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ObjectiveTo verify the anti-oxidative stress effect of Huangqintang based on the nuclear factor E2-related factor 2 (Nrf2) signaling pathway by using Caco-2 cells as a carrier and RNA interference (RNAi) technology with in vitro experiments. MethodThe Caco-2 cells in the logarithmic growth phase were transfected with siRNA to construct siRNA Caco-2 cells. After normal Caco-2 cells and siRNA Caco-2 cells were incubated with Huangqintang of different doses, RNA and protein were extracted. Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) and Western blot were used to detect the mRNA and protein expression of heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GST), Kelch-like ECH-associated protein 1 (Keap1), and Nrf2. Meanwhile, the activities of superoxide dismutase (SOD) and GSH-Px, as well as the expression levels of malondialdehyde (MDA) and reactive oxygen species (ROS), were detected by the colorimetric method and the probe method. ResultCompared with the results in the normal group, only the 400 mg·L-1 Huangqintang group and the sulforaphane (SFN) group could reduce the content of ROS and MDA in Caco-2 cells (P<0.01), while the activities of SOD and GSH-Px in the cells of the Huangqintang groups and the SFN group showed an upward trend. Furthermore, there were significant differences in the 400 mg·L-1 Huangqintang group/the SFN group and the normal group (P<0.01). Meanwhile, the protein and mRNA expression levels of HO-1, GST, Keap1, NQO1, and Nrf2 showed an upward trend in all groups (P<0.05, P<0.01). After transfection, compared with the normal group, the model group showed increased content of MDA and ROS, blunted activities of GSH-Px and SOD, and reduced protein and mRNA expression of HO-1, GST, Keap1, and NQO1 (P<0.05, P<0.01). After drug incubation, compared with the model group, the SFN group showed potentiated SOD activity, and the SFN group and the Huangqintang groups showed enhanced GSH-Px activity (P<0.01). Moreover, the activities of SOD and GSH-Px in the 400 and 200 mg·L-1 Huangqintang groups and the SFN group showed an upward trend (P<0.01), and the content of MDA in the 400 mg·L-1 Huangqintang group and the SFN group showed a downward trend. ROS decreased in all groups with drug intervention (P<0.01), and the protein and mRNA expression of HO-1, GST, Keap1, NQO1, and Nrf2 increased to varying degrees (P<0.05, P<0.01). ConclusionHuangqintang can play an anti-oxidative stress role by regulating the Nrf2 pathway.
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ObjectiveTo explore the neuroprotective mechanism of Buyang Huanwutang (BYHW) on diabetic peripheral neuropathy (DPN) rats based on oxidative stress and investigate the dosage of Astragali Radix (AR). MethodNinety SD rats were randomly divided into a normal group, a model group, an α-lipoic acid group (60 mg·kg-1·d-1), and BYHW groups with high- (15 g·kg-1·d-1), medium- (8.75 g·kg-1·d-1), and low-dose (5.625 g·kg-1·d-1) AR groups. The diabetes model was induced in rats except for those in the normal group by the high-sugar/high-fat diet and intraperitoneal injection of streptozotocin (STZ). Drug intervention lasted for 12 weeks. The paw withdrawal threshold (PWT) and sensory nerve conduction velocity (SNCV) were detected after drug intervention. Gonad-stimulating hormone (GSH) and malondialdehyde (MDA) were determined. The mitochondrial morphology and structure in sensory neurons of L4-5 dorsal root ganglion (DRG) of rats were observed by electron microscopy. Respiratory chain complex Ⅰ, Ⅱ, Ⅲ, and Ⅳ activities and the mitochondrial membrane potential were detected. The main proteins in the adenosine monophosphate-activated protein kinase (AMPK)/nuclear factor-related factor-2 (Nrf2) pathway, such as phosphorylated AMPK (p-AMPK), phosphorylated Nrf2(p-Nrf2), heme oxygenase-1 (HO-1), and quinone NADH dehydrogenase 1 (NQO1), were detected by immunohistochemistry and Western blot. ResultCompared with the normal group, the model group showed increased fasting blood glucose (P<0.01), decreased content of SNCV, PWT, and GSH (P<0.01), elevated MDA content (P<0.01), obvious mitochondrial damage with vacuolations, reduced activities of respiratory chain complex Ⅰ, Ⅱ, Ⅲ, and Ⅳ and mitochondrial membrane potential (P<0.01), and declining p-AMPK, p-Nrf2, HO-1, and NQO1 (P<0.01). Compared with the model group, the α-lipoic acid group and BYHW high-dose group showed increased SNCV, PWT, and GSH, decreased MDA (P<0.05, P<0.01), alleviated mitochondrial structural damage, increased respiratory chain complex Ⅰ, Ⅱ, Ⅲ, and Ⅳ activities and mitochondrial membrane potential (P<0.01), and elevated p-AMPK, p-Nrf2, HO-1, and NQO1 (P<0.05, P<0.01). ConclusionBYHW regulates oxidative stress through the AMPK/Nrf2 pathway to treat DPN. The therapeutic effect of BYHW is related to the dosage of AR. The BYHW group with high-dose AR is superior to the BYHW groups with medium- and low-dose AR groups in inhibiting oxidative stress.
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Objective:To investigate the effect of miR-141 down-regulation on the damage of renal tubular epithelial cell, and further to explore its mechanism.Methods:The renal tubular epithelial cell line HK-2 cells were divided into normal (5.5 mmol/L D-glucose) group, hypertonic group, high glucose (30 mmol/L D-glucose) group, negative control+high glucose group (transfected with NC inhibitor vector) and si-miR-141+high glucose group (transfected with miR-141 inhibitor vector) . Real time quantitative polymerase chain reaction (qRT-PCR) was used to detect the relative expression of miR-141. The production of reactive oxygen species (ROS) , cell viability and apoptosis were detected by DCFH-DA fluorescence staining, CCK-8 method and flow cytometry. The expression of Sirt1/Nrf2 signaling pathway related proteins was detected by Western blot. Luciferase reporter gene assay verified the targeting relationship between miR-141 and Sirt1 mRNA.Results:①Compared with the normal group, after transfection with si-miR-141, the relative expression of miR-141 decreased (1.00±0.03 vs 0.52±0.06) , the difference was statistically significant ( F=278.104, P<0.05) ; ② Compared with the normal group [DCFH-DA fluorescence intensity (7.18±0.59) %], the high glucose group [DCFH-DA fluorescence intensity (84.95±3.21) %] cell ROS level was significantly increased, and compared with the high glucose group [DCFH-DA fluorescence intensity (84.95±3.21) %] Compared with the si-miR-141+ high glucose group [DCFH-DA fluorescence intensity (45.10±4.29) %] cell ROS levels were significantly reduced, the difference was statistically significant (all P<0.05) ; ③compared with the normal group (5.13%±0.78) % Compared with the hypertonic group (5.96±0.81) %, the high glucose group (32.76±2.95) % cell apoptosis rate was significantly increased, while the si-miR-141+ high glucose group (17.54%± 2.79) % apoptosis rate was significantly lower in the higher glucose group and the negative control+ high glucose group (33.40%±3.14) %, the difference was statistically significant ( F=221.419, P<0.05) ; ④compared with the normal group (100±3.98) % Compared with the hypertonic group (95.68±5.14) %, the high glucose group (67.24±5.18) % HK-2 cell survival rate was significantly reduced; at the same time, compared with the high glucose group (67.24±5.18) % and Compared with the negative control+ high glucose group (65.33±3.10) %, the si-miR-141+ high glucose group (83.55±5.10) % cell survival rate increased significantly, and the difference was statistically significant ( F=93.008, P<0.05) ; ⑤ Compared with the normal group and the hypertonic group, the expression of Cleaved Caspase 3 protein in the high glucose group increased, while the expression of Sirt1, Nrf2 and HO-1 protein was down-regulated; however, compared with the high glucose group, si- In the miR-141+ high glucose group, Cleaved caspase 3 protein expression decreased, while Sirt1, Nrf2 and HO-1 protein expression increased, the difference was statistically significant (all P<0.05) . Conclusions:Down-regulation of miR-141 can ameliorate high glucose-induced renal tubular epithelial cell damage induced oxidative stress by activating Sirt1/Nrf2 signaling pathway.
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Objective: The study was designed to assess the beneficial role of mangiferin (MGN) in lead (Pb)-induced neurological damages in the activation of Nrf2-governed enzymes, genes and proteins. Methods: A total of 96 weaned Wistar rats (48 males and 48 females, 26- to 27-day-old), weighing 50−80 g were used. The experiment was performed in six groups: normal group (control, n = 16), model group (chronic Pb exposed, n = 16), Dimercaptosuccinic acid (DMSA)-treated group (positive control, Pb + DMSA, n = 16), three MGN-treated groups with different doses (Pb + MGN, n = 48). Normal group freely had access to purified water. DMSA-treated group was given DMSA, which was clinically used as the standard treatment for moderate Pb poisoning, at 50 mg/kg (2 mL suspension with purified water) by intragastric gavage (ig) 4 continual days a week for 4 weeks, MGN-treated groups were given MGN at 50, 100, or 200 mg/kg (2 mL suspension with purified water) by ig daily for 4 weeks. At the end of the treatment, all rats were sacrificed and the brain samples were collected. The haematoxylin and eosin (H&E) staining was used for observation of histopathology. Commercial kit, real-time quantitative polymerase chain reaction (RT-qPCR), Western-blot and immunohistochemistry (IHC) detection were used to detect the mRNA and protein expression. Results: Eight weeks exposure to Pb-containing water resulted in pathological alterations, anti-oxidative system disorder in the brain, all of which were blocked by MGN in a Nrf2-dependent manner. Nrf2 downstream enzymes such as HO-1, NQO1, γ-GCS were activated. Nrf2, GCLC, GCLM, HO-1 mRNA and total Nrf2, Nuclear Nrf2, γ-GCS, HO-1 protein expression were affected too. Conclusion: MGN ameliorated morphological damage in the hippocampus. Its neuroprotective effects were achieved by the activation of the Nrf2 downstream genes. The data from this in vitro study indicates that MGN targeting Nrf2 activation is a feasible approach to reduce adverse health effects associated with Pb exposure. Thus, MGN could be an effective candidate agent for the Pb-induced oxidative stress and neurotoxicity in the human body.
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Lignosus rhinocerotis (tiger milk mushroom) is widely used by the indigenous people of Malaysia as a traditional remedy. The present study was carried out in order to evaluate the antioxidant, cytotoxic and anti-neuroinflammatory activities of L. rhinocerotis extract on brain microglial cells (BV2). The antioxidant activity was evaluated by 2,2-diphenyl-1-picryhydrazyl (DPPHâ¢), 2,2'-azinobis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTSâ¢+) scavenging assays, and ferric reducing antioxidant power (FRAP). The FRAP, DPPH and ABTSâ¢+ scavenging capacities of the TE3 fraction were 420.77 mg FE/g, 58.01%, and 7%, respectively. The cytotoxic activity was determined by MTS assay. The in vitro model of anti-neuroinflammatory property was evaluated by measuring the production of nitric oxide (NO) in lipopolysaccharide (LPS)-induced BV2 cells. The TE3 fraction showed a significant NO reduction at 1 to 100 µg/mL. The TE3 fraction down-regulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) genes while it upregulated heme oxygenase (HO-1) and NADPH quinone acceptor oxidoreductase-1 (NQO-1) genes. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription was also activated. The chemical component of the active fraction (TE3) was identified by gas chromatography-mass spectrometry (GCMS). Overall, the BV2 in vitro model anti-neuroinflammatory activity of L. rhinocerotis may be caused by the lipid constituents identified in the fraction
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Técnicas In Vitro/métodos , Células/classificação , Agaricales/classificação , Inflamação/tratamento farmacológico , Lipídeos/efeitos adversos , Cromatografia Gasosa-Espectrometria de Massas/instrumentação , Antioxidantes/farmacologiaRESUMO
Objective: To observe the therapeutic effect of Xiaochaihu granule on acute liver injury (ALI) induced by thioacetamide (TAA) in rats, and to explore the role of transcription factor (NF) -E2 related factor 2 (Nrf2) in the pathway of oxidative damage. Method: SD rats were randomly divided into control group, model group, Xiaochaihu granule low, middle and high dose groups (1, 2, 4 g·kg-1). 250 mg·kg-1 TAA was given to the rats by ip. administration for 2 days to prepare the liver injury model, and from the 3rd day, same amount of double distilled water or different doses of Xiaochaihu granule was given to corresponding groups by ig. administration for 2 weeks. 24 hours after the last administration, liver tissues were taken and stained with hematoxylin-eosin(HE) staining. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were measured by colorimetry. The activities of total superoxide dismutase (T-SOD) and malondialdehyde (MDA) in liver tissues were measured by colorimetry. Real-time PCR was used to detect Nrf2 pathway related mRNA expression. Result:As compared with control group, ALT, AST and MDA in model group were significantly increased,while T-SOD was significantly decreased (PPPPPPPConclusion:Xiaochaihu granule may play a therapeutic role in TAA induced ALI in rats by up-regulating the expression of downstream molecules in Nrf2 signaling pathway.
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Objective To investigate the effects and mechanisms of glutamine (Gln) supplementation on oxidative stress,autophagy response and neurobehavioral outcome after traumatic brain injury (TBI) in rats.Methods TBI animal models were established using Feeney's method.Eighty SD rats were randomly divided into 4 groups:sham operation group (group Sham),Sham + glutamine supplementation group (group Sham+ GLN),traumatic brain injury group (group TBI),and TBI + glutamine supplementation group (group TBI+ GLN).We measured rat behavioral outcomes by modified neurologic severity score (mNSS) tests at day 1,3,7 and 14 after TBI.The apoptosis neurons in TBI cerebral cortex were determined by TUNEL staining.The expression of reactive oxygen species (ROS) was tested by ROS kits.Oxidative stress and autophagy related cytokines (HO-1,NQO1,Nrf2,LC3-Ⅱ and Beclin-1) were tested with Western blotting.Results Compared with the TBI group,the neurological function was improved [(9.79±0.43) vs.(8.43±0.30),F =6.775,P =0.010] and the apoptosis rate decreased (19.88% ± 1.60% vs.15.35% ± 1.28%,P =0.013) in the TBI+ GLN group after 7-day treatment.Compared with the Sham group,the protein expression of ROS increased (P=0.000),and the expression of anti-oxidative stress factors (HO-1,NQO1) and Nrf2 pathway significantly decreased in the TBI group.After glutamine supplementation was given,the expression of ROS decreased and the expressions of HO-1 and NQO1 increased.The Nrf2 pathway and autophagy response also were activated with the expressions of Nrf2,LC3-Ⅱ and Beclin-1 increasing.Conclusion Glutamine supplementation can markedly reduce neuron apoptosis and improve neurological outcomes after TBI,thus has the protective effect on nerves by inhibiting TBI-induced oxidative stress response,activating Nrf2 pathway and autophagy response.
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Objective:To investigate the role of NRF2 pathway in the drug resistance to sorafenib in hepatocellular carcinoma (HCC).Methods:Two sorafenib-resistant cells HepG2-SR and Huh7-SR were established by incubating human HCC HepG2 and Huh7 cells at a increasing concentration of sorafenib,and verify resistant cell properties by detecting the cell apoptosis.The levels of NRF2 were detected by Western blot and Real-time PCR.SiRNA was used to silence NRF2,then the cell apoptosis were detected by flow cytometry to explore the effect of reversing the drug resistance and synergy in combination with sorafenib.Results:Sorafenib induced pro-apoptosis effect was significantly in Huh7 and HepG2 cells than the corresponding Huh7-SR and HepG2-SR cells.The NRF2 expression levels were significantly higher in sorafenib-resistant cells and the parental cells treated with sorafenib than the corresponding untreated parental cells,while the NFR2 mRNA expression levels were no significant.When in combination with sorafenib,NRF2 siRNA showed the synergistic effect in inducing cell apoptosis in sorafenib-resistant cells and parental cells.Conclusion:NRF2,activated by post-transcriptional level after sorafenib exposure,is responsible for the drug resistance to sorafenib in HCC.Inhibiting NRF2 could reverse the drug resistance to sorafenib in HCC.
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OBJECTIVE Oxidative sress is one of the key factor responsible for occurrence and development of hepatic fibrosis, a common consequence of chronic liver injury of multiple etiology. Nuclear factor erythroid 2-related factor 2 (Nrf2) serves as a major regulator of a celular defense system against oxidative stress. Xiaochaihutang (XCHT), a compound of seven botanical extracts used for liver diseases traditionally in East Asia. However, few studies have investigated its anti-hepatic fibrosis effects and pathophysiological mechanism of action. The present study was designed to confirm the anti-hepatic fibrosis effects and explore its potential mechanism of action by investigating the intervention of Nrf2 pathway. METHODS Liver fibrosis was induced by repeated injection of Carbon tetrachloride (CCl4) over a period of 9 weeks. Starting from the 6th week, the animals in treatment groups were given the appropriate dose of XCHT granules and silybin. Biochemical parameters, histological changes of the liver and alpha-smooth muscle actin (α-SMA) were determined. The expressions of Nrf2, Keap1, Nqo1, HO-1, Gclc and Gclm were assessed by RT-PCR and Western blot. RESULTS CCl4 caused a significant fibrosis damage in the rat liver and the liver functions and fibrosis degree were significantly improved by XCHT (5 g·kg- 1 and 10 g·kg- 1). XCHT (5 g·kg- 1 and 10 g·kg- 1) treatment significantly decreased the number of cells labeled with α-SMA antibodies. Moreover, XCHT (5 g·kg-1 and 10 g·kg-1) significantly increase Nqo1, HO-1, Gclc and Gclm expressions in the liver. CONCLUSION These studies establish XCHT is a potentially useful therapeutic agent for treatment of hepatic fibrosis and it might be via regulation of Nrf2 pathway in rats against oxidative stress, making further efforts to inhibiting the activated HSCs. Activation or up-regulation of Nrf2 pathway may be an alternative treatment strategy for liver fibrosis.
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Aim To investigate the roles of intracellu-lar reactive oxygen species ( ROS ) and Nrf2 pathway in shikonin-induced A549 cell apoptosis. Methods The cytotoxicity was analyzed by MTT assay. The ap-optosis of A549 cells was analyzed by both cellular morphological and biochemical methods. The relative changes of the redox marks ( ROS/GSH) were studied by fluorescence assay in the shikonin-treated A549 cells in accompany with the changes of the intracellular redox homeostasis by GSH/GSSG ratio. ROS inhibitor was also employed in the treatment to find the role of ROS in shikonin-induced A549 cell apoptosis. Real-time PCR analysis and ELISA assay were performed as well to determine the role of Nrf2 pathway in the shiko-nin-induced A549 cell apoptosis. Results The IC50 of shikonin on A549 cells was 3. 2 mg·L-1 . The cellu-lar redox homeostasis shifted toward oxidation signifi-cantly in shikonin treatment in a time-dependent man-ner. The expression of the Nrf2 pathway related genes was up-regulated by shikonin ( 3 . 2 mg · L-1 , 8 h ) . The expression of the anti-apoptotic genes was down-regulated , and proapoptotic genes were up-regulated by shikonin (3. 2 mg·L-1, 24h). Futhermore, the inhi-bition of intracellular ROS alleviated the cytotoxicity of shikonin in A549 cells. Conclusion The critical role of shikonin-induced redox imblance in A549 cell, coped with the secondary produced ROS and Nrf2 path-way antioxidants, result in A549 cell apoptosis.
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Objective To explore the effects of pyrrolidine dithiocarbamate (PDTC) on the acute lung injury and the activation of Nrf2 pathway after Paraquat (PQ) induced lung injury.Methods Fortyeight adult male SD rats with lung injury induced by PQ were randomly (random number) divided into control group and PDTC group.Three animals were sacrificed at every 1-week interval,7d,14d and 21 days after PQ intoxication,and the lungs of rats were removed for acute lung injury score after HE staining,and for lung fibrosis assessment after Masson staining,and the levels of reduced glutathione (GSH) and malondialdehyde (MDA) in the lung tissue homogenate were assayed and the phosphorylation of Nrf2 (nuclear-E2-related factor 2) was detected by Weston blot.The mean values of detected variables between two groups were compared by t test,and survival curve was tested by Wilcoxon (Gehan) test.Results The intoxication symptoms of rats were obvious,and 4 rats in control group and 9 rats in PDTC group survived until 21days.The survival time of animals in PDTC group was longer than that in control group (Wilcoxon (Gehan) =10.17023,P =0.001).The levels of MDA in control group were higher than those in PDTC group,while the levels of GSH in control group were lower than those in PDTC group.The levels of phosphorylation of Nrf2 in PDTC group were higher than those in control group at 1-week intervals,1-week:(0.32±0.04) vs.(0.23±0.05),P=0.003; 2-week:(0.62±0.06) vs.(0.33±0.03),P<0.001; 3-week:(0.61 ±0.04) vs.(0.33±0.05),P<0.001.The acute lung injury (ALI) scores in PDTC group were lower than those in control group,1-week:(5 ± 0.95) vs.(8 ± 1.23),P =0.002 ; 2-week:(9±1.18) vs.(11±1.02),P=0.019; 3-week:(11±1.33) vs.(12±1.42),P=0.002.The percentages of lung fibrosis at 1-week intervals after PQ intoxication were (40.87 ± 7.25) %,(43.38 ±5.71)% and (45.91 ± 3.97)% in control group,and they were higher than those in PDTC group (32.92±2.34)%,(33.45 ±3.04)% and (35.27 ±3.81)% in PDTC group,P=0.017,0.001 and 0.001 respectively.Conclusions Attenuation of acute lung injury and lung fibrosis,and prolongation of survival time of SD rats by PDTC were associated with activation of Nrf2 pathway.