Grape seed procyanidin extract ameliorates lead-induced liver injury via miRNA153 and AKT/GSK-3β/Fyn-mediated Nrf2 activation

Lead-induced hepatotoxicity is characterized by an extensive oxidative stress. Grape seed procyanidin extract (GSPE) possesses abundant biological activities. Herein, we investigated the protective role of GSPE against lead-induced liver injury and determined the potential molecular mechanisms. In vivo, rats were treated with/without lead acetate (PbAc) (0.05%, w/v) in the presence/absence of GSPE (200 mg/kg). In vitro, hepatocytes were pretreated with/without GSPE (100 μg/ml) in the presence/absence of PbAc (100 μM). PbAc administration to rats resulted in anemia, liver dysfunction, lead accumulation in the bone and liver, oxidative stress, DNA damage and apoptosis. GSPE significantly attenuated these adverse effects, except lead accumulation in liver. GSPE also decreased the expression of miRNA153 and increased the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and levels of its downstream protein, and protein kinase B (AKT) phosphorylation in PbAc-induced liver injury. In primary hepatocytes treated with PbAc, GSPE increased hepatocyte viability and decreased lactate dehydrogenase release and reactive oxygen species levels. Dietary GSPE attenuated PbAc-induced liver injury in rats via an integrated mechanism associated with the miRNA153 and AKT/glycogen synthase kinase 3 beta/Fyn-mediated Nrf2 activation.     

Dietary supplementation with sulforaphane ameliorates skin aging through activation of the Keap1-Nrf2 pathway

Visible impairments in skin appearance, as well as a subtle decline in its functionality at the molecular level, are hallmarks of skin aging. Activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-pathway, which is important in controlling inflammation and oxidative stress that occur during aging, can be triggered by sulforaphane (SFN), an isothiocyanate found in plants from the Brassicaceae family. This study aimed to assess the effects of SFN intake on age-related skin alterations. Male C57BL6 young (2 months) and old (21 months) mice were treated for 3 months with SFN diet (442.5 mg per kg) or control diet. The antioxidant capacities of the skin were increased in old SFN-treated animals as measured by mRNA levels of Nrf2 (P<.001) and its target genes NQO1 (P<.001) and HO1 (P<.01). Protein expression for Nrf2 was also increased in old SFN fed animals (P<.01), but not the protein expression of NQO1 or HO1. Additionally, ROS and MMP9 protein levels were significantly decreased (P<.05) in old SFN fed animals. Histopathological analysis confirmed that there was no difference in epidermal thickness in old, when compared to young, SFN treated animals, while the dermal layer thickness was lower in old vs. young, treated animals (P<.05). Moreover, collagen deposition was improved with SFN treatment in young (P<.05) and structurally significantly improved in the old mice (P<.001). SFN dietary supplementation therefore ameliorates skin aging through activation of the Nrf2-pathway.     

Effect of Lead on Lipid Peroxidation, Phospholipids Composition, and Methylation in Erythrocyte of Human

Lead (Pb) is one of the most abundant heavy metals on earth considered as number one environmental persistent toxin and health hazard affecting millions of people in all age groups. After entering bloodstream, 99 % of Pb is accumulated in erythrocytes and causes poisoning. Toxic Pb effects on erythrocytes membrane’s composition of phosphatidyl serine (PS), phosphatidyl ethanolamine (PE), phosphatidyl choline (PC), and sphingomyelin (SM), and phospholipids transmethylation were determined. Lipid peroxidation in Pb-exposed erythrocytes was evaluated as malondialdehyde (MDA) formation in presence of Fe and vitamin E to understand severity of Pb toxicity and its mitigation. Pb (0.5–5.0 μM) degraded PS (12 to 31 %, P?<?0.05–0.001) and elevated SM (19–51 %, P?<?0.05–0.001). Composition of PC and PE were diminished (22 %) and elevated (29 %), respectively, with higher Pb exposure (5.0 μM, P?<?0.001). Pb toxicity suppressed (P?<?0.001) transmethylation of phospholipids in membranes (34, 41, and 50 %, respectively, with 0.5, 2.5, and 5.0 μM). Pb-induced dose-related MDA production (P?<?0.05–0.001) in erythrocytes was obtained, which was accentuated in presence of Fe (P?<?0.05–0.001). The vitamin E mitigated (P?<?0.05–0.01) the severity of Pb-induced lipid peroxidation. The ratio PS/SM showed maximum change of ?27 (P?<?0.01), ?30 (P?<?0.01), and ?54 % (P?<?0.001), respectively at 0.5, 2.5, and 5.0 μM Pb exposures. Ratios PC/SM and PS/PE were at the second, whereas PE/PS at the third order. The study suggests that the mechanisms underlying distortion of compositional phospholipids, inhibition of transmethylation, and exasperated phospholipid peroxidative damage are the active phenomena of Pb toxicity in erythrocytes.

Prenatal choline supplementation attenuates spatial learning deficits of offspring rats exposed to low-protein diet during fetal period

Prenatal intake of choline has been reported to lead to enhanced cognitive function in offspring, but little is known about the effects on spatial learning deficits. The present study examined the effects of prenatal choline supplementation on developmental low-protein exposure and its potential mechanisms. Pregnant female rats were fed either a normal or low-protein diet containing sufficient choline (1.1 g/kg choline chloride) or supplemented choline (5.0 g/kg choline chloride) until delivery. The Barnes maze test was performed at postnatal days 31–37. Choline and its metabolites, the synaptic structural parameters of the CA1 region in the brain of the newborn rat, were measured. The Barnes maze test demonstrated that prenatal low-protein pups had significantly greater error scale values, hole deviation scores, strategy scores and spatial search strategy and had lesser random search strategy values than normal protein pups (all P<.05). These alterations were significantly reversed by choline supplementation. Choline supplementation increased the brain levels of choline, betaine, phosphatidylethanolamine and phosphatidylcholine of newborns by 51.35% (P<.05), 33.33% (P<.001), 28.68% (P<.01) and 23.58% (P<.05), respectively, compared with the LPD group. Prenatal choline supplementation reversed the increased width of the synaptic cleft (P<.05) and decreased the curvature of the synaptic interface (P<.05) induced by a low-protein diet. Prenatal choline supplementation could attenuate the spatial learning deficits caused by prenatal protein malnutrition by increasing brain choline, betaine and phospholipids and by influencing the hippocampus structure.     

Mitigation of Lead-Induced Neurotoxicity by the Naringin: Erythrocytes as Neurons Substitute Markers

This study aimed to investigate the effect of lead (Pb) on neuronal nitric oxide synthase (nNOS) activity using erythrocytes as neurons surrogate markers. Moreover, the protective effect of naringin (NAR) against lead acetate (PbAc)-induced neurotoxicity was investigated. Human erythrocytes were incubated with l-arginine (l-Arg), N ^ω-nitro-l-Arginine methyl ester ( l-NAME), NAR, PbAc, PbAc + l-Arg, PbAc + NAR, or PbAc + l-Arg +NAR. The present results revealed that incubation of erythrocytes with PbAc inhibited NOS activity and decreased nitrite levels as an index for nitric oxide (NO) production to values similar that of l-NAME as known NOS inhibitor. Likewise, PbAc induced a significant decrease in activities of ATPases and acetylcholinesterase compared to control cells. Furthermore, PbAc exposure significantly increased protein carbonyl content (PCC) and malondialdehyde (MDA) levels while significantly decrease the levels of reduced glutathione (GSH). On the contrary, incubation of erythrocytes with PbAc in the presence of l-Arg + NAR synergistically ameliorated the investigated parameters compared to erythrocytes incubated with PbAc alone. These data suggest that NAR can restore NO bioavailability in a situation of Pb-induced cellular damage. This attributed to antioxidant activity and restoration NOS activity.     

Adequate but not supplemental folic acid combined with soy isoflavones during early life improves bone health at adulthood in male mice

Previous investigations from our laboratory have demonstrated that neonatal exposure to soy isoflavones (ISO) improves bone outcomes in CD-1 mice at adulthood with greater benefits in females than males. This study determined whether early-life exposure to supplemental folic acid (FA) — that may enhance DNA methylation of target genes — in combination with ISO provides greater benefits to male bone development than ISO alone. CD-1 dams were randomized to a low (0 mg/kg diet), adequate (2 mg/kg diet) or supplemental (8 mg/kg diet) level of FA during pregnancy and lactation. Offspring received corn oil or ISO (7 mg/kg of body weight per day) from postnatal day 1–10. From weaning, males were fed adequate FA and studied to age 4 months. Offspring exposed to adequate FA+ISO had multiple benefits to bone health: higher (P<.05) bone mineral density (BMD) and greater (P<.05) resistance to fracture at the femur and lumbar spine than mice exposed to adequate FA alone. Exposure to supplemental FA+ISO resulted in higher (P<.05) serum osteoprotegerin (OPG), and a higher ratio of OPG to receptor activator for nuclear factor κβ ligand (RANKL) but did not result in greater BMD or strength at the femur or lumbar spine than supplemental FA alone. In conclusion, early-life exposure to adequate FA+ISO provided functional benefits to male bone development, while improvements induced by supplemental FA+ISO were limited to a higher level of serum OPG. Mechanistic studies are needed to better understand how FA and ISO improve bone development in male offspring.     

Adiponectin ameliorates hyperglycemia-induced cardiac hypertrophy and dysfunction by concomitantly activating Nrf2 and Brg1

Hyperglycemia-induced oxidative stress is implicated in the development of cardiomyopathy in diabetes that is associated with reduced adiponectin (APN) and heme oxygenase-1 (HO-1). Brahma-related gene 1 (Brg1) assists nuclear factor-erythroid-2-related factor-2 (Nrf2) to activate HO-1 to increase myocardial antioxidant capacity in response to oxidative stress. We hypothesized that reduced adiponectin (APN) impairs HO-1 induction which contributes to the development of diabetic cardiomyopathy, and that supplementation of APN may ameliorate diabetic cardiomyopathy by activating HO-1 through Nrf2 and Brg1 in diabetes. Control (C) and streptozotocin-induced diabetic (D) rats were untreated or treated with APN adenovirus (1×10⁹ pfu) 3 weeks after diabetes induction and examined and terminated 1 week afterward. Rat left ventricular functions were assessed by a pressure–volume conductance system, before the rat hearts were removed to perform histological and biochemical assays. Four weeks after diabetes induction, D rats developed cardiac hypertrophy evidenced as increased ratio of heart weight to body weight, elevated myocardial collagen I content, and larger cardiomyocyte cross-sectional area (all P<0.05 vs C). Diabetes elevated cardiac oxidative stress (increased 15-F_2t-isoprostane, 4-hydroxynonenal generation, 8-hydroxy-2′-deoxyguanosine, and superoxide anion generation), increased myocardial apoptosis, and impaired cardiac function (all P<0.05 vs C). In D rats, myocardial HO-1 mRNA and protein expression were reduced which was associated with reduced Brg1 and nuclear Nrf2 protein expression. All these changes were either attenuated or prevented by APN. In primarily cultured cardiomyocytes (CMs) isolated from D rats or in the embryonic rat cardiomyocytes cell line H9C2 cells incubated with high glucose (HG, 25 mM), supplementation of recombined globular APN (gAd, 2 μg/mL) reversed HG-induced reductions of HO-1, Brg1, and nuclear Nrf2 protein expression and attenuated cellular oxidative stress, myocyte size, and apoptotic cells. Inhibition of HO-1 by ZnPP (10 μM) or small interfering RNA (siRNA) canceled all the above gAd beneficial effects. Moreover, inhibition of Nrf2 (either by the Nrf2 inhibitor luteolin or siRNA) or Brg1 (by siRNA) canceled gAd-induced HO-1 induction and cellular protection in CMs and in H9C2 cells incubated with HG. In summary, our present study demonstrated that APN reduced cardiac oxidative stress, ameliorated cardiomyocyte hypertrophy, and prevented left ventricular dysfunction in diabetes by concomitantly activating Nrf2 and Brg1 to facilitate HO-1 induction.     

Apigenin protects from hepatorenal damage caused by lead acetate in rats

Exposure to lead (Pb) is associated with serious health problems including hepatorenal toxicity. Apigenin is a natural-sourced flavonoid with promising antioxidant and anti-inflammatory effects. In this research, we investigated the potential protective role of apigenin against lead acetate (PbAc)-induced hepatorenal damage. Thus, this experiment studied the exposure of male Wistar Albino rats to apigenin and/or PbAc and their effects in comparison to the control rats. Apigenin administration decreased the levels of Pb and prevented the histopathological deformations in liver and kidney tissues following PbAc exposure. This was confirmed by the normalized levels of liver and kidney function markers. Additionally, apigenin inhibited significantly oxidative reactions through upregulating Nrf2 and HO-1, and activating their downstreamed antioxidants accompanied by a marked depletion of pro-oxidants. Moreover, apigenin decreased the elevated pro-inflammatory cytokines and inhibited cell loss in liver and kidney tissues in response to PbAc intoxication in both tissues. The obtained results demonstrated that apigenin could be used to attenuate the molecular, biochemical, and histological alterations associated with Pb exposure due to its potent antioxidant, anti-inflammatory, and antiapoptotic effects.     

Increasing vitamin A in post-weaning diets reduces food intake and body weight and modifies gene expression in brains of male rats born to dams fed a high multivitamin diet

High multivitamin gestational diets (HV, 10-fold AIN-93G levels) increase body weight (BW) and food intake (FI) in rat offspring weaned to a recommended multivitamin (RV), but not to a HV diet. We hypothesized that high vitamin A (HA) alone, similar to HV, in post-weaning diets would prevent these effects of the HV maternal diet consistent with gene expression in FI and reward pathways. Male offspring from dams fed HV diets were weaned to a high vitamin A (HA, 10-fold AIN-93G levels), HV or RV diet for 29 weeks. BW, FI, expression of genes involved in regulation of FI and reward and global and gene-specific DNA methylation of pro-opiomelanocortin (POMC) in the hypothalamus were measured. Both HV and HA diets slowed post-weaning weight gain and modified gene expression in offspring compared to offspring fed an RV post-weaning diet. Hypothalamic POMC expression in HA offspring was not different from either HV or RV, and dopamine receptor 1 was 30% (P<.05) higher in HA vs. HV, but not different from RV group. Hippocampal expression of serotonin receptor 1A (40%, P<.01), dopamine receptor 2 (40%, P<.05) and dopamine receptor 5 (70%, P<.0001) was greater in HA vs. RV fed pups and is 40% (P<.01), 50% (P<.05) and 40% (P<.0001) in HA vs. HV pups, respectively. POMC DNA methylation was lower in HA vs. RV offspring (P<.05). We conclude that high vitamin A in post-weaning diets reduces post-weaning weight gain and FI and modifies gene expression in FI and reward pathways.     

The Involvement of Copper Transporter in Lead-induced Oxidative Stress in Astroglia

Lead (Pb), depositing primarily in astroglia in the brain, is a well-known neurotoxicant and a risk factor for neurologic disorders. Pb has been reported to induce oxidative stress by probably the disturbance of copper (Cu) homeostasis in astroglia. Thus, we hypothesized that Pb-induced oxidative stress is initiated by interfering with Cu transporter in astroglia. In this study, we observed Pb-induced oxidative stress as indicated by reactive oxygen species (ROS) augmentation and GRP78 and GRP94 protein induction, and it was parallel to Cu accumulation intracellularly by Pb. To further address Cu transporter as a potential Pb target, a heavy metal-binding (HMB) domain of Cu-transporting ATPase (Atp7a) was overexpressed and purified. Evidence showed that one molecule of HMB chelated 11 Pb ions or seven Cu ions and that Pb competed with Cu for binding to HMB. These findings suggest that Pb-induced oxidative stress results from the impairment of Cu metabolism by Pb targeting of Atp7a.     

Resveratrol attenuates oxidative stress and prevents steatosis and hypertension in obese rats programmed by early weaning

We hypothesized that resveratrol, a natural phytoalexin found in grapes, can prevent oxidative stress, obesity and its related disturbances in obese rats programmed by early weaning. Lactating Wistar rats were separated into two groups: early weaning (EW) — dams who were wrapped with a bandage to interrupt the lactation in the last 3 days of lactation; control — dams whose pups had free access to milk during all lactation. At the 150th day, EW offspring were randomly subdivided into EW+resveratrol (EW+Res) — resveratrol (30 mg/kg/day); EW+vehicle (EW) — rats that received 0.5% (w/v) aqueous methylcellulose. The control group received vehicle. Rats were treated by gavage daily for 30 days. EW offspring developed hyperphagia, higher body weight, visceral obesity, higher systolic (SBP) and diastolic blood pressure (DBP) (+15% and +20%, respectively; P<.05) and higher serum triglycerides (TG) and low-density lipoprotein but lower high-density lipoprotein (+55%, +33% and ?13%, respectively; P<.05). Resveratrol normalized food intake, SBP and DBP and prevented obesity and dyslipidemia in EW+Res. EW rats had higher plasma and liver thiobarbituric-acid-reactive substances (TBARS) and lower plasma superoxide dismutase (SOD) and liver glutathione peroxidase activities (+51%, +18%, ?58%, ?31%, respectively; P<.05), and resveratrol normalized both plasma and liver TBARS and increased the activity of SOD and catalase in plasma. EW rats presented liver steatosis and higher liver TG, and resveratrol prevented these hepatic alterations. In conclusion, this study demonstrated a potential therapeutic use of resveratrol in preventing obesity and oxidative stress and reducing the risk of hypertension, dyslipidemia and steatosis in adult rats programmed by early weaning.     

虎杖苷对老年小鼠术后认知功能障碍及海马氧化应激和神经炎症的影响

摘要 目的：探讨虎杖苷对老年小鼠术后认知功能障碍及海马氧化应激和神经炎症的影响。方法：C57BL/6J雄性老年小鼠,18月龄,体重24~28 g,随机分为4组：假手术组（Sham组）、术后认知功能障碍组（POCD组）、低剂量虎杖苷组（PD1组）、高剂量虎杖苷组（PD2组）。Sham组老年小鼠仅接受水合氯醛麻醉,不行外科手术;POCD组老年小鼠接受腹部手术以制备POCD老年动物模型;PD1组小鼠制备POCD模型,并于术后即刻、24 h和48 h分别腹腔注射低剂量虎杖苷25 mg/kg;PD2组小鼠制备POCD模型,并于术后即刻、24 h和48 h分别腹腔注射高剂量虎杖苷50 mg/kg。使用Morris水迷宫行为学实验评估老年小鼠术后认知功能,使用Western blot法检测术后小鼠海马Nrf2、HO-1、HMGB1、Iba-1蛋白水平,检测海马活性氧（ROS）、丙二醛（MDA）、超氧化物歧化酶（SOD）含量以反映术后海马氧化应激水平。结果：（1）与Sham组比较,POCD组老年小鼠术后逃离潜伏期显著增加（P<0.05）,穿越平台次数和目标象限停留时间下降（P<0.05）,海马Nrf2和HO-1蛋白表达水平明显降低（P<0.05）,氧化应激产物ROS和MDA含量增加（P<0.05）,抗氧化酶SOD活性降低（P<0.05）,Iba-1和HMGB1蛋白表达水平增加（P<0.05）;（2）与POCD组比较,PD1组和PD2组小鼠术后逃离潜伏期缩短（P<0.05）,穿越平台次数和目标象限停留时间增多（P<0.05）,海马Nrf2和HO-1蛋白表达水平升高（P<0.05）,ROS和MDA含量减少（P<0.05）,SOD活性增加（P<0.05）,Iba-1和HMGB1蛋白表达减少（P<0.05）。结论：虎杖苷可减轻老年小鼠术后认知功能损伤,缓解术后海马氧化应激和神经炎症,其机制可能与促进Nrf2/HO-1信号通路有关。     

Transcription Factor Nrf1 Negatively Regulates the Cystine/Glutamate Transporter and Lipid-Metabolizing Enzymes

Liver-specific Nrf1 (NF-E2-p45-related factor 1) knockout mice develop nonalcoholic steatohepatitis. To identify postnatal mechanisms responsible for this phenotype, we generated an inducible liver-specific Nrf1 knockout mouse line using animals harboring an Nrf1^flox allele and a rat CYP1A1-Cre transgene (Nrf1^flox/flox::CYP1A1-Cre mice). Administration of 3-methylcholanthrene (3-MC) to these mice (Nrf1^flox/flox::CYP1A1-Cre+3MC mice) resulted in loss of hepatic Nrf1 expression. The livers of mice lacking Nrf1 accumulated lipid, and the hepatic fatty acid (FA) composition in such animals differed significantly from that in the Nrf1^flox/flox::CYP1A1-Cre control. This change was provoked by upregulation of several FA metabolism genes. Unexpectedly, we also found that the level of glutathione was increased dramatically in livers of Nrf1^flox/flox::CYP1A1-Cre+3MC mice. While expression of glutathione biosynthetic enzymes was unchanged, xCT, a component of the cystine/glutamate antiporter system x_c⁻, was significantly upregulated in livers of Nrf1^flox/flox::CYP1A1-Cre+3MC mice, suggesting that Nrf1 normally suppresses xCT. Thus, stress-inducible expression of xCT is a two-step process: under homeostatic conditions, Nrf1 effectively suppresses nonspecific transactivation of xCT, but when cells encounter severe oxidative/electrophilic stress, Nrf1 is displaced from an antioxidant response element (ARE) in the gene promoter while Nrf2 is recruited to the ARE. Thus, Nrf1 controls both the FA and the cystine/cysteine content of hepatocytes by participating in an elaborate regulatory network.