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.

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.

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.

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.

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 icariin on microglia-mediated neuro-inflammation through Nrf2 signaling pathway

OBJECTIVE To investigate the protective effect of icariin(ICA) on lipopolysaccharide(LPS)-induced BV2 microglia injury,and to clarify the role of nuclear factor erythroid 2-related factor 2(Nrf2) signaling pathway in BV2 microglia-mediated neuroinflammation.METHODS BV2 microglia were randomly divided into control,ICA(0.1 μmol·L^(-1)),LPS(1 mg·L^(-1)),LPS + ICA(0.01 μmol·L^(-1)),and LPS + ICA(0.1 μmol·L^(-1))groups.BV2 microglia were treated with ICA for 30 min and then treated with LPS for 24 h.MTT assay was used to determine the cells survival rate,Griess kit and ELISA kits were used to detect the contents of NO,IL-1β and IL-18 in the culture supernatant,Western blotting was used to detect the expression of Nrf2,HO-1 and NQO1.Real time RT-PCR was used to detect the expression of Nrf2,HO-1 and NQO1 after ICA addition for 2,6 and 24 h.And immunofluorescence was used to observe the activation of Nrf2.RESULTS ICA reduced LPS-induced NO,IL-1β and IL-18 production in the culture supernatant,and ICA increase LPS-induced mRNA and protein expression of Nrf2 signaling pathway.CONCLUSION ICA protects LPS induced neuroinflammation by regulating Nrf2 signaling pathway.

An exploration on the protective mechanism of Xuduan Zhongzi prescription against epididymis oxidative damage in oligoasthenospermia model rats based on Nrf2-NQO1/γ-GCS signaling pathway

Objective:To investigate the protective mechanism of Xuduan Zhongzi prescription against epididymal oxidative damage in oligoasthenospermia model rats.Methods:Forty SD rats were randomly divided into blank group,model group,Xuduan Zhongzi prescription group(10g/kg)and L-carnitine group(0.1g/kg).Except blank group,all induced oligoasmospermia.The blank group and model group were given normal saline intragastric administration,the Xuduan Zhongzi prescription group was given Xuduan Zhongzi prescription solution intragastric administration,and the L-carnitine group was given L-carnitine intragastric administration.HE staining was used to observe the epididymis structure after 8 weeks.The concentration and activity rate of epididymis sperm were measured by sperm quality.MRNA and protein expression levels of Nrf2,NQO1 andγ-GCs in epididymis were detected by RT-qPCR and immunohistochemistry.Results:①HE staining:in the blank group,the epididymis tubes were arranged tightly and regularly,the tissue structure was complete,the epithelial cells were arranged orderly,and the lumen sperm were numerous and evenly distributed.The epididymis of model group showed structural atrophy,loose arrangement,enlarged mesenchyme,increased cell debris and significantly reduced sperm cells.Compared with the model group,the lumen lesions of epididymis in Xuduan Zhongzi prescription group and L-carnitine group were significantly improved,and the amount of normal sperm in lumen was increased and the distribution was uniform.②Results of sperm quality comparison among each group:sperm density and sperm motility rate:compared with blank group,sperm density and sperm motility rate in other groups were significantly decreased(P<0.05),and sperm density and sperm motility rate in model group were significantly decreased(P<0.05);Compared with model group,the sperm density and motility rate in Xuduan Zhongzi prescription group and L-carnitine group were significantly increased(P<0.05).③RT-qPCR and immunohistochemistry:Compared with the blank group,the mRNA and protein levels of Nrf2,NQO1 andγ-GCs in epididymal rats in model group were significantly decreased(P<0.05),while the mRNA and protein levels of Nrf2,NQO1 andγ-GCs were significantly increased in L-carnitine group and Continua seed formula group(P<0.05).Conclusion:Xuduan Zhongzi prescription can reduce oxidative stress damage and improve sperm quality of oligoasthenospermia.The mechanism may related to promoting the activation of Nrf2-NQO1/γ-GCS pathway in epididymis of oligoasthenospermia rats,and up-regulate the expressions of Nrf2,NQO1 andγ-GCS proteins.

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).

Rosmarinic acid elicits neuroprotection in ischemic stroke via Nrf2 and heme oxygenase 1 signaling

Rosmarinic acid（RA） can elicit a neuroprotective effect against ischemic stroke, but the precise molecular mechanism remains poorly understood. In this study, an experimental ischemic stroke model was established in CD-1 mice（Beijing Vital River Laboratory Animal Technology, Beijing, China） by occluding the right middle cerebral artery for 1 hour and allowing reperfusion for 24 hours. After intraperitoneally injecting model mice with 10, 20, or 40 mg/kg RA, functional neurological deficits were evaluated using modified Longa scores. Subsequently, cerebral infarct volume was measured using TTC staining and ischemic brain tissue was examined for cell apoptosis with TUNEL staining. Superoxide dismutase activity and malondialdehyde levels were measured by spectrophometry. Expression of heme oxygenase-1（HO-1）, nuclear factor erythroid 2-related factor 2（Nrf2）, Bcl-2, Bax, Akt, and phospho-Ser473 Akt proteins in ischemic brain tissue was detected by western blot, while mRNA levels of Nrf2, HO-1, Bcl-2, and Bax were analyzed using real time quantitative PCR. In addition, HO-1 enzyme activity was measured spectrophotometrically. RA（20 and 40 mg/kg） greatly improved neurological function, reduced infarct volume, decreased cell apoptosis, upregulated Bcl-2 protein and mRNA expression, downregulated Bax protein and mRNA expression, increased HO-1 and Nrf2 protein and mRNA expression, increased superoxide dismutase activity, and decreased malondialdehyde levels in ischemic brain tissue of model mice. However, intraperitoneal injection of a HO-1 inhibitor（10 mg/kg zinc protoporphyrin IX） reversed the neuroprotective effects of RA on HO-1 enzyme activity and Bcl-2 and Bax protein expression. The PI3 K/Akt signaling pathway inhibitor LY294002（10 mM） inhibited Akt phosphorylation, as well as Nrf2 and HO-1 expression. Our findings suggest that RA has anti-oxidative and anti-apoptotic properties that protect against ischemic stroke by a mechanism involving upregulation of Nrf2 and HO-1 expression via the PI3 K/Akt signaling pathway.

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.

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.

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.

Photothermal therapy with regulated Nrf2/NF-κB signaling pathway for treating bacteria-induced periodontitis

Periodontitis is an inflammatory disease initiated by bacterial infection,developed by excessive immune response,and aggravated by high level of reactive oxygen species(ROS).Hence,herein,a versatile metal-organic framework(MOF)-based nanoplatform is prepared using mesoporous Prussian blue(MPB)nanoparticles to load BA,denoted as MPB-BA.The established MPB-BA nanoplatform serves as a shelter and reservoir for vulnerable immunomodulatory drug BA,which possesses antioxidant,anti-inflammatory and anti-bacterial effects.Thus,MPB-BA can exert its antioxidant,anti-inflammatory functions through scavenging intracellular ROS to switch macrophages from M1 to M2 phenotype so as to relieve inflammation.The underlying molecular mechanism lies in the upregulation of phosphorylated nuclear factor erythroid 2-related factor 2(Nrf2)to scavenge ROS and subsequently inhibit the nuclear factor kappa-B(NF-κB)signal pathway.Moreover,MPB-BA also exhibited efficient photothermal antibacterial activity against periodontal pathogens under near-infrared(NIR)light irradiation.In vivo RNA sequencing results revealed the high involvement of both antioxidant and anti-inflammatory pathways after MPB-BA application.Meanwhile,micro-CT and immunohistochemical staining of p-Nrf2 and p-P65 further confirmed the superior therapeutic effects of MPB-BA than minocycline hydrochloride.This work may provide an insight into the treatment of periodontitis by regulating Nrf2/NF-κB signaling pathway through photothermal bioplatform-assisted immunotherapy.

Arsenic Trioxide Combining Leflunomide Activates Nrf2-ARE-HO-1 Signaling Pathway and Protects Heart Xenografts

Objective:To investigate the molecular mechanisms underlying the effects of arsenic trioxide(As_(2)0_(3))in combination with leflunomide on the hamster-to-rat heart xenotransplant.Methods:Transplantation of LVG hamster hearts to Lewis rats was performed by anastomosis of vessels in the neck using end-to-end anastomosis with a non-suture cuff technique.Four groups of recipient rats(n=6 in each)were treated with normal saline(control),As_(2)0_(3)[5 mg/(kg*day)intraperitoneally],leflunomide[5 mg/(kg*d)orally],or leflunomide[5 mg/(kg.d)+As_(2)0_(3)5 mg/(kg.d)]in combination.Donor hearts and/or rat spleens were harvested and analyzed 4 days after transplantation.Quantitative reverse-transcription polymerase chain reaction and Western blot analysis were performed to detect the expression of the nuclear factor erythroid-derived factor 2-related factor(Nrf2)and its target gene heme oxygenase-1(HO-1),Treg cell marker fork-head Box P3(FOXP3),apoptosis-associated proteins Bcl-2,Bax,and cleaved caspase-3.Immunohistochemical staining was used to detect the levels of inflammatory natural killer cell and macrophage infiltration,intercellular cell adhesion molecule-1(ICAM-1)and complement C3.Results:Expression of Nrf2-ARE-HO-1 signaling pathway was upregulated in heart xenografts in rats treated with As_(2)0_(3) plus leflunomide compared with control rats or rats treated with either drug alone(P<0.01),and this was accompanied by an increased Treg cells in the recipient rat spleen(P<0.01).In contrast,the expressions of Bax,cleaved caspase-3,ICAM-1,and complement C3,and infiltration of inflammatory cells in the xenografts were inhibited by As_(2)0_(3) plus leflunomide treatment(P<0.01).Conclusion:Combination treatment with As_(2)0_(3) and leflunomide protected hamster heart-xenografts in recipient rats.

Apigenin alleviates neomycin-induced oxidative damage via the Nrf2 signaling pathway in cochlear hair cells

Oxidative stress plays an important role in the pathogenesis of aminoglycoside-induced hearing loss and represents a promising target for treatment.We tested the potential effect of apigenin,a natural flavonoid with anticancer,anti-inflammatory,and antioxidant activities,on neomycin-induced ototoxicity in cochlear hair cells in vitro.Results showed that apigenin significantly ameliorated the loss of hair cells and the accumulation of reactive oxygen species upon neomycin injury.Further evidence suggested that the nuclear factor erythroid 2-related factor 2(Nrf2)signaling pathway was activated by apigenin treatment.Disruption of the Nrf2 axis abolished the effects of apigenin on the alleviation of oxidative stress and subsequent apoptosis of hair cells.This study provided evidence of the protective effect of apigenin on cochlear hair cells and its underlying mechanism.

Over-expression of Toll-like receptor 2 up-regulates heme oxygenase-1 expression and decreases oxidative injury in dairy goats

Background： Mastitis, an infection caused by Gram-positive bacteria, produces udder inflammation and oxidative injury in milk-producing mammals. Toll-like receptor 2（TLR2） is important for host recognition of invading Grampositive microbes. Over-expression of TLR2 in transgenic dairy goats is a useful model for studying various aspects of infection with Gram-positive bacteria, in vivo.Methods： We over-expressed TLR2 in transgenic dairy goats. Pam3CSK4, a component of Gram-positive bacteria,triggered the TLR2 signal pathway by stimulating the monocytes-macrophages from the TLR2-positive transgenic goats, and induced over-expression of activator protein-1（AP-1）, phosphatidylinositol 3-kinase（PI3K） and transcription factor nuclear factor kappa B（NF-κB） and inflammation factors downstream of the signal pathway.Results： Compared with wild-type controls, measurements of various oxidative stress-related molecules showed that TLR2, when over-expressed in transgenic goat monocytes-macrophages, resulted in weak lipid damage, high level expression of anti-oxidative stress proteins, and significantly increased m RNA levels of transcription factor NF-E2-related factor-2（Nrf2） and the downstream gene, heme oxygenase-1（HO-1）. When Pam3CSK4 was used to stimulate ear tissue in vivo the HO-1 protein of the transgenic goats had a relatively high expression level.Conclusions： The results indicate that the oxidative injury in goats over-expressing TLR2 was reduced following Pam3CSK4 stimulation. The underlying mechanism for this reduction was increased expression of the anti-oxidation gene HO-1 by activation of the Nrf2 signal pathway.

2-羟基-3-甲基蒽醌抑制结直肠癌的作用及机制研究

为观察2-羟基-3-甲基蒽醌(HMA)对结直肠癌细胞增殖的影响并探讨其作用机制,本研究采用CCK8法检测HMA对结直肠癌细胞增殖的影响;Heochst-33343/PI染色检测细胞凋亡情况;同时检测细胞内ROS及GSH含量变化并应用Western blot检测细胞凋亡相关蛋白及Keap-1/Nrf-2/ARE信号途径相关蛋白的表达。实验结果显示,HMA给予结直肠癌细胞后,细胞内ROS含量升高,GSH含量减少;HMA通过抑制Keap-1/Nrf-2/HO-1通路,诱导细胞发生凋亡。综上表明HMA具有抑制结肠癌细胞增殖的作用,其机制可能与破坏细胞内氧化还原平衡,抑制Keap-1/Nrf-2/HO-1通路有关。

麝香对Aβ_(1-42)诱导PC12细胞氧化应激损伤的保护作用研究

目的:探讨麝香对阿尔兹海默症(AD)细胞模型氧化应激损伤及凋亡的保护作用。方法:使用LC-MS/MS法分析麝香化学成分。通过β-淀粉样蛋白1-42(Aβ_(1-42))诱导PC12细胞建立体外AD细胞模型,采用CCK-8法测定PC12细胞存活率,考察Aβ_(1-42)对PC12的细胞毒性及麝香对Aβ_(1-42)诱导PC12细胞损伤的保护作用;采用Annexin V-FITC/PI双染色法检测麝香对Aβ_(1-42)致PC12细胞凋亡的影响,JC-1法检测麝香对Aβ_(1-42)致PC12细胞线粒体膜电位的影响;采用探针DCFH-DA检测麝香对Aβ_(1-42)致PC12细胞内活性氧(ROS)的影响;采用超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)试剂盒检测麝香对Aβ_(1-42)致PC12细胞内抗氧化酶活性水平的影响;采用Western blot法检测PC12细胞内相关凋亡蛋白BAX、BCL-2和Cleaved caspase-3的表达以及核因子红细胞2相关因子2(Nrf-2)、血红素加氧酶-1(HO-1)和Kelch样ECH相关蛋白1(Keap 1)的表达。结果:通过LC-MS/MS法鉴定出麝香的7种化学成分分别为:次黄嘌呤、雄烯二酮、表睾酮、十六碳酰胺、麝香酮、油酸酰胺、硬脂酰胺。与空白对照组比较,模型对照组PC12细胞存活率显著降低(P<0.01),细胞内ROS水平、细胞凋亡率显著升高(P<0.01),线粒体膜电位明显降低(P<0.05),SOD和GSH-Px活力明显降低(P<0.05或P<0.01),BAX、Cleaved caspase-3和Keap 1蛋白表达明显上调,BCL-2、Nrf-2和HO-1蛋白表达明显下调(P<0.05或P<0.01);与模型对照组相比,麝香0.025、0.05、0.1 mg/mL组细胞存活率明显升高(P<0.05或P<0.01),细胞内ROS水平、细胞凋亡显著减少(P<0.01),SOD活力明显升高(P<0.05或P<0.01);其中0.05和0.1 mg/mL组中线粒体膜电位显著升高(P<0.01),BAX、Cleaved caspase-3和Keap 1蛋白表达明显下调(P<0.05或P<0.01);麝香0.1 mg/mL组GSH-Px活力显著升高(P<0.01),BCL-2、Nrf-2和HO-1蛋白表达明显上调(P<0.05或P<0.01)。结论:麝香通过Nrf-2/HO-1通路显著改善Aβ_(1-42)诱导的AD细胞模型的氧化应激和凋亡。