Natural astaxanthin enhanced antioxidant capacity and improved semen quality through the MAPK/Nrf2 pathway in aging layer breeder roosters

Background:Natural astaxanthin(ASTA)has strong antioxidant properties and has been widely used as a health product to improve human health.However,the effects of ASTA on the reproductive performance of aging roosters have been poorly studied.We aimed to investigate the effects of dietary ASTA on semen quality and antioxidant capacity in aging roosters and to explore the potential mechanism of semen quality change via antioxidation defense system.Methods:In the present study,9653-week-old Jinghong No.1 layer breeder roosters were fed a corn-soybean meal basal diet containing 0,25,50,or 100 mg/kg ASTA for 6 weeks.Results:Semen quality in the ASTA groups remarkably improved than that in the control group,and antioxidant activities,the abilities to scavenge hydroxyl radicals and superoxide anions,increased gradually with ASTA addition(P<0.05).In addition,the mRNA levels of antioxidant enzymes as well as the mRNA and protein levels of the mitogen-activated protein kinase(MAPK)and nuclear factor-erythroid 2-related factor 2(Nrf2)were markedly increased in the 50-100 mg/kg ASTA group(P<0.05).Conclusions:Collectively,these results demonstrate that dietary ASTA may improve semen quality by increasing antioxidant enzyme activities and the ability to scavenge hydroxyl radicals,which may be related to upregulation of the MAPK/Nrf2 pathway.

Xiaochaihutang attenuates liver fibrosis in rats through activation of Nrf2 pathway

OBJECTIVE Oxidative sress is one of the key factor responsible for occurrence and development of hepatic fibrosis,a common consequence of chronic liver injury of multiple etiology.Nuclear factor erythroid 2-related factor 2(Nrf2)serves as a major regulator of a celular defense system against oxidative stress.Xiaochaihutang(XCHT),a compound of seven botanical extracts used for liver diseases traditionally in East Asia.However,few studies have investigated its anti-hepatic fibrosis effects and pathophysiological mechanism of action.The present study was designed to confirm the anti-hepatic fibrosis effects and explore its potential mechanism of action by investigating the intervention of Nrf2 pathway.METHODS Liver fibrosis was induced by repeated injection of Carbon tetrachloride(CCl4) over a period of 9 weeks.Starting from the 6 th week,the animals in treatment groups were given the appropriate dose of XCHT granules and silybin.Biochemical parameters,histological changes of the liver and alpha-smooth muscle actin(α-SMA) were determined.The expressions of Nrf2,Keap1,Nqo1,HO-1,Gclc and Gclm were assessed by RT-PCR and Western blot.RESULTS CCl4 caused a significant fibrosis damage in the rat liver and the liver functions and fibrosis degree were significantly improved by XCHT(5 g·kg^(-1) and 10 g·kg^(-1)).XCHT(5 g·kg^(-1) and 10 g·kg^(-1)) treatment significantly decreased the number of cells labeled with α-SMA antibodies.Moreover,XCHT(5 g·kg^(-1) and 10 g·kg^(-1))significantly increase Nqo1,HO-1,Gclc and Gclm expressions in the liver.CONCLUSION T hese studies establish XCHT is a potentially useful therapeutic agent for treatment of hepatic fibrosis and it might be via regulation of Nrf2 pathway in rats against oxidative stress,making further efforts to inhibiting the activated HSCs.Activation or up-regulation of Nrf2 pathway may be an alternative treatment strategy for liver fibrosis.

Nrf2 Pathway Involvement in the Beneficial Skin Effects of Moderate Ionic Osmotic Stress—The Case of The Dead Sea Water

Objectives: Exposing skin to moderate ionic osmotic stress (MIOS) triggers several biochemical responses. The objective of this work is to reveal the mechanism triggered by MIOS on the skin surface. Furthermore, this work aims to study the involvement of the Nrf2 (nuclear factor erythroid-2-related factor 2) pathway, activated by MIOS, and its beneficial effect in protecting skin against stress via the stimulation of phase II enzymes. Methods: HaCaT cells and human skin organ culture were exposed to Dead Sea Water (DSW) as MIOS inducers and the induction of internal ROS elevation, Nrf2 translocation, mRNA gene expressions of the phase II enzymes, heme-oxygenase 1 (HO1), and Catalase (CAT) were determined. Results: Skin exposure to MIOS increases Nrf2 translocation to the nucleus, leading to increased levels of ROS, HO1, and CAT. Furthermore, exposing skin to MIOS promotes protection against UVB-related risks. This is demonstrated by attenuation of the expression of biomarkers, related to UVB-induced damage, Caspase-3, IL-8, and IL-1β. Conclusions: Skin exposure to MIOS leads to the activation of Nrf2 skin defense pathway and, therefore, could present beneficial advantages to human skin health, as demonstrated on human skin models. The beneficial effects of MIOS, induced by DSW are significantly superior to eq. NaCl brine, suggests that MIOS protection of skin against stress is partially related to specific mineral combinations.

Reduction of the oxidative damage to H_(2)O_(2)-induced HepG2 cells via the Nrf2 signalling pathway by plant flavonoids Quercetin and Hyperoside

Hyperoside and quercetin are similar in molecular structures.In this study,the antioxidant regulatory targets of hyperoside and quercetin are mainly in the nuclear factor(erythroid-2-derived)-related factor 2(Nrf2)pathway predicted by network pharmacology.And the antioxidant effect and mechanism of hyperoside and quercetin were measured and compared in H_(2)O_(2)-induced Hep G2 cells and Caenorhabditis elegans.The findings indicated that quercetin was more effective than hyperoside in reducing oxidative damage,which was proved by improved cell viability,decreased reactive oxygen species(ROS)production,decreased cellular apoptosis,and alleviated mitochondrial damage.In addition,quercetin was more efficient than hyperoside in enhancing the expression of Nrf2-associated m RNAs,increasing the activities of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),and catalase(CAT),and reducing the cellular malondialdehyde(MDA)content.Quercetin was superior to hyperoside in prolonging the lifespan of worms,decreasing the accumulation of lipofuscin,inhibiting ROS production,and increasing the proportion of skn-1 in the nucleus.With the Nrf2 inhibitor ML385,we verified that quercetin and hyperoside primarily protected the cells against oxidative damage via the Nrf2 signalling pathway.Furthermore,molecular docking and dynamics simulations demonstrated that the quercetin-Kelch-like ECH-associated protein 1(Keap1)complex was more stable than the hyperoside-Keap1 complex.The stable structure of the complex might hinder the binding of Nrf2 and Keap1 to release Nrf2 and facilitate its entry into the nucleus to play an antioxidant role.Overall,quercetin had a better antioxidant than hyperoside.

Hesperidin ameliorates H_(2)O_(2)-induced bovine mammary epithelial cell oxidative stress via the Nrf2 signaling pathway

Background Hesperidin is a citrus flavonoid with anti-inflammatory and antioxidant potential. However, its protective effects on bovine mammary epithelial cells(b MECs) exposed to oxidative stress have not been elucidated.Results In this study, we investigated the effects of hesperidin on H_(2)O_(2)-induced oxidative stress in b MECs and the underlying molecular mechanism. We found that hesperidin attenuated H_(2)O_(2)-induced cell damage by reducing reactive oxygen species(ROS) and malondialdehyde(MDA) levels, increasing catalase(CAT) activity, and improving cell proliferation and mitochondrial membrane potential. Moreover, hesperidin activated the Keap1/Nrf2/ARE signaling pathway by inducing the nuclear translocation of Nrf2 and the expression of its downstream genes NQO1 and HO-1, which are antioxidant enzymes involved in ROS scavenging and cellular redox balance. The protective effects of hesperidin were blocked by the Nrf2 inhibitor ML385, indicating that they were Nrf2 dependent.Conclusions Our results suggest that hesperidin could protect b MECs from oxidative stress injury by activating the Nrf2 signaling pathway, suggesting that hesperidin as a natural antioxidant has positive potential as a feed additive or plant drug to promote the health benefits of bovine mammary.

Enhancement of porcine in vitro embryonic development through luteolin‑mediated activation of the Nrf2/Keap1 signaling pathway

Background Oxidative stress,caused by an imbalance in the production and elimination of intracellular reactive oxygen species(ROS),has been recognized for its detrimental effects on mammalian embryonic development.Luteolin(Lut)has been documented for its protective effects against oxidative stress in various studies.However,its specific role in embryonic development remains unexplored.This study aims to investigate the influence of Lut on porcine embryonic development and to elucidate the underlying mechanism.Results After undergoing parthenogenetic activation(PA)or in vitro fertilization,embryos supplemented with 0.5μmol/L Lut displayed a significant enhancement in cleavage and blastocyst formation rates,with an increase in total cell numbers and a decrease in the apoptosis rate compared to the control.Measurements on D2 and D6 revealed that embryos with Lut supplementation had lower ROS levels and higher glutathione levels compared to the control.Moreover,Lut supplementation significantly augmented mitochondrial content and membrane potential.Intriguingly,activation of the Nrf2/Keap1 signaling pathway was observed in embryos supplemented with Lut,leading to the upregulation of antioxidant-related gene transcription levels.To further validate the relationship between the Nrf2/Keap1 signaling pathway and effects of Lut in porcine embryonic development,we cultured PA embryos in a medium supplemented with brusatol,with or without the inclusion of Lut.The positive effects of Lut on developmental competence were negated by brusatol treatment.Conclusions Our findings indicate that Lut-mediated activation of the Nrf2/Keap1 signaling pathway contributes to the enhanced production of porcine embryos with high developmental competence,and offers insight into the mechanisms regulating early embryonic development.

Water Extract of Rice False Smut Balls Activates Nrf2/HO-1 and Apoptosis Pathways,Causing Liver Injury

Ustiloxins are vital cyclopeptide mycotoxins originally isolated from rice false smut balls that form in rice spikelets infected by the fungal pathogen Ustilaginoidea virens.The toxicity of the water extract of rice false smut balls(RBWE) remains to be investigated.Studies have shown that RBWE may be toxic to animals,but toxicological evidence is still lacking.In this study,we found that the IC50 values of RBWE to BNL CL.2 cells at 24 and 48 h were 40.02 and 30.11 μg/m L,respectively,with positive correlations with dose toxicity and time toxicity.After treatment with RBWE,the number of BNL CL.2 cells decreased significantly,and the morphology of BNL CL.2 cells showed atrophy and wall detachment.RBWE induced DNA presynthesis phase arrest of BNL CL.2 cells,increased the proportion of apoptotic cells and inhibited cell proliferation.RBWE up-regulated reactive oxygen species(ROS) levels and lowered mitochondrial membrane potentials.Additionally,Western blot and q RT-PCR results suggested that RBWE exerted the above effects by promoting the Nrf2/HO-1 and caspase-induced apoptosis pathways in vitro and in vivo.The contents of alanine aminotransferase,aspartate aminotransferase,alkaline phosphatase,and total bile acids in the serum of mice from Institute of Cancer were significantly up-regulated by RBWE.At the same time,RBWE can lead to increases in ROS and malondialdehyde contents,decreases in contents of oxidized glutathione,glutathione and reduced glutathione,as well as decrease in catalase and superoxide dismutase activities in mouse liver tissues,demonstrating that oxidative stress occurred in mice.Moreover,liver damage was further detected by haematoxylin-eosin staining and electron microscopy to verify the damage to the mice caused by RBWE.In general,RBWE may cause hepatotoxicity in vivo and in vitro via the apoptosis pathway,which provides a reference for hepatotoxicity and its mechanism of action.

Scutellarin alleviates complete freund’s adjuvant-induced rheumatoid arthritis in mice by regulating the Keap1/Nrf2/HO-1 pathway

Scutellarin(SCU)is a herbal flavonoid glucuronide with multiple pharmacological activities,including antioxidant,anti-inflammation,vascular relaxation,anti-platelet,and myocardial protection.However,the effect of SCU on complete Freund’s adjuvant(CFA)-induced rheumatoid arthritis(RA)had not been studied.In this study,we investigated the beneficial effects of SCU in the CFA-induced RA mice model and the anti-arthritic activity was evaluated by paw edema.Enzyme-linked immunosorbent assay(ELISA)was carried out to evaluate the plasma levels of immunoglobulin(Ig)G,IgE,tumor necrosis factor(TNF)-α,interleukin(IL)-1β,IL-6,receptor activator of nuclear factor-κB ligand(RANKL),and osteoprotegerin(OPG).Histological slides were prepared from the harvested paws of mice to determine the pathological changes in the joints.The proportions of T helper type 1(Th1)and T helper type 2(Th2)cells of CD4+T lymphocyte subsets were analyzed by flow cytometry.The expression of Kelch-like ECHassociated protein 1(Keap1),nuclear factor erythroid 2-related factor 2(Nrf2),and heme oxygenase-1(HO-1)was analyzed using real-time quantitative PCR(RT-qPCR)and western blotting assays.The present study demonstrated that SCU prevented CFA-induced RA,and inhibited the expression of inflammation factors,IgG,IgE,TNF-α,IL-1β,and IL-6.While SCU also reduced the RANKL level,it increased OPG expression in RA mice.The Th1/Th2 ratio was significantly lower in mice treated with SCU.Additionally,HO-1 expression was reduced while the expression of Keap1 and Nrf2 was elevated following SCU treatment.Results provide preliminary evidence to employ SCU in arthritis treatment which might be related to the regulation of Th1/Th2 balance and the Keap1/Nrf2/HO-1 pathway.

Natural products for cancer prevention associated with Nrf2–ARE pathway

Cancer chemoprevention involves the application of natural or synthetic compounds to reduce the risk of cancer development.One of the most effective strategies for preventing human cancers might involve inducing phase II detoxifying enzymes and antioxidant enzymes via natural dietary compounds.The regulatory regions of these inducible genes encode the antioxidant response element(ARE).Nuclear factor-erythroid 2-related factor 2(Nrf2),as a transcription factor,plays a key role in the expression of ARE-mediated genes.Similarly,Nrf2 performs an essential function in the up-regulation of these genes in response to oxidative stress and treatment with dietary phytochemicals.In this article,we discuss the current state of knowledge regarding the Nrf2/ARE pathway as a potential molecular target for cancer chemoprevention and its molecular regulation mechanisms,and highlight Nrf2/ARE inducers derived from natural products,which may be used as chemopreventive agents for cancer patients.©2013 Beijing Academy of Food Sciences.Production and hosting by Elsevier B.V.All rights reserved.

The protective effects of peptides from Chinese baijiu on AAPH-induced oxidative stress in HepG2 cells via Nrf2 signaling pathway

Antioxidant peptides have been widely reported.However,only a few reports have been published examining the antioxidant peptides derived from Chinese baijiu.In this study,6 novel peptides derived from Chinese baijiu were identified successfully using high-performance liquid chromatography-quadrupoletime-of-flight mass spectrometry(HPLC-QTOF-MS)with a concentration of 0.835–24.540μg/L.The underlying molecular mechanisms were investigated,and their cytoprotective effects were examined against 2,2’-azobis(2-methylpropanimidamidine)dihydrochloride(AAPH)-induced oxidative stress in Hep G2 cells.The results showed that these peptides exerted protective effects by suppressing reactive oxygen species(ROS)generation,preventing malondialdehyde(MDA)formation,and upregulating cellular antioxidant enzyme activities(SOD,CAT,and GSH-Px)in a dose-dependent manner.Further experiments proved that these peptides exerted antioxidant effects via Nrf2/ARE-mediated signaling pathway by promoting Nrf2 nuclear translocation,inhibiting ubiquitination,and enhancing transcription capacity of Nrf2 in Hep G2 cells.These findings provide the molecular basis for the effects of antioxidant peptides derived from Chinese baijiu,which is important for a deeper understanding of the relationship between human health and moderate drinking.

Effect of pestle intervention in type 2 diabetic peripheral neuropathy on Keap1/Nrf2/ARE pathway and the relationship with oxidative stress

Objective:To investigate the effect of pestle needle treatment on Nrf2 pathway and the relationship with oxidative stress in diabetic peripheral neuropathy.Methods:Patients with DPN who met the inclusion criteria were randomly divided into control and test groups with 30 patients in each group in a 1:1 allocation ratio.Both groups were given basic treatment,and the pestle group was treated with needle pestle therapy 5 times a week for a total of 4 weeks of intervention.Serum SOD and GSH PX levels were examined by colorimetry before and after intervention;Serum Keap1/Nrf2/ARE signaling pathway related factors expression levels were measured by ELISA;Keap1 and Nrf2 mRNA expression was determined by RT-PCR.Results:Compared with the control group,SOD and GSH-Px in the test group were significantly increased,Keap1 expression was decreased,Nrf2 expression was increased,Keap1 mRNA expression was significantly decreased,and Nrf2 mRNA expression was significantly increased.Conclusions:the pestle needle may enhance the body's antioxidant capacity by modulating the Keap1/Nrf2/ARE signaling pathway to enhance the production of its downstream antioxidant enzymes SOD and GSH Px,thereby protecting and repairing the damaged peripheral nerves in DPN patients.

Mechanism of hesperidin improving myocardial ischemia/reperfusion injury in type 2 diabetic rats through SIRT1/Nrf2/HO-1 signaling pathway

Objective:To observe the protective effect of hesperidin on myocardial ischemia/reperfusion injury in type 2 diabetes mellitus and its effect on SIRT1/Nrf2/HO-1 signaling pathway.Methods:50 Sprague-Dawley(SD)rats were randomly assigned to the normal control group(NC),model group,ischemia-reperfusion group(IR),hesperidin group,SIRT1 inhibitor group and hesperidin plus SIRT1 inhibitor group.In addition to NC,the rats in the remaining groups were replicated by intraperitoneal of high-fat diet combined with injection of streptozotocin for type 2 diabetic rats.After then,the myocardial ischemia/reperfusion injury(MIRI)rat model was established by LAd for 30 minutes with 2 hours reperfusion.He staining was used to observe the pathological changes of myocardial tissue,and the levels of serum LDH,CK-MB and SOD,GSH and MDA in myocardial tissue were detected by kit methods,and the expression abundance of related proteins in 4-HNE and SIRT1/Nrf2/HO-1 signal pathway were detected by immunohistochemistry and Western blot;Results:Hesperidin could significantly inhibit cardiomyocyte necrosis and inflammatory cell infiltration,reduce LDH activity,CK-MB and MDA level,and increase SOD activity,GSH and 4-HNE level,the differences were statistically significant when compared with IR group(P<0.01).In addition,compared with the ischemia-reperfusion group,the expressions of SIRT1,Nrf2 and HO-1 proteins in hesperidin group were significantly up-regulated,the differences were statistically significant(P<0.01);Conclusion:Hesperidin inhibits oxidative stress by activating SIRT1/Nrf2/HO-1 signaling pathway,and play a protective effect of myocardial ischemia reperfusion injury in diabetic rats.

Effects of nuciferine on Nrf2/HO-1 signaling pathway in adipose tissue of obesity model rats

Objective:This study aimed to explore the therapeutic effect of nuciferine on high-fat diet-induced obesity in rats and the influence of nuciferine on nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)signaling pathway in the adipose tissue.Methods:A total of 40 male Sprague Dawley(SD)rats were evenly divided into the normal,model,positive control,and nuciferine groups,using the random number table method.Except for the normal group,rats in the other groups were fed with high-fat diet for 12 weeks to establish the obesity model.During the model establishment,rats in the positive control group received atorvastatin calcium 2 mg/kg,rats in the nuciferine group received nuciferine 20 mg/kg,and rats in the normal and model groups received normal saline 2 mL,daily through intragastric administration for 12 consecutive weeks.After model establishment and administration,the body weight,Lee’s index,and blood lipids of rats in each group were measured,and hematoxylin and eosin(HE)staining was performed on the liver and adipose tissues to evaluate the therapeutic effect of nuciferine on obesity rat model.Additionally,the levels of superoxide dismutase(SOD),malondialdehyde(MDA),and glutathione peroxidase(GSH-Px)in the serum of rats in each group were determined,and the gene expressions of Nrf2 and HO-1 in the adipose tissue of rats in each group were detected through quantitative polymerase chain reaction(qPCR)to investigate the mechanism of action of nuciferine in the treatment of obesity.Results:After 12 weeks of model establishment and administration,we observed that compared with the model group,nuciferine could significantly reduce the body weight,Lee’s index,and serum triglyceride(TG),total cholesterol(TC),and low-density lipoprotein cholesterol(LDL-C)levels and increase the serum high-density lipoprotein cholesterol(HDL-C)level in obesity rat model(P<0.05 or P<0.01).HE staining revealed that nuciferine could significantly alleviate liver steatosis in obesity rat model and improve the cell morphology in epididymal adipose tissue.Moreover,nuciferine could elevate serum SOD and GSH-Px activities in obesity rat model and lower the serum MDA level(P<0.05 or P<0.01).The qPCR indicated that nuciferine could upregulate the gene expression of Nrf2 and HO-1 in the adipose tissue of obesity rat model(P<0.05 or P<0.01).

Berberine Inhibits Ferroptosis and Stabilizes Atherosclerotic Plaque through NRF2/SLC7A11/GPX4 Pathway

Objective:To investigate potential mechanisms of anti-atherosclerosis by berberine(BBR)using ApoE-/-mice.Methods:Eight 8-week-old C57BL/6J mice were used as a blank control group(normal),and 568-week-old AopE-/-mice were fed a high-fat diet for 12 weeks,according to a completely random method,and were divided into the model group,BBR low-dose group(50 mg/kg,BBRL),BBR medium-dose group(100 mg/kg,BBRM),BBR high-dose group(150 mg/kg,BBRH),BBR+nuclear factor erythroid 2-related factor 2(NRF2)inhibitor group(100 mg/kg BBR+30 mg/kg ML385,BBRM+ML385),NRF2 inhibitor group(30 mg/kg,ML385),and positive control group(2.5 mg/kg,atorvastatin),8 in each group.After 4 weeks of intragastric administration,samples were collected and serum,aorta,heart and liver tissues were isolated.Biochemical kits were used to detect serum lipid content and the expression levels of malondialdehyde(MDA)and superoxide dismutase(SOD)in all experimental groups.The pathological changes of atherosclerosis(AS)were observed by aorta gross Oil Red O,aortic sinus hematoxylin-eosin(HE)and Masson staining.Liver lipopathy was observed in mice by HE staining.The morphology of mitochondria in aorta cells was observed under transmission electron microscope.Flow cytometry was used to detect reactive oxygen species(ROS)expression in aorta of mice in each group.The content of ferrous ion Fe^(2+)in serum of mice was detected by biochemical kit.The mRNA and protein relative expression levels of NRF2,glutathione peroxidase 4(GPX4)and recombinant solute carrier family 7 member 11(SLC7A11)were detected by quantitative real time polymerase chain reaction(RT-q PCR)and Western blot,respectively.Results:BBRM and BBRH groups delayed the progression of AS and reduced the plaque area(P<0.01).The characteristic morphological changes of ferroptosis were rarely observed in BBR-treated AS mice,and the content of Fe^(2+)in BBR group was significantly lower than that in the model group(P<0.01).BBR decreased ROS and MDA levels in mouse aorta,increased SOD activity(P<0.01),significantly up-regulated NRF2/SLC7A11/GPX4 protein and mRNA expression levels(P<0.01),and inhibited lipid peroxidation.Compared with the model group,the body weight,blood lipid level and aortic plaque area of ML385 group increased(P<0.01);the morphology of mitochondria showed significant ferroptosis characteristics;the serum Fe^(2+),MDA and ROS levels increased(P<0.05 or P<0.01),and the activity of SOD decreased(P<0.01).Compared with BBRM group,the iron inhibition effect of BBRM+ML385 group was significantly weakened,and the plaque area significantly increased(P<0.01).Conclusion:Through NRF2/SLC7A11/GPX4 pathway,BBR can resist oxidative stress,inhibit ferroptosis,reduce plaque area,stabilize plaque,and exert anti-AS effects.

大鼠脊髓Nrf2/HO-1通路参与不同部位电针调节神经性疼痛效应的机制探讨

Objective:This study aimed to assess the effects of electroacupuncture(EA)at the contralateral,ipsilateral,or bilateral"Zusanli(ST36)"and"Yanglingquan(GB34)"on neuropathic pain caused by chronic contractile injury(CCI)and to explore the role of the nuclear factor erythroid 2-related factor 2(Nrf2)pathway in the effects of EA.Methods:Male Sprague-Dawley rats were subjected to the CCI model to induce neuropathic pain.A total of 45 rats were randomly divided into five groups(n=9):sham,CCI,EA-Co(CCI+EA at contralateral acupoints),EA-Ip(CCI+EA at ipsilateral acupoints),and EA-Bi(CCI+EA at bilateral acupoints).The rats received EA treatment on day 8 after CCI,once every alternate day,for a total of eight times.The time courses of mechanical pain threshold(MWT),hind paw withdrawal latency(HWL),and sciatic functional index(SFI)were determined.The expression levels of 8-hydroxydeoxyguanosine(8-OHdG),glutathione(GSH),superoxide dismutase(SOD)activity,interleukin-6(IL-6),interleukin-1 beta(IL-1β),and tumor necrosis factor factor-alpha(TNF-α)in the spinal cord were measured.The distribution of Nrf2,its expression of Nrf2 in both the cytosol and nucleus,and the protein levels of its downstream target genes,NQO1 and HO-1,were detected via double immunofluorescence staining and western blotting,respectively.Results:Following CCI,both MWT and HWL in the CCI group significantly decreased from day 14 after surgery(P<0.001).EA treatment exhibited significant antinociceptive effects induced by CCI by increasing the MWT and HWL values,especially bilateral EA(P<0.05).The SFI of the CCI group was significantly lower than that of the sham group(P<0.001).Only bilateral EA improved the SFI scores compared to the CCI group(P<0.05).8-OHdG levels in the spinal cord of the CCI group were significantly higher than those in the sham group(P<0.05),whereas GSH levels and SOD activity in the spinal cord of the CCI group were significantly lower than those in the sham group(P<0.001 and P<0.01,respectively).Bilateral EA administration significantly downregulated 8-OHdG levels(P<0.01)and upregulated GSH levels and SOD activity in the spinal cord(P<0.01).CCI significantly enhanced the production of IL-1β,IL-6,and TNF-αin the spinal cord compared with that in the sham group(all P<0.001).Meanwhile,the effects of EA were also accompanied by markedly decreased expression of IL-1βand IL-6 in the spinal cord(P<0.05).TNF-αlevels were only decreased in the EA-Ip and EA-Bi groups compared with those in the CCI group(P<0.001).Confocal microscopy revealed that Nrf2 was mainly localized in the neurons of the spinal cord.Notably,EA treatment enhanced nuclear translocation of Nrf2 in neurons.CCI significantly decreased the production of Nrf2,HO-1,and NQO1 in the spinal cord compared to the sham group(P<0.001),and bilateral EA up-regulated the protein levels of Nrf2 and its target genes HO-1 and NQO1(all P<0.001).Conclusion:Our results suggest that bilateral EA is an optimal therapeutic strategy for neuropathic pain.The effects of EA on neuropathic pain may be mediated by the restoration of the Nrf2 pathway in the spinal cord.

Fucoxanthin suppresses OxLDL-induced inflammation via activation of Nrf2 and inhibition of NF-κB signaling

Objective:To explore the impact of fucoxanthin on oxidized low-density lipoprotein(OxLDL)-induced stress and inflammation in human endothelial cells and its underlying mechanisms.Methods:HUVECs were treated with OxLDL and/or fucoxanthin for a range of time points and concentrations.We evaluated the effects of fucoxanthin on OxLDL-induced HUVECs using the MTT assay,reactive oxygen species accumulation assay,ELISA,RT-PCR,immunofluorescence,and Western blotting.Results:Fucoxanthin enhanced the cell viability in a dose dependent manner after OxLDL exposure.Furthermore,fucoxanthin pretreatment significantly decreased OxLDL-induced reactive oxygen species production and prevented the activation of the nuclear factor kappa-B pathway,which led to substantial suppression of pro-inflammatory gene expressions.OxLDL-induced upregulation of interleukin-6,intercellular adhesion molecule-1,vascular cell adhesion molecule-1,interleukin-1β,monocyte chemotactic protein-1,cyclooxygenase-1,and tumor necrosis factor-αwas significantly reduced by fucoxanthin.Conclusions:Fucoxanthin can inhibit OxLDL-induced vascular inflammation and oxidative stress in HUVECs by targeting Nrf2 signaling pathways.

Hesperetin induces glyoxalase 1 enhancement in SH-SY5Y cells cultured with high glucose via Nrf2/ARE activation

OBJECTIVE To investigate the neuroprotective effects of hesperetin on central neurons under chronic high glucose,and the relationship to glyoxalase 1(Glo-1),a cytoprotective enzyme.METHODS The human neuroblas⁃toma SH-SY5Y cells were divided into 5 groups:normal glucose,high glucose(HG),HG plus low,middle,or high concentra⁃tion of hesperetin(1,5,25μmol·L^-1).After treatment for 72 h,neuron damages,Glo-1 expressions and functions,as well as Nrf2/ARE pathway and its regulating mechanisms were examined.RESULTS Hesperetin increased cell viability and decreased lactate dehydrogenase release,which was accompanied by the elevated activity,protein,and mRNA levels of Glo-1 as well as the enhanced Glo-1 functions in SH-SY5Y cells cultured with HG.Moreover,hesperetin activated Nrf2/ARE pathway as evidenced by the raised Nrf2 and p-Nrf2 levels in nucleus and up-regulation of γ-glutamycysteine synthase(γ-GCS),a well-known target gene of Nrf2/ARE pathway.Nevertheless,pretreatment with a PKC inhibitor(Go 6983)or an Akt inhibitor(MK-22062HCl,reflecting GSK-3β activation)abolished the effect of hesperetin on protein expressions of Glo-1 and γ-GCS.CONCLUSION Hesperetin exerted the neuroprotection by promoting Glo-1 function in central neurons in long-term HG condition,which was mediated by activation of Nrf2/ARE pathway;moreover,the increased Nrf2 phosphorylation and nuclear translocation mediated by PKC activation and/or GSK-3β inhibition were involved in the activation of Nrf2/ARE pathway by hesperetin.

Liaoqiao aqueous extract inhibits B16 melanoma growth involving MAPKs/Nrf2/HO-1 mediated anti-oxidation and anti-inflammation

Aim Forsythia suspensa （Thunb.） Vahl, Lianqiao in Chinese, is one of the most fundamental herbs in traditional Chinese medicine （TCM） with heat-clearing and detoxicating properties. In this study, we aimed to study the antitumor activity of Lianqiao aqueous extract against melanoma using cancer cell line-based in vitro and mouse allografl tumor in vivo models. Furthermore, we also investigated the underlying molecular mechanisms, par- ticularly the involvement of anti-inflammation and anti-oxidation properties in its antitumor activity. Methods The proliferation of cancer cells was measured by MTT assay. The transplanted B16-F10 melanoma in C57BL/6 mice were established and used for the evaluation of in vivo antitumor effect of LQ. Tumor growth was monitored twice a week. Ki67 and CD31 were used to detect cancer cell proliferation and angiogenesis in tumor, respectively. The anti-oxidative property of LQ was determined by measuring the levels of ROS, MDA and GSH. The anti-inflamma- tory effect of LQ was evaluated by measuring TNF-α and IL-6 using ELISA kits. Other protein expression was deter- mined by Western Blot. Results LQ strongly inhibited the growth of B16-F10 cells in vitro and the tumor growth in vivo. The survival time of tumor-bearing mice was significantly prolonged by LQ. LQ inhibited cancer cell prolif- eration and angiogenesis in tumor as evidenced by decreased expressions of Ki67 and CD31. Levels of ROS, MDA TNF-α and IL-6 decreased, while GSH increased in LQ treatment group, indicating a strong anti-oxidative and an- ti-inflammatory activity of LQ. The expression of antioxidant proteins Nff-2 and HO-1, tumor suppressors P53 and p-PTEN, and the MAPK pathways in tumor tissues were upregulated by LQ treatment. Conclusions LQ exhibited strong antitumor activity against B16-F10 murine melanoma both in vitro and in vivo. The antitumor effect of LQ in- volved the decreased oxidative stress and inflammation in tumor, which is closely related to the heat-clearing and detoxicating properties of LQ.

Protective effects of peptide KSPLY derived from Hericium erinaceus on H_(2)O_(2)-induced oxidative damage in HepG2 cells

Reactive oxygen species(ROS)-induced oxidative damage is strongly associated with the pathogenesis of chronic diseases,and natural antioxidant peptides have good abilities of scavenging ROS.The antioxidant activity of peptide Lys-Ser-Pro-Leu-Tyr(KSPLY)derived from Hericium erinaceus remains unclear.In the present study,the antioxidant effect and mechanism of KSPLY on H_(2)O_(2)-induced oxidative damage in HepG2 cells were investigated.The results indicated that KSPLY exhibited the antioxidant capacity in H_(2)O_(2)-induced HepG2 cells by enhancing superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),and catalase(CAT)activities.In comparison with the H_(2)O_(2)-treated damage group,the apoptosis rate,ROS level,and malondialdehyde(MDA)content of HepG2 cells treated with KSPLY were significantly decreased.The H.erinaceus-derived peptide KSPLY pretreatment promoted the expression of detoxification and antioxidant enzymes via the Keap1/Nrf2 signal pathway,thereby inhibiting the generation of ROS and MDA.In conclusion,the H.erinaceus-derived peptide KSPLY effectively protected HepG2 cells against H_(2)O_(2)-induced oxidative damage,and it provided a theoretical basis for the further development of new natural antioxidants.

Engineered Bacillus subtilis alleviates intestinal oxidative injury through Nrf2-Keap1 pathway in enterotoxigenic Escherichia coli(ETEC) K88-infected piglet

Engineered probiotics can serve as therapeutics based on their ability of produce recombinant immune-stimulating properties.In this study,we built the recombinant Bacillus subtilis WB800 expressing antimicrobial peptide KR32(WB800-KR32)using genetic engineering methods and investigated its protective effects of nuclear factor-E2-related factor 2(Nrf2)-Kelch-like ECH-associated protein 1(Keap1)pathway activation in intestinal oxidative disturbance induced by enterotoxigenic Escherichia coli(ETEC)K88 in weaned piglets.Twenty-eight weaned piglets were randomly distributed into four treatment groups with seven replicates fed with a basal diet.The feed of the control group(CON)was infused with normal sterilized saline;meanwhile,the ETEC,ETEC+WB800,and ETEC+WB800-KR32 groups were orally administered normal sterilized saline,5×10^(10)CFU(CFU:colony forming units)WB800,and 5×10^(10)CFU WB800-KR32,respectively,on Days 1-14 and all infused with ETEC K881×10^(10)CFU on Days 15-17.The results showed that pretreatment with WB800-KR32 attenuated ETEC-induced intestinal disturbance,improved the mucosal activity of antioxidant enzyme(catalase(CAT),superoxide dismutase(SOD),and glutathione peroxidase(GPx))and decreased the content of malondialdehyde(MDA).More importantly,WB800-KR32 downregulated genes involved in antioxidant defense(GPx and SOD1).Interestingly,WB800-KR32 upregulated the protein expression of Nrf2 and downregulated the protein expression of Keap1 in the ileum.WB800-KR32 markedly changed the richness estimators(Ace and Chao)of gut microbiota and increased the abundance of Eubacterium_rectale_ATCC_33656 in the feces.The results suggested that WB800-KR32 may alleviate ETEC-induced intestinal oxidative injury through the Nrf2-Keap1 pathway,providing a new perspective for WB800-KR32 as potential therapeutics to regulate intestinal oxidative disturbance in ETEC K88 infection.