracts Inhibit LDL Oxidation and Macrophage-Derived Foam Cell Formation Induced by Oxidative LDL

ABSTRACT:  Low-density lipoprotein (LDL) oxidation plays a role in atherosclerosis; therefore the lower the formation of oxidative LDL (oxLDL), the lower the occurrence of coronary heart diseases (CHD). Mulberry, the fruit of Morus alba L., is used effectively in Chinese medicines for prevention of CHD. However, the mechanism of this action is unclear. Two extracts, MWEs (mulberry water extracts) and MACs (mulberry anthocyanin-rich extracts), which exhibit antioxidative and anti-atherosclerogensis abilities in vitro . Data showed that MWEs and MACs were able to inhibit ( P < 0.05) the relative electrophoretic mobility (REM), ApoB fragmentation, and thiobarbituric acid reaction substances (TBARS) formation in Cu²⁺-mediated oxidation LDL. MWEs and MACs also had the ability of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging for reducing the formation of free radicals mediated by copper ions. Furthermore, we observed that MWEs and MACs could decrease ( P < 0.05) macrophage death induced by oxLDL. In addition, the MWEs and MACs also could inhibit ( P < 0.05) the formation of foam cells. Both MWEs and MACs showed a great ability of scavenging radicals, inhibition of LDL oxidation, and decrease in atherogenic stimuli in macrophages, while the efficacy of MACs is 10-fold greater than that of MWEs. It also demonstrated that anthocyanin components in mulberry extracts were regarded as the prevention of atherosclerosis.     

Cardiovascular disease and nutrient antioxidants: Role of low‐density lipoprotein oxidation

Increasing evidence indicates that oxidative modification of low‐density lipoprotein (LDL) is causally related to atherosclerosis. Oxidatively modified LDL (oxLDL), in contrast to native LDL, is taken up avidly by macrophages, leading to formation of lipid‐laden foam cells. Foam cells are pathognomonic of the atherosclerotic fatty streak. Modified LDL may also promote atherosclerosis by many other mechanisms, such as recruitment and retention of monocyte‐macrophages, T‐lymphocytes, and smooth muscle cells in the arterial intima, and cytotoxicity toward endothelial cells and macrophage‐derived foam cells. The “oxidation hypothesis of atherosclerosis” is supported by a number of in vivo findings, such as the presence of oxLDL in atherosclerotic lesions, and increased titers of autoantibodies against modified LDL in patients with atherosclerosis.

As a corollary of the oxidation hypothesis of atherosclerosis, antioxidants that can inhibit LDL oxidation may act as antiatherogens. This conception is supported by animal studies showing that antioxidants such as probucol, butylated hydroxytoluene, and α‐tocopherol can slow the progression of atherosclerosis. Epidemiological and clinical data indicate a protective role of dietary antioxidants against cardiovascular disease, including vitamin E, β‐carotene, and vitamin C. Likewise, basic research studies on LDL oxidation have demonstrated a protective role for antioxidants, present either in the aqueous environment of LDL or associated with the lipoprotein itself. More studies are needed to establish the effectiveness and determine the required doses of specific antioxidants to prevent and possibly treat cardiovascular disease.     

Bax induces activation of the unfolded protein response by inducing HAC1 mRNA splicing in Saccharomyces cerevisiae

Bax, a multidomain pro‐apoptotic Bcl‐2 protein, localizes to the endoplasmic reticulum (ER), where it regulates ER stress‐induced apoptosis. Adaptation to ER stress depends on the activation of an integrated signal transduction pathway known as the unfolded protein response (UPR). This study examined the death‐inducing activity of Bax and its ability to induce UPR signalling pathways in yeast. We observed that inhibition of global translation in yeast cells expressing Bax correlated with Bax‐induced cell death. Using a lacZ reporter containing several UPR cis‐activating regulatory elements, we also found that Bax directly activated the UPR. Furthermore, this correlated with the splicing of HAC1 mRNA, a gene involved in UPR activation. Bax induced expression of representative UPR target genes such as KAR2, DER1 and GCN4. Finally, we found that Ire1p function is critical for Bax‐induced cell death. Copyright © 2012 John Wiley & Sons, Ltd.     

Bovine milk phospholipid fraction protects Neuro2a cells from endoplasmic reticulum stress via PKC activation and autophagy

Endoplasmic reticulum stress commonly causes neuronal damage in a lot of neurodegenerative diseases. In this study, we examined neuroprotective effect of bovine milk phospholipid fraction (mPL) on mouse neuroblastoma Neuro2a cells from endoplasmic reticulum (ER) stress induced cell death. Neuro2a cells were induced cell death by ER stressor tunicamycin (TM) or thapsigargin (TG), and studied whether mPL could attenuate the toxicity. By preincubation with mPL, the cell viabilities were significantly increased in TM or TG treated cells, and caspase-12 activated cells induced by TM or TG treatment were significantly decreased. Protein kinase C inhibitor GF109203x significantly reduced the protective effect on TM induced cell death, and autophagy inhibitor 3-methyladenine reduced the protective effect on TM or TG induced cell death. Moreover, preincubation with mPL significantly stimulated autophagosomes formation observed by dansylcadaverine staining. Our data suggest that mPL will be applicable to prevent neurodegenerative diseases caused by ER stress.     

Suppressive effect of ethyl acetate extract of Teucrium polium on cellular oxidative damages and apoptosis induced by 2-deoxy-d-ribose: Role of de novo synthesis of glutathione

The effect of ethyl acetate (EtOAc) extract of Teucrium polium on 2-deoxy-d-ribose (dRib)-induced oxidative stress and apoptosis in erythroleukemic K562 cells was investigated. K562 cells exposed to dRib (50 mM) exhibited abnormal properties such as overproduction of reactive oxygen species, lipid peroxidation, glutathione (GSH) depletion and biochemical features of apoptosis. Treatment with EtOAc extract (25 and 50 μg/ml) reduced oxidative stress and apoptosis among the dRib-treated K562 cells. To disclose the mode of action, the effect of the extract on the cellular GSH and its synthesis by γ-glutamylcysteine synthetase (γ-GCS), a key rate-limiting enzyme of the GSH synthetic pathway, were evaluated. The results demonstrated that the antioxidant property of EtOAc extract mainly results from increasing the level of cellular GSH by inducing the activity of γ-GCS. It can be concluded that the EtOAc extract of T. polium prevents dRib-induced oxidative stress and apoptosis mostly through antioxidative mechanisms.     

Soy extract has different effects compared with the isolated isoflavones on the proteome of homocysteine-stressed endothelial cells

Epidemiological studies suggest that soy consumption may provide a protection in the development and progression of atherosclerosis. It is under debate, however, whether the soy isoflavones or other compounds are the "active principle". As apoptosis is a driving force in the process of atherosclerosis, we tested whether a soy extract or a combination of the two predominant isoflavones genistein and daidzein, in concentrations as found in the extract, exert similar or different effects on apoptosis in EA.hy 926 endothelial cells after exposure to the endothelial stressor homocysteine. Plasma membrane disintegration and nuclear fragmentation served as relevant apoptosis markers. To assess whether the extract and the genistein/daidzein mixture differently affect cellular target proteins changed in amount by homocysteine treatment, proteome analysis was performed by two-dimensional gel-electrophoresis and peptide mass fingerprinting of regulated protein spots. Homocysteine induced apoptosis in the cells, and both extract and genistein/daidzein inhibited apoptosis to a comparable extent. Whereas the extract prevented for 10 proteins the changes in expression levels as caused by homocysteine, the genistein/daidzein mixture reversed the homocysteine effects on the proteome for 13 proteins. The cytoskeletal protein matrin 3 and a U5 snRNP-specific 40-kDa protein were the only protein entities where both extract and genistein/daidzein reversed the homocysteine-induced changes in a common way. In conclusion, our studies provide evidence that an isoflavone containing soy extract and isolated isoflavones, despite similar effects on inhibition of homocysteine-induced apoptosis in endothelial cells, affect a quite different spectrum of cellular target proteins.     

黄酮类化合物保护低密度脂蛋白氧化作用和减少动脉粥样硬化作用

食用含黄酮类物质食物将导致它们在血浆和组织中出现,摄入量和心血管的疾病成反比关系可能是与黄酮类减少了低密度脂蛋白氧化作用。黄酮类对动脉低密度脂蛋白细胞间接氧化作用可通过对他们在脂蛋白动脉的细胞累积来测定,如巨噬细胞。黄酮类可清除反应的氧、氮种类,螯合金属离子和节制低密度脂蛋白联合抗氧化作用,减少低密度脂蛋白氧化作用。通过抑制细胞氧合酶(如烟酰胺、腺嘌呤、二核甙酸、磷酸盐,减少氧化酶的形成)或通过启动细胞抗氧化剂(如谷胱甘肽系统)也能减少巨噬细胞氧化压力。 因此,黄酮类植物是有效的天然的抗氧化剂,既能保护反抗动脉细胞和脂蛋白类脂过氧化作用,又能有效的减少动脉粥样硬化的形成。     

Evaluation of Equol Function on Anti- or Prooxidant Status in vivo

ABSTRACT:  The present study was performed to investigate the effects of equol on oxidative stress and the antioxidant defense system in the livers of mice. Mice were orally administered equol at either 5 or 25 mg/kg body weight/day for 1, 3, or 7 wk. Equol administration significantly inhibited biomarkers of oxidative stress (thiobarbituric acid-reactive substances value, carbonyl content, and serum 8-OH-dG) at all doses and for all durations of administration, and this phenomenon was most pronounced at 3 wk. Moreover, catalase and total superoxide dismutase (SOD) activities and their mRNA expression were significantly increased by equol. Although equol increased the glutathione peroxidase (GSH-px) activity in mice treated with equol for 1 wk, long-term administration of equol (7 wk) caused a decrease in the ratio of reduced/oxidized glutathione (GSH/GSSG) and the activities of GSH-px and glutathione reductase (GR). Taken together, these results suggest that equol may act as an antioxidant through an inhibition of oxidative stress and stimulation of catalase and SOD, but can also cause prooxidant effects such as reduction of the GSH/GSSG ratio, depending on the treatment period.     

Chemoprotective action of l-(+)-selenomethionine on the modulation of genes involved in oxidative stress and in the UPR pathway

The installation of oxidative process arises from an imbalance between oxidants and antioxidants compounds, in favor of excessive generation of free radicals. This process can affect cellular components, like endoplasmic reticulum (ER). The ER is extremely sensitive to changes in homeostasis, where, on different stimuli, may result in adaptation to survival or induction of apoptosis (unfolded protein response, “UPR” pathway). The oxidative damage caused by endogenous or exogenous agents or a dysregulation of the UPR pathway can lead to adverse conditions, whose chronicity has important implications for the etiology of chronic diseases, including cancer. Therefore, it becomes important to search for chemoprotective agents aiming their role in preventive medicine. Between these substances, selenium’s antioxidant activity seems to be effective in treating diseases which have the oxidative stress as its development. We evaluated the modulating action of l-(+)-selenomethionine (SeMet) in HepG2 cells against cellular stress induced by H₂O₂ through MTT assay, comet assay, and gene expression by qRT-PCR of genes related to oxidative stress, UPR pathway, and apoptosis. In MTT assay, the lower concentrations of SeMet showed a cytoprotective action against the damage caused by H₂O₂. Likewise, it was verified in the comet assay that the concentration of 50 ng/mL reduced the genotoxic damage caused by H₂O₂. SeMet at 50 ng/mL regulated the genes tested in the qRT-PCR, showing an antiapoptotic and antioxidant effect. These results suggest that SeMet positively modulates the genes of oxidative stress and ER, leading to a chemoprotective and antioxidant effect, becoming an alternative in preventive medicine.     

莲壳多酚对T-BHP致HepG2氧化应激损伤的保护作用

目的：研究莲子壳多酚对叔丁基过氧化氢（tert-butylhydro-peroxide,T-BHP）诱导的HepG2氧化应激损伤的保护作用。方法：选取存活率接近50%的T-BHP浓度作为氧化损伤模型建立的浓度。以细胞活力、活性氧水平（reactive oxygen species,ROS）、丙二醛水平（malondialdehyde,MDA）、乳酸脱氢酶水平（Lactic dehydrogenase,LDH）、谷胱甘肽（Glutathione,GSH）水平及抗氧化酶相关基因表达量为评价指标,以茶多酚为阳性对照,评价不同浓度（2.5、5、7.5μg/mL）莲子壳多酚抗氧化应激活性水平。结果：当120μmol/L浓度的T-BHP造模4 h后,细胞存活率达49.18%±7.55%,符合模型构建要求,故选择120μmol/L为氧化损伤模型的建模浓度进行后续实验。莲子壳多酚能极显著抑制T-BHP造成的细胞存活率降低（P<0.01）,7.5μg/mL时,ROS水平降低45.99%,MDA下降58.77%,LDH下降71.61%,GSH提高206.60%(P<0.05),抗氧化酶相关基因...     

Oxidized phospholipids, isolevuglandins, and atherosclerosis

Autoxidation of polyunsaturated phosphatidylcholines (PCs) generates isolevuglandins (isoLGs) through rearrangements of isoprostanoid endoperoxides. Within seconds, isoLGs are sequestered by covalent adduction with proteins. Murine plasma isoLG-protein levels increased at least 2.5-fold in response to inflammation. IsoLG-protein adducts accumulate in vivo providing a convenient dosimeter of oxidative stress. Elevated blood isoLG-protein levels present in atherosclerosis (AS) patients point to an independent defect that is not associated with total cholesterol levels, which results in an abnormally high level of oxidative injury in AS. Protein adduction and cross-linking caused by isoLGs can obstruct protein function. For example, it interferes with proteosomal degradation of proteins and, consequently, may result in apoptotic death of smooth muscle cells and destabilization of atherosclerotic plaques. Phospholipid autoxidation also generates biologically active oxidatively truncated PCs through fragmentation of dihydroperoxydienes that can be promoted by alpha-tocopherol. The oxidatively truncated PCs in oxidized low-density lipoprotein (oxLDL) contribute to the etiology of AS by inhibiting enzymatic activities required for normal processing of oxLDL by macrophages. They promote interactions of monocytes with endothelial cells that may foster migration of monocytes into the subendothelial space. They are also ligands for unregulated receptor-mediated uptake of oxLDL by monocyte macrophages leading to foam cell formation.     

草苁蓉多糖对氧化应激所致血管内皮细胞凋亡的抑制作用

目的：研究草苁蓉多糖（Boschniakia rossica polysaccharides,BRPS）对氧化应激所致血管内皮细胞凋亡 的抑制作用。方法：利用叔丁基过氧化氢（tert-butyl hydroperoxide,tBHP）损伤人脐静脉血管内皮细胞制备细胞 氧化应激损伤模型,采用四甲基偶氮唑盐（methylthiazolyldiphenyl-tetrazolium bromide,MTT）比色法检测细胞存 活率,分光光度法检测细胞内丙二醛（malondialdelyde,MDA）水平、超氧化物歧化酶（superoxide dismutase, SOD）活力和还原型谷胱甘肽（reduced glutathione,GSH）水平。采用二氯荧光乙酰乙酸盐（dichlorofluorescein diacetate,DCFH-DA）法检测细胞内活性氧（reactive oxygen species,ROS）水平,采用JC-1荧光染色法观察 细胞线粒体跨膜电位（mitochondrial membrane potentials,ΔΨm）水平,采用Hoechst染色和原位末端标记（TdTmediated dUTP nick end labeling,TUNEL）法观察细胞凋亡情况。Western blot法检测细胞Bax、Bcl-2、细胞色素c （cytochrome c,Cyt c）、Bid片段（truncated Bid,tBid）、Caspase-3、Caspase-8和Caspase-9蛋白的表达情况。 结果：BRPS提高tBHP损伤的内皮细胞存活率,降低细胞ROS水平,减少MDA生成,提高SOD活性与GSH水 平,升高ΔΨm水平,抑制细胞凋亡。Western blot结果显示,BRPS降低胞浆Cyt c、线粒体tBid水平,减小细胞 Bax/Bcl-2比值,抑制Caspase-3、Caspase-8和Caspase-9的活化。结论：BRPS对氧化应激所致血管内皮细胞凋亡具有 抑制作用,机制可能与线粒体凋亡途径和死亡受体途径均有关。     

Zearalenone induces apoptosis in bovine mammary epithelial cells by activating endoplasmic reticulum stress

Zearalenone (ZEA) is a common mycotoxin produced by fungi within the genus Fusarium. However, few studies have examined the direct effects of the toxin on the mammary glands. In the present study, the effects of ZEA treatment on bovine mammary epithelial cells (MAC-T) from dairy cows were investigated. The cells were treated with different concentrations of ZEA to evaluate the effect of the toxin on cell viability, intracellular reactive oxygen species (ROS) concentrations, mitochondrial membrane potential, endoplasmic reticulum (ER) stress, and the expression of apoptosis-related genes. The results indicated that different concentrations (5, 10, 15, 20, 25, 30, 50, 60, or 100 μM) of ZEA were able to inhibit growth of MAC-T cells. After exposing the MAC-T cells to 30 μM ZEA, compared with the control group, ROS levels increased, mitochondrial membrane potential decreased, and mRNA expression of the ER-specific stress-related genes GRP78, HSP70, ATF6, EIF2A, ASK1, and CHOP was upregulated in the ZEA-treated group. Further, we analyzed the increase in apoptotic rate by flow cytometry. At the mRNA level, compared with the control group, the expression of the apoptosis-promoting gene BAX was increased in the ZEA-treated group, the expression of the inhibitory gene BCL2 decreased, and the expression of the gene CASP3 increased. We observed a significant increase in caspase-3 activity in ZEA-treated MAC-T cells. Furthermore, the apoptotic rate of the cells in the ZEA group treated with 4-phenylbutyric acid (ER stress inhibitor) decreased and the mRNA expression levels of ER stress markers GRP78 and CHOP decreased. Compared with the ZEA treatment group, the mRNA expression level of the apoptosis-related gene BAX was decreased and the expression level of BCL2 was increased in the ZEA + 4-phenylbutyric acid cotreatment group. These findings indicate that ZEA-induced ER stress increases apoptosis in MAC-T cells. The treatment of MAC-T cells with ZEA reduced cell viability, increased ROS content, decreased mitochondrial membrane potential, increased ER stress marker expression, and induced apoptosis.     

Effect of frying oils on the postprandial endoplasmic reticulum stress in obese people

The addition of antioxidants to frying oil reduces postprandial oxidative stress and the inflammatory response. ER stress may trigger both inflammation and oxidative stress processes. We aimed to determine the biological effects of the intake of four models of frying oils on postprandial ER stress in peripheral blood mononuclear cells. Twenty obese people received four breakfasts following a randomized crossover design, consisting of muffins made with different oils (virgin olive oil (VOO), sunflower oil (SFO), and a mixture of seed oils (SFO/canola oil) with either dimethylpolysiloxane (SOD) or natural antioxidants from olives (SOP) added), which were previously subjected to 20 heating cycles. ER stress was assessed by measuring the mRNA levels of sXBP1, BiP, CRT, and CNX in peripheral blood mononuclear cells. Our study showed that the intake of the muffins made with SFO induced the postprandial increase of the mRNA levels of the ER stress‐sensor sXBP1, and the ER stress related chaperones BiP and CRT (all p‐values <0.05). The harmful effects associated with the use of SFO as frying oil, in terms of inflammatory response and postprandial oxidative stress, may be partially mediated by the induction of postprandial ER stress.     

Effect of coffee melanoidin on human hepatoma HepG2 cells. Protection against oxidative stress induced by tert-butylhydroperoxide

Soluble high-molecular weight fraction (named melanoidin) from coffee brew was isolated by ultrafiltration, subsequently digested by simulating a gastric plus pancreatic digestive condition and partly characterized by CZE, gel-filtration and browning. The objective of the present study was to investigate the potential protective effect of the coffee melanoidin submitted to gastrointestinal digestion on cell viability (lactate dehydrogenase leakage) and redox status of cultured human hepatoma HepG2 cells submitted to oxidative stress induced by tert-butylhydroperoxide (t-BOOH). Concentration of reduced glutathione (GSH) and malondialdehyde (MDA), generation of reactive oxygen species (ROS) and activity of antioxidant enzymes glutathione peroxidase (GPx) and reductase (GR) were used as markers of cellular oxidative status. Pretreatment of cultured HepG2 cells with 0.5-10 microg/mL digested coffee melanoidin (DCM) for 2 or 20 h completely prevented the increase in cell damage and GR and partly prevented the decrease of GSH and the increase of MDA and GPx evoked by t-BOOH in HepG2 cells. In contrast, increased ROS generation induced by t-BOOH was not prevented when cells were pretreated with DCM. The results show that treatment of HepG2 cells with concentrations of DCM within the expected physiological range confers the cells a significant protection against an oxidative insult.     

Effects of Long‐Chain and Medium‐Chain Fatty Acids on Apoptosis and Oxidative Stress in Human Liver Cells with Steatosis

Nonalcoholic fatty liver disease (NAFLD) is closely associated with obesity‐related metabolic complications, which caused by excess energy intake and physical inactivity apart from genetic defects. The mechanisms that promote disease progression from NAFLD to further liver injury are still unclear. We hypothesize that the progression involved “2nd hit” is strongly influenced by the type of fatty acids in diets. Flow cytometric analysis showed that medium‐chain fatty acid (MCFA) markedly decreased the percentage of late apoptotic and necrotic cells compared with long‐chain fatty acid (LCFA), and MCFA inhibited the activities of caspase‐3 and ‐9 in human liver cells with steatosis. Western blot analysis found that the levels of inflammatory markers (interleukin [IL]‐6, IL‐1‐β, and tumor necrosis factor‐α) were substantially reduced by MCFA compared with LCFA. Proteomic analysis further showed that LCFA inhibited the expression of antioxidant enzymes, and increased the expression of proteins associated with oxidative stress. It was found that LCFA (palmitate), not MCFA induced apoptosis, oxidative stress and chronic inflammatory responses in the hepatic cells with steatosis. In conclusion, reasonable selection of dietary fats has potential to translate therapeutically by ameliorating disease progression in patients with NAFLD.