OBJECTIVE Nuclear factor erythroid 2-related factor 2(Nrf2) is found to be ubiquitiously expressed in many tissues,and works as the key regulator against oxidative stress damage in cells and organs,which makes Nrf2 a ...OBJECTIVE Nuclear factor erythroid 2-related factor 2(Nrf2) is found to be ubiquitiously expressed in many tissues,and works as the key regulator against oxidative stress damage in cells and organs,which makes Nrf2 a widely concerned drug target.Recent research has identified that Nrf2 is involved in the pathology of Alzheimer disease(AD),whereas the mechanism is unknown.The purpose of this study is to figure out the role of Nrf2 in the pathologic process of AD through Nrf2-Keap1-ARE pathway and the effects of Keap1-Nrf2 inhibitor in AD mice models.METHODS Amyloid β^(1-42)(Aβ^(1-42))was injected into the bilateral hippocampus to induce the cognitive dysfunction in eight-week old male mice.The mice were treated with Keap1-Nrf2 inhibitor NXPZ of three doses as well as donepezil as a positive control by intragastric administration one time a day for one week.Several behavior tests were used to analyze the mice learning and memory ability.Additionally,we detected Nrf2 and Aβ in the plasma in mice with ELISA kits,as well as some factors related to oxidative stress in the hippocampus and cortex.The expression levels of Nrf2,Keap1,Tau and p-Tau were measured in the murine brain tissue with Western blotting.SH-SY5 Y cells were studied as an in vitro model to further clarify the mechanism.RESULTS The treatment of NXPZ ameliorated learning and memory dysfunction in AD mice in a dose-dependent manner,and the high dose group recovered better than the positive drug group.The plasma Nrf2 level was increased in a dose-dependent manner in the treatment groups;however,the plasma Aβ was decreased.What′ s more,superoxide dismutase(SOD) and glutathione reductase(GSSH) in the hippocampus and cortex were increased in the treatment group,while the malondialdehyde(MDA) was decreased,meaning that NXPZ treatment promoted expression of the anti-oxidative factors and inhibited the expression of the oxidative factors in the down-stream.Western blotting analysis of hippocampus and cortex showed up-regulated Nrf2,decreased Keap1 and decreased p-Tau in NXPZ treatment mice.In ex vivo experiments,when SH-SY5 Y cells were treated with Aβ,Nrf2 in the cytoplasm was increased,as well as the expression Nrf2 in the nuclear was decreased.The treatment of NXPZ increased nuclear Nrf2,decreased cytoplasm Nrf2,and decreased the expression of p-Tau.CONCLUSION Nrf2 has an important role in neuron function.Nrf2 activation by selective Keap1-Nrf2 inhibitor NXPZ may contribute to improve cognitive function in AD mice.The mechanism may be related to increased generation and release of Nrf2 induced by more disaggregation with Keap1,leading to more expression of anti-oxidative molecules to protect the damage caused by Aβ.These results indicates that Nrf2 may be a novel therapeutic target of AD and Keap1-Nrf2 inhibitor may be a novel medication for protecting the loss of learning and memory ability.展开更多
OBJECTIVE To investigate the neuroprotective effects of quercetin on central neurons against chronic high glucose in central neurons,in relation to Nrf2/ARE/Glo-1 activation.METHODS SH-SY5Y cells were cultured with hi...OBJECTIVE To investigate the neuroprotective effects of quercetin on central neurons against chronic high glucose in central neurons,in relation to Nrf2/ARE/Glo-1 activation.METHODS SH-SY5Y cells were cultured with high glucose(HG,70 mmol·L^(-1)),4-fold of the normal glucose(17.5 mmol·L^(-1)).Quercetin was set three concentrations(5,10,20μmol·L^(-1)),with Nrf2 activator sulforaphane(SFN)as a positive group(2.5μmol·L^(-1)).After 72 h,cells were collected for glyoxalase 1(Glo-1)activity and GSH level were by spectrophotometry;advanced glycation end-products(AGEs)as well as nuclear Nrf2 and p-Nrf2 levels by immunofluorescence;Glo-1,γ-glutamycysteine synthase(γ-GCS),Nrf2 and p-Nrf2 protein levels by Western blotting,and Glo-1 andγ-GCS m RNA levels by real-time qP CR.RESULTS Quercetin increased the cell viability of SH-SY5Y cells,and upregulated the levels of Glo-1 activity,protein,and m RNA in SH-SY5Y cells cultured with HG,accompanied by the elevated levels of glutathione,a cofactor of Glo-1 activity,and the reduced levels of AGEs.Meanwhile,quercetin could increase p-Nrf2 and Nrf2 levels in nucleus as well as p-Nrf2 levels in cytosol of SH-SY5Y cells exposed to chronic HG,accompanied by the elevated protein expression and m RNA levels ofγ-GCS,a known target gene of Nrf2/ARE signaling.Moreover,a PKC activator or a p38MAPK inhibitor pretreatment could significantly increase the protein expression ofγ-GCS in HG condition,but an alkylating agent for sulfydryl of cysteine in Keap 1,a negative regulator of Nrf2,pretreatment only showed an increased tendency ofγ-GCS protein,compared with without pretreatment;however,after pretreatment with those tool drugs,co-treatment with quercetin and HG had similar results to those of single tool drug pretreatment followed by HG exposure.CONCLUSION Firstly,quercetin can enhance Glo-1 function in central neurons,which is mediated by activation of Nrf2/ARE pathway,then exerts the neuroprotection against HG induced damage;moreover,PKC and p38 MAPK pathways may be involved in Nrf2 inactivation in chronic HG condition.展开更多
OBJECTIVE The present study aimed to investigate the relationship between Wnt/β-catenin and Nrf2 signaling pathways,and understanding the mechanisms underlying the process of inflammatory in chronic obstructive pulmo...OBJECTIVE The present study aimed to investigate the relationship between Wnt/β-catenin and Nrf2 signaling pathways,and understanding the mechanisms underlying the process of inflammatory in chronic obstructive pulmonary disease(COPD),which was a serious disease of respiratory system.METHODS We duplicate the emphysema model with porcine pancreatic elastase(PPE)in Nrf2-/-and WT mouse for 21d,and intraperitoneal injection of Li Cl,the activator of Wnt/β-catenin signaling pathway from 14 d to the end.Hematoxylin and eosin(H&E)staining was performed to assess the histopathologic level,and immunohistochemistry(IHC)for Mac-3(the marker of macrophagocyte)and Ly6G(the marker of neutrophil)was used to observe the inflammatory infiltrate,while the levels of Wnt/β-catenin and Nrf2 signaling pathways related proteins heme oxygenase-1(HO-1),NAD(P)H:quinone oxidoreductase 1(NQO1),and the expression of inflammatory cytokine interleukin-6(IL-6)were detected by Western blotting of lung tissues.In vitro,cigarette smoke extract(CSE)-treated normal human bronchial epithelial(NHBE)cells,cell viability was examined by MTT assay,and then we treated recombinant human Wnt3a,si Nrf2 and si Wnt3a to measure the expression of Wnt3a,β-catenin,Nrf2,HO-1,NQO-1,and IL-6.Cellular immunofluorescence staining was employed to identify the nuclear translocation of Nrf2.RESULTS We found that the Li Cl-treated group has markedly decreased the damage of alveolar structure and inflammatory signs than the model group of WT mice rather than Nrf2-/-group.It also seen that Li Cl not only increasedβ-catenin,but it also led to a comparable increase in Nrf2,HO-1,NQO1,and decrease of IL-6 compared with WT model groups but except to Nrf2-/-group in vivo.And it showed that Wnt3atreatment has significantly increased the nuclear translocation of Nrf2 and the expression of HO-1 and NQO1,reduced the IL-6 release,while there has no significance when Nrf2 was blocked in CSE-induced NHBE cells.CONCLUSION Our results demonstrated that Wnt3a/β-catenin significantly balanced oxidative stress and attenuated inflammation reaction by promoting Nrf2 nuclear translocation and activity.展开更多
OBJECTIVE Epithelial-mesenchymal transition(EMT)is a phenotype conversion that plays a critical role in the development of pulmonary fibrosis(PF).It is known that a transcription factor snail could regulate the progre...OBJECTIVE Epithelial-mesenchymal transition(EMT)is a phenotype conversion that plays a critical role in the development of pulmonary fibrosis(PF).It is known that a transcription factor snail could regulate the progression of EMT.Nuclear factor erythroid 2 related factor 2(Nrf2),a key regulator of antioxidant defense system,protects cells and tissues against oxidative stress.However,it is not known whether Nrf2 regulates snail thereby modulating the development of PF.MEHODS Bleomycin(BLM)was intratracheally injected into both Nrf2-knockout(Nrf2-/-)and wild-type mice to compare the development of PF.Rat type II alveolar epithelial cells(AECs)RLE-6TN were treated with a specific Nrf2activator sulforaphane,or transfected with Nrf2 and snail si RNAs to determine their effects on transforming growth factorβ1(TGF-β1)-induced EMT.RESULTS BLM-induced EMT and lung fibrosis were more severe in Nrf2-/-mice compared to wild-type mice.In vitro,sulforaphane treatment attenuated TGF-β1-induced EMT,accompanied by the down-regulation of snail.Inversely,silencing Nrf2 by si RNA enhanced TGF-β1-induced EMT along with the expression of snail.Interestingly,silencing snail by si RNA reduced TGF-β1-induced EMT even in the presence of sulforaphane in RLE-6TN cells.CONCLUSION These findings suggested that Nrf2 may attenuate EMT and fibrosis process through regulating the expression of snail in PF.展开更多
OBJECTIVE To investigate icariside(ICS)Ⅱ protects against PC12 cel damage induced by oxygen-glucose deprivation and reoxygenation and explore its mechanism.METHODS The oxidative stress injury model was induced by oxy...OBJECTIVE To investigate icariside(ICS)Ⅱ protects against PC12 cel damage induced by oxygen-glucose deprivation and reoxygenation and explore its mechanism.METHODS The oxidative stress injury model was induced by oxygen-glucose deprivation/reoxygenation(OGD/R) 2 h/24 h in PC12 cells.N-acetyl-lcysteine(NAC),a classical anti-oxidant,was used as positive control.Pharmacodynamic experimental study groups as follows:control,control+ICS Ⅱ50 μmol·L^(-1),OGD/R,OGD/R+ICSⅡ 12.5 μmol·L^(-1),OGD/R + ICS Ⅱ 25 μmol·L^(-1),OGD/R + ICS Ⅱ50 μmol·L^(-1),and OGD/R+NAC 100 μmol·L^(-1) groups.Cell viability and lactate dehydrogenase(LDH) leakage rate were measured by MTT assay and LDH ELISA kit,respectively.Moreover,reactive oxygen species(ROS) ELISA kit was used for detection of intracellular ROS generation,Mito-SOX fluorescence staining was used for detecting production of ROS in mitochondria and mitochondrial membrane potential(MMP)was detected by rhodamine 123 dye.In addition,PC12 cells apoptosis was detected by one-step TUNEL assay.Furthermore,the expressions of nuclear factor erythroid 2-related factors(Nrf2),Keap1,HO^(-1),NQO^(-1),silent information regulator 3(SIRT3),IDH2,Bax,Bcl-2 and caspase 3 were detected by Western blotting analysis.RESULTS The results of MTT and LDH assay showed that OGD/R reduced the cell viability and improved LDH release compared with the control or ICSⅡ 50 μmol·L^(-1) alone(P<0.01).Meanwhile,OGD/R not only increased intracellular and mitochondrial ROS generation,but also elevated the fluorescence intensity of TUNEL staining,at the same time,the MMP was declined when challenged by OGD/R.Furthermore,the Western blotting results showed that OGD/R induced the increase in the expression of cytoplasm-Nrf2,Keap1,Bax and cleaved-caspase 3 level,while the decrease in the expression of nucleus-Nrf2,HO^(-1),NQO^(-1),SIRT3,IDH2 and Bcl-2(P<0.05).However,ICS Ⅱ significantly increased the viability of PC12 cells and reduced LDH leakage(P<0.01).Notably,ICS Ⅱ also suppressed ROS generation both in the intracellular and mitochondria,as well as restored MMP.It was also worthy to note that ICS Ⅱ decreased the expressions of cytoplasmNrf2,Keap1,Bax and the level of cleaved-caspase3,whereas,it increased the expressions of nucleus-Nrf2,HO^(-1),NQO^(-1),SIRT3,IDH2 and Bcl-2(P<0.05).CONCLUSION ICSⅡ reduced OGD/Rinduced oxidative damage in PC12 cells under the laboratory conditions,and its underlying mechanism may be related to the regulation of Nrf2/SIRT3 signaling pathway.展开更多
OBJECTIVE Ginsenoside Rg1(Rg1),a purified compound from Panax ginseng,has been well documented to be effective against ischemia/reperfusion(I/R) neurotoxicity.However,the underlying mechanism is stil obscure.METHODS T...OBJECTIVE Ginsenoside Rg1(Rg1),a purified compound from Panax ginseng,has been well documented to be effective against ischemia/reperfusion(I/R) neurotoxicity.However,the underlying mechanism is stil obscure.METHODS The anti-I/R effect of Rg1 were investigated in vitro and in vivo,and the dynamics of nuclear accumulation and the transcriptional activity of NF-E2-related factor 2(Nrf2) determined by Western blotting and Dual Luciferase Reporter Assay,respectively.Nrf2 siRNA was employed to investigate Nrf2′s role in the protective effect of Rg1 against I/R.Furthermore,the role of miR-144,which could regulate post-translational Nrf2 levels,was investigated in the anti-I/R effect of Rg1 by injection of AAV-hypoxia-inducible factor miR-144-shRNA in the predicted ischemic penumbra.RESULTS It was found that the anti-I/R effect of Rg1 was related to its anti-oxidative capacity,which is mainly regulated by the Nrf2/antioxidant response element(ARE) pathway.Further study suggested that Rg1 contributes to the enhancement of the Nrf2/ARE pathway,as manifested by increasing the dynamic peak content of Nrf2,which prolonged the maintenance stage,and promoting the expression of ARE-target genes after oxygen glucose deprivation/reperfusion(OGD/R) in PC12 cells.Nrf2-siRNA application significantly reduced these changes.Furthermore,the enhancement of the Nrf2/ARE pathway by Rg1 was independent of disassociation from Keap1;rather it was a result of posttranslational regulations.It was found that Rg1 significantly reduced the expression of miR-144,which down-regulates Nrf2 production by targeting its 3′-untranslated region,after OGD/R.Knockdown of Nrf2 showed no effect on the expression of miR-144,indicating that miR-144 is an upstream regulator of Nrf2.Moreover,direct binding between Nrf2 and miR-144 in the PC12 cells was identified.Application of anti-miR-144 significantly reduced Rg1′s anti-OGD/R capacity.Final y,the role of miR-144 in Rg1′ s anti-I/R effect was tested by inhibiting miR-144 in the predicted ischemic penumbra when hypoxia-inducible-factor was activated.The results showed that loss of miR-144 abolished the anti-I/R effect of Rg1,which included reduced infarct volume,improved neurological scores,attenuated oxidative impairment,as well as activation of the Nrf2/ARE pathway.CONCLUSION Oxidative stress after I/R is alleviated by Rg1 through inhibition of miR-144 activity and subsequent promotion of the Nrf2/ARE pathway at the post-translational level.展开更多
OBJECTIVE To investigates the effects of imperatorin on the oxidative stress in the cerebral cortex and hippocampus after focal cerebral ischemia/reperfusion injury.METHODS Transient focal cerebral ischemia/reperfusio...OBJECTIVE To investigates the effects of imperatorin on the oxidative stress in the cerebral cortex and hippocampus after focal cerebral ischemia/reperfusion injury.METHODS Transient focal cerebral ischemia/reperfusion model in male Sprague-Dawley rats was induced by 2 h middle cerebral artery occlusion followed by 24 h reperfusion.Imperatorin(1.25 and 2.5 mg·kg-1)or vehicle were administered intraperitoneally at 1,5 and 9 h after the onset of ischemia.At 24 h after reperfusion,the biomarkers of oxidative stress such as the levels of reactive oxygen species(ROS),lipid peroxidation products malondialdehyde(MDA),nitric oxide(NO)and total antioxidant capacity(T-AOC),the activities of inducible nitric oxide synthase(iN OS),superoxide dismutase(SOD)and catalase(CAT)in the cerebral cortex and hippocampus were observed.We also assessed the nuclear factor erythroid 2-related factor 2(Nrf2),heme oxygenase-1(HO-1),and the NAD(P)H-quinone oxidoreductase 1(NQO-1)protein expression by Western blot.RESULTS As compared to vehicle-treated animals,imperatorin treatment significantly reduced the ROS,MDA,NO levels and i NOS activity,increased T-AOC and the activities of SOD and CAT.Furthermore,imperatorin treatment also significantly induced the nuclear translocation of Nrf2,enhanced the protein expression of HO-1 and NQO-1 in the cerebral cortex and hippocampus.CONCLUSION Our findings indicate that imperatorin can protect the brain against the excessive oxidative stress induced by cerebral ischemia/reperfusion through activation of Nrf2 signaling pathway.展开更多
20C,a bibenzyl compound isolated from Gastrodia elata,possesses antioxidative properties in PC12 cells,but its in-depth molecular mechanisms against rotenone-induced neurotoxicity remains unknown.Recent studies indica...20C,a bibenzyl compound isolated from Gastrodia elata,possesses antioxidative properties in PC12 cells,but its in-depth molecular mechanisms against rotenone-induced neurotoxicity remains unknown.Recent studies indicate that without intact DJ-1,nuclear factor erythroid 2-related factor(Nrf2)protein becomes unstable,and the activity of Nrf2-mediated downstream antioxidant enzymes are thereby suppressed.Therefore,increasing the nuclear translocation of Nrf2 by DJ-1 may present a helpful means for the prevention and treatment of chronic diseases related to oxidative stress.Our results showed that 20C clearly protected PC12 and SH-SY5Y cells against rotenone-induced oxidative injury in a concentration-dependent manner.Furthermore,20C markedly up-regulated the levels of DJ-1,which in turn activated phosphoinositide-3-kinase(PI3K)/Akt signaling and inhibited glycogen synthase kinase 3β(GSK3β)activation,eventually promoting Nrf2 nuclear translocation and inducing the expression of Nrf2-mediated downstream antioxidative enzymes such as HO-1.The antioxidative effects of 20C could be partially blocked by ShR NA-mediated knockdown of DJ-1 and inhibition of the PI3K/Akt pathways with Akt1/2 kinase inhibitor in PC12 and SH-SY5Y cells,respectively.Conclusively,our findings confirm that DJ-1 is necessary for 20C-mediated protection against rotenone-induced oxidative damage,at least in part,by activating PI3K/Akt signaling,and subsequently enhancing the nuclear accumulation of Nrf2.The findings from our investigation suggest that 20C should be developed as a novel candidate for preventing or alleviating the consequences of PD in the future.展开更多
基金National Natural Science Foundation of China(81703520).
文摘OBJECTIVE Nuclear factor erythroid 2-related factor 2(Nrf2) is found to be ubiquitiously expressed in many tissues,and works as the key regulator against oxidative stress damage in cells and organs,which makes Nrf2 a widely concerned drug target.Recent research has identified that Nrf2 is involved in the pathology of Alzheimer disease(AD),whereas the mechanism is unknown.The purpose of this study is to figure out the role of Nrf2 in the pathologic process of AD through Nrf2-Keap1-ARE pathway and the effects of Keap1-Nrf2 inhibitor in AD mice models.METHODS Amyloid β^(1-42)(Aβ^(1-42))was injected into the bilateral hippocampus to induce the cognitive dysfunction in eight-week old male mice.The mice were treated with Keap1-Nrf2 inhibitor NXPZ of three doses as well as donepezil as a positive control by intragastric administration one time a day for one week.Several behavior tests were used to analyze the mice learning and memory ability.Additionally,we detected Nrf2 and Aβ in the plasma in mice with ELISA kits,as well as some factors related to oxidative stress in the hippocampus and cortex.The expression levels of Nrf2,Keap1,Tau and p-Tau were measured in the murine brain tissue with Western blotting.SH-SY5 Y cells were studied as an in vitro model to further clarify the mechanism.RESULTS The treatment of NXPZ ameliorated learning and memory dysfunction in AD mice in a dose-dependent manner,and the high dose group recovered better than the positive drug group.The plasma Nrf2 level was increased in a dose-dependent manner in the treatment groups;however,the plasma Aβ was decreased.What′ s more,superoxide dismutase(SOD) and glutathione reductase(GSSH) in the hippocampus and cortex were increased in the treatment group,while the malondialdehyde(MDA) was decreased,meaning that NXPZ treatment promoted expression of the anti-oxidative factors and inhibited the expression of the oxidative factors in the down-stream.Western blotting analysis of hippocampus and cortex showed up-regulated Nrf2,decreased Keap1 and decreased p-Tau in NXPZ treatment mice.In ex vivo experiments,when SH-SY5 Y cells were treated with Aβ,Nrf2 in the cytoplasm was increased,as well as the expression Nrf2 in the nuclear was decreased.The treatment of NXPZ increased nuclear Nrf2,decreased cytoplasm Nrf2,and decreased the expression of p-Tau.CONCLUSION Nrf2 has an important role in neuron function.Nrf2 activation by selective Keap1-Nrf2 inhibitor NXPZ may contribute to improve cognitive function in AD mice.The mechanism may be related to increased generation and release of Nrf2 induced by more disaggregation with Keap1,leading to more expression of anti-oxidative molecules to protect the damage caused by Aβ.These results indicates that Nrf2 may be a novel therapeutic target of AD and Keap1-Nrf2 inhibitor may be a novel medication for protecting the loss of learning and memory ability.
基金supported by National Natural Science Foundation of China(81371210)Qing Lan Project of Jiangsu Province(2014)Priority Academic Program Development of Jiangsu Higher Education Institutions
文摘OBJECTIVE To investigate the neuroprotective effects of quercetin on central neurons against chronic high glucose in central neurons,in relation to Nrf2/ARE/Glo-1 activation.METHODS SH-SY5Y cells were cultured with high glucose(HG,70 mmol·L^(-1)),4-fold of the normal glucose(17.5 mmol·L^(-1)).Quercetin was set three concentrations(5,10,20μmol·L^(-1)),with Nrf2 activator sulforaphane(SFN)as a positive group(2.5μmol·L^(-1)).After 72 h,cells were collected for glyoxalase 1(Glo-1)activity and GSH level were by spectrophotometry;advanced glycation end-products(AGEs)as well as nuclear Nrf2 and p-Nrf2 levels by immunofluorescence;Glo-1,γ-glutamycysteine synthase(γ-GCS),Nrf2 and p-Nrf2 protein levels by Western blotting,and Glo-1 andγ-GCS m RNA levels by real-time qP CR.RESULTS Quercetin increased the cell viability of SH-SY5Y cells,and upregulated the levels of Glo-1 activity,protein,and m RNA in SH-SY5Y cells cultured with HG,accompanied by the elevated levels of glutathione,a cofactor of Glo-1 activity,and the reduced levels of AGEs.Meanwhile,quercetin could increase p-Nrf2 and Nrf2 levels in nucleus as well as p-Nrf2 levels in cytosol of SH-SY5Y cells exposed to chronic HG,accompanied by the elevated protein expression and m RNA levels ofγ-GCS,a known target gene of Nrf2/ARE signaling.Moreover,a PKC activator or a p38MAPK inhibitor pretreatment could significantly increase the protein expression ofγ-GCS in HG condition,but an alkylating agent for sulfydryl of cysteine in Keap 1,a negative regulator of Nrf2,pretreatment only showed an increased tendency ofγ-GCS protein,compared with without pretreatment;however,after pretreatment with those tool drugs,co-treatment with quercetin and HG had similar results to those of single tool drug pretreatment followed by HG exposure.CONCLUSION Firstly,quercetin can enhance Glo-1 function in central neurons,which is mediated by activation of Nrf2/ARE pathway,then exerts the neuroprotection against HG induced damage;moreover,PKC and p38 MAPK pathways may be involved in Nrf2 inactivation in chronic HG condition.
基金The project supported by National Natural Science Foundation of China(81274172,81473267,30801535,81470003)
文摘OBJECTIVE The present study aimed to investigate the relationship between Wnt/β-catenin and Nrf2 signaling pathways,and understanding the mechanisms underlying the process of inflammatory in chronic obstructive pulmonary disease(COPD),which was a serious disease of respiratory system.METHODS We duplicate the emphysema model with porcine pancreatic elastase(PPE)in Nrf2-/-and WT mouse for 21d,and intraperitoneal injection of Li Cl,the activator of Wnt/β-catenin signaling pathway from 14 d to the end.Hematoxylin and eosin(H&E)staining was performed to assess the histopathologic level,and immunohistochemistry(IHC)for Mac-3(the marker of macrophagocyte)and Ly6G(the marker of neutrophil)was used to observe the inflammatory infiltrate,while the levels of Wnt/β-catenin and Nrf2 signaling pathways related proteins heme oxygenase-1(HO-1),NAD(P)H:quinone oxidoreductase 1(NQO1),and the expression of inflammatory cytokine interleukin-6(IL-6)were detected by Western blotting of lung tissues.In vitro,cigarette smoke extract(CSE)-treated normal human bronchial epithelial(NHBE)cells,cell viability was examined by MTT assay,and then we treated recombinant human Wnt3a,si Nrf2 and si Wnt3a to measure the expression of Wnt3a,β-catenin,Nrf2,HO-1,NQO-1,and IL-6.Cellular immunofluorescence staining was employed to identify the nuclear translocation of Nrf2.RESULTS We found that the Li Cl-treated group has markedly decreased the damage of alveolar structure and inflammatory signs than the model group of WT mice rather than Nrf2-/-group.It also seen that Li Cl not only increasedβ-catenin,but it also led to a comparable increase in Nrf2,HO-1,NQO1,and decrease of IL-6 compared with WT model groups but except to Nrf2-/-group in vivo.And it showed that Wnt3atreatment has significantly increased the nuclear translocation of Nrf2 and the expression of HO-1 and NQO1,reduced the IL-6 release,while there has no significance when Nrf2 was blocked in CSE-induced NHBE cells.CONCLUSION Our results demonstrated that Wnt3a/β-catenin significantly balanced oxidative stress and attenuated inflammation reaction by promoting Nrf2 nuclear translocation and activity.
基金The project supported by National Natural Science Foundation of China(81274172,81473267,30801535,and 81470003)
文摘OBJECTIVE Epithelial-mesenchymal transition(EMT)is a phenotype conversion that plays a critical role in the development of pulmonary fibrosis(PF).It is known that a transcription factor snail could regulate the progression of EMT.Nuclear factor erythroid 2 related factor 2(Nrf2),a key regulator of antioxidant defense system,protects cells and tissues against oxidative stress.However,it is not known whether Nrf2 regulates snail thereby modulating the development of PF.MEHODS Bleomycin(BLM)was intratracheally injected into both Nrf2-knockout(Nrf2-/-)and wild-type mice to compare the development of PF.Rat type II alveolar epithelial cells(AECs)RLE-6TN were treated with a specific Nrf2activator sulforaphane,or transfected with Nrf2 and snail si RNAs to determine their effects on transforming growth factorβ1(TGF-β1)-induced EMT.RESULTS BLM-induced EMT and lung fibrosis were more severe in Nrf2-/-mice compared to wild-type mice.In vitro,sulforaphane treatment attenuated TGF-β1-induced EMT,accompanied by the down-regulation of snail.Inversely,silencing Nrf2 by si RNA enhanced TGF-β1-induced EMT along with the expression of snail.Interestingly,silencing snail by si RNA reduced TGF-β1-induced EMT even in the presence of sulforaphane in RLE-6TN cells.CONCLUSION These findings suggested that Nrf2 may attenuate EMT and fibrosis process through regulating the expression of snail in PF.
基金National Natural Science Foundation of China(81560666)Program for Excellent Young Talents of Zunyi Medical Uiverstity(15zy-002)+1 种基金Science and Technology Innovation Talent Team of Guizhou Province(20154023)the ″Hundred″Level of High-level Innovative Talents in Guizhou Province(QKHRCPT 20165684)；and Program forChangjiang Scholars and Innovative ResearchTeam in University of China(IRT一17R113).
文摘OBJECTIVE To investigate icariside(ICS)Ⅱ protects against PC12 cel damage induced by oxygen-glucose deprivation and reoxygenation and explore its mechanism.METHODS The oxidative stress injury model was induced by oxygen-glucose deprivation/reoxygenation(OGD/R) 2 h/24 h in PC12 cells.N-acetyl-lcysteine(NAC),a classical anti-oxidant,was used as positive control.Pharmacodynamic experimental study groups as follows:control,control+ICS Ⅱ50 μmol·L^(-1),OGD/R,OGD/R+ICSⅡ 12.5 μmol·L^(-1),OGD/R + ICS Ⅱ 25 μmol·L^(-1),OGD/R + ICS Ⅱ50 μmol·L^(-1),and OGD/R+NAC 100 μmol·L^(-1) groups.Cell viability and lactate dehydrogenase(LDH) leakage rate were measured by MTT assay and LDH ELISA kit,respectively.Moreover,reactive oxygen species(ROS) ELISA kit was used for detection of intracellular ROS generation,Mito-SOX fluorescence staining was used for detecting production of ROS in mitochondria and mitochondrial membrane potential(MMP)was detected by rhodamine 123 dye.In addition,PC12 cells apoptosis was detected by one-step TUNEL assay.Furthermore,the expressions of nuclear factor erythroid 2-related factors(Nrf2),Keap1,HO^(-1),NQO^(-1),silent information regulator 3(SIRT3),IDH2,Bax,Bcl-2 and caspase 3 were detected by Western blotting analysis.RESULTS The results of MTT and LDH assay showed that OGD/R reduced the cell viability and improved LDH release compared with the control or ICSⅡ 50 μmol·L^(-1) alone(P<0.01).Meanwhile,OGD/R not only increased intracellular and mitochondrial ROS generation,but also elevated the fluorescence intensity of TUNEL staining,at the same time,the MMP was declined when challenged by OGD/R.Furthermore,the Western blotting results showed that OGD/R induced the increase in the expression of cytoplasm-Nrf2,Keap1,Bax and cleaved-caspase 3 level,while the decrease in the expression of nucleus-Nrf2,HO^(-1),NQO^(-1),SIRT3,IDH2 and Bcl-2(P<0.05).However,ICS Ⅱ significantly increased the viability of PC12 cells and reduced LDH leakage(P<0.01).Notably,ICS Ⅱ also suppressed ROS generation both in the intracellular and mitochondria,as well as restored MMP.It was also worthy to note that ICS Ⅱ decreased the expressions of cytoplasmNrf2,Keap1,Bax and the level of cleaved-caspase3,whereas,it increased the expressions of nucleus-Nrf2,HO^(-1),NQO^(-1),SIRT3,IDH2 and Bcl-2(P<0.05).CONCLUSION ICSⅡ reduced OGD/Rinduced oxidative damage in PC12 cells under the laboratory conditions,and its underlying mechanism may be related to the regulation of Nrf2/SIRT3 signaling pathway.
基金The project supported by National Natural Science Foundation of China(81603315 81730096+4 种基金 81373551 81730093U1402221)CAMS Innovation Fund for Medical Sciences(CIFMS)(2016-I2M-1-004)the Opening Program of Shanxi Key Laboratory of Chinese Medicine Encephalopathy(CME-OP-2017001)
文摘OBJECTIVE Ginsenoside Rg1(Rg1),a purified compound from Panax ginseng,has been well documented to be effective against ischemia/reperfusion(I/R) neurotoxicity.However,the underlying mechanism is stil obscure.METHODS The anti-I/R effect of Rg1 were investigated in vitro and in vivo,and the dynamics of nuclear accumulation and the transcriptional activity of NF-E2-related factor 2(Nrf2) determined by Western blotting and Dual Luciferase Reporter Assay,respectively.Nrf2 siRNA was employed to investigate Nrf2′s role in the protective effect of Rg1 against I/R.Furthermore,the role of miR-144,which could regulate post-translational Nrf2 levels,was investigated in the anti-I/R effect of Rg1 by injection of AAV-hypoxia-inducible factor miR-144-shRNA in the predicted ischemic penumbra.RESULTS It was found that the anti-I/R effect of Rg1 was related to its anti-oxidative capacity,which is mainly regulated by the Nrf2/antioxidant response element(ARE) pathway.Further study suggested that Rg1 contributes to the enhancement of the Nrf2/ARE pathway,as manifested by increasing the dynamic peak content of Nrf2,which prolonged the maintenance stage,and promoting the expression of ARE-target genes after oxygen glucose deprivation/reperfusion(OGD/R) in PC12 cells.Nrf2-siRNA application significantly reduced these changes.Furthermore,the enhancement of the Nrf2/ARE pathway by Rg1 was independent of disassociation from Keap1;rather it was a result of posttranslational regulations.It was found that Rg1 significantly reduced the expression of miR-144,which down-regulates Nrf2 production by targeting its 3′-untranslated region,after OGD/R.Knockdown of Nrf2 showed no effect on the expression of miR-144,indicating that miR-144 is an upstream regulator of Nrf2.Moreover,direct binding between Nrf2 and miR-144 in the PC12 cells was identified.Application of anti-miR-144 significantly reduced Rg1′s anti-OGD/R capacity.Final y,the role of miR-144 in Rg1′ s anti-I/R effect was tested by inhibiting miR-144 in the predicted ischemic penumbra when hypoxia-inducible-factor was activated.The results showed that loss of miR-144 abolished the anti-I/R effect of Rg1,which included reduced infarct volume,improved neurological scores,attenuated oxidative impairment,as well as activation of the Nrf2/ARE pathway.CONCLUSION Oxidative stress after I/R is alleviated by Rg1 through inhibition of miR-144 activity and subsequent promotion of the Nrf2/ARE pathway at the post-translational level.
基金supported by National Natural Science Foundation of China(81060269 and81360492)Natural Science Foundation of Jiangxi Province of China(20122BAB205036)
文摘OBJECTIVE To investigates the effects of imperatorin on the oxidative stress in the cerebral cortex and hippocampus after focal cerebral ischemia/reperfusion injury.METHODS Transient focal cerebral ischemia/reperfusion model in male Sprague-Dawley rats was induced by 2 h middle cerebral artery occlusion followed by 24 h reperfusion.Imperatorin(1.25 and 2.5 mg·kg-1)or vehicle were administered intraperitoneally at 1,5 and 9 h after the onset of ischemia.At 24 h after reperfusion,the biomarkers of oxidative stress such as the levels of reactive oxygen species(ROS),lipid peroxidation products malondialdehyde(MDA),nitric oxide(NO)and total antioxidant capacity(T-AOC),the activities of inducible nitric oxide synthase(iN OS),superoxide dismutase(SOD)and catalase(CAT)in the cerebral cortex and hippocampus were observed.We also assessed the nuclear factor erythroid 2-related factor 2(Nrf2),heme oxygenase-1(HO-1),and the NAD(P)H-quinone oxidoreductase 1(NQO-1)protein expression by Western blot.RESULTS As compared to vehicle-treated animals,imperatorin treatment significantly reduced the ROS,MDA,NO levels and i NOS activity,increased T-AOC and the activities of SOD and CAT.Furthermore,imperatorin treatment also significantly induced the nuclear translocation of Nrf2,enhanced the protein expression of HO-1 and NQO-1 in the cerebral cortex and hippocampus.CONCLUSION Our findings indicate that imperatorin can protect the brain against the excessive oxidative stress induced by cerebral ischemia/reperfusion through activation of Nrf2 signaling pathway.
基金supported by National Natural Science Foundation of China(U1402221,81573640,81603316)Beijing Natural Science Foundation(7161011)+3 种基金CAMS Innovation Fund for Medical Sciences(CIFMS)(2016-I2M-1-004)Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study(BZ0150)Key Research and Development Project of Hunan Province(2015SK2029-1)Scientific Research Foundation of the Higher Education Institutions of Hunan Province(15K091)
文摘20C,a bibenzyl compound isolated from Gastrodia elata,possesses antioxidative properties in PC12 cells,but its in-depth molecular mechanisms against rotenone-induced neurotoxicity remains unknown.Recent studies indicate that without intact DJ-1,nuclear factor erythroid 2-related factor(Nrf2)protein becomes unstable,and the activity of Nrf2-mediated downstream antioxidant enzymes are thereby suppressed.Therefore,increasing the nuclear translocation of Nrf2 by DJ-1 may present a helpful means for the prevention and treatment of chronic diseases related to oxidative stress.Our results showed that 20C clearly protected PC12 and SH-SY5Y cells against rotenone-induced oxidative injury in a concentration-dependent manner.Furthermore,20C markedly up-regulated the levels of DJ-1,which in turn activated phosphoinositide-3-kinase(PI3K)/Akt signaling and inhibited glycogen synthase kinase 3β(GSK3β)activation,eventually promoting Nrf2 nuclear translocation and inducing the expression of Nrf2-mediated downstream antioxidative enzymes such as HO-1.The antioxidative effects of 20C could be partially blocked by ShR NA-mediated knockdown of DJ-1 and inhibition of the PI3K/Akt pathways with Akt1/2 kinase inhibitor in PC12 and SH-SY5Y cells,respectively.Conclusively,our findings confirm that DJ-1 is necessary for 20C-mediated protection against rotenone-induced oxidative damage,at least in part,by activating PI3K/Akt signaling,and subsequently enhancing the nuclear accumulation of Nrf2.The findings from our investigation suggest that 20C should be developed as a novel candidate for preventing or alleviating the consequences of PD in the future.
