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BACKGROUND:Oxidative stress plays a critical role in intervertebral disc degeneration.As a reducing material with good biocompatibility,black phosphorus quantum dots have the potential to resist oxidative stress and retard intervertebral disc degeneration.OBJECTIVE:To evaluate the effect of black phosphorus quantum dots on scavenging reactive oxygen species in the microenvironment of an intervertebral disc through in vivo and in vitro experiments,and further explore the role of black phosphorus quantum dots in Nrf2/ARE pathway and intervertebral disc inflammation.METHODS:Black phosphorus quantum dots were prepared by a liquid exfoliation technique.(1)In vitro experiment:The nucleus pulposus cells of SD rats were isolated and extracted,and the passages 2-4 nucleus pulposus cells were cocultured with different solutions,including F12-DMEM medium(blank group),black phosphorus quantum dot solution,hydrogen peroxide solution,hydrogen peroxide+black phosphorus quantum dot solution,hydrogen peroxide+black phosphorus quantum dot+Nrf2 specific inhibitor ML385 solution.Cell live/dead staining and intracellular reactive oxygen species,mitochondrial membrane potential and western blot assay were performed respectively.(2)In vivo experiment:Thirty SD rats were randomly divided into sham operation,puncture and puncture + black phosphorus groups,with 10 rats in each group.A Co7-10 intervertebral disc degeneration model was established using intervertebral disc puncture in the puncture group and the puncture+black phosphorus group.Black phosphorus quantum dot solution was injected in the intervertebral disc after a puncture in the puncture+black phosphorus group.The intervertebral disc tissue imaging and histological staining were evaluated at 4 and 8 weeks after surgery.RESULTS AND CONCLUSION:(1)In vitro experiment:Live/dead staining revealed that the black phosphorus quantum dots had good biocompatibility,were non-toxic to cells,and had a protective effect on nucleus pulposus cells under oxidative stress.Intracellular reactive oxygen species and JC-1 fluorescent probes showed that black phosphorus quantum dots could regulate the reduction of mitochondrial membrane potential caused by oxidative stress in nucleus pulposus cells and protected cells from hydrogen peroxidation-induced intracellular oxidative stress.Western blot analysis showed that compared with the blank group,the protein expressions of Nrf2,heme oxygenase 1,quinone oxidoreductase and type Ⅱ collagen were decreased in the hydrogen peroxide group(P<0.05),while the protein expressions of tumor necrosis factor α,interleukin 1β,matrix metalloproteinase 13 and p65 were increased(P<0.05).The addition of black phosphorus quantum dots could reverse the inhibitory effect of hydrogen peroxide on the Nrf2 pathway and reduce the inflammatory response caused by oxidative stress,but NrF2-specific inhibitors could cancel this effect.(2)In vivo experiment:X-ray and MRI demonstrated that at 4 and 8 weeks after surgery,the intervertebral disc height and water content of nucleus pulposus in the puncture group were lower than those in the sham operation group(P<0.05),and the intervertebral disc height and water content of nucleus pulposus in the puncture+black phosphorus group were higher than those in the puncture group(P<0.05).Histological staining exhibited that the degree of intervertebral disc degeneration in the puncture+black phosphorus group was less than that in the puncture group,and the expression of heme oxygenase 1 protein was higher than that in the puncture+black phosphorus group.(3)Our results have indicated that black phosphorus quantum dots can exert an antioxidant effect and delay intervertebral disc degeneration by regulating Nrf2/ARE pathway.
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Aim To explore the effects of Lycium berry seed oil on Nrf2/ARE pathway and oxidative damage in testis of subacute aging rats. Methods Fifty out of 60 male SD rats, aged 8 weeks, were subcutaneously injected with 125 mg • kg"D-galactosidase in the neck for 8 weeks to establish a subacute senescent rat model. The presence of senescent cells was observed using P-galactosidase ((3-gal), while testicular morphology was examined using HE staining. Serum levels of testosterone (testosterone, T), follicle-stimulating hormone ( follicle stimulating hormone, FSH ) , luteinizing hormone ( luteinizing hormone, LH ) , superoxide dis-mutase ( superoxide dismutase, SOD ) , glutathione ( glutathione, GSH) and malondialdehyde ( malondial-dehyde, MDA) were measured through ELISA, and the expressions of factors related to aging, oxidative damage, and the Nrf2/ARE pathway were assessed via immunohistochemical analysis and Western blotting. Results After successfully identifying the model, the morphology of the testis was improved and the intervention of Lycium seed oil led to a down-regulation in the expression of [3-gal and -yH2AX. The serum levels of SOD, GSH, T, and FSH increased while MDA and LH decreased (P 0. 05) . Additionally, there was an up-regulated expression of Nrf2, GCLC, NQOl, and SOD2 proteins in testicular tissue ( P 0. 05 ) and nuclear expression of Nrf2 in sertoli cells. Conclusion Lycium barbarum seed oil may reduce oxidative damage in testes of subacute senescent rats by activating the Nrf2/ARE signaling pathway.
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Objective:To investigate the effects of casticin(CAS)on lipopolysaccharide-induced injury of human normal lung epithelial cells(BEAS-2B)and NF-κB-Keap1-Nrf2/ARE pathway.Methods:BEAS-2B cells were pretreated with different concentra-tions of casticin for 1 h,2 h and 4 h,and then treated with 1 μg/ml liposolysaccharide to construct cell damage model.The contents of inflammatory factors in cell supernatant was detected by ELISA to screen the optimal concentration and time of casticin.The cells were divided into normal group,model group,Casticin group,ML385 group,Casticin+ML385 group and dexamethasone group.Cell apoptosis was detected by flow cytometry,the concentrations of inflammatory factors were detected by ELISA,and the levels of NF-κB p65,p-NF-κB p65,Keap1,Nrf2 and Nrf2 protein in cell nucleus were detected by Western blot.Results:The optimal concentration of CAS was 10 μmol/L and the optimal time was 2 h.Compared with normal group,the apoptosis rate,the contents of inflammatory fac-tors,p-NF-κB p65 and Keap1 protein expression levels in model group were significantly increased(P<0.01),and Nrf2 protein expression levels in cells and nuclei were significantly decreased(P<0.01).Compared with model group,apoptosis rate and contents of inflammatory factors in casticin group and dexamethasone group were significantly decreased(P<0.01),protein expression levels of p-NF-κB p65 and Keap1 in Casticin group were significantly decreased(P<0.01).The expression level of Nrf2 protein in cells and nuclei was significantly increased(P<0.01).Compared with ML385 group,apoptosis rate,inflammatory factors contents,p-NF-κB p65 and Keap1 protein expression levels in Casticin+ML385 group were significantly decreased(P<0.01),the expression level of Nrf2 protein in cells and nuclei were significantly increased(P<0.01).Conclusion:CAS can inhibit cell inflammation induced by lipo-polysaccharide by regulating NF-κB-Keap1-Nrf2/ARE signaling pathway.
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Objective:To explore the mechanism of gypenoside ⅩⅦ against cerebral ischemia/reperfusion (I/R) through nuclear factor erythroid 2-related factor 2/antioxidant responsive element (Nrf2/ARE) signaling pathway.Methods:Forty SPF Sprague Dawley (SD) rats were randomly divided into sham operated group, I/R model group, 25, 50 and 100 mg/kg gypenoside ⅩⅦ groups ( n = 8). Gypenoside ⅩⅦ groups were administered 25, 50 or 100 mg/kg (0.01 mL/g) gypenoside ⅩⅦ by intragastric administration for 14 days; the other two groups received the same dose of saline. Rat cerebral I/R model was established by modified line bolt method; rats in the sham operated group underwent the same procedure without producing substantial embolization. After 24 hours of reperfusion, the neurological deficit scores of the rats in each group were assessed. Rat abdominal aortic whole blood was collected and the serum reactive oxygen species (ROS), heme oxygenase-1 (HO-1), γ-glutamylcysteine synthase (γ-GCS), superoxide dismutase (SOD), quinone NADH oxidoreductase 1 (NQO1), and malondialdehyde (MDA) were detected. Then whole brain tissue was harvested and penumbra tissue was isolated from cerebral cortex, the general condition of rat brain tissue and the volume of cerebral infarction were evaluated, the histopathological changes in the brain were observed under light microscopy, the mRNA expressions of Nrf2 and Keap1 were measured by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR), the protein expressions of Nrf2 and Keap1 were determined by Western blotting. Results:After 24 hours of reperfusion, compared with the sham operated group, the score of neurological deficit and infarct volume were significantly increased, the NQO1, SOD and γ-GCS levels in serum were significantly decreased, MDA, HO-1 and ROS levels in serum were significantly increased, the Nrf2 and Keap1 mRNA and protein expressions in the ischemic penumbra were significantly increased in rats from I/R model group. Compared with the I/R model group, the neurological deficit scores (1.50±0.53, 1.37±0.52 vs. 2.75±0.46) and brain infarct volume [(19.8±5.1)%, (21.4±6.4)% vs. (42.3±5.8)%] were significantly reduced, serum NQO1, SOD, HO-1 and γ-GCS were significantly increased [NQO1 (ng/L): 186.05±10.38, 220.75±16.22 vs. 131.36±5.95, SOD (kU/L): 63.23±5.30, 72.70±8.62 vs. 36.75±6.55, HO-1 (ng/L): 60.57±7.93, 60.35±4.72 vs. 42.72±4.95, γ-GCS (kU/L): 8.81±0.53, 8.72±0.69 vs. 6.80±0.56], serum MDA and ROS levels were significantly reduced [MDA (μmol/L): 5.94±0.66, 5.61±0.53 vs. 10.88±1.34, ROS (kU/L): 69.11±4.23, 67.12±4.52 vs. 104.43±7.54], the mRNA and protein expressions of Nrf2 and Keap1 in the ischemic penumbra were significantly increased in rats from 50 mg/kg and 100 mg/kg gypenoside ⅩⅦ groups [Nrf2 mRNA (2 -△△Ct): 1.90±0.13, 2.13±0.18 vs. 1.48±0.11, Keap1 mRNA (2 -△△Ct): 1.78±0.11, 1.85±0.10 vs. 1.43±0.10, Nrf2/β-actin: 0.73±0.04, 0.79±0.03 vs. 0.60±0.03, Keap1/β-actin: 0.71±0.01, 0.76±0.03 vs. 0.61±0.01], all the comparative differences were statistically significant (all P < 0.01); 25 mg/kg gypenoside ⅩⅦ had no significant effect. Conclusion:Gypenoside ⅩⅦ (50 mg/kg and 100 mg/kg) may play a role in anti-cerebral I/R injury by regulating NQO1, SOD, HO-1, γ-GCS, ROS and MDA through Nrf2/ARE signaling pathway.
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Objective To investigate the effect and mechanism of miR-141-3p on pulmonary fibrosis in rats with acute respiratory distress syndrome(ARDS).Methods Rats were divided into the control group,the model group,the agomir negative control group and the miR-141-3p agomir group according to random number table,with 10 rats in each group.In addition to the control group,the ARDS rat model was established by lipopolysaccharide(LPS)infusion.Rat alveolar typeⅡepithelial cells RLE-6TN cells were divided into the NC group,the LPS group,the miR-NC group,the miR-141-3p mimics group,the miR-141-3p mimics+pcDNA group and the miR-141-3p mimics+NRF2 and Kelch-like ring associated protein 1(Keap1)group.LPS cell model was established in all groups except the NC group.The mRNA expression levels of miR-141-3p and Keap1 in lung tissue and cells were detected by qPCR.Western blot assay was used to analyze lung tissue and cell epithelial cadherin(E-cadherin),neural cadherin(N-cadherin),microtubule associated protein light chain 3B(LC3B),autophagy associated gene Beclin-1,α-smooth muscle actin(α-SMA),type I collagen(Col-Ⅰ),Keap1 and nuclear factors E2 related factor 2(NRF2)and heme oxygenase 1(HO-1).HE staining and Masson staining were used to observe pathological changes of lung tissue and to estimate the area of lung tissue injury and pulmonary fibrosis.Hydroxyproline(Hyp)in lung tissue was detected by the kit.Levels of inflammatory factor interleukin-1β,tumor necrosis factor(TNF-α)and oxidative stress index malondialdehyde(MDA)and superoxide dismutase(SOD)were detected by ELISA.Dual luciferase reporting experiment was used to verify the targeting relationship between miR-141-3p and Keap1.Results The expression of miR-141-3p was down-regulated and the expression of Keap1 was up-regulated in lung tissue and cells(P＜0.05).Overexpression of miR-141-3p can reduce the degree of pathological damage and fibrosis of lung tissue in rats,Hyp content,and up-regulate expression levels of SOD,E-cadherin,LC3B,Beclin-1,NRF2 and HO-1 in lung tissue and cells,and down-regulate the expression levels of IL-1β,TNF-α,MDA,N-cadherin,α-SMA,Col-I and Keap1(P＜0.05).Overexpression of Keap1 was able to reverse the improvement effect of overexpression of miR-141-3p on alveolar epithelial cell damage in ARDS rats(P＜0.05).Double Luciferase reporter gene experiment confirmed that miR-141-3p and Keap1 may have a targeted regulatory relationship.Conclusion Overexpression of miR-141-3p may activate the Keap1-NRF2/ARE signaling pathway,activate autophagy,inhibit inflammatory response,oxidative stress,and EMT progression,and improve pulmonary fibrosis in ARDS rats.
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Objective:To investigate the neuroprotective effect of synthetic triterpenoid(CDDO-Im)on ischemic stroke rats and its influence on inflammatory response by activating the nuclear factor 2-related factor 2(Nrf2)/antioxidant response elements(ARE)signaling pathway.Methods:SD rats were divided into sham group(S group),MCAO group(M group)and CDDO-Im inter-vention group(M+C group).The middle cerebral artery occlusion(MCAO)was used in M group and M+C group to establish ischemic stroke model in rats,and sham operation was used in S group to control.After operation,M+C group was injected with CDDO-Im(64 μg/300 g)every12 hours via caudal vein,S group and M group were given the same amount of normal saline.After 3 days,the nerve function of rats was measured by Longa score,and the area of cerebral infarction was evaluated by 2,3,5-triphenyltetrazolium chlo-ride(TTC)staining.The expressions of Nrf2,heme oxygenated-1(HO-1),ionic calcium binding adaptor molecule 1(Iba1),IL-1β and IL-4 protein were detected by Western blot.Results:Compared with S group,M group rats showed significant neurologic deficit and cerebral infarction area,and the expression of Nrf2 protein had no significant difference,and the expression levels of HO-1,Iba1,IL-1β and IL-4 protein were increased significantly(P<0.05).Compared with M group,the neurological deficit score,cerebral infarction area,the expression levels of Iba1 and IL-1β protein were decreased significantly(P<0.05),while Nrf2,HO-1 and IL-4 pro-tein expression increased significantly in M+C group(P<0.05).Conclusion:CDDO-Im may activate the Nrf2/ARE signaling pathway and play a neuroprotective role,which may be related to the modulation of microglia to M2 and the regulation of inflammatory response.
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Objective:To investigate the effects and potential mechanism of vitamin D supplementation on testicular function in aging rats induced by D-galactose.Methods:The aging rats were induced by D-galactose with subcutaneous injection. The animals were randomly divided into 6 groups: aging rats (DG), aging rats with low-dose vitamin D supplementation (LD), aging rats with high-dose vitamin D supplementation (HD), normal control rats(NC), normal rats with low-dose vitamin D supplementation(LN), normal rats with high-dose vitamin D supplementation (HN). The body weight, testicular weight, serum testosterone concentrations and sperm quality of the rats in each group were measured. The testis morphological changes were detected using light microscopy. The activity of superoxide dismutase (SOD) and level of malondialdehyde(MDA) were determined with spectrophotometer. The expression levels of Nrf2, GCLC, SOD2 and VDR in testis were detected by western blot.Results:At baseline, compared with NC group, testicular weight, serum testosterone level, SOD activity, Nrf2, GCLC and SOD2 expression levels were significantly decreased in DG group, while MDA level was significantly increased. After vitamin D supplementation, testicular weight, testosterone levels and SOD activity in both of HD and LD groups were significantly increased, while the MDA level was significantly decreased. The expression levels of Nrf2, GCLC, SOD2 and VDR were significantly increased.Conclusion:Vitamin D supplementation may enhance the testicular antioxidant capacity through activating Nrf2-ARE signaling pathway, and improve the testicular function in D-galactose-induced aging rats.
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Objective:To investigate protective effect of Pinus massoniana needle extract (PMNE) against oxidative stress in human dermal papilla cells (HDPC) , and to explore its mechanisms. Methods:As research objects, some cultured HDPC were treated with H 2O 2 at different concentrations of 0 (control group) , 0.1, 0.2, 0.4, 0.8 and 1.0 mmol/L, in order to establish the optimal condition for in vitro oxidative stress in HDPC; some other HDPC were transfected with nuclear factor-erythroid 2-related factor 2 (Nrf2) specific small interfering RNAs (siRNA1, siRNA2, siRNA3) or a Nrf2-overexpressing plasmid (pCMV6-XL5-Nrf2) , the HDPC transfected with a scrambled-siRNA and an empty plasmid pCMV6-XL5 served as the control siRNA group and control plasmid group respectively, and HDPC subjected to conventional culture served as the blank group; after the above treatment, real-time fluorescence-based quantitative PCR and Western blot analysis were performed to determine Nrf2 mRNA and protein expression, respectively; cell viability and apoptosis were detected in the above transfected cells after the treatment with H 2O 2 at an optimal concentration. In the subsequent experiment, some HDPC were divided into several groups: control group subjected to conventional culture, dihydrotestosterone group treated with 0.03 μg/ml dihydrotestosterone, proanthocyanidin group treated with 0.03 μg/ml dihydrotestosterone and 6.00 μg/ml proanthocyanidin B2, PMNE groups treated with 0.03 μg/ml dihydrotestosterone and PMNE at different concentrations of 1, 5, 25 and 100 μg/ml; after the above treatment, cell viability and apoptosis were detected, relative fluorescence intensity of intracellular reactive oxygen species (ROS) , malondialdehyde (MDA) content, mRNA and protein expression of Nrf2, quinone oxidoreductase 1 (NQO1) , heme oxygenase 1 (HO-1) , Kelch-like ECH-related protein 1 (Keap1) , transforming growth factor (TGF) -β1, Sma- and Mad-related protein 2/3 (Smad2/3) , phosphorylated Smad2/3 (p-Smad2/3) were determined in HDPC. One-way analysis of variance was used for comparisons among multiple groups, and least significant difference- t test for multiple comparisons. Results:The viability of HDPC ranged from 75% to 85% after the treatment with 0.4 mmol/L H 2O 2, which was selected as the optimal condition for in vitro oxidative stress in HDPC. Compared with the blank group and control siRNA group, the Nrf2-siRNA1, Nrf2-siRNA2, Nrf2-siRNA3 groups showed significantly decreased Nrf2 mRNA and protein expression (all P < 0.05) , but significantly increased apoptosis rate (Nrf2-siRNA1, Nrf2-siRNA2, Nrf2-siRNA3 groups, blank group and control group: 12.50% ± 0.05%, 26.07% ± 0.05%, 58.44% ± 1.03%, 10.38% ± 0.64%, 13.05% ± 0.12%, respectively; all P < 0.05) . Nrf2 protein expression was the lowest in the Nrf2-siRNA2 group, so Nrf2-siRNA2 was selected as the optimal interfering fragment for subsequent experiments. Compared with the blank group and control plasmid group, the Nrf2 overexpression group showed significantly increased Nrf2 mRNA and protein expression (both P < 0.05) , but a significantly decreased apoptosis rate (all P < 0.05) . After the treatment with 0.4 mmol/L H 2O 2, the Nrf2 overexpression group showed a significantly decreased apoptosis rate, but significantly increased cell viability compared with the empty vector group ( t = 3.66, 40.40, respectively, both P < 0.001) ; the Nrf2-siRNA2 group showed a significantly increased apoptosis rate, but significantly decreased cell viability compared with the control group ( t = 13.13, 67.37, respectively, both P < 0.001) . In the PMNE treatment experiment, the proanthocyanidin group and PMNE groups showed significantly increased cell viability, but significantly decreased apoptosis rates compared with the dihydrotestosterone group (all P < 0.01) ; proanthocyanidin and PMNE at different concentrations could significantly inhibit dihydrotestosterone-induced overexpression of ROS and MDA in HDPC (all P < 0.01) ; the protein expression of Nrf2, NQO1 and HO-1 was significantly higher in the proanthocyanidin group, 5-, 25- and 100-μg/ml PMNE groups than in the dihydrotestosterone group (all P < 0.05) , while the protein expression of Keap1 and TGF-β1, and the Smad2/3 phosphorylation level were significantly lower in the proanthocyanidin group, 25- and 100-μg/ml PMNE groups than in the dihydrotestosterone group (all P < 0.05) . Conclusion:Nrf2 plays an important role in protecting against oxidative damage in HDPC, and PMNE may exert marked protective effect on HDPC by activating the Nrf2-antioxidant responsive element signaling pathway.
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OBJECTIVE@#To investigate the molecular mechanisms underlying the effects of arsenic trioxide (As@*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@*RESULTS@#Expression of Nrf2-ARE-HO-1 signaling pathway was upregulated in heart xenografts in rats treated with As@*CONCLUSION@#Combination treatment with As
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Animales , Cricetinae , Ratas , Trióxido de Arsénico , Trasplante de Corazón , Hemo-Oxigenasa 1/metabolismo , Xenoinjertos , Leflunamida , Factor 2 Relacionado con NF-E2/metabolismo , Ratas Endogámicas Lew , Transducción de SeñalRESUMEN
Aim To investigate the effects of astragalus polysaccharide (APS) on depressive behaviors, hippocampal damage and Nrf2-ARE signaling pathway in rats. Methods Wistar rats were randomly divided into control group, depression group, APS low dose group and APS high dose group. Rats (except the control group) underwent chronic unpredictable mild stress (CUMS) for 28 days. The depressive behaviors were assessed by tail suspension test, forced swim test and sucrose preference test. The histopathological changes of the hippocampus were valuated by HE staining. Levels of nuclear factor erythroid 2-related factor 2 (Nrf2) protein and Nrf2 mRNA were measured. The hippocampal levels of oxidative stress were evaluated. Results Compared with the control group, the depression group showed significant depressive behaviors and hippocampal damage. The depression group had higher levels of Nrf2 and MDA, but lower levels of HO-1, SOD, CAT and GSH-Px than the control group. However, APS does-dependently attenuated the hippocampal damage and depressive behaviors, increased hippocampal levels of Nrf2, HO-1, SOD, CAT and GSH-Px, but decreased hippocampal levels of MDA in rats. Conclusions APS can attenuate CUMS-induced hippocampal damage and depressive behaviors in rats, and the effects may be associated with the activation of Nrf2-ARE signaling pathway.
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OBJECTIVES: To investigate the molecular mechanism of edaravone (EDA) in improving the post-traumatic brain injury (TBI) dysfunction in learning and memory. METHODS: In vitro and in vivo TBI models were established using hydrogen peroxide (H2O2) treatment for hippocampal nerve stem cells (NSCs) and surgery for rats, followed by EDA treatment. WST 1 measurement, methylthiazol tetrazolium assay, and flow cytometry were performed to determine the activity, proliferation, and apoptosis of NSCs, and malondialdehyde (MDA), lactic dehydrogenase (LDH), and reactive oxygen species (ROS) detection kits were used to analyze the oxides in NSCs. RESULTS: Following EDA pretreatment, NSCs presented with promising resistance to H2O2-induced oxidative stress, whereas NSCs manifested significant increases in activity and proliferation and a decrease in apoptosis. Meanwhile, for NSCs, EDA pretreatment reduced the levels of MDA, LDH, and ROS, with a significant upregulation of Nrf2/antioxidant response element (ARE) signaling pathway, whereas for EDA-treated TBI rats, a significant reduction was observed in the trauma area and injury to the hippocampus, with improvement in memory and learning performance and upregulation of Nrf2/ARE signaling pathway. CONCLUSIONS: EDA, by regulating the activity of Nrf2/ARE signal pathway, can improve the TBI-induced injury to NSCs and learning and memory dysfunction in rats.
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Animales , Ratas , Elementos de Respuesta Antioxidante , Lesiones Traumáticas del Encéfalo/fisiopatología , Lesiones Traumáticas del Encéfalo/metabolismo , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Edaravona/farmacología , Aprendizaje/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Apoptosis/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Memoria/efectos de los fármacosRESUMEN
ObjectiveTo investigate the effects of pentoxifylline (PTX) on oxidative stress in brains of epileptic (EP) rats based on the nuclear factor E2 related factor 2 (Nrf2) antioxidant response element (ARE) signal pathway.MethodsThirty-six healthy and adult male Wistar rats were included in the experiment and were divided into blank control group (peritoneal injection of isotonic saline), EP control group (induced EP episode), and PTX group (induced EP episode ＋ PTX pretreatment) according to a completely random method, then 12 rats in each group. The behavioral changes of rats in each group were monitored, and the EP attack rate and seizure latency were recorded. The rats were sacrificed to collect substantia nigra and hippocampus for testing oxidative stress indicators and expression levels of Nrf2 ARE signaling pathway-related proteins.ResultsNo abnormal reaction was observed in the control group after treatment. The EP attack rate in the EP control group reached 83.33%. The EP attack rate (33.3%) and the attack level ((2.14±0.40) vs (3.09±0.58)) in the PTX group were significantly lower than those in the EP control group, and the seizure latency was significantly longer than that in the EP control group (P0.05) ). The expression of substantia nigra tissue was significantly higher than that of the blank control group (P<0.05).ConclusionPTX can inhibit EP seizure and improve the oxidative stress in the brain of rats at the early stage of EP. The possible mechanism is that PTX can specifically activate Nrf2 ARE signaling pathway.
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OBJECTIVE@#To observe the effect of moxibustion on oxidative stress injury of nigrostriatal system in rats with Parkinson's disease (PD) based on nuclear factor erythroid 2-related factor (Nrf2)/antioxidant response element (ARE) pathway, and to explore its mechanism.@*METHODS@#A total of 48 SD rats were randomized into a blank group, a sham-operation group, a model group and a moxibustion group, 12 rats in each group. Unilateral two-point injection with 6-hydroxydopamine (6-OHDA) was adopted in the model group and the moxibustion group to establish the PD model; the operation manipulation in the sham-operation group was the same as the model group and the moxibustion group, and the same volume of 0.9% sodium chloride solutions was given by unilateral two-point injection. Moxibustion was adopted at "Baihui" (GV 20) and "Sishencong" (EX-HN 1) in the moxibustion group for 20 min, once a day, 6 times a week for 6 weeks. No intervention was given in the other 3 groups. Morphology of right mesencephalon substantia nigra was observed by HE staining, the expression of tyrosine hydroxylase (TH) in right mesencephalon substantia nigra was detected by immunohistochemistry method, the expression of reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), and glutathione peroxidase (GSH-Px) in corpus striatum was detected by colorimetry method, and the expression of Nrf2 and heme oxygenase-1 (HO-1) proteins was detected by Western blot in the 4 groups.@*RESULTS@#Clear tissue structure and complete dopaminergic neurons of right mesencephalon substantia nigra were observed in the blank group and the sham-operation group; unclear tissue structure, decreased and swelling dopaminergic neurons were observed in the model group; compared with the model group, more neurons were observed and the swelling of cyton was reduced in the moxibustion group. Compared with the sham-operation group, the expression of TH in right mesencephalon substantia nigra was decreased in the model group (<0.01); compared with the model group, the expression of TH in right mesencephalon substantia nigra was increased in the moxibustion group (<0.05). Compared with the sham-operation group, the expression of ROS, MDA was increased (<0.01), the expression of GSH, GSH-Px, Nrf2 and HO-1 was decreased in the model group (<0.01, <0.05); compared with the model group, the expression of ROS, MDA was decreased (<0.05, <0.01), the expression of GSH, GSH-Px, Nrf2 and HO-1 was increased in the moxibustion group (<0.05, <0.01).@*CONCLUSION@#Moxibustion can alleviate oxidative stress injury of nigrostriatal system in rats with Parkinson's disease by activating the Nrf2/ARE pathway, and protect the dopamine neurons.
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ABSTRACT Schisandra chinensis (Turcz.) Baill., Schisandraceae, is a well-known traditional Chinese medicine used mainly as a recipe for hepatoprotection. Modern researches have revealed that the hepatoprotection is related to its lignans and crude polysaccharide. In this study, we examined the effect and mechanism of S. chinensis total lignans on the liver injury induced by alcoholic. S. chinensis total lignans were extracted with ethanol extraction. The liver index, alanine aminotransferase and aspartate aminotransferase levels in serum of the rat culture supernatant were examined. The malondialdehyde level and superoxide dismutase activities in serum and liver tissue, and triacylglyceride content in liver tissue were tested. Western blot was conducted to determine cytochrome P450 2E1 expression in liver tissue of rats. The results showed that S. chinensis total lignans administration significantly inhibited alcohol-induced liver injury. In exploring the underlying mechanisms of S. chinensis total lignans action, we found that it significantly decreased Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), Glutamyl transpeptidase (γ-GT), reactive oxygen species (ROS) and malondialdehyde (MDA) level in livers/serum and inhibited the gene expression level of CYP2E1 in rat livers. The Nuclear factor erythroid-2 related factor 2 (Nrf2) gene expression and Nuclear factor erythroid-2 related factor 2 (Nrf2) protein nuclear transfer increased significantly, and significantly increased the expression of downstream target gene and protein heme oxygenase-1 (HO-1), Glutamate--cysteine ligase regulatory subunit (GCLM), NAD(P)H:quinine oxidoreductase 1 (NQO1). Moreover, S. chinensis total lignans decreased production of pro-inflammatory markers including Tumor Necrosis Factor-α (TNF-α), Interleukin-1beta (IL-1β) and Interleukin-6 (IL-6). In conclusion, these results suggested that the inhibition of alcohol-induced liver injury by S. chinensis total lignans is associated with its ability to inhibiting CYP2E1 activation and activating the Nrf2/Antioxidant response element(ARE) signaling pathway.
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Scavenging reactive oxygen species (ROS) by antioxidants is the important therapy to cerebral ischemia-reperfusion injury (CIRI) in stroke. The antioxidant with novel dual-antioxidant mechanism of directly scavenging ROS and indirectly through antioxidant pathway activation may be a promising CIRI therapeutic strategy. In our study, a series of chalcone analogues were designed and synthesized, and multiple potential chalcone analogues with dual antioxidant mechanisms were screened. Among these compounds, the most active not only conferred cytoprotection of HO-induced oxidative damage in PC12 cells through scavenging free radicals directly and activating NRF2/ARE antioxidant pathway at the same time, but also played an important role against ischemia/reperfusion-related brain injury in animals. More importantly, in comparison with mono-antioxidant mechanism compounds, exhibited higher cytoprotective and neuroprotective potential and Overall, our findings showed compound could emerge as a promising anti-ischemic stroke drug candidate and provided novel dual-antioxidant mechanism strategies and concepts for oxidative stress-related diseases treatment.
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The chemical constituents from methanol extract of Dichroa hirsuta were separated by silica gel and Sephadex LH-20 column chromatography,high pressure preparative liquid chromatography( HPLC) and recrystallization. Their structures were elucidated by NMR and MS. Nine compounds were obtained and their structures were identified as 3β,21α-O-diacetyl-lup-9( 11)-en-7β-ol( 1),( Z)-methyl p-hydroxycinnamate( 2),cis-p-coumaric acid ethyl ester( 3),( E)-methyl p-hydroxycinnamate( 4),trans-p-coumaric acid ethyl ester( 5),4( 3 H)-quinazolinone( 6),7-hydroxycoumarin( 7),hydrangenol( 8) and thunberginol C( 9). Compound 1 is a new lupane-type triterpenoid,and compounds 1-5,8-9 were firstly isolated from this plant. Dual reporter assay results showed that compounds 2-5 could activate the Nrf2-ARE signaling pathway.
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Cromatografía Líquida de Alta Presión , Medicamentos Herbarios Chinos , Hydrangea , Química , Fitoquímicos , Farmacología , Triterpenos , FarmacologíaRESUMEN
OBJECTIVE: To investigate the anti-oxidant mechanism of andrographolide on HaCaT cells via Nrf2/ARE signal pathway. METHODS: The effect of andrographolide on the viability of HaCaT cells and the effect of H2O2-induced cell viability were measured by CCK-8. HaCaT cells were pretreated with andrographolide of different concentration for 24 h. The protein and mRNA expression levels of Nrf2, HO-1, AKR1C1 and NQO1 in HaCaT cells were detected by Western blot and RT-qPCR, respectively. The expression of Nrf2 protein in the nucleus was analyzed by nuclear cytoplasmic separation and immunofluorescence. RESULTS: Andrographolide had no significant effect on cell viability and dose-dependently decreased H2O2-induced cell death, the difference was statistically significant. Andrographolide significantly enhanced the expression of protein and mRNA of antioxidant enzymes Nrf2, HO-1, AKR1C1, NQO1, increased the distribution of Nrf2 in the nucleus, and up-regulated the expression of ARE. Besides, andrographolide upregulated the phosphorylation level of the upstream protein kinase AMPKα (p-AMPKα). CONCLUSION: Andrographolide could decrease H2O2-induced cell death, and its mechanism may be through the activation of Nrf2/ARE signaling pathway, thereby regulating the expression levels of HO-1, AKR1C1, and NQO1.
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OBJECTIVE@#To study the role of Nrf2/ARE signaling pathway in cypermethrin-induced oxidative stress and apoptosis of cerebral cortex neurons in C57BL/6 mice.@*METHODS@#The cortical neurons of C57BL/6 mice were cultured and identified, and a cypermethrin-induced cell injury model was established by treating the cells with 0, 25, 50 and 100 μmol/L of cypermethrin for 48 h. CCK-8 assay was used to analyze the effects of cypermethrin on the cell viability, and the fluorescence probe DCFH-DA was used for detecting intracellular reactive oxygen species (ROS); flow cytometry was performed for determining the apoptosis rate of the cells. The mRNA and protein expression levels of Nrf2 and its downstream genes HO-1 and NQO1 were detected using qPCR and Western blotting.@*RESULTS@#Exposure to cypermethrin at different doses inhibited the viability of the cultured cortical neurons. With the increase of cypermethrin dose, the viability of the neurons decreased progressively, the intracellular ROS and the cell apoptosis rate increased, and the neuronal injury worsened. At the dose of 50 and 100 μmol/L, cypermethrin significantly down-regulated the expressions of HO-1, NQO1 and Nrf2 at both the mRNA and protein levels in the cells ( < 0.01).@*CONCLUSIONS@#Cypermethrin exposure shows a dose-dependent neurotoxicity by inhibiting Nrf2/ARE signaling pathway, down-regulating the expression of Nrf2 and its downstream genes HO-1, NQO1 mRNA and protein, and inducing oxidative damage and apoptosis in primary mouse cortical neurons, .
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Animales , Ratones , Hidrolasas de Éster Carboxílico , Corteza Cerebral , Ratones Endogámicos C57BL , Factor 2 Relacionado con NF-E2 , Neuronas , Piretrinas , Transducción de SeñalRESUMEN
Objective: To investigate the reproductive protective effect of Duzhong Butiansu Capsule (DBC) by using cyclophosphamide induced spermatogenic disorder model, and explore its mechanism. Methods: The model of spermatogenic disorder was established by intraperitoneal injection of cyclophosphamide (60 mg/kg) for 5 d. Drug intervention at high and low doses (1.388 g/kg and 0.694 g/kg) was given for 4 weeks from the 8th day. The body weight and organ index of each group were measured. The pathological structure of testis was detected by HE staining. ELISA method was used to detect the levels of T, FSH, LH, MDA, SOD, GSH-Px. Western blotting and immunohistochemistry were used to analyze the expression of Nrf2/ARE signaling pathway related factors Nrf2, HO-1, NQO1, HDAC2, and p-PKC in testicular tissue. Results: Compared with the model group, DBC significantly reduced the weight of mice, increased the index of testis, epididymis and kidney, improved the pathological morphology of testis, increased the number of spermatozoa, increased the motility of sperm, decreased the rate of abnormal sperm, increased the level of T and decreasd the level of LH and FSH, increased the content of MDA, decreased the content of SOD and GSH-Px, increased the expression of Nrf2, HO-1, NQO1, HDAC2, and p-PKC protein, and increased the area of positive expression of Nrf2, HO-1 protein (P < 0.05 or P < 0.01). Conclusion: DBC can obviously improve the spermatogenic disorder induced by cyclophosphamide, and the mechanism may be related to the regulation of Nrf2/ARE signal pathway associated with oxidative stress.
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Aim: To establish ARE dual-luciferase reporter assay system and used it to identify the antioxidant substance of Scutellaria baicalensis Georgi. Methods: 293T cells were transiently co-transfected with ARE luciferase reporter plasmid PGL 4. 37 and sea kidney luciferase reporter plasmid PRL-TK. Three major active ingredients of Scutellaria baicalensis Georgi such as scutellarin, baicalein, baicalin and/or estrogen receptor (ER) specific inhibitor were added to Nrf2-ARE luciferase reporter assay system to detect whether they exerted antioxidant effect through the estrogen receptor affecting the Nrf2-ARE signaling pathway. Results: Baicalin (100 μmol · L-1) could obviously activate Nrf2-ARE pathway in 293T cells, and the induced expression was(1. 56 ±0. 01) times that of blank group (P < 0. 01). After pre-administration of ER specific inhibitor, the induced expression decreased to (1. 02 ±0. 23) times, and the antioxidant effect disappeared. After pre-administration of ER and Nrf2-ARE pathway specific inhibitor respectively, ROS in HaCaT cells injured by UVB significantly increased and and SOD was markedly down-regulated by baicalin. Conclusion Baicalin plays antioxidant activity through mediating Nrf2-ARE signaling pathway based on estrogen receptor.