The antioxidant effect of preischemic dexmedetomidine in a rat model: increased expression of Nrf2/HO-1 via the PKC pathway

Abstract Background The precise underlying mechanism of antioxidant effects of dexmedetomidine-induced neuroprotection against cerebral ischemia has not yet been fully elucidated. Activation of Nuclear factor erythroid 2-related factor (Nrf2) and Heme Oxygenase-1 (HO-1) represents a major antioxidant-defense mechanism. Therefore, we determined whether dexmedetomidine increases Nrf2/HO-1 expression after global transient cerebral ischemia and assessed the involvement of Protein Kinase C (PKC) in the dexmedetomidine-related antioxidant mechanism. Methods Thirty-eight rats were randomly assigned to five groups: sham (n = 6), ischemic (n = 8), chelerythrine (a PKC inhibitor; 5 mg.kg-1 IV administered 30 min before cerebral ischemia) (n = 8), dexmedetomidine (100 µg.kg-1 IP administered 30 min before cerebral ischemia (n = 8), and dexmedetomidine + chelerythrine (n = 8). Global transient cerebral ischemia (10 min) was applied in all groups, except the sham group; histopathologic changes and levels of nuclear Nrf2 and cytoplasmic HO-1 were examined 24 hours after ischemia insult. Results We found fewer necrotic and apoptotic cells in the dexmedetomidine group relative to the ischemic group (p< 0.01) and significantly higher Nrf2 and HO-1 levels in the dexmedetomidine group than in the ischemic group (p< 0.01). Additionally, chelerythrine co-administration with dexmedetomidine attenuated the dexmedetomidine-induced increases in Nrf2 and HO-1 levels (p< 0.05 and p< 0.01, respectively) and diminished its beneficial neuroprotective effects. Conclusion Preischemic dexmedetomidine administration elicited neuroprotection against global transient cerebral ischemia in rats by increasing Nrf2/HO-1 expression partly via PKC signaling, suggesting that this is the antioxidant mechanism underlying dexmedetomidine-mediated neuroprotection.

Preliminary exploration of detoxification mechanism of processing methods on cardiotoxicity induced by radix Tripterygium wilfordii in mice via Nrf2/HO-1 pathway / 中国中药杂志

This study aims to investigate the detoxification effects of different processing methods on the cardiotoxicity induced by radix Tripterygium wilfordii, and preliminarily explore the detoxification mechanism via the nuclear factor E2-related factor 2(Nrf2)/heme oxygenase 1(HO-1) pathway. The raw and processed products [stir-fried product, product stir-fried with Lysimachiae Herba(JQC), product stir-fried with Phaseoli Radiati Semen(LD), product stir-fried with Paeoniae Radix Alba(BS), product stir-fried with Glycyrrhizae Radix et Rhizoma(GC), and product stir-fried with vinegar(CZ)] of radix T. wilfordii were administrated to mice by gavage at a dose of 2 g·kg~(-1)(based on crude drugs) for 28 days. Twenty-four hours after the last administration, we measured the serum biochemical indexes of mice to evaluate the detoxification effect. Furthermore, we determined the expression of key proteins of Nrf2/HO-1 pathway in mouse heart tissue by Western blot and some oxidation/antioxidation-related indexes by corresponding kits to explore the detoxification mechanism. The administration of the raw product elevated the levels of serum creatine kinase, lactate dehydrogenase, and malondialdehyde, a product of cardiac lipid peroxidation(P<0.01), down-regulated the protein levels of Nrf2 and HO-1(P<0.01), and reduced the levels of total superoxide dismutase, glutathione, glutathione peroxidase, and glutathione S-transferase(P<0.01). However, after the administration of the products stir-fried with JQC, LD, BS, GC, and CZ, the abnormalities of the above indexes induced by the raw product were recovered(P<0.05 or P<0.01). In particular, the product stir-fried with JQC showed the best performance. Taken all together, the cardiotoxicity induced by radix T. wilfordii could be attenuated by stir-frying with JQC, LD, BS, GC, and CZ, and the stir-frying with JQC showed the best detoxification effect. The mechanism might be associated with the cardiac antioxidant defense and oxidative damage mitigation mediated by the up-regulated Nrf2.

Effects of butyphthalide on microglia polarization after intracerebral hemorrhage and the underlying mechanisms / 中南大学学报(医学版)

OBJECTIVES@#Because intracerebral hemorrhage (ICH) has high morbidity, disability and mortality, it is significant to find new and effective treatments for ICH. This study aims to explore the effect of butyphthalide (NBP) on neuroinflammation secondary to ICH and microglia polarization.@*METHODS@#A total of 48 healthy male SD rats were randomly divided into 6 groups: a sham 24 h group, a sham 72 h group, an ICH 24 h group, an ICH 72 h group, an ICH+NBP 24 h group, and an ICH+NBP 72 h group (8 rats per group). After operation, the neurological deficiencies were assessed based on improved Garcia scores and corner test. The expressions of Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB), nuclear factor erythroid 2-related factor 2 (Nrf2), aquaporin-4 (AQP4), zonula occludens-1 (ZO-1), occludin, CD68, CD86, and CD206 were observed by Western blotting. Inflammatory cytokines were detected by ELISA. The immunofluorescence was to detect the polarization of microglia.@*RESULTS@#1) Compared with the sham groups, the expression of TLR4 (24 h: P<0.05; 72 h: P<0.01), NF-κB (both P<0.01) and Nrf2 (both P<0.01) in the perihematoma of the ICH group was increased, leading to microglia activation (P<0.01). The expressions of IL-6 (24 h: P<0.05; 72 h: P<0.01) and TNF-α (both P<0.01), the pro-inflammatory cytokines were up-regulated, and the expression of anti-inflammatory cytokine IL-4 was down-regulated (both P<0.01). Besides, the expression of AQP4 was enhanced (both P<0.01). The protein level of tightly connected proteins (including ZO-1, occludin) was decreased (all P<0.01). The neurological function of the rats in the ICH group was impaired in the 2 time points (both P<0.01). 2) Compared with the sham group at 24 h and 72 h after the intervention of NBP, the expressions of TLR4 (both P<0.05) and NF-κB (both P<0.01) were significantly declined, and the expression of Nrf2 was further enhanced (both P<0.05) in the perihematoma of the ICH+NBP group. Furthermore, the expression of M1 microglia marker was inhibited (P<0.05), and the polarization of microglia to the M2 phenotype was promoted (P<0.01). 3) In terms of inflammation after ICH, the IL-4 expression in the ICH+NBP group was increased compared with the ICH group (24 h: P<0.05; 72 h: P<0.01); the expression of IL-6 was decreased significantly in the ICH+NBP 72 h group (P<0.01); the level of AQP4 was declined significantly in the ICH+NBP 24 h group (P<0.05), there was a downward trend in the 72-hour intervention group but without significant statistical difference. 4) Compared with the ICH group, the ZO-1 protein levels were increased (24 h: P<0.05; 72 h: P<0.01), and the symptoms of nerve defect were improved eventually (both P<0.05) in the ICH+NBP groups.@*CONCLUSIONS@#After ICH, the TLR4/NF-κB pathway is activated. The M1 microglia is up-regulated along with the release of detrimental cytokines, while the anti-inflammatory cytokines are down-regulated. The expression of AQP4 is increased, the tight junction proteins from the blood-brain barrier (BBB) is damaged, and the neurological function of rats is impaired. On the contrary, NBP may regulate microglia polarization to M2 phenotype and play a role in the neuroprotective effect mediated via inhibiting TLR4/NF-κB and enhancing Nrf2 pathways, which relieves the neuroinflammation, inhibits the expression of AQP4, repairs BBB, and improves neurological functional defects.

Protective Effects of Anthocyanins Extracted from Vaccinium Uliginosum on 661W Cells Against Microwave-Induced Retinal Damage / 中国结合医学杂志

OBJECTIVE@#To study the protective effect of anthocyanins extracted from Vaccinium Uliginosum (VU) on retinal 661W cells against microwave radiation induced retinal injury.@*METHODS@#661W cells were divided into 6 groups, including control, model [661W cells radiated by microwave (30 mW/cm2, 1 h)] and VU groups [661W cells pretreated with anthocyanins extracted from VU (25, 50, 100 and 200 µg/mL, respectively) for 48 h, and radiated by microwave 30 mW/cm2, 1 h]. After treatment with different interventions, the cell apoptosis index (AI) was determined using Heochst staining; contents of malonaldehyde (MDA), glutataione (GSH), and activity of superoxide dismutase (SOD) were measured. mRNA expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1(HO-1) were detected by real time quantitative polymerase chain reaction, and the expression of HO-1 protein was examined by Western blot analysis. Nucleus and cytoplasm were separated and Nrf2 protein expression was further verified by Western blot analysis.@*RESULTS@#There was significant difference in AI among the groups (F=322.83, P<;0.05). Compared with the control group, AI was significantly higher in the model group and was lower in 4 VU-pretreated groups (P<;0.05). Linear regression analysis showed the decline of AI was in a dose-dependent manner with VU treatment (r=0.8419, P<;0.05). The MDA and GSH contents of 661W cells in VU-treated groups were significantly lower than the model group (P<;0.05). Compared with the model group, the SOD activity in the VU-treated groups (50, 100 and 200 µg/mL) was significantly higher (all P<;0.05). The Nrf2 and HO-1 mRNA expressions were slightly increased after irradiation, and obviously increased in 100 µg/mL VU-treated group. After irradiation, the relative expressions of HO-1 and Nrf2 proteins in nucleus were slightly increased (P<;0.05), and the changes in cytoplasm were not obvious, whereas it was significantly increased in both nucleus and cytoplasm in the VU treatment groups.@*CONCLUSIONS@#Anthocyanins extracted from VU could reduce apoptosis, stabilize cell membrane, and alleviate oxidant injury of mouse retinal photoreceptor 661W cells. The mechanism might be through activating Nrf2/HO-1 signal pathway and inducing HO-1 transcription and translation.

Fucoxanthin regulates Nrf2/Keap1 signaling to alleviate myocardial hypertrophy in diabetic rats / 南方医科大学学报

OBJECTIVE@#To investigate the protective effect of fucoxanthin (FX) against diabetic cardiomyopathy and explore the underlying mechanism.@*METHODS@#Rat models of diabetes mellitus (DM) induced by intraperitoneal injection of streptozotocin (60 mg/kg) were randomized into DM model group, fucoxanthin treatment (DM+FX) group and metformin treatment (DM+ Met) group, and normal rats with normal feeding served as the control group. In the two treatment groups, fucoxanthin and metformin were administered after modeling by gavage at the daily dose of 200 mg/kg and 230 mg/kg, respectively for 12 weeks, and the rats in the DM model group were given saline only. HE staining was used to examine the area of cardiac myocyte hypertrophy in each group. The expression levels of fibrotic proteins TGF-β1 and FN proteins in rat hearts were detected with Western blotting. In the cell experiment, the effect of 1 μmol/L FX on H9C2 cell hypertrophy induced by exposure to high glucose (HG, 45 mmol/L) was evaluated using FITC-labeled phalloidin. The mRNA expression levels of the hypertrophic factors ANP, BNP and β-MHC in H9C2 cells were detected using qRT-PCR. The protein expressions of Nrf2, Keap1, HO-1 and SOD1 proteins in rat heart tissues and H9C2 cells were determined using Western blotting. The DCFH-DA probe was used to detect the intracellular production of reactive oxygen species (ROS).@*RESULTS@#In the diabetic rats, fucoxanthin treatment obviously alleviated cardiomyocyte hypertrophy and myocardial fibrosis, increased the protein expressions of Nrf2 and HO-1, and decreased the protein expressions of Keap1 in the heart tissue (P < 0.05). In H9C2 cells with HG exposure, fucoxanthin significantly inhibited the enlargement of cell surface area, lowered the mRNA expression levels of ANP, BNP and β-MHC (P < 0.05), promoted Nrf2 translocation from the cytoplasm to the nucleus, and up-regulated the protein expressions its downstream targets SOD1 and HO-1 (P < 0.05) to enhance cellular antioxidant capacity and reduce intracellular ROS production.@*CONCLUSION@#Fucoxanthin possesses strong inhibitory activities against diabetic cardiomyocyte hypertrophy and myocardial fibrosis and is capable of up-regulating Nrf2 signaling to promote the expression of its downstream antioxidant proteins SOD1 and HO-1 to reduce the level of ROS.

Hyperoside protects mouse spermatocytes GC-2 cells from oxidative damage by activating the Keap1/Nrf2/HO-1 pathway / 南方医科大学学报

OBJECTIVE@#To study the protective effect of hyperoside (Hyp) against ydrogen peroxide (H2O2)- induced oxidative damage in mouse spermatocytes GC-2 cells and explore the role of the Keap1/Nrf2/HO-1 pathway in this protective mechanism.@*METHODS@#GC-2 cells were treated with 2.5 mmol/L azaacetylcysteine (NAC), 50, 100, and 200 μmol/L hyperoside, or the culture medium for 48 h before exposure to H2O2 (150 μmol/L) for 2 h. CCK-8 assay was used to detect the changes in cell viability, and cell apoptosis was analyzed using flow cytometry. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) activity and malondialdehyde (MDA) in the culture medium. Western blotting and RT-qPCR were used to detect the protein and mRNA expression levels of nuclear factor erythroid 2-related factor2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), and heme oxygenase-1 (HO-1); the nuclear translocation of Nrf2 was detected using immunofluorescence assay.@*RESULTS@#Exposure to H2O2 significantly lowered the proliferation rate, reduced the activities of SOD, GSH and CAT, and obviously increased MDA content, cell apoptosis rate, and the expressions of Keap1 and Nrf2 mRNA and Keap1 protein in GC-2 cells (P < 0.05 or 0.01). Treatment of the cells prior to H2O2 exposure with either NAC or 200 μmol/L hyperoside significantly increased the cell proliferation rate, enhanced the activities of SOD, GSH-PX and CAT, and lowered MDA content and cell apoptosis rate (P < 0.05). Treatment with 200 μmol/L hyperoside significantly decreased the mRNA and protein expressions of Keap1 and increased the expressions of HO-1 mRNA and the protein expressions of Nrf2 and HO-1 (P < 0.05 or 0.01). Hyperoside also caused obvious nuclear translocation of Nrf2 in the cells (P < 0.05).@*CONCLUSION@#Hyperoside protects GC-2 cells against H2O2- induced oxidative damage possibly by activation of the Keap1/Nrf2/HO-1 signaling pathway.

Berberine alleviates programmed necrosis of metabolic-associated fatty liver disease via activating Nrf2 pathway in mice / 中华肝脏病杂志

Objective: To investigate the effect of berberine on programmed necrosis of hepatocytes induced by metabolic-associated fatty liver disease (MAFLD) in mice and its related molecular mechanism. Methods: Twenty male C57BL/6N mice were randomly divided into four groups (n=5 in each group): control group (S), fatty liver group (H), berberine group(B), nuclear factor erythroid 2-related factor 2 inhibitor group (Nrf2), and all-trans-retinoic acid (ATRA) group (A). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), triglycerides (TG), total cholesterol (TC), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) concentrations were detected at the end of week 12 to calculate fatty liver index (liver mass/body mass ratio). Liver tissue was stained with HE, Masson and Oil Red O, and SAF score was used to evaluate the degree of liver injury. The expression levels of hepatic programmed necrosis-related proteins, namely receptor-interacting protein kinase 3 (RIPK3), phosphorylated mixed series protease-like domain (p-MLKL) and Nrf2 were detected by Western blot method. One-way ANOVA was used for intragroup comparisons and LSD-t tests were used for intergroup comparisons. Results: Compared with S group, H group serum ALT, AST, LDH, TG, TC, TNF-α, IL-1β levels and fatty liver index were significantly increased. The liver tissue was filled with vacuolar-like changes and inflammatory cell infiltration. Numerous red lipid droplets were observed with oil red O staining. Collagen fiber hyperplasia was evident with Masson staining. SAF scores (6.60 ± 0.55 and 0.80 ± 0.45) were significantly increased. The expressions of RIPK3 and p-MLKL were up-regulated. Nrf2 level was relatively increased, and the differences were statistically significant (P < 0.05). Compared with H group, berberine intervention group liver biochemical indexes, lipid levels, pro-inflammatory mediator expression, fatty liver index, and SAF score were significantly reduced, and the expression of RIPK3 and p-MLKL were down-regulated, while Nrf2 levels were further increased, and the differences were statistically significant (P<0.05). Compared with B group, treatment with Nrf2 inhibitor had antagonized the protective effect of berberine on fatty liver. Serum ALT, AST, LDH, TG, TC and TNF-α, IL-1β levels, fatty liver index, and SAF scores were significantly increased and the expressions of RIPK3 and p-MLKL were relatively increased, and the differences were statistically significant (P < 0.05). Conclusion: Berberine can significantly improve the metabolic-associated fatty liver disease injury in mice, and its mechanism is related to activation of Nrf2 and inhibition of programmed necrosis of hepatocytes.

Four-Octyl itaconate ameliorates periodontal destruction via Nrf2-dependent antioxidant system / 国际口腔科学杂志·英文版

Periodontitis is a widespread oral disease characterized by continuous inflammation of the periodontal tissue and an irreversible alveolar bone loss, which eventually leads to tooth loss. Four-octyl itaconate (4-OI) is a cell-permeable itaconate derivative and has been recognized as a promising therapeutic target for the treatment of inflammatory diseases. Here, we explored, for the first time, the protective effect of 4-OI on inhibiting periodontal destruction, ameliorating local inflammation, and the underlying mechanism in periodontitis. Here we showed that 4-OI treatment ameliorates inflammation induced by lipopolysaccharide in the periodontal microenvironment. 4-OI can also significantly alleviate inflammation and alveolar bone loss via Nrf2 activation as observed on samples from experimental periodontitis in the C57BL/6 mice. This was further confirmed as silencing Nrf2 blocked the antioxidant effect of 4-OI by downregulating the expression of downstream antioxidant enzymes. Additionally, molecular docking simulation indicated the possible mechanism under Nrf2 activation. Also, in Nrf2-/- mice, 4-OI treatment did not protect against alveolar bone dysfunction due to induced periodontitis, which underlined the importance of the Nrf2 in 4-OI mediated periodontitis treatment. Our results indicated that 4-OI attenuates inflammation and oxidative stress via disassociation of KEAP1-Nrf2 and activation of Nrf2 signaling cascade. Taken together, local administration of 4-OI offers clinical potential to inhibit periodontal destruction, ameliorate local inflammation for more predictable periodontitis.

A Network Pharmacology-Based Study on Antidepressant Effect of Salicornia europaea L. Extract with Experimental Support in Chronic Unpredictable Mild Stress Model Mice / 中国结合医学杂志

OBJECTIVE@#To investigate the pharmacodynamic material basis, mechanism of actions and targeted diseases of Salicornia europaea L. (SE) based on the network pharmacology method, and to verify the antidepressant-like effect of the SE extract by pharmacological experiments.@*METHODS@#Retrieval tools including Chinese medicine (CM), PubMed, PharmMapper, MAS 3.0 and Cytoscape were used to search the components of SE, predict its targets and related therapeutic diseases, and construct the "Component-Target-Pathway" network of SE for central nervous system (CNS) diseases. Further, protein-protein interaction (PPI) network, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) function annotation of depression-related targets were analyzed to predict the antidepressant mechanism of SE. Chronic unpredictable mild stress (CUMS) model was used to construct a mouse model with depression-like symptoms. And the animals were randomly divided into 6 groups (n=10) including the normal group (nonstressed mice administered with distilled water), the CUMS group (CUMS mice administered with distilled water), the venlafaxine group (CUMS mice administered with venlafaxine 9.38 mg/kg), SE high-, medium-, and low-dose groups (CUMS mice administered with SE 1.8, 1.35 and 0.9 g/kg, respectively). Then some relevant indicators were determined for experimental verification by the forced swim test (FST), the tail suspension test (TST) and open-field test (OFT). Dopamine (DA) concentration in hippocampus and cerebral cortex, IL-2 and corticosterone (CORT) levels in blood, and nuclear factor E2 related factor 2 (Nrf2), kelch-like epichlorohydrin related protein 1 (Keap1), NAD(P) H dehydrogenase [quinone] 1 (NQO1) and heme oxygenase-1 (HO-1) levels in mice were measured by enzyme linked immunosorbent assay (ELISA) and Western blot respectively to explore the possible mechanisms.@*RESULTS@#The "target-disease" network diagram predicted by network pharmacology, showed that the potential target of SE involves a variety of CNS diseases, among which depression accounts for the majority. The experimental results showed that SE (1.8, 1.35 g/kg) significantly decreased the immobility period, compared with the CUMS group in FST and TST in mice after 3-week treatment, while SE exhibited no significant effect on exploratory behavior in OFT in mice. Compared with CUMS group, the SE group (0.9 g/kg) showed significant differences (P<0.05) in DA levels in the hippocampus and cerebral cortex. In addition, compared with CUMS control group, SE (1.8 g/kg) group showed a significant effect on decreasing the activities of CORT (P<0.05), and serum IL-2 level with no statistical significance. Finally, Western blot results showed that compared with the model group, Nrf2, Keap1, NQO1 and HO-1 protein expressions in SE group (1.8 g/kg) were up-regulated (all P<0.01).@*CONCLUSION@#The SE extract may have an antidepressant effect, which appeared to regulate Nrf2-ARE pathway and increased levels of DA and CORT in the hippocampus and cortex.

Sagittaria sagittifolia polysaccharides regulates Nrf2/HO-1 to relieve liver injury caused by multiple heavy metals in vivo and in vitro / 中国中药杂志

This study explored whether Sagittaria sagittifolia polysaccharides(SSP) activates the nuclear factor erythroid-2-related factor2(Nrf2)/heme oxygenase-1(HO-1) signaling pathway to protect against liver damage jointly induced by multiple heavy metals. First, based on the proportion of dietary intake of six heavy metals in rice available in Beijing market, a heavy metal mixture was prepared for inducing mouse liver injury and HepG2 cell injury. Forty male Kunming mice were divided into five groups: control group, model group, glutathione positive control group, and low-and high-dose SSP groups, with eight mice in each group. After 30 days of intragastric administration, the liver injury in mice was observed by HE staining. In the in vitro experiment, MTT assay was conducted to detect the effects of SSP at 0.25, 0.5, 1, and 2 mg·mL~(-1) on HepG2 cell survival at different time points. The content of alanine transaminase(ALT) and aspartate aminotransferase(AST) in the 48-h cell culture fluid was measured using micro-plate cultivation method, followed by the detection of the change in reactive oxygen species(ROS) content by flow cytometry. The mRNA expression levels of Nrf2 and HO-1 in cells were determined by RT-PCR, and their protein expression by Western blot. HE staining results showed that compared with the model group, the SSP administration groups exhibited significantly alleviated inflammatory cell infiltration and fatty infiltration in the liver, with better outcomes observed in the high-dose SSP group. In the in vitro MTT assay, compared with the model group, SSP at four concentrations all significantly increased the cell survival rate, decreased the ALT, AST, and ROS content(P<0.05), and down-regulated Nrf2 and HO-1 mRNA and protein expression(P<0.05). SSP significantly improves inflammatory infiltration in the liver tissue of mice exposed to a variety of heavy metals and corrects the liver fat degeneration, which may be related to its regulation of the Nrf2/HO-1 signaling pathway, reduction of ROS, and alleviation of oxidative damage.

Preparation and characterization of fenugreek leaf flavonoids and their protective effects against oxidative damage to hepatocytes / 中国中药杂志

The present study investigated the main components of fenugreek(Trigonella foenum-graecum L.) leaf flavonoids(FLFs) and their antioxidant activity. FLFs were prepared and enriched by solvent extraction, and the flavonoids were characterized by high-performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry(HPLC-Q-TOF-MS/MS). The protective effect of FLFs against H_2O_2-induced stress damage to L02 hepatocytes was also investigated. Firstly, the cell viability was measured by MTT assay. The oxidative stress injury model was induced by H_2O_2 in L02 cells. The release of lactate dehydrogenase(LDH), the content of reduced glutathione(GSH) and malondialdehyde(MDA), and the activities of superoxide dismutase(SOD) and catalase(CAT) were measured by assay kits. Hoechst fluorescence staining was performed to observe the cell apoptosis. The expression levels of c-Jun N-terminal kinase(JNK), extracellular signal-regulated kinase 1/2(ERK1/2), nuclear factor erythroid-2 related factor 2(Nrf2), heme oxygenase 1(HO-1), and their phosphorylated proteins were detected by Western blot. Based on the MS fragment ion information and data in databases, FLFs contained eight flavonoids with quercetin and kaempferol as the main aglycons. The cell viabi-lity assay revealed that as compared with the conditions in the H_2O_2 treatment group, 3.125-25 μg·mL~(-1) FLFs could increase the viability of L02 cells, reduce LDH release and MDA content in a dose-dependent manner, potentiate the activities of SOD, CAT, and GSH, decrease the phosphorylation of JNK and ERK1/2 proteins, and up-regulate the expression of Nrf2 and HO-1. The results of fluorescence staining showed that the nucleus of the H_2O_2 treatment group showed concentrated and dense strong blue fluorescence, while the blue fluorescence intensity of the FLFs group decreased significantly. FLFs showed a protective effect against H_2O_2-induced oxidative damage in L02 cells, and the underlying mechanism is associated with the enhancement of cell capability in clearing oxygen free radicals and the inhibition of apoptosis by the activation of the MAPKs/Nrf2/HO-1 signaling pathway. The antioxidant effect of fenugreek leaf is related to its rich flavonoids.

Mechanism of Tibetan medicine Ershiwuwei Shanhu Pills on scopolamine-induced learning and memory impairment in mice based on Keap1/Nrf2/HO-1 signaling pathway / 中国中药杂志

This study aims to investigate the mechanism of the Tibetan medicine Ershiwuwei Shanhu Pills(ESP) in improving scopolamine-induced learning and memory impairment in mice based on Keap1/Nrf2/HO-1 signaling pathway. ICR mice were randomized into blank group, model group, low-dose(200 mg·kg~(-1)), medium-dose(400 mg·kg~(-1)), and high-dose(800 mg·kg~(-1)) ESP groups, and donepezil hydrochloride group. The learning and memory impairment was induced in mice by intraperitoneal injection of scopola-mine. The learning and memory abilities of mice were detected by Morris water maze test, and the damage of hippocampal neurons and cortical neurons was detected based on Nissl staining. The expression of neuron specific nuclear protein(NeuN) in hippocampus and cortex of mice was determined by immunofluorescence assay, and the content of acetylcholine(Ach) and the activity of acetylcholines-terase(AchE) in hippocampus of mice by kits. Moreover, the content of superoxide dismutase(SOD), malondialdehyde(MDA), catalase(CAT), and total antioxidant capacity(T-AOC) in serum of mice was detected. The content of Kelch-like ECH-associated protein 1(Keap1), nuclear factor erythroid 2-related factor 2(Nrf2), and heme oxygenase 1(HO-1) in hippocampus was determined by Western blot. The results showed that there were significant differences in the trajectory map of mice among different groups in the behavioral experiment. Moreover, the latency of ESP groups decreased significantly compared with that in the model group. The hippocampal neurons in the high-dose ESP group were significantly more than those in the model group and the cortical neurons in the high-dose and medium-dose ESP groups were significantly more than those in the model group. The expression of NeuN in the model group was significantly decreased compared with that in the blank group, and the expression in the ESP groups was significantly higher than that in the model group. The AchE activity and MDA level were significantly decreased, and Ach content and levels of SOD, CAT, and T-AOC in the ESP groups were significantly increased in the ESP groups compared with those in the model group. The expression of Keap1 in the model group was significantly increased compared with that in the blank group, and the Keap1 expression increased insignificantly in ESP groups compared with that in the model group. The expression of Nrf2 and HO-1 was significantly lower in the model group than in the blank group, and the expression was significantly higher in the medium-dose ESP group than in the model group. In conclusion, ESP protected mice against the scopolamine-induced learning and memory impairment by regulating the Keap1/Nrf2/HO-1 signaling pathway.

Mechanism of Tibetan medicine Ershiwuwei Songshi Pills against liver injury induced by acetaminophen in mice based on Keap1/Nrf2 and TLR4/NF-κB p65 signaling pathways / 中国中药杂志

The present study investigated the mechanism of the Tibetan medicine Ershiwuwei Songshi Pills(ESP) against the liver injury induced by acetaminophen(APAP) in mice based on the kelch-like ECH-associated protein 1(Keap1)/nuclear transcription factor E2 related factor 2(Nrf2) and Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) p65 signaling pathways. Kunming mice were randomly divided into a blank control group, a model group, an N-acetyl-L-cysteine(NAC) group, and high-(400 mg·kg~(-1)), medium-(200 mg·kg~(-1)), and low-dose(100 mg·kg~(-1)) ESP groups. After 14 days of continuous administration, except for those in the control group, the mice were intraperitoneally injected with 200 mg·kg~(-1) APAP. After 12 h, the serum and liver tissues of mice were collected. Hematoxylin-eosin(HE) staining was performed on pathological sections of the liver, and the levels of aspartate aminotransferase(AST) and alanine aminotransferase(ALT) in the serum and the levels of glutathione(GSH), malondialdehyde(MDA), superoxide dismutase(SOD), catalase(CAT), myeloperoxidase(MPO), and total antioxidant capacity(T-AOC) in liver tissue homogenate were detected to observe and analyze the protective effect of ESP on APAP-induced liver injury in mice. The serum levels of tumor necrosis factor-alpha(TNF-α), interleukin-1 beta(IL-1β), and interleukin-6(IL-6) were determined by enzyme-linked immunosorbent assay(ELISA). The protein expression of Nrf2, Keap1, TLR4, and NF-κB p65 in the liver was determined by Western blot. Quantitative real-time was used to determine the mRNA expression of glutamate-cysteine ligase catalytic subunit(GCLC), glutamate-cysteine ligase regulatory subunit(GCLM), heme oxygenase-1(HO-1), and NAD(P)H dehydrogenase quinone 1(NQO-1) in the liver to explore the mechanism of ESP in improving APAP-induced liver damage in mice. As revealed by results, compared with the model group, the ESP groups showed improved liver pathological damage, decreased ALT and AST levels in the serum and MDA and MPO content in the liver, increased GSH, SOD, CAT, and T-AOC in the liver, reduced TNF-α and IL-6 levels in the serum, down-regulated expression of Keap1 in the liver cytoplasm and NF-κB p65 in the liver nucleus, up-regulated expression of Nrf2 in the liver nucleus, insignificant change in TLR4 expression, and elevated relative mRNA expression levels of antioxidant genes GCLC, GCLM, HO-1, and NQO-1. ESP can reduce the oxidative damage and inflammation caused by APAP, and the mechanism may be related to the Keap1/Nrf2 signaling pathway and the signal transduction factors on the TLR4/NF-κB p65 pathway.

Di'ao Xinxuekang alleviates non-alcoholic steatohepatitis in mice by up-regulating Nrf2/HO-1 signaling pathway / 中国中药杂志

The present study investigated the therapeutic effect and mechanism of Di'ao Xinxuekang(DXXK) on non-alcoholic steatohepatitis(NASH) in mice. Sixty-five C57 BL/6 J mice were randomly divided into a normal group and an experimental group for model induction with the high-fat diet for 16 weeks. Then the mice in the experimental group were randomly divided into a model group, an atorvastatin group(4 mg·kg~(-1)·d~(-1)), and high-(200 mg·kg~(-1)·d~(-1)), medium-(60 mg·kg~(-1)·d~(-1)), and low-dose(20 mg·kg~(-1)·d~(-1)) DXXK groups, with 10 mice in each group. Drugs were administered by gavage for eight weeks. Serum lipid, liver lipid, serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), malondialdehyde(MDA), superoxide dismutase(SOD), and glutathione reductase(GSH-Px) were determined. Interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) were measured by enzyme-linked immunosorbent assay(ELISA). The liver index was calculated. The liver pathological change and lipid accumulation were observed by HE and oil red O staining. The liver ultrastructure was observed by the transmission electron microscope. The mRNA and protein expression of nuclear factor-erythroid 2 related factor 2(Nrf2) and heme oxygenase-1(HO-1) was detected by real-time fluorescence-based quantitative PCR and Western blot, respectively. The results showed that compared with the normal group, the model group displayed serum lipid and liver lipid metabolism disorders, elevated transaminase, lipid deposition, steatosis, and inflammation, suggesting that the NASH model in mice was properly induced. Compared with the model group, the DXXK groups showed decreased serum lipid, liver lipid, ALT, AST, MDA, IL-1β, and TNF-α, increased SOD and GSH-Px, alleviated hepatic steatosis, ballooning, and inflammation, and up-regulated Nrf2 and HO-1 gene and protein expression. In conclusion, DXXK can significantly alleviate NASH in mice, which is related to the inhibition of oxidative stress and inflammatory damage by up-regulating the Nrf2/HO-1 signaling pathway.

Berberine inhibits erastin-induced ferroptosis of mouse hippocampal neuronal cells possibly by activating the Nrf2-HO-1/GPX4 pathway / 南方医科大学学报

OBJECTIVE@#To explore the mechanism by which berberine inhibits ferroptosis of mouse hippocampal neuronal cells (HT22).@*METHODS@#Cultured HT22 cells were pretreated with 30 or 60 μmol/L berberine for 2 h before exposure to 0.5 μmol/L erastin for 8 h, and the cell proliferation, intracellular ferric iron level, changes in intracellular reactive oxygen species (ROS) and cell apoptosis were detected using CCK-8, Fe2+ fluorescent probe, fluorescent dye (DAPI) and fluorescent probe (H2DCFH-DA). RT-qPCR and Western blotting were used to detect the mRNA and protein expressions of Nrf2, HO-1 and GPX4 in the cells. We further tested the effects of treatments with 2 μmol/L ML385 (a Nrf2 inhibitor), 60 μmol/L berberine and erastin in the cells to explore the protective mechanism of berberine against erastin-induced ferroptosis in the neuronal cells.@*RESULTS@#Treatment with 0.5 μmol/L erastin significantly lowered the viability of HT22 cells (P < 0.05) and increased the production of ROS, cell apoptosis rate and ferric iron level (P < 0.05). Pretreatment with 30 and 60 μmol/L berberine both significantly increased the vitality of erastin-exposed cells (P < 0.05) and lowered the levels of intracellular ROS and ferric iron content (P < 0.05). RT-qPCR and Western blotting showed that berberine obviously promoted the expressions of Nrf2, HO-1 and GPX4 in the cells (P < 0.05), and treatment with ML385 significantly inhibited the Nrf2-HO-1/GPX4 pathway, increased intracellular ROS and ferric iron contents and mitigated the protective effect of berberine against erastin-induced ferroptosis (P < 0.05).@*CONCLUSION@#Berberine can inhibit erastin-induced ferroptosis in HT22 cells possibly by activating the Nrf2-HO-1/ GPX4 pathway.

Effect of moxibustion on Nrf2/HO-1 signaling pathway in rats with diminished ovarian reserve / 中国针灸

OBJECTIVE@#To observe the effect of moxibustion on Nrf2/HO-1 signaling pathway in rats with diminished ovarian reserve (DOR), and to explore the protective mechanism of moxibustion on ovarian reserve function.@*METHODS@#Forty SD rats were randomly divided into a blank group, a model group, a moxibustion group and a hormone group, 10 rats in each group. The rats in the model group, moxibustion group and hormone group were treated with intragastric administration of tripterysium glycosides turbid liquid to prepare DOR model. The rats in the blank group were treated with intragastric administration of sodium chloride solution with the same volume, once a day for 14 days. The rats in the hormone group were treated with hormone sequential therapy for 14 days from the day of modeling; the rats in the moxibustion group were treated with moxibustion at bilateral "Shenshu" (BL 23) or "Guanyuan" (CV 4) and "Zhongwan" (CV 12) from the day of modeling, and the two groups acupoints were alternated every other day, 10 min each time, for 14 consecutive days. The estrus cycle was observed every day by vaginal exfoliated cell smear, and the estrus cycle disorder rate in each group was calculated. After the intervention, the HE staining was used to observe the histological morphology of ovaries; ELISA was used to detect the contents of follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E@*RESULTS@#Compared with the blank group, the rate of estrus cycle disorder in the model group was increased (@*CONCLUSION@#Moxibustion could reduce the rate of estrus cycle disorder, improve the level of serum sex hormones and antioxidant stress in DOR rats, and the mechanism may be related to the regulation of Nrf2/HO-1 signaling pathway.

Research advance of Nrf2 on atherosclerosis by regulating vascular smooth muscle cell / 浙江大学学报·医学版

Atherosclerosis is a common pathological change in cardiovascular disease. Vascular smooth muscle cell is the main source of plaque cell and extracellular matrix, and nuclear factor-erythroid 2-related factor 2 (Nrf2) is a key transcription factor regulating the function of vascular smooth muscle cell. In the process of atherosclerosis, Nrf2 signaling pathway has the following regulatory effects on vascular smooth muscle cell: regulating the phenotype of vascular smooth muscle cell to change to the direction conducive to the alleviation of disease progression; inhibiting the proliferation and migration of vascular smooth muscle cell; mitigating the level of blood lipid; alleviating vascular smooth muscle cell calcification, aging and apoptosis process. This article reviews the specific mechanisms of Nrf2 regulating atherosclerosis, such as phenotypic transformation, proliferation and migration, lipid metabolism, calcification, aging and apoptosis in atherosclerosis, in order to provide a basis for understanding the molecular mechanism of atherosclerosis development and finding therapeutic targets.

Effect of Nrf2/HO-1 signaling pathway in intestinal protection by Sishen Pills against ulcerative colitis in mice / 中国中药杂志

The present study aimed to explore the effect of nuclear factor erythroid 2 related factor 2(Nrf2)/heme oxygenase-1(HO-1) signaling pathway in intestinal protection by Sishen Pills against ulcerative colitis(UC). After the UC model was induced by 3% dextran sodium sulfate(DSS), experimental animals were randomly divided into control group, model group, salazosulfapyridine(SASP) group, and low-and high-dose Sishen Pills groups. Drug intervention(ig) was performed for seven consecutive days during modeling. On the 7 th day, the mice were euthanized. The body weight and colon length were recorded, and the histopathological changes of the colon were observed by HE staining. Serum interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), total antioxidant capacity(T-AOC), malondialdehyde(MDA), and reactive oxygen species(ROS) were detected by ELISA. The protein and mRNA expression of Nrf2, HO-1, and NADPH quinine oxidoreductase-1(NQO-1) was determined by Western blot and reverse transcription-polymerase chain reaction(RT-PCR). Compared with the normal group, the model group exhibited reduced body weight, colon length, and T-AOC, increased IL-6, TNF-α, MDA, and ROS, and diminished protein and mRNA expression of Nrf2, HO-1, and NQO-1 in the colon tissues. Compared with the model group, the SASP group and high-dose Sishen Pills group showed elevated body weight, colon length, and T-AOC, lowered IL-6, TNF-α, MDA, and ROS levels, and increased protein and mRNA expression of Nrf2, HO-1, and NQO-1 in the colon tissues. As assessed by HE staining, Sishen Pills could improve the pathological changes of the colon. The findings suggested that Sishen Pills could protect the colon against UC induced by 3% DSS. The specific mechanism of action may be related to the anti-inflammatory and anti-oxidative stress effects by the activation of the Nrf2/HO-1 signaling pathway.

Shenlian extract attenuates TNF-α-induced ECV304 injury by regulating Nrf2/Keap1 signaling pathway / 中国中药杂志

This study aimed to investigate the effect and the possible mechanism of Shenlian( SL) extract on tumor necrosis factor-α( TNF-α)-induced ECV304 injury. After the establishment of TNF-α-induced ECV304 cells injure model,MTT assay was used to detect cell viability and the level of reactive oxygen species( ROS) was measured by flow cytometry. The contents of superoxide dismutase( SOD),malondialdehyde( MDA),nitric oxide( NO),endothelin-1( ET-1) and interleukin-1β( IL-1β) in the supernatant were detected by biochemical method and enzyme linked immunosorbent assay( ELISA). The expression levels of apoptosis-related proteins B-lymphoma-2 gene( Bcl-2),Bcl-2 associated X protein( Bax),caspase-3,caspase-9 and nuclear factor E2 associated factor2( Nrf2)/Kelch like epichlorohydrin associated protein-1( Keap1) signaling pathway related proteins Nrf2,Keap1,quinone oxidoreductase( NQO1) and heme oxygenase 1( HO-1) were detected by Western blot. The results showed that 50 μg·L-1 TNF-α significantly damaged ECV304 cells,induced the impairment of cell viability( P<0. 01),the increase of ROS production,the decrease of SOD activity,and the increase of MDA,NO,ET-1 and IL-1β( P<0. 01),meanwhile,it caused the up-regulation of Keap1,caspase-9 and Bax protein expression,and down-regulation of NQO1 and Bcl-2 protein expression( P<0. 05) compared with the control group.Compared with the model group,SL extract reduced the damage of ECV304 cells induced by TNF-α,improved cell viability,reduced ROS production,increased SOD activity and decreased MDA,NO,ET-1,IL-1β content( P<0. 01 or P<0. 05). In addition,SL extract also down-regulated the protein expression levels of Keap1,caspase-3,caspase-9 and Bax,and increased the protein expressions of Nrf2,NQO1,HO-1 and Bcl-2( P<0. 01 or P<0. 05). The above results indicate that SL extract can provide protective effect on ECV304 cells injury induced by TNF-α,alleviate oxidative stress injury,inflammation and apoptosis,and its mechanism may be related to regulating Nrf2/Keap1 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