Lycium barbarian seed oil activates Nrf2/ARE pathway to reduce oxidative damage in testis of subacute aging rats / 中国药理学通报

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.

Mechanism of gypenoside ⅩⅦ against cerebral ischemia/reperfusion injury based on nuclear factor erythroid 2-related factor 2/antioxidant responsive element signaling pathway / 中华危重病急救医学

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.

Vitamin D supplementation improves testicular function in aging rats induced by D-galactose / 中华内分泌代谢杂志

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.

Edaravone improves the post-traumatic brain injury dysfunction in learning and memory by modulating Nrf2/are signal pathway

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.

Effects of astragalus polysaccharide on depressive behaviors and hippocampal Nrf2-ARE signaling pathway in rats / 中国药理学通报

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.

Protective effect of Pinus massoniana needle extract against oxidative stress in human dermal papilla cells via the nuclear factor-erythroid 2-related factor 2-antioxidant responsive element signaling pathway / 中华皮肤科杂志

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.

Arsenic Trioxide Combining Leflunomide Activates Nrf2-ARE-HO-1 Signaling Pathway and Protects Heart Xenografts / 中国结合医学杂志

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

Effect and mechanism of moxibustion on oxidative stress injury in rats with Parkinson's disease by activating Nrf2/ARE pathway / 中国针灸

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.

Analysis of the effect of pentoxifylline on oxidative stress in brains of epileptic rats based on Nrf2-ARE signaling pathway / 医学研究生学报

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.

Protective effect of Schisandra chinensis total lignans on acute alcoholic-induced liver injury related to inhibiting CYP2E1 activation and activating the Nrf2/ARE signaling pathway

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.

Anti-Oxidation Mechanism of Andrographolide on HaCaT Cells Via Nrf2/ARE Pathway / 中国药学杂志

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.

Effect of Duzhong Butiansu Capsule on improving spermatogenesis obstacle in mice induced by cyclophosphamide / 中草药

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.

A new lupane-type triterpenoid from Dichroa hirsuta / 中国中药杂志

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.

Design, synthesis and biological evaluation of chalcone analogues with novel dual antioxidant mechanisms as potential anti-ischemic stroke agents

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.

Mrp1 is up-regulated in lung tissues of mildly stable COPD mice induced by smoke, Nrf2 pathway may be involved / 中国药理学通报

Aim: To investigate the effect of Nrf2 pathway on the expression of MRP1 in mildly stable COPD mice. Methods: The mild COPD mouse model was established by passive cigarette smoking. The pathological changes of lung tissues were examined by HE staining. Immunohistochemistry and Western blot were used to detect the protein expression of MRP1, Nrf2 and HO-1. Results: Compared with normal group, each lung function index of the mild-moderate COPD model group was significantly lower, but compared with wide type(WT) model group, the reduction was more significant in Nrf2-/- model group. HE results showed diffuse inflammatory reaction and alveolar bronchial structure damage in alveolar of WT and Nrf2-/- model mice, and it was more pronounced in Nrf2-/- mice. Immunohistochemistry and Western blot results showed that the expression of MRP1 in lung tissue of Nrf2-/- normal mice was significantly reduced compared with the normal WT group. After passive cigarette smoking, The expression of MRP1, Nrf2 and HO-1 in WT model group increased significantly, but compared with Nrf2-/- normal mice, there was no significant change in the expression of MRP1 in Nrf2-/- model group. Conclusions: Mildly stable COPD mice may counteract the xenobiotic damage caused by cigarette smoke through up-regulating the expression of MRP1 protein, which may be associated with Nrf2 signaling activation.

Sodium selenite induced human lung cancer A549 cells apoptosis through Keap1/Nrf2/ARE signaling pathway / 中国药理学通报

Aim: To study the induction of apoptotic effect of sodium selenite on human lung cancer A549 cells and its mechanisms. Methods: A549 cells were exposed to different concentrations of sodium selenite for 24 h. MTT assay was applied to determine A549 cell proliferation. Inverted fluorescence microscope was used to investigate the morphological changes in A549 cells. Flow cytometry analysis was applied to assess the apoptotic rates of A549 cells. Laser confocal microscope was employed to measure the reactive oxygen species (ROS) fluorescence intensity. A multi-detection reader was used to determine the antioxidant parameter. Western blot was utilized to detect the expression of Keapl, Nrf2, HO-1 and Nrf2 in cytoplasm and nucleus. Results: MTT results showed that sodium selenite inhibited the proliferation of A549 cells in a concentration-dependent manner. After treatment with sodium selenite for 24 h, the apoptotic rate of A549 cells was markedly increased through Hoechst 33342 staining and flow cytometry measurement. Sodium selenite significantly up-regulated ROS and malondialdehyde (MDA) content and down-regulated the levels of superoxide dismutase (SOD) and glutathione (GSH). Meanwhile, sodium selenite treatment also reduced the expressions of Keapl, Nrf2 and HO-1 at protein levels and inhibited Nrf2 protein nuclear translocation in A549 cells. Conclusions: Treatment with sodium selenite induces A549 cells apoptosis, which may contribute to the anti-proliferation activity, induction of apoptosis and regulation of oxidative stress reaction and Keapl/Nrf2/ARE antioxidative signaling pathway expression.

Screening of antioxidant components in Scutellariae Radix through mediating Nrf2-ARE signaling pathway based on estrogen receptor / 中国药理学通报

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.

Cypermethrin induces cell injury in primary cortical neurons of C57BL/6 mice by inhibiting Nrf2/ARE signaling pathway / 南方医科大学学报

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, .

Synthesis and cytotoxicity evaluation of 3-amino-2-hydroxypropoxygenistein derivatives / 中国天然药物

Soy isoflavones exhibit various biological activities, such as antioxidant, anti-tumor, anti-inflammatory, and cardiovascular protective effects. The present study was designed to investigate the effects of sixteen synthesized 3-amino-2-hydroxypropoxy genistein derivatives on cell proliferation and activation of Nrf2 (Nuclear factor erythroid 2-related factor 2)/ARE (antioxidant response elements) pathway in human cancer cell lines. Most of the tested compounds exerted greater cytotoxic activity than genistein, as measured by MTT assay. Moreover, compound 8c showed the highest ARE-luciferase reporter activity among the test compounds. It strongly promoted Nrf2 nuclear translocation and up-regulated the expression of total Nrf2 and downstream targets NQO-1 and HO-1 at protein level. The present study may provide a basis for the application of isoflavone derivatives as Nrf2/ARE pathway inducers for cancer therapy and cancer prevention.

Lipoic acid protects the lungs of rats against acute injury induced by paraquat poisoning through Nrf2-ARE signaling pathway / 中华急诊医学杂志

Objective To investigate the nuclear factor-erythroid 2-related factor (Nrf-2),and heme oxidase 1 (HO-1) expression in acute lung injury induced by paraquat poisoning in rats and explore the mechanism of lipoic acid acting on protection of lung from paraquat poisoning.Methods Seventy-two adult healthy male Sprague Dawley (SD) rats were randomly divided into three groups with different treatments designated as:control group (control group,n =12),paraquat group (PQ group,n =30) and paraquat + lipoic acid group (LA group,n =30).PQ group and LA group were randomly divided into five subgroups (n =6 in each) according to 6 h,12 h,24 h,48 h and 72 h after modeling and treatment.The rats in PQ group and PQ + LA group were treated with intra-peritoneal injection (ip) of PQ (25 mg/kg),while the rats in control group were treated with the equal volume of saline instead.Half an hours after intra-peritoneal injection of PQ,lipoic acid (100 mg/kg) was injected into caudal vein of rats once a day until they were sacrificed.The body weight was measured everyday.The rats of each group were sacrificed at the given intervals,and lung tissues were harvested to measure lung coefficient of rats.The same part of left lung of rats in each group was taken for HE staining and immunohistochemistry in order to detect the expressions of Nrf2 and HO-1.The right lung of rats in each group was taken for the detection of GSH-Px and SOD activity.All data were analyzed by using the One-way analysis of variance (ANOVA) and SNK-q test.Results The body weight reduction in LA group (191.02 ± 0.82) g,(183.37 ± 7.74) g was significantly less than that in PQ group (183.85 ± 2.07) g,(173.13 ± 4.34) g at 48 h and 72 h after PQ poisoning,respectively (P ＜ 0.01,P ＜ 0.05).The lung coefficient in LA group (6.83 ± 0.48) mg/g,(7.61 ±0.28) mg/g,(8.29 ±0.36) mg/g was less compared with PQ group (7.39 ±0.53) mg/g,(8.48±0.23) mg/g,(9.06±0.10) mg/g at 24 h,48 h,and 72 h,respectively (P＜0.01,P＜ 0.05).The immunohistochemical expressions of Nrf-2 in LA group (3.99 ±0.50),(3.51 ±0.12) were higher than those in PQ group (1.33 ±0.22),(1.62 ±0.41) at 48 h and 72 h.The immunohistochemical expression of HO-1 in LA group (1.76 ±0.17) was higher than that in PQ group (1.31 ±0.15) at 72 h.The levels of GSH-Px activity in LA group were significantly higher in comparison with PQ group at 24h,48h,and 72h (P ＜0.01,P ＜0.05).The levels of SOD activity in the LA group were significantly higher in comparison with PQ group at 6 h,12 h,24 h,48 h,and 72 h after PQ administration (P ＜ 0.01).Conclusions Nrf2-ARE (antioxidant response element) signaling pathway is involved in the pathogenesis of acute lung injury induced by paraquat poisoning,and lipoic acid may protect acute lung injury in rats induced by paraquat poisoning through Nrf2-ARE signaling pathway.