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ObjectiveTo explore the mechanism of plumbagin as a novel ferroptosis inducer in bladder cancer inhibition. MethodBladder cancer T24 cells were used in this study. The effect of different concentrations of plumbagin （0.1， 1， 2， 3， 6， 12， 24， 48 μmol·L-1） on the viability of T24 cells was detected by cell counting kit-8 （CCK-8）. The effect of different concentrations of plumbagin （1.5， 3， 6 μmol·L-1） on the apoptosis of T24 cells was detected by annexin V-fluorescein isothiocyanate （Annexin V FITC）/PI apoptosis kit. Different inhibitors （ferroptosis inhibitor Fer-1， apoptosis inhibitor VAD， and necroptosis inhibitor Nec-1） were used in combination with plumbagin （6 μmol·L-1）. Reactive oxygen species （ROS） fluorescent probe （DCFH-DA）， malonaldehyde （MDA）， and glutathione （GSH） kits were used to detect the effects of different concentrations of plumbagin （1.5， 3， 6 μmol·L-1） on the level of ROS and the content of MDA and GSH in T24 cells， respectively. The effect of different concentrations of plumbagin （1.5， 3， 6 μmol·L-1） on peroxide levels in T24 cells was detected by C11-BODIPY fluorescent probe. Western blot was used to detect the effect of different concentrations of plumbagin （1.5， 3， 6 μmol·L-1） on the protein expression of solute carrier family 7 member 11 （SLC7A11）， glutathione peroxidase 4 （GPX4）， nuclear factor E2-related factor-2 （Nrf-2）， and Kelch-like ECH-associated protein 1 （Keap1）. ResultCompared with the blank group， plumbagin could inhibit the activity of T24 cells （P<0.05） with IC50 of 3.52 μmol·L-1. At the concentrations of 1.5， 3， 6 μmol·L-1， plumbagin significantly promoted the apoptosis of T24 cells （P<0.05） as compared with the blank group. Compared with the plumbagin group at 6 μmol·L-1， the ferroptosis inhibitor and apoptosis inhibitor groups could reverse the inhibitory effect of 6 μmol·L-1 plumbagin on the proliferation of T24 cells （P<0.05）. Compared with the blank group， the plumbagin groups at 1.5， 3， 6 μmol·L-1 showed increased content of ROS， MDA， and lipid peroxides in T24 cells， decreased GSH level， and reduced SLC7A11， GPX4， and Nrf-2/Keap1 （P<0.05）. Conclusionplumbagin can induce ferroptosis， and its mechanism is related to the Nrf-2/Keap1 signaling pathway.
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The pathological manifestations of neurodegenerative diseases， such as Alzheimer's disease， Parkinson's disease， and multiple sclerosis， are abnormal protein aggregation and accumulation， microglia activation， and mitochondrial dysfunction， which eventually lead to the gradual loss of neuronal structure or function and deteriorate over time. These pathological processes are related to the production of reactive oxygen species （ROS）， which can cause oxidative stress and damage proteins， lipids， and DNA， leading to cell and tissue injuries. The Kelch-like ECH-associated protein 1 （Keap1）/nuclear factor E2-related factor 2 （Nrf2）/antioxidant response element （ARE） signaling pathway is the main mechanism to maintain the redox balance of the body and defend against oxidative stress injury. Nrf2 activates the expression of a series of antioxidant genes related to ARE through the dissociation of Keap1 and nuclear transfer in the cytoplasm to protect the body from oxidative damage. Therefore， the discovery and study of the Keap1/Nrf2/ARE signaling pathway activator is of great significance for the prevention and treatment of neurodegenerative diseases. Because of the remarkable biological activity and slight side effects， natural products are a treasure trove for new drug research and development. Studies have shown that a variety of natural products can activate the Keap1/Nrf2/ARE signaling pathway and play a neuroprotective role. According to the structural characteristics， natural products can be divided into flavonoids， terpenoids， volatile oils， polyphenols， and phenylpropanoids. This study summarized the underlying mechanism of the Keap1/Nrf2/ARE signaling pathway in regulating diseases and reviewed the research progress on natural products based on this signaling pathway in neuroprotection to provide references for the development of clinical drugs for the prevention and treatment of neurodegenerative diseases.
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Engineered probiotics can serve as therapeutics based on their ability of produce recombinant immune-stimulating properties. In this study, we built the recombinant Bacillus subtilis WB800 expressing antimicrobial peptide KR32 (WB800-KR32) using genetic engineering methods and investigated its protective effects of nuclear factor-E2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway activation in intestinal oxidative disturbance induced by enterotoxigenic Escherichia coli (ETEC) K88 in weaned piglets. Twenty-eight weaned piglets were randomly distributed into four treatment groups with seven replicates fed with a basal diet. The feed of the control group (CON) was infused with normal sterilized saline; meanwhile, the ETEC, ETEC+WB800, and ETEC+WB800-KR32 groups were orally administered normal sterilized saline, 5×1010 CFU (CFU: colony forming units) WB800, and 5×1010 CFU WB800-KR32, respectively, on Days 1‒14 and all infused with ETEC K88 1×1010 CFU on Days 15‒17. The results showed that pretreatment with WB800-KR32 attenuated ETEC-induced intestinal disturbance, improved the mucosal activity of antioxidant enzyme (catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx)) and decreased the content of malondialdehyde (MDA). More importantly, WB800-KR32 downregulated genes involved in antioxidant defense (GPx and SOD1). Interestingly, WB800-KR32 upregulated the protein expression of Nrf2 and downregulated the protein expression of Keap1 in the ileum. WB800-KR32 markedly changed the richness estimators (Ace and Chao) of gut microbiota and increased the abundance of Eubacterium_rectale_ATCC_33656 in the feces. The results suggested that WB800-KR32 may alleviate ETEC-induced intestinal oxidative injury through the Nrf2-Keap1 pathway, providing a new perspective for WB800-KR32 as potential therapeutics to regulate intestinal oxidative disturbance in ETEC K88 infection.
Subject(s)
Animals , Swine , Enterotoxigenic Escherichia coli , Kelch-Like ECH-Associated Protein 1 , Bacillus subtilis , NF-E2-Related Factor 2 , Antioxidants , Oxidative StressABSTRACT
Oxidative stress(OS)is a major reason for body damage. Studies have shown that a variety of factors, such as ischemia and hypoxia, excessive light and hyperglycemia can cause the increase of reactive oxygen species and free radicals in the retina, thus inducing OS, damaging retina and affecting the normal visual function. Kelch-like ECH-associated protein 1(KEAP1)and nuclear factor erythroid 2 related factor 2(NRF2), which together constitute the main antioxidant stress signaling pathway in the body, play an antioxidant role by regulating retinal energy metabolism and cell proliferation, apoptosis and autophagy through various ways, so as to reduce retinal damage caused by OS. In this paper, the role and mechanism of the KEAP1-NRF2 signaling pathway regulation of OS in the retinal are briefly reviewed, aiming to provide ideas for subsequent research.
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Oxidative stress plays a crucial role in the emergence of numerous neurodegenerative diseases, with protein accumulation and mitochondrial damage, which result in neurological disorders. To minimize oxidative stress, several defensive mechanisms protect nerve cells by releasing antioxidants such as nuclear erythroid factor2 (Nrf2)-Kelch-like ECH-associated protein1 (Keap1) signaling pathway activation has been proved to be a prospective treatment to reduce oxidative stress and neuroinflammation for protection of neurons in a variety of neurological disorders. In this review, we focus beneficial role of Nrf2 in Alzheimer’s and Parkinson’s diseases. Nrf2 is proved to be a master regulator of antioxidants by releasing over 250 cytoprotective genes aimed at oxidative stress and neuroinflammation. In animal studies Nrf2 activation is proved to improve autophagy, mitochondrial biogenesis, and Suppression of inflammatory cytokinin which protects neuronal cells and inhibit progressive neurodegeneration.
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ObjectiveTo study the effect of Buyang Huanwutang on Kelch-like Ech-related protein 1 （Keap1）/nuclear factor E2-related factor 2 （Nrf2）/heme oxygenase-1 （HO-1） antioxidant signaling pathway in rats with idiopathic pulmonary fibrosis （IPF） and explore the mechanism of this prescription in the treatment of IPF. MethodForty SPF-grade male SD rats were assigned into a sham operation group， a model group， a Buyang Huanwutang group， and a nintedanib group according to random number table method， with 10 rats in each group. IPF rat model was established by intratracheal infusion of bleomycin （0.005 g·kg-1） in other groups except the sham operation group. Buyang Huanwutang group was administrated with Buyang Huanwutang (14.84 g·kg-1)，intragastric administration of nitedanib suspension (0.1 g·kg-1)，sham operation group and model group were given equal volume of normal saline， for 28 days. After lung function test， serum and lung tissue samples were collected. Hematoxylin-eosin （HE） staining and Masson trichrome staining were employed to observe the pathological changes of the lung tissue. The content of hydroxyproline （HYP） in lung tissue was detected. The levels of malondialdehyde （MDA） in serum and lung tissue， and the activities of superoxide dismutase （SOD）， glutathione peroxidase （GSH-Px） and catalase （CAT） were determined. The mRNA and protein levels of Keap1， Nrf2， and HO-1 was determined by Real-time fluorescent quantitative polymerase chain reaction， immunohistochemical staining， and Western blot. ResultCompared with the sham operation group， the modeling increased the resistance and elasticity and decreased the compliance of respiratory system （P<0.01）， elevated the lung index， pathological score， and HYP content in lung tissue （P<0.01）， and enriched MDA in serum and lung tissue， while it decreased the activities of SOD， GSH-Px， and CAT （P<0.01）. Furthermore， the modeling down-regulated the mRNA and protein levels of Keap1 and up-regulated those of Nrf2 and HO-1 in lung tissue （P<0.01）. Compared with the model group， Buyang Huanwutang decreased the resistance and elasticity and increased the compliance of respiratory system （P<0.01）， lowered the lung index， pathological score， and HYP content in lung tissue （P<0.01）， and reduced MDA in serum and lung tissue， while it increased the activities of SOD， GSH-Px， and CAT （P<0.01）. Additionally， Buyang Huanwutang down-regulated the expression of Keap1 and up-regulated that of Nrf2 and HO-1 in lung tissue （P<0.05， P<0.01）. ConclusionBuyang Huanwutang can activate Keap1/Nrf2/HO-1 signaling pathway to enhance the antioxidant capacity and slow down the pathological process of IPF in rats.
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ObjectiveTo study the protective effect of total flavonoids of lavender on skin photoaging induced by ultraviolet B （UVB） in mice and to explore its mechanism from the perspective of nuclear factor E2-related factor 2 （Nrf2） antioxidant pathway. MethodEighty-four female KM mice were randomly divided into seven groups， namely blank group， model group， solvent group， vitamin E （0.013 g·kg-1） group， as well as low-， middle-， and high-dose （0.25， 1.25， 2.50 g·kg-1） groups of total flavonoids of lavender. The naked skin on the back of mice was irradiated with UVB for inducing optical damage. Thirty minutes before irradiation， the skin was coated with the total flavonoids of lavender. After continuous irradiation for one week， the skin moisture and elasticity on the back of mice were evaluated， and the effects of total flavonoids of lavender on histopathological changes in mouse skin were investigated by hematoxylin-eosin （HE） and Van Gieson （VG） staining. The levels of malondialdehyde （MDA）， superoxide dismutase （SOD）， total antioxidant capacity （T-AOC）， nitric oxide synthase （NOS）， and glutathione peroxidase （GSH-Px） after skin homogenization were detected by colorimetry， the inflammatory factors interleukin-1 （IL-1）， interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in skin tissue by enzyme-linked immunosorbent assay （ELISA）， and the mRNA expression levels of Nrf2， Kelch-like epichlorohydrin-associated protein 1 （Keap1）， BTB-CNC homology 1 （Bach1）， heme oxygenase-1 （HO-1）， quinone oxidoreductase 1 （NQO1）， and glutamate-cysteine ligase catalytic subunit （GCLC） by real-time polymerase chain reaction （Real-time PCR）. ResultCompared with the blank group， the model group exhibited significantly increased appearance score （P<0.01）， reduced skin moisture and elasticity （P<0.01）， pronounced pathological changes in the skin tissue like epidermal thickening， scabbing， small abscess， and severe injury， elevated MDA， NOS， IL-1， IL-6 and TNF-α （P<0.05， P<0.01）， lowered SOD， T-AOC， Nrf2， Keap1， NQO1 and GCLC mRNA expression （P<0.05，P<0.01）， and up-regulated Bach1 mRNA expression （P<0.01）. Compared with the model group， the total flavonoids of lavender at the low， middle， and high doses all remarkably reduced the appearance score （P<0.01）， enhanced the skin moisture and elasticity （P<0.01）， diminished the MDA， NOS， IL-1， IL-6， and TNF-α （P<0.05， P<0.01）， increased SOD， T-AOC， Nrf2， Keap1， NQO1， HO-1 and GCLC mRNA expression （P<0.05， P<0.01）， and down-regulated the expression of Bach1 mRNA （P<0.01）. ConclusionThe protective effect of the total flavonoids of lavender against skin photoaging in mice is significant， which may be related to its activation of Keap1/Nrf2/ARE signaling pathway， regulation of oxidative stress， and improvement of inflammatory response.
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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.
Subject(s)
Animals , Rats , Antioxidants/metabolism , Atrial Natriuretic Factor/pharmacology , Cardiomegaly , Diabetes Mellitus, Experimental/metabolism , Fibrosis , Kelch-Like ECH-Associated Protein 1/metabolism , Metformin , NF-E2-Related Factor 2/metabolism , Oxidative Stress , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolism , Superoxide Dismutase-1/pharmacology , XanthophyllsABSTRACT
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.
Subject(s)
Animals , Male , Mice , Antioxidants/metabolism , Heme Oxygenase-1/metabolism , Hydrogen Peroxide/pharmacology , Kelch-Like ECH-Associated Protein 1/metabolism , NF-E2-Related Factor 2/metabolism , Oxidative Stress , Quercetin/analogs & derivatives , RNA, Messenger/metabolism , Spermatocytes/metabolism , Superoxide Dismutase/metabolismABSTRACT
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.
Subject(s)
Animals , Mice , Kelch-Like ECH-Associated Protein 1/metabolism , Medicine, Tibetan Traditional , Mice, Inbred ICR , NF-E2-Related Factor 2/metabolism , Oxidative Stress , Plant Extracts , Scopolamine/adverse effects , Signal Transduction , Superoxide Dismutase/metabolismABSTRACT
Modern pharmacological studies have shown that Cistanche deserticola (C. deserticola) has a protective effect on the liver, but its active fraction and mechanism are not clear. In order to identify the effective fraction of C. deserticola Y. C. Ma, an acute alcoholic liver injury model in mice was established with 56-proof Erguotou and different fractional extracts of C. deserticola Y. C. Ma (total glycosides, polysaccharides, and oligosaccharides) were administered. After 14 days of oral administration, liver pathology and lipid deposition were measured and the expression of nuclear factor E2-related factor (Nrf-2), kelch-like ECH-associated protein-1 (Keap-1), and plasmalemma vesicle-associated protein-1 (PV1) were measured by immunofluorescence. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), endotoxin (ET), diamine oxidase (DAO), and D-lactic acid (D-LA) in serum, and superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and malondialdehyde (MDA) in liver were measured by ELISA. All animal experiments were carried out with approval of the Experimental Animal Welfare Ethics Committee of the Peking University Health Science Center. The results show that the total glycosides of C. deserticola Y. C. Ma (400 mg·kg-1) could decrease liver pathology, decrease serum endotoxin, diamine oxidase, and D-lactic acid, and reduce hepatic lipid deposition. Total glycosides also promoted Nrf-2 transfer into the nucleus and decreased the expression of Keap-1 and PV1. In summary, the total glycosides of C. deserticola Y. C. Ma had a protective effect in acute alcoholic liver injury and the mechanism may be related to the activation of the Nrf-2/Keap-1 pathway, improvement of intestinal wall integrity, and inhibition of the transport of harmful substances into the liver.
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Objective:To explore the possible mechanism of Kaixinsan in improving cognitive impairment in Alzheimer's disease （AD） model rats based on the epichlorohydrin associated protein-1 （Keap-1）/nuclear factor E2 related factor （Nrf2）/manganese superoxide dismutase （MnSOD） signaling pathway. Method:The AD model was established by injecting Amyloid <italic>β</italic><sub>1-42</sub> （A<italic>β</italic><sub>1-42</sub>， 5 μL） into the lateral ventricle. After modeling， the experimental rats were randomly divided into model group， donepezil group， and Kaixinsan low dose， medium dose and high dose groups. Another normal control group was also established. The donepezil group received donepezil tablets （1.8 g·kg<sup>-1</sup>·d<sup>-1</sup>）， Kaixinsan low dose， medium dose and high dose groups received corresponding doses of Kaixinsan （10， 20， 40 g·kg<sup>-1</sup>·d<sup>-1</sup>， respectively）， and the normal control group and model group were given with equal volume of pure water. Morris water maze was used to test the learning and memory ability of rats. The pathological morphology of hippocampal CA3 area was observed by Nissl staining. The expression levels of myeloperoxidase （MPO）， inducible nitric oxide synthase （iNOS） and superoxide dismutase （SOD） in serum were detected by colorimetry， and the protein expression levels of Keap-1， Nrf2 and MnSOD in hippocampus were detected by immunohistochemistry （IHC） and Western bolt. Result:Compared with the normal control group， the escape latency， total swimming distance and first arrival time of the plateau in the model group increased （<italic>P</italic><0.01）， while the times of crossing the plateau and the time in target quadrant decreased （<italic>P</italic><0.01）. Compared with the model group， the rats in donepezil group and Kaixinsan groups showed less latency， lower total swimming distance and first arrival time on the platform （<italic>P</italic><0.05， <italic>P</italic><0.01）， while the times of crossing the platform and time in target quadrant increased （<italic>P</italic><0.05， <italic>P</italic><0.01）. Compared with the normal control group， the expression levels of MPO and iNOS in serum of the model group increased （<italic>P</italic><0.01）， while the expression levels of SOD decreased （<italic>P</italic><0.01）. Compared with model group， the expression of MPO and iNOS in serum of donepezil group and Kaixinsan groups decreased （<italic>P</italic><0.05， <italic>P</italic><0.01）， while the expression of SOD increased （<italic>P</italic><0.05， <italic>P</italic><0.01）. In the normal control group， the neurons in the hippocampal CA3 of the rats were arranged neatly， without obvious Nissl body shrinkage. The neurons in the CA3 of the hippocampus of the model group were not arranged neatly， with obvious neuron loss and pyknosis of Nissl body. The neurons in the CA3 of the hippocampus of the rats in the donepezil group and Kaixinsan groups were arranged neatly， with increased number of neurons and decreased Nissl body shrinkage. Compared with the normal control group， the integrated optical density （<italic>IA</italic>） and protein level of Keap-1 in the hippocampus of the model group decreased（<italic>P</italic><0.01）， while the <italic>IA</italic> and protein level of Nrf2 and MnSOD increased （<italic>P</italic><0.01）. Compared with model group， <italic>IA</italic> and protein levels of Keap-1 and MnSOD in hippocampus of rats in donepezil group and Kaixinsan groups increased （<italic>P</italic><0.05， <italic>P</italic><0.01）， while <italic>IA</italic> and protein levels of Nrf2 decreased （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:Kaixinsan could alleviate memory impairment in AD rats， and its mechanism may be related to its regulation of Keap-1/Nrf2/MnSOD signaling pathway.
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Objective:To explore the mechanism of Banxia Xiexintang （BXXX） in preventing and treating chronic atrophic gastritis （CAG） through Kelch-like ECH-associated protein 1 （Keap1）/nuclear factor erythroid 2-related factor 2 （Nrf2）/antioxidant response element （ARE） signaling pathway. Method:SD rats were divided into a normal group （<italic>n</italic>=12） and an experimental group for CAG model induction. The model rats were then randomly divided into a model group， a vatacoenayme （VG） group （60 mg·kg<sup>-1</sup>）， and high- （280 mg·kg<sup>-1</sup>）， medium- （140 mg·kg<sup>-1</sup>）， and low-dose （70 mg·kg<sup>-1</sup>） BXXX groups. The doses in the BXXX groups were equivalent to 28， 14， and 7 g·kg<sup>-1</sup> crude drugs. The rats in the normal group and the model group received distilled water at an equal volume， and those in the VG group and the BXXX groups were treated correspondingly by gavage. After 12 weeks of treatment， hematoxylin-eosin （HE） staining was carried out to observe pathological changes in the gastric mucosa of CAG rats. Western blot and real-time fluorescence-based quantitative PCR was used to detect the protein and mRNA expression levels of Nrf2， glutathione S-transferase （GST）， and NAD （P）H：quinone oxidoreductase 1 （NQO1） in the gastric mucosa of CAG rats. Result:Compared with the normal group， the model group showed increased protein and mRNA expression levels of Nrf2， NQO1， and GST in the gastric mucosa of the rats （<italic>P</italic><0.05）， atrophic gastric mucosa， and even intestinal metaplasia. The protein and mRNA expression levels of Nrf2， NQO1， and GST in the VG group and the high- and medium-dose BXXX groups were lower than those in the model group （<italic>P</italic><0.05）， and gastric mucosa atrophy and intestinal metaplasia were significantly improved， especially in the high-dose BXXX group. However， the effect in the low-dose BXXX group was not significant. Conclusion:BXXX can blunt the transcriptional activity of Nrf2， shut down Nrf2 signaling pathway， and reduce the expression levels of NQO1 and GST to achieve normal oxidation-anti-oxidation balance， which may be one of its action mechanisms in the treatment of CAG.
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Objective:To confirm the protective effect of Xiangsha Yuyang decoction on acetic acid-induced gastric ulcer model rats and explore its mechanism， so as to provide experimental basis for clinical drug use. Method:The 60 SPF Wistar rats were randomly divided into 6 groups： group， model group， high， middle and low dose groups of Xiangsha Yuyang decoction and omeprazole control group. The rat model of gastric ulcer was induced by acetic acid. The rats in the high， middle and low dose groups of Xiangsha Yuyang decoction were intragastrically administered at the dose of 28，14，7 g·kg<sup>-1</sup>， and with omeprazole at the dose of 4.17 g·kg<sup>-1</sup>in normal saline， respectively. The rats in the blank group and model group were intragastrically infused with the same volume of normal saline once a day. After 14 days of continuous treatment， the rats were killed， the blood was collected， the area and inhibition rate of gastric ulcer were measured and calculated， the histopathological sections of gastric mucosa were made and the state of gastric mucosal injury was observed， and the changes of gastric mucosal repair factor， gastric tissue related protein， oxidative stress factor and inflammatory factor in serum were detected by enzyme-linked immunosorbent assay（ELISA）. Detected the expression of p62 Kelch-like epichlorohydrin-related protein 1 （Keap1）， nuclear transcription factor E2 related factor 2 （Nrf2） and heme oxygenase-1 （HO-1） signal pathway-related proteins in gastric mucosa by Western blot. Result:Compared with control group， the gastric mucosa of the model group showed obvious pathological changes and a large number of leukocytes infiltrated. In model group， the ulcer area was significantly increased（<italic>P</italic><0.01）， the contents of mucin mucoprotein 5AC （MUC5AC）， epidermal growth factor （EGF）， superoxide dismutase （SOD） and increased prostaglandin E<sub>2</sub> （PGE<sub>2</sub>） were significantly decreased（<italic>P</italic><0.01）， the gastrin （GAS）， 8-hydroxydeoxyguanosine （8-OHdG）， malondialdehyde （MDA）， tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>）， cyclooxygenase-2 （COX-2） were significantly increased. The expression of HO-1 and Nrf2 protein decreased significantly（<italic>P</italic><0.01）， the content of Keap1 increased significantly （<italic>P</italic><0.05）， and the expression of p62 protein decreased. Compared with model group， the hierarchical structure of cells in Xiangsha Yuyang decoction high dose group and omeprazole group were clearer and regular， middle and low dose groups could also repair gastric mucosa to a certain extent. The high and middle dose groups of Xiangsha Yuyang decoction could significantly reduce the gastric ulcer area of acetic acid-induced gastric ulcer rat model （<italic>P</italic><0.01） and increase the ulcer inhibition rate. It can effectively promote the expression of MUC5AC and EGF in gastric mucosa， decrease the level of GAS（<italic>P</italic><0.05，<italic>P</italic><0.01）， decrease the level of 8-OHdG and MDA， increase the activity of SOD（<italic>P</italic><0.01）， decrease the expression level of TNF-<italic>α</italic> and COX-2， increase the content of PGE<sub>2</sub>， and significantly increase the amount of Nrf2 and HO-1 protein in gastric mucosa（<italic>P</italic><0.01）. The high dose group of Xiangsha Yuyang decoction could decrease the protein expression of Keap1（<italic>P</italic><0.05） and increase the expression of p62 protein. Conclusion:Xiangsha Yuyang decoction is effective in the treatment of acetic acid-induced gastric ulcer model rats， which can effectively reduce the ulcer area， increase the ulcer inhibition rate and protect the ulcer tissue. Its mechanism may be related to activating p62/Keap1/Nrf2 signal pathway and regulating the expression of related genes so as to improve inflammatory response and regulate oxidative stress response.
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BACKGROUND: N-Acetyl-L-cysteine is a precursor of glutathione, which can directly scavenge oxygen free radicals. However, it is not entirely clear whether N-acetyl-L-cysteine can alleviate smoking-induced lung injury by reducing oxidative stress. OBJECTIVE: To investigate the effects of N-acetylcysteine on oxidative stress in lung tissue of smoked rats, and to clarify its possible mechanism of action. METHODS: Thirty male rats were randomly divided into control, smoked and smoked+N-acetylcysteine groups. The rats in the smoked and smoked+N-acetylcysteine groups were placed in the passive smoking animal exposure system, with smoking of 20 cigarettes, twice/d, 1 hour/times, for 8 consecutive weeks. The rats in the smoked+N-acetylcysteine group were given 200 mg/kg N-acetylcysteine via gavage before passive smoking daily, for 8 consecutive weeks. The control rats were fed normally for 8 consecutive weeks. The study was approved by the Laboratory Animal Ethical Committee of Shenyang Medical College in October 2018, approval No. (2018)85. RESULTS AND CONCLUSION: (1) Pathological observation showed that the pulmonary tissue was disordered, alveolar septal thickening, inflammatory cell infiltration and interstitial fibrosis in the smoking group, while the pulmonary hemorrhage, the number of inflammatory cells and the degree of fibrosis in the smoking+N-acetylcysteine group were significantly reduced. (2) Compared with the control group, the expression levels of malondialdehyde-5 and inositol-alpha genes were significantly increased, and superoxide dismutase-1 gene expression was significantly decreased in the smoked group, while N-acetylcysteine could inhibit the above changes. (3) Immunofluorescence staining and western blot assay results revealed that the expression levels of malondialdehyde-5 and inositol-alpha in lung tissue of rats in the smoked+N-acetylcysteine group was significantly lower than those in the smoked group, and the expression level of superoxide dismutase-1 was significantly higher than that in the smoked group. In addition, the expression levels of Nrf2 and Keap1 mRNA and protein were significantly increased, and the expression levels of Bach1 mRNA and protein were significantly decreased in the smoking+N-acetylcysteine group. (4) These results suggest that N-acetylcysteine can protect smoking-induced lung injury by reducing oxidative stress, and which might be through activating Nrf2/Keap1 signaling pathway.
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BACKGROUND: Oxidative stress plays an important role in femoral head necrosis. Platelet-rich plasma (PRP) contains growth factors that can accelerate fracture healing. PRP combined with core decompression can promote recovery from non-traumatic femoral head necrosis. OBJECTIVE: To investigate whether PRP combined with core decompression can inhibit oxidative stress in steroid-induced avascular necrosis of the femoral head model via Keap1/Nrf2/HO-1 signaling pathway. METHODS: Forty New Zealand rabbits were randomly divided into normal group, model group, control group and PRP group, with 10 rabbits in each group. In the model and PRP groups, a model of steroid-induced femoral head necrosis was established in a sterile environment. At 4 weeks after operation, the rabbits in the PRP group were injected with 0.4 mL of 3% PRP after core decompression. The control group received core decompression treatment, and the control and model groups were raised normally. After 14 weeks, the experimental animals were sacrificed. Hematoxylin-eosin staining was used to observe the pathological changes of bone marrow cavity and the vacancy rate of bone lacunae in the femoral head of each group. Total antioxidant capacity, superoxide dismutase, glutathione peroxidase, reduced glutathione, and malondialdehyde were detected. TUNEL was used to detect bone cell apoptosis in the femoral head. Immunofluorescence staining was used to determine the distribution of Keapl and Nrf2. Western blot was used to measure Keapl, Nrf2, and HO-1 protein expression in the femoral head. Approval was obtained from the Animal Ethics Committee of the Affiliated Hospital of Qinghai University, approval No. qhdx-201908374. RESULTS AND CONCLUSION: (1) Compared with the normal group, the trabecular bone in model group was thinned with structure disorder. Compared with the model group, the trabecular bone structure in control group was restored, and the number of vacant bone lacunae was reduced (P 0.05). (2) The total antioxidant capacity and serum levels of superoxide dismutase, glutathione peroxidase, and reduced glutathione in the model group were significantly lower than those in normal animals (P 0.05), while these indexes were significantly improved in the PRP group than the model and control groups (P < 0.05). (3) The expression of Keapl in the model group was significantly lower than that of the normal group (P < 0.05), and the expression of Nrf2 and HO-1 protein was significantly higher than that of the normal group (P < 0.05). The expression of Keapl in the PRP group was lower than that of the model and control groups (P < 0.05), and the expression of Nrf2 and HO-1 was significantly higher than that of the model and control groups (P < 0.05). Therefore, PRP can effectively inhibit oxidative stress in the process of steroid-induced femoral head necrosis, which may be caused by activating the Keapl/Nrf2/HO-1 signaling pathway.
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Objective::To study whether long-term administration of Gastrodiae Rhizoma powder can improve the learning and memory ability of APPswe/PSldE9 double transgenic (APP/PS1) Alzheimer' s disease(AD) model mice and delay the progress of AD whether these effects are related to the regulation of antioxidant stress pathway in Kelch-like epoxylopropylamine-related protein 1(Keap1)-nuclear factor E2 related factor 2 (Nrf2)/heme oxygenase(HO)-1, and further explore the neuroprotective mechanism of Gastrodiae Rhizoma powder and its role in the prevention and treatment of AD. Method::APP/PS1 double transgenic mice model, the mice consisted of five groups: normal, normal administration group, model group, Gastrodiae Rhizoma powder prevention group, Gastrodiae Rhizoma powder treatment group.The mice in the normal administration group and the Gastrodiae Rhizoma powder prevention group were given the same dose of Gastrodiae Rhizoma powder (1.5 g·kg-1) daily at the age of 8 weeks.The normal group and model group were given the same amount of normal saline at the same time, until 24 weeks old, Morris water maze was used to test the learning and memory ability of mice, and the treatment group was treated with Gastrodiae Rhizoma powder at 22 weeks old.The mice were given the same dose of Gastrodiae Rhizoma powder (1.5 g·kg-1) every day for 2 weeks.The number of crossing platform, escape latency and platform residence time of mice were detected by Morris water maze from 24 weeks old to 24 weeks old.RNA, Real-time PCR was extracted from mouse hippocampus to detect the mRNA level of Nrf2, HO-1, Keap1, and Western blot was used to detect the expression of Nrf2, HO-1, Keap1 protein in mouse hippocampus. Result::Compared with normal group, the water maze test showed that the learning and memory ability of model group was lower than that of the model group (P<0.01), and the learning and memory ability of Gastrodiae Rhizoma powder prevention group and Gastrodiae Rhizoma powder treatment group was significantly higher than that of model group (P<0.01). Compared with normal group, the levels of Nrf2, HO-1 and protein in the hippocampus in model group decreased in varying degrees (P<0.05). Compared with model group, Gastrodiae Rhizoma powder prevented Nrf2, in the hippocampus of mice in model group.The level of HO-1 in mRNA and protein increased in different degrees (P<0.05, P<0.01). Levels of Nrf2, HO-1 mRNA in Gastrodiae Rhizoma powder treatment group was significantly higher than that in Gastrodiae Rhizoma powder group (P<0.05). There was no significant difference in the expression of Nrf2, HO-1 protein.There was no significant difference in mRNA and protein levels of Keap1 among different groups. Conclusion::Morris water maze test and other results showed that Gastrodiae Rhizoma powder could improve the learning and memory ability of APP/PS1 mice, and it may enhance the expression of downstream antioxidant genes by regulating Keap1-Nrf2/HO-1 pathway.And then improve the learning and memory ability of APP/PS1 mice.
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Objective:To investigate the protective effect of Wutou Chishizhi Wan on myocardial ischemia reperfusion injury （MIRI） in rats， and observe its effect on such mechanisms as coagulation function， vascular endothelial cells and oxidative stress in rats. Method:A total of 40 SD rats were randomly divided into normal group， model group， positive drug group （Urokinase group） and Wutou Chishizhi Wan group， with 10 rats in each group. Except for the normal group， rat myocardial ischemia-reperfusion injury models were established. The changes of heart rate （HR） at 10 min before ischemia， 30 min after ischemia and 30， 60， 120 min （T0，T1，T2，T3，T4）， and the change of electrocardiogram （ECG） J point after modeling in rats were observed. The pathological changes of rat myocardial tissue were observed by hematoxylin-eosin （HE） staining. The changes of four indexes of coagulation ［prothrombin time （PT）， activated partial thromboplastin time （APTT）， thrombin time （TT）， fibrinogen content decreased significantly （FIB）］ in rats were observed. The contents of endothelin-1 （ET-1）， thromboxane A2 （TXA2） and prostacyclin （PGI2） in serum and myocardium levels of superoxide dismutase （SOD）， malondialdehyde （MDA） and glutathione peroxidase （GSH-Px） of MIRI rats were observed. Western blot assay was used for the detection of oxidative stress protein Keap1 and transcription factor-E2-related factor （Nrf2） expression levels in rat myocardial tissue. Result:Compared with the normal group， the ECG of MIRI rats showed significant myocardial ischemic injury-like changes， ST segment was significantly elevated， J point was significantly increased， and the incidences of HR in T1， T2， T3 and T4 were significantly reduced （P<0.05， P<0.01）. Compared with the model group， Wutou Chishizhi Wan significantly reduced ECG J-point changes in MIRI rats， while increased the incidence of HR in T1， T2， T3 and T4 （P<0.05， P<0.01）. Compared with the normal group， PT， APTT and TT in the model group were significantly shortened （P<0.01）， FIB content was significantly increased （P<0.01）， and the serum PGI2 level decreased and TXA2 and ET-1 levels increased significantly in the model group （P<0.01）. SOD content and GSH-Px activities of myocardial tissue in the model group were significantly reduced （P<0.01）， whereas the MDA content was increased （P<0.01）. Compared with the model group， PT of the Wutou Chishizhi Wan group was prolonged （P<0.05） and APTT slightly prolonged， TT significantly prolonged （P<0.01）， FIB content decreased （P<0.05）， serum PGI2 increased （P<0.05）， TXA2 and ET-1 decreased significantly in the Wutou Chishizhi Wan group （P<0.01）， myocardial MDA content decreased， and SOD content and GSP-Px activity increased significantly （P<0.01）. Meanwhile， the Wutou Chishizhi Wan group was able to activate the Keap1/Nrf2 signaling pathway， which significantly increased Nrf2 expression and significantly decreased Keap1 expression （P<0.01）. Conclusion:Wutou Chishizhi Wan group can protect myocardial injury in MIRI rats. The specific mechanism is to protect MIRI by regulating vascular endothelial cell homeostasis and oxidative stress levels and activating Keap1/Nrf2 signaling pathway.
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@#AIM: To investigate the protective effect and mechanism of quercetin on age-related macular degeneration in mice through Nrf2/Keap1/ARE pathway. <p>METHODS: Kunming mice were used as research objects, which were divided into control group, model group and quercetin group. Fundus examination was showed whether yellow-white like glassy sputum substances appeared in the fundus of each group of mice; OCT was used to examine the retinal thickness of each group of mice; HE staining was used to observe the changes of retinal morphology in each group of mice; FFA was observed the fundus vascular integrity of each group of mice. The activities of SOD, GSH-Px, CAT and the contents of ROS and MDA in serum were detected by ELISA; Western blot was used to detect the expression of Nrf2/Keap1/ARE related proteins in the retina of each group. <p>RESULTS: Quercetin can reduce the yellow and white glassy wart substance in the fundus of mice and increase the thickness of the retina(<i>P</i><0.05), and the points of retinal vascular leakage is significantly reduced. Compared with the model group, the a-wave amplitude and b-wave amplitude of the quercetin group were significantly higher than those of the model group(<i>P</i><0.01); Quercetin can make the retinal structure of mice clearer, necrosis and shed part of the outer nuclear layer, and reduce the content of ROS and MDA in mouse serum(all <i>P</i><0.05), and increase the activities of SOD, GSH-Px, and CAT(all <i>P</i><0.05). Compared with the model group, the expression of Nrf2 protein in the retinal cytoplasm of mice in the quercetin group was up-regulated(<i>P</i><0.05), and the expression of Nrf2 protein in the nucleus was down-regulated(<i>P</i><0.05), GCL protein expression was down-regulated(<i>P</i><0.05).<p>CONCLUSION: Quercetin improved the oxidative stress state after retinal photodamage through the Nrf2/Keap1/ARE pathway, protected the retinal function, and protected against age-related macular degeneration.
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The objective of this study was to examine the expression pattern of Kelch-like ECH-associated protein 1 (Keap1) in the maxillary 2(nd) molar germs of rats. We used the maxillary 2(nd) molar germs in rats' pup at postnatal day 3 (bell stage), 6 (crown formation stage) and 9 (root formation stage). The investigation on mRNA and protein levels were done using reverse transcription - polymerase chain reaction and western blot. Localization of Keap 1 in the maxillary 2(nd) molar germs were revealed through immunofluorescence staining. Keap1 from the maxillary 2nd molar germs were mostly manifested on postnatal day 3 and dramatically decreased on postnatal day 6 and 9 at mRNA and protein levels, while amelogenin and ameloblastin increased during the development of maxillary 2nd molar germs. During immunofluorescence analysis, the strong immunoreactivity against Keap1 was detected in the apical side of ameloblasts at the presecretory and secretory stages. However, Keap1 expression was hardly observed in the ameloblasts at the maturation stage. These results shows that Keap1 is strongly expressed in the presecretory and secretory ameloblasts of amelogenesis, and suggest that Keap1 may be a crucial molecule for the regulatory mechanisms tasked with the formation of enamel layer.