X-Paste improves wound healing in diabetes via NF-E2-related factor/HO-1 signaling pathway

BACKGROUND Diabetic foot ulcers(DFU),as severe complications of diabetes mellitus(DM),significantly compromise patient health and carry risks of amputation and mortality.AIM To offer new insights into the occurrence and development of DFU,focusing on the therapeutic mechanisms of X-Paste(XP)of wound healing in diabetic mice.METHODS Employing traditional Chinese medicine ointment preparation methods,XP combines various medicinal ingredients.High-performance liquid chromatography(HPLC)identified XP’s main components.Using streptozotocin(STZ)-induced diabetic,we aimed to investigate whether XP participated in the process of diabetic wound healing.RNA-sequencing analyzed gene expression differences between XP-treated and control groups.Molecular docking clarified XP’s treatment mechanisms for diabetic wound healing.Human umbilical vein endothelial cells(HUVECs)were used to investigate the effects of Andrographolide(Andro)on cell viability,reactive oxygen species generation,apoptosis,proliferation,and metastasis in vitro following exposure to high glucose(HG),while NF-E2-related factor-2(Nrf2)knockdown elucidated Andro’s molecular mechanisms.RESULTS XP notably enhanced wound healing in mice,expediting the healing process.RNA-sequencing revealed Nrf2 upregulation in DM tissues following XP treatment.HPLC identified 21 primary XP components,with Andro exhibiting strong Nrf2 binding.Andro mitigated HG-induced HUVECs proliferation,metastasis,angiogenic injury,and inflammation inhibition.Andro alleviates HG-induced HUVECs damage through Nrf2/HO-1 pathway activation,with Nrf2 knockdown reducing Andro’s proliferative and endothelial protective effects.CONCLUSION XP significantly promotes wound healing in STZ-induced diabetic models.As XP’s key component,Andro activates the Nrf2/HO-1 signaling pathway,enhancing cell proliferation,tubule formation,and inflammation reduction.

Low- and high-dose hydrogen peroxide regulation of transcription factor NF-E2-related factor 2

Background Reactive oxygen species （ROS） may play both physiological and pathophysiological roles. Transcription factor NF-E2-related factor 2 （Nrf2） regulates antioxidant response element （ARE）-mediated genes expression and coordinates induction of chemoprotective proteins in response to physical and chemical stresses. The exact role of Nrf2 in cellular responses to different levels of oxidative stresses remains unknown. Methods Rat pulmonary microvascular endothelial cells were cultured and treated with 0 mmol/L, 0.125 mmol/L, 0.25 mmol/L, 0.5 mmol/L, 1.0 mmol/L and 2.0 mmol/L hydrogen peroxide solution for 2 hours. Nrf2 gene expression was assayed by reverse transcription-PCR, Nrf2-ARE binding activity was assayed with electrophoretic mobility shift assay （EMSA）, and localization of Nrf2 was detected with immunohistochemistry. Results Low and moderate （0.125 mmol/L, 0.25 mmol/L and 0.5 mmol/L） doses hydrogen peroxide exposure of rat pulmonary microvascular endothelial cells led to the nuclear accumulation of Nrf2, increased activity of transcription regulation and up-regulation of ARE-medicated gene expression. In contrast, high doses of hydrogen peroxide （1 mmol/L 2 mmol/L） exposure of the cells led to the nuclear exclusion of Nrf2, decreased activity transcription regulation and down-regulation of ARE-mediated gene expression. Conclusion Low and moderate doses of hydrogen peroxide play protective roles by increasing transcription activity of Nrf2, whereas high- dose hydrogen peroxide plays a deleterious role by decreasing transcription activity of Nrf2.

Emerging trends and hotspots of Nuclear factor erythroid 2-related factor 2 in nervous system diseases

BACKGROUND The Nuclear factor erythroid 2-related factor 2(NRF2)transcription factor has attracted much attention in the context of neurological diseases.However,none of the studies have systematically clarified this field's research hotspots and evolution rules.AIM To investigate the research hotspots,evolution patterns,and future research trends in this field in recent years.METHODS We conducted a comprehensive literature search in the Web of Science Core Collection database using the following methods:(((((TS=(NFE2 L2))OR TS=(Nfe2 L2 protein,mouse))OR TS=(NF-E2-Related Factor 2))OR TS=(NRF2))OR TS=(NFE2L2))OR TS=(Nuclear factor erythroid2-related factor 2)AND(((((((TS=(neurological diseases))OR TS=(neurological disorder))OR TS=(brain disorder))OR TS=(brain injury))OR TS=(central nervous system disease))OR TS=(CNS disease))OR TS=(central nervous system disorder))OR TS=(CNS disorder)AND Language=English from 2010 to 2022.There are just two forms of literature available:Articles and reviews.Data were processed with the software Cite-Space(version 6.1.R6).RESULTS We analyzed 1884 articles from 200 schools in 72 countries/regions.Since 2015,the number of publications in this field has increased rapidly.China has the largest number of publications,but the articles published in the United States have better centrality and H-index.Among the top ten authors with the most published papers,five of them are from China,and the author with the most published papers is Wang Handong.The institution with the most articles was Nanjing University.To their credit,three of the top 10 most cited articles were written by Chinese scholars.The keyword co-occurrence map showed that"oxidative stress","NRF2","activation","expression"and"brain"were the five most frequently used keywords.CONCLUSION Research on the role of NRF2 in neurological diseases continues unabated.Researchers in developed countries published more influential papers,while Chinese scholars provided the largest number of articles.There have been numerous studies on the mechanism of NRF2 transcription factor in neurological diseases.NRF2 is also emerging as a potentially effective target for the treatment of neurological diseases.However,despite decades of research,our knowledge of NRF2 transcription factor in nervous system diseases is still limited.Further studies are needed in the future.

Neuroprotective effects of salidroside on focal cerebral ischemia/reperfusion injury involve the nuclear erythroid 2-related factor 2 pathway

Salidroside,the main active ingredient extracted from Rhodiola crenulata,has been shown to be neuroprotective in ischemic cerebral injury,but the underlying mechanism for this neuroprotection is poorly understood.In the current study,the neuroprotective effect of salidroside on cerebral ischemia-induced oxidative stress and the role of the nuclear factor erythroid 2-related factor 2（Nrf2）pathway was investigated in a rat model of middle cerebral artery occlusion.Salidroside（30 mg/kg）reduced infarct size,improved neurological function and histological changes,increased activity of superoxide dismutase and glutathione-S-transferase,and reduced malon-dialdehyde levels after cerebral ischemia and reperfusion.Furthermore,salidroside apparently increased Nrf2 and heme oxygenase-1 expression.These results suggest that salidroside exerts its neuroprotective effect against cerebral ischemia through anti-oxidant mechanisms and that activation of the Nrf2 pathway is involved.The Nrf2/antioxidant response element pathway may become a new therapeutic target for the treatment of ischemic stroke.

Interplay between nuclear factor erythroid 2-related factor 2 and inflammatory mediators in COVID-19-related liver injury

Coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus 2 is a global pandemic and poses a major threat to human health worldwide.In addition to respiratory symptoms,COVID-19 is usually accompanied by systemic inflammation and liver damage in moderate and severe cases.Nuclear factor erythroid 2-related factor 2(NRF2)is a transcription factor that regulates the expression of antioxidant proteins,participating in COVID-19-mediated inflammation and liver injury.Here,we show the novel reciprocal regulation between NRF2 and inflammatory mediators associated with COVID-19-related liver injury.Additionally,we describe some mechanisms and treatment strategies.

Nuclear factor erythroid 2-related factor 2-mediated signaling and metabolic associated fatty liver disease

Oxidative stress is a key driver in the development and progression of several diseases,including metabolic associated fatty liver disease(MAFLD).This condition includes a wide spectrum of pathological injuries,extending from simple steatosis to inflammation,fibrosis,cirrhosis,and hepatocellular carcinoma.Excessive buildup of lipids in the liver is strictly related to oxidative stress in MAFLD,progressing to liver fibrosis and cirrhosis.The nuclear factor erythroid 2-related factor 2(NRF2)is a master regulator of redox homeostasis.NRF2 plays an important role for cellular protection by inducing the expression of genes related to antioxidant,anti-inflammatory,and cytoprotective response.Consistent evidence demonstrates that NRF2 is involved in every step of MAFLD development,from simple steatosis to inflammation,advanced fibrosis,and initiation/progression of hepatocellular carcinoma.NRF2 activators regulate lipid metabolism and oxidative stress alleviating the fatty liver disease by inducing the expression of cytoprotective genes.Thus,modulating NRF2 activation is crucial not only in understanding specific mechanisms underlying MAFLD progression but also to characterize effective therapeutic strategies.This review outlined the current knowledge on the effects of NRF2 pathway,modulators,and mechanisms involved in the therapeutic implications of liver steatosis,inflammation,and fibrosis in MAFLD.

Keap1-nuclear factor rythroid 2-related factor 2 inhibitor NXPZ ameliorates Aβ1-42-induced cognitive dysfunction in mice

OBJECTIVE Nuclear factor erythroid 2-related factor 2(Nrf2) is found to be ubiquitiously expressed in many tissues,and works as the key regulator against oxidative stress damage in cells and organs,which makes Nrf2 a widely concerned drug target.Recent research has identified that Nrf2 is involved in the pathology of Alzheimer disease(AD),whereas the mechanism is unknown.The purpose of this study is to figure out the role of Nrf2 in the pathologic process of AD through Nrf2-Keap1-ARE pathway and the effects of Keap1-Nrf2 inhibitor in AD mice models.METHODS Amyloid β^(1-42)(Aβ^(1-42))was injected into the bilateral hippocampus to induce the cognitive dysfunction in eight-week old male mice.The mice were treated with Keap1-Nrf2 inhibitor NXPZ of three doses as well as donepezil as a positive control by intragastric administration one time a day for one week.Several behavior tests were used to analyze the mice learning and memory ability.Additionally,we detected Nrf2 and Aβ in the plasma in mice with ELISA kits,as well as some factors related to oxidative stress in the hippocampus and cortex.The expression levels of Nrf2,Keap1,Tau and p-Tau were measured in the murine brain tissue with Western blotting.SH-SY5 Y cells were studied as an in vitro model to further clarify the mechanism.RESULTS The treatment of NXPZ ameliorated learning and memory dysfunction in AD mice in a dose-dependent manner,and the high dose group recovered better than the positive drug group.The plasma Nrf2 level was increased in a dose-dependent manner in the treatment groups;however,the plasma Aβ was decreased.What′ s more,superoxide dismutase(SOD) and glutathione reductase(GSSH) in the hippocampus and cortex were increased in the treatment group,while the malondialdehyde(MDA) was decreased,meaning that NXPZ treatment promoted expression of the anti-oxidative factors and inhibited the expression of the oxidative factors in the down-stream.Western blotting analysis of hippocampus and cortex showed up-regulated Nrf2,decreased Keap1 and decreased p-Tau in NXPZ treatment mice.In ex vivo experiments,when SH-SY5 Y cells were treated with Aβ,Nrf2 in the cytoplasm was increased,as well as the expression Nrf2 in the nuclear was decreased.The treatment of NXPZ increased nuclear Nrf2,decreased cytoplasm Nrf2,and decreased the expression of p-Tau.CONCLUSION Nrf2 has an important role in neuron function.Nrf2 activation by selective Keap1-Nrf2 inhibitor NXPZ may contribute to improve cognitive function in AD mice.The mechanism may be related to increased generation and release of Nrf2 induced by more disaggregation with Keap1,leading to more expression of anti-oxidative molecules to protect the damage caused by Aβ.These results indicates that Nrf2 may be a novel therapeutic target of AD and Keap1-Nrf2 inhibitor may be a novel medication for protecting the loss of learning and memory ability.

High glucose reduces Nrf2-dependent cRAGE release and enhances inflammasome-dependent IL-1βproduction in monocytes:the modulatory effects of EGCG

Soluble receptor for advanced glycation end products(sRAGE)acts as a decoy sequestering of RAGE ligands,thus preventing the activation of the ligand-RAGE axis linking human diseases.However,the molecular mechanisms underlying sRAGE remain unclear.In this study,THP-1 monocytes were cultured in normal glucose(NG,5.5 mmol/L)and high glucose(HG,15 mmol/L)to investigate the effects of diabetesrelevant glucose concentrations on sRAGE and interleukin-1β(IL-1β)secretion.The modulatory effects of epigallocatechin gallate(EGCG)in response to HG challenge were also evaluated.HG enhanced intracellular reactive oxygen species(ROS)generation and RAGE expression.The secretion of sRAGE,including esRAGE and cRAGE,was reduced under HG conditions,together with the downregulation of a disintegrin and metallopeptidase 10(ADAM10)and nuclear factor erythroid 2-related factor 2(Nrf2)nuclear translocation.Mechanistically,the HG effects were counteracted by siRAGE and exacerbated by siNrf2.Chromatin immunoprecipitation results showed that Nrf2 binding to the ADAM10 promoter and HG interfered with this binding.Our data reinforce the notion that RAGE and Nrf2 might be sRAGE-regulating factors.Under HG conditions,the treatment of EGCG reduced ROS generation and RAGE activation.EGCG-stimulated cRAGE release was likely caused by the upregulation of the Nrf2-ADAM10 pathway.EGCG inhibited HG-mediated NLRP3 inflammasome activation at least partly by stimulating sRAGE,thereby reducing IL-1βrelease.

Hydrogen sulfide reduces oxidative stress in Huntington's disease via Nrf2

The pathophysiology of Huntington's disease involves high levels of the neurotoxin quinolinic acid. Quinolinic acid accumulation results in oxidative stress, which leads to neurotoxicity. However, the molecular and cellular mechanisms by which quinolinic acid contributes to Huntington's disease pathology remain unknown. In this study, we established in vitro and in vivo models of Huntington's disease by administering quinolinic acid to the PC12 neuronal cell line and the striatum of mice, respectively. We observed a decrease in the levels of hydrogen sulfide in both PC12 cells and mouse serum, which was accompanied by down-regulation of cystathionine β-synthase, an enzyme responsible for hydrogen sulfide production. However, treatment with NaHS(a hydrogen sulfide donor) increased hydrogen sulfide levels in the neurons and in mouse serum, as well as cystathionine β-synthase expression in the neurons and the mouse striatum, while also improving oxidative imbalance and mitochondrial dysfunction in PC12 cells and the mouse striatum. These beneficial effects correlated with upregulation of nuclear factor erythroid 2-related factor 2 expression. Finally, treatment with the nuclear factor erythroid 2-related factor 2inhibitor ML385 reversed the beneficial impact of exogenous hydrogen sulfide on quinolinic acid-induced oxidative stress. Taken together, our findings show that hydrogen sulfide reduces oxidative stress in Huntington's disease by activating nuclear factor erythroid 2-related factor 2,suggesting that hydrogen sulfide is a novel neuroprotective drug candidate for treating patients with Huntington's disease.

Lactobacillus plantarum J26 alleviates alcohol-induced oxidative liver injury by regulating the Nrf2 signaling pathway

Oxidative stress is one of the main ways to cause alcohol-induced liver injury,and alcoholic liver disease(ALD)has been a common health problem worldwide.Lactic acid bacteria(LAB)is also considered as a potential treatment to alleviate alcohol-induced liver injury.Lactobacillus plantarum J26 is a LAB isolated from Chinese traditional fermented dairy products with excellent probiotic effects.This study aimed to establish a mice model of alcoholic liver injury through acute-on-chronic alcohol feeding and to study the alleviating effect of pre-intake of L.plantarum J26 on alcohol-induced oxidative liver injury and focus on its potential mechanism of alleviating effect.The results showed that pre-intake of L.plantarum J26 could improve liver pathological changes,reduce lipid accumulation,increase mitochondrial ATP and mitochondrial(mtDNA)levels,and alleviate liver injury.In addition,pre-intake L.plantarum J26 can improve the level of short-chain fatty acids(SCFAs)in the intestines in mice,short chain fatty acids can be used as a signaling molecule activation of nuclear factor E2-related factor 2(Nrf2)signaling pathway to alleviate liver oxidative stress,and maintain mitochondrial homeostasis by regulating the expression of genes related to mitochondrial dynamics and autophagy,thereby reducing cell apoptosis to alleviate alcohol-induced oxidative liver injury.

Procyanidin A_1 and its digestive products alleviate acrylamide-induced IPEC-J2 cell damage through regulating Keap1/Nrf2 pathway

Our previous study has revealed that procyanidin A_(1)(A_(1))and its simulated digestive product(D-A,)can alleviate acrylamide(ACR)-induced intestine cell damage.However,the underlying mechanism remains unknown.In this study,we elucidated the molecular mechanism for and D-A_(1) to alleviate ACR-stimulated IPEC-J2 cell damage.ACR slightly activated nuclear factor erythroid 2-related factor 2(Nrf2)signaling and its target genes,but this activation could not reduce intestine cell damage.A_(1) and D-A_(1) could alleviate ACR-induced cell damage,but the effect was abrogated in cells transiently transfected with Nrf2 small interfering RNA(siRNA).Further investigation confirmed that A_(1) and D-A_(1) interacted with Ketch-like ECH-associated protein 1(Keapl),which boosted the stabilization of Nrf2,subsequently promoted the translocation of Nrf2 into the nucleus,and further increased the expression of antioxidant proteins,thereby inhibiting glutathione(GSH)consumption,maintaining redox balance and eventually alleviating ACR-induced cell damage.Importantly,there was no difference between A_(1) and D-A_(1) treated groups,indicating that A_(1) can tolerate gastrointestinal digestion and may be a potential compound to limit the toxicity of ACR.

Functionalized selenium nanoparticles ameliorated acetaminophen-induced hepatotoxicity through synergistically triggering PKCδ/Nrf2 signaling pathway and inhibiting CYP 2E1

Selenium nanoparticles(SeNPs)have been demonstrated potential for use in diseases associated with oxidative stress.Functionalized SeNPs with lower toxicity and higher biocompatibility could bring better therapeutic activity and clinical application value.Herein,this work was conducted to investigate the protective effect of Pleurotus tuber-regium polysaccharide-protein complex funtionnalized SeNPs(PTR-SeNPs)against acetaminophen(APAP)-induced oxidative injure in HepG2 cells and C57BL/6J mouse liver.Further elucidation of the underlying molecular mechanism,in particular their modulation of Nrf2 signaling pathway was also performed.The results showed that PTR-SeNPs could significantly ameliorate APAP-induced oxidative injury as evidenced by a range of biochemical analysis,histopathological examination and immunoblotting study.PTR-SeNPs could hosphorylate and activate PKCδ,depress Keap1,and increase nuclear accumulation of Nrf2,resulting in upregulation of GCLC,GCLM,HO-1 and NQO-1 expression.Besides,PTR-SeNPs suppressed the biotransformation of APAP to generate intracellular ROS through CYP 2E1 inhibition,restoring the mitochondrial morphology.Furthermore,the protective effect of PTR-SeNPs against APAP induced hepatotoxicity was weakened as Nrf2 was depleted in vivo,indicating the pivotal role of Nrf2 signaling pathway in PTR-SeNPs mediated hepatoprotective efficacy.Being a potential hepatic protectant,PTR-SeNPs could serve as a new source of selenium supplement for health-promoting and biomedical applications.

Neferine inhibits the progression of diabetic nephropathy by modulating the miR-17-5p/nuclear factor E2-related factor 2 axis

OBJECTIVE:To investigate the effect of Neferine(Nef)on diabetic nephropathy(DN)and to explore the mechanism of Nef in DN based on miRNA regulation theory.METHODS:A DN mouse model was constructed and treated with Nef.Serum creatinine(Crea),blood urea(UREA)and urinary albumin were measured in mice by kits,and renal histopathological changes and fibrosis were observed by hematoxylin-eosin staining and Masson staining.Renal tissue superoxide dismutase(SOD),malondialdehyde(MDA)and glutathione peroxidase(GSH-Px)activities were measured by enzyme-linked immunosorbent assay(ELISA).Western blotting was used to detect the expression of nuclear factor E2-related factor 2(Nrf2)/heme oxygenase 1(HO-1)signaling pathway-related proteins in kidney tissues.Quantitative reverse transcription-polymerase chain reaction(q RT-PCR)was used to detect the expression of miR-17-5p in kidney tissues.Subsequently,a DN in vitro model was constructed by high glucose culture of human mesangial cells(HMCs),cells were transfected with miR-17-5p mimic and/or treated with Nef,and we used q RTPCR to detect cellular miR-17 expression,flow cytometry to detect apoptosis,ELISAs to detect cellular SOD,MDA,and GSH-Px activities,Western blots to detect Nrf2/HO-1 signaling pathway-related protein expression,and dual luciferase reporter gene assays to verify the targeting relationship between Nrf2 and miR-17-5p.RESULTS:Administration of Nef significantly reduced the levels of blood glucose,Crea,and UREA and the expression of miR-17-5p,improved renal histopathology and fibrosis,significantly reduced MDA levels,elevated SOD and GSH-Px activities,and activated Nrf2 expression in kidney tissues from mice with DN.Nrf2 is a post-transcriptional target of miR-17-5p.In HMCs transfected with miR-17-5p mimics,the m RNA and protein levels of Nrf2 were significantly suppressed.Furthermore,miR-17-5p overexpression and Nef intervention resulted in a significant increase in high glucose-induced apoptosis and MDA levels in HMCs and a significant decrease in the protein expression of HO-1 and Nrf2.CONCLUSION:Collectively,these results indicate that Nef has an ameliorative effect on DN,and the mechanism may be through the miR-17-5p/Nrf2 pathway.

Effects of blueberry on hepatic fibrosis and transcription factor Nrf2 in rats

AIM:To investigate the effects of blueberry on hepatic fibrosis and NF-E2-related factor 2(Nrf2) transcription factor in rats.METHODS:Forty-five male Sprague-Dawley rats were randomly divided into control group(A);CCl4-induced hepatic fibrosis group(B);blueberry prevention group(C);Dan-shao-hua-xian capsule(DSHX) prevention group(D);and blueberry + DSHX prevention group(E).Liver fibrosis was induced in rats by subcutaneous injection of CCl4 and a high-lipid/low-protein diet for 8 wk(except the control group).The level of hyaluronic acid(HA) and alanine aminotransferase(ALT) in serum was examined.The activity of superoxide dismutase(SOD),glutathione-S-transferase(GST) and malondialdehyde(MDA) in liver homogenates was determined.The degree of hepatic fibrosis was evaluated by hematoxylin and eosin and Masson staining.Expression of Nrf2 and NADPH quinone oxidoreductase 1(Nqo1) was detected by real-time reversed transcribed-polymerase chain reaction,immunohistochemical techniques,and western blotting.RESULTS:Compared with group B,liver indices,levels of serum HA and ALT of groups C,D and E were reduced(liver indices:0.038 ± 0.008,0.036 ± 0.007,0.036 ± 0.005 vs 0.054 ± 0.009,P<0.05;HA:502.33 ± 110.57 ng/mL,524.25 ± 255.42 ng/mL,499.25 ± 198.10 ng/mL vs 828.50 ± 237.83 ng/mL,P<0.05;ALT:149.44 ± 16.51 U/L,136.88 ± 10.07 U/L,127.38 ± 11.03 U/L vs 203.25 ± 31.62 U/L,P<0.05),and SOD level was significantly higher,but MDA level was lower,in liver homogenates(SOD:1.36 ± 0.09 U/mg,1.42 ± 0.13 U/mg,1.50 ± 0.15 U/mg vs 1.08 ± 0.19 U/mg,P<0.05;MDA:0.294 ± 0.026 nmol/mg,0.285 ± 0.025 nmol/mg,0.284 ± 0.028 nmol/mg vs 0.335 ± 0.056 nmol/mg,P<0.05).Meanwhile,the stage of hepatic fibrosis was significantly weakened(P<0.05).Compared with group A,the activity of GST liver homogenates and expression levels of Nrf2 and Nqo1 in group B were elevated(P<0.05).The expression level of Nrf2 and Nqo1 in groups C,D,and E were increased as compared with group B,but the difference was not significant.CONCLUSION:Blueberry has preventive and protective effects on CCl4-induced hepatic fibrosis by reducing hepatocyte injury and lipid peroxidation.However,these effects may not be related to the activation of Nrf2 during long-term of CCl4.

Effect of miR-27b-3p and Nrf2 in human retinal pigment epithelial cell induced by high-glucose

AIM:To determine whether the microRNA-27b-3p(miR-27b-3p)/NF-E2-related factor 2(Nrf2)pathway plays a role in human retinal pigment epithelial(hRPE)cell response to high glucose,how miR-27b-3p and Nrf2 expression are regulated,and whether this pathway could be specifically targeted.METHODS:hRPE cells were cultured in normal glucose or high glucose for 1,3,or 6d before measuring cellular proliferation rates using cell counting kit-8 and reactive oxygen species(ROS)levels using a dihydroethidium kit.miR-27b-3p,Nrf2,NAD(P)H quinone oxidoreductase 1(NQO1)and heme oxygenase-1(HO-1)mRNA and protein levels were analyzed using reverse transcription quantitative polymerase chain reaction(RT-qPCR)and immunocytofluorescence(ICF),respectively.Western blot analyses were performed to determine nuclear and total Nrf2 protein levels.Nrf2,NQO1,and HO-1 expression levels by RT-qPCR,ICF,or Western blot were further tested after miR-27b-3p overexpression or inhibitor lentiviral transfection.Finally,the expression level of those target genes was analyzed after treating hRPE cells with pyridoxamine.RESULTS:Persistent exposure to high glucose gradually suppressed hRPE Nrf2,NQO1,and HO-1 mRNA and protein levels and increased miR-27b-3p mRNA levels.High glucose also promoted ROS release and inhibited cellular proliferation.Nrf2,NQO1,and HO-1 mRNA levels decreased after miR-27b-3p overexpression and,conversely,both mRNA and protein levels increased after expressing a miR-27b-3p inhibitor.After treating hRPE cells exposed to high glucose with pyridoxamine,ROS levels tended to decreased,proliferation rate increased,Nrf2,NQO1,and HO-1 mRNA and protein levels were upregulated,and miR-27b-3p mRNA levels were suppressed.CONCLUSION:Nrf2 is a downstream target of miR-27b-3p.Furthermore,the miR-27b-3p inhibitor pyridoxamine can alleviate high glucose injury by regulating the miR-27b-3p/Nrf2 axis.

Oxidative stress in retinal pigment epithelium degeneration:from pathogenesis to therapeutic targets in dry age-related macular degeneration

Age-related macular degeneration is a primary cause of blindness in the older adult population. Past decades of research in the pathophysiology of the disease have resulted in breakthroughs in the form of anti-vascular endothelial growth factor therapies against neovascular age-related macular degeneration;however, effective treatment is not yet available for geographical atrophy in dry agerelated macular degeneration or for preventing the progression from early or mid to the late stage of age-related macular degeneration. Both clinical and experimental investigations involving human agerelated macular degeneration retinas and animal models point towards the atrophic alterations in retinal pigment epithelium as a key feature in age-related macular degeneration progression. Retinal pigment epithelium cells are primarily responsible for cellular-structural maintenance and nutrition supply to keep photoreceptors healthy and functional. The retinal pigment epithelium constantly endures a highly oxidative environment that is balanced with a cascade of antioxidant enzyme systems regulated by nuclear factor erythroid-2-related factor 2 as a main redox sensing transcription factor. Aging and accumulated oxidative stress triggers retinal pigment epithelium dysfunction and eventually death. Exposure to both environmental and genetic factors aggravates oxidative stress damage in aging retinal pigment epithelium and accelerates retinal pigment epithelium degeneration in age-related macular degeneration pathophysiology. The present review summarizes the role of oxidative stress in retinal pigment epithelium degeneration, with potential impacts from both genetic and environmental factors in age-related macular degeneration development and progression. Potential strategies to counter retinal pigment epithelium damage and protect the retinal pigment epithelium through enhancing its antioxidant capacity are also discussed, focusing on existing antioxidant nutritional supplementation, and exploring nuclear factor erythroid-2-related factor 2 and its regulators including REV-ERBα as therapeutic targets to protect against age-related macular degeneration development and progression.

Upregulation of CDGSH iron sulfur domain 2 attenuates cerebral ischemia/reperfusion injury

CDGSH iron sulfur domain 2 can inhibit ferroptosis,which has been associated with cerebral ischemia/reperfusion,in individuals with head and neck cancer.Therefore,CDGSH iron sulfur domain 2 may be implicated in cerebral ischemia/reperfusion injury.To validate this hypothesis in the present study,we established mouse models of occlusion of the middle cerebral artery and HT22 cell models of oxygen-glucose deprivation and reoxygenation to mimic cerebral ischemia/reperfusion injury in vivo and in vitro,respectively.We found remarkably decreased CDGSH iron sulfur domain 2 expression in the mouse brain tissue and HT22 cells.When we used adeno-associated virus and plasmid to up-regulate CDGSH iron sulfur domain 2 expression in the brain tissue and HT22 cell models separately,mouse neurological dysfunction was greatly improved;the cerebral infarct volume was reduced;the survival rate of HT22 cells was increased;HT22 cell injury was alleviated;the expression of ferroptosis-related glutathione peroxidase 4,cystine-glutamate antiporter,and glutathione was increased;the levels of malondialdehyde,iron ions,and the expression of transferrin receptor 1 were decreased;and the expression of nuclear-factor E2-related factor 2/heme oxygenase 1 was increased.Inhibition of CDGSH iron sulfur domain 2 upregulation via the nuclear-factor E2-related factor 2 inhibitor ML385 in oxygen-glucose deprived and reoxygenated HT22 cells blocked the neuroprotective effects of CDGSH iron sulfur domain 2 up-regulation and the activation of the nuclear-factor E2-related factor 2/heme oxygenase 1 pathway.Our data indicate that the up-regulation of CDGSH iron sulfur domain 2 can attenuate cerebral ischemia/reperfusion injury,thus providing theoretical support from the perspectives of cytology and experimental zoology for the use of this protein as a therapeutic target in patients with cerebral ischemia/reperfusion injury.

Nrf2基因敲除对小鼠蛛网膜下腔出血后脑损伤的作用

目的蛛网膜下腔出血(subarachnoid hemorrhage,SAH)是一种致死率较高的危重疾病,文中研究氧化应激调节因子Nrf2在SAH后脑损伤作用及机制。方法实验选取雄性ICR野生型(wild type,WT)小鼠及来源于ICR的Nrf2基因敲除(knockout,KO)小鼠,采用视交叉自体血注射建立小鼠SAH模型,实验动物分为WT假手术组、KO假手术组、WT SAH组和KO SAH组4个组,检测SAH后24 h氧化应激产物丙二醛(malondialdehyde,MDA)及GSH/GSSG,炎症因子TNF-α和IL-1β,脑组织含水量和伊文思蓝含量,TUNEL和尼氏染色,活动评分及大脑前和大脑中动脉血管痉挛情况。结果与假手术组比较,SAH组MDA、TNF-α、IL-1β表达量上升,而GSH/GSSG下降(P<0.01);与WT SAH组比较,MDA、TNF-α、IL-1β表达量上升(P<0.05),而GSH/GSSG下降(P<0.05)。SAH组前脑脑组织含水量、伊文思蓝含量较假手术组增加(P<0.01),与WT SAH组比较,KO SAH组脑组织含水量、伊文思蓝含量均升高[(0.808±0.004)vs(0.819±0.004)、(7.230±1.192)μg/g vs(11.628±1.040)μg/g,P<0.05]。SAH后24 h,与假手术组比较,SAH组神经细胞凋亡率上升(P<0.01),而神经元数量、ACA比值、血管半径/壁厚值、活动评分下降(P<0.01),与WT SAH组比较,KO SAH组细胞凋亡率上升[(23.733±8.204)%vs(36.267±10.612)%],而神经元数、ACA比值、血管半径/壁厚值、活动评分下降[(70.833±8.750)vs(51.767±13.006),(8.024±2.780)vs(6.861±2.702),(6.337±3.993)vs(5.107±3.805),(1.967±0.928)vs(1.433±0.679),P<0.05]。结论 Nrf2 KO加重了SAH后氧化应激和炎性反应,从而导致了SAH继发性脑损伤加重。Nrf2对SAH后继发性脑损伤具有保护作用。

血管紧张素-(1-7)/血管紧张素-Ⅱ通过调节Nrf2/HO-1通路在糖尿病肾脏缺血再灌注损伤中的作用及机制

目的探讨血管紧张素-(1-7)/血管紧张素-Ⅱ[Ang-(1-7)/Ang-Ⅱ]调节NF-E2-related factor-2(Nrf2)/血红素氧合酶1(HO-1)通路在糖尿病肾脏缺血再灌注损伤中的作用及机制。方法 60只成年雄性SD大鼠随机分为正常假手术组(Sham组)、正常缺血再灌注组(IR组)、糖尿病假手术组(DS组)、糖尿病缺血再灌注组(DIR组),每组各15只。检测血尿素氮(BUN)、肌酐(Cr)和中性粒细胞明胶酶相关载脂蛋白(NGAL),观察肾脏病理学改变,检测肾组织超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量,Western blot检测Ang-(1-7)和Ang-Ⅱ蛋白以及Nrf2和HO-1蛋白表达。结果 Ang-(1-7)蛋白在DS组中表达较Sham组增加(P<0.05),在IR组中表达较Sham组减少(P<0.05),而在DIR组中较IR组进一步减少(P<0.05)。IR组和DS组中Ang-Ⅱ蛋白表达显著高于Sham组(P<0.05),而DIR组中Ang-Ⅱ蛋白表达进一步增高(P<0.05)。IR组和DS组中Nrf2和HO-1蛋白表达显著高于Sham组(P<0.05),而DIR组中Nrf2和HO-1蛋白表达较IR组和DS组降低(P<0.05)。与Sham组比较,IR组和DS组中肾脏损伤程度显著增高(P<0.05),血清BUN、Cr和NGAL水平、肾脏组织MDA含量明显升高而SOD活性显著降低(P<0.05)。与IR组和DS组比较,DIR组中肾脏损伤程度进一步增高(P<0.05),血清BUN、Cr和NGAL水平、肾脏组织MDA含量明显增高,而SOD活性明显降低(P<0.05)。结论缺血再灌注可能通过抑制Ang-(1-7),激活Ang-Ⅱ,下调Nrf2/HO-1通路的抗氧化应激能力,加重了糖尿病肾脏的损伤。

D-GalN/LPS诱导大鼠急性肝衰竭中髓过氧化物酶和 Nrf2/HO-1信号通路的变化

目的 探讨D-氨基半乳糖(D-GalN)/脂多糖(LPS)诱导急性肝衰竭(acute liver failure,ALF)大鼠中髓过氧化物酶(myeloperoxidase,MPO)和核因子E2相关因子2(Nrf2)/血红素氧合酶-1(HO-1)信号通路的变化及ALF的发生机制。方法 SD大鼠随机分为对照组和ALF组。ALF组:将D-GalN 800 mg/kg和LPS 8μg/只同时腹腔注射,于注射后6、12和24 h检测血清总胆红素(TBiL)、丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)含量。ELISA法检测血清MPO、肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)水平,比色法测定MPO活性,RT-PCR检测肝组织Nrf2和HO-1的mRNA水平,Western blot法检测肝组织MPO、Nrf2和HO-1蛋白水平。结果 与对照组比较,ALF组血清MPO水平于造模后6 h升高,12 h为最高,24 h开始下降(23.33±2.06 vs.33.00±3.16,65.75±7.02,53.92±5.63,P<0.05);血清TNF-α(11.30±3.26 vs.102.17±14.80,83.33±11.22,64.25±9.29,P<0.01)和IL-6(10.83±2.92 vs.89.25±10.86,77.33±8.02,65.58±7.31,P<0.01)于造模后6 h升高最明显,12 h后逐渐下降,差异均有统计学意义。ALF组6、12、24 h肝组织MPO活性高于对照组,差异均有统计学意义(15.5±1.51,28.08±4.65,22.92±1.93 vs.12.17±1.27,P<0.05);ALF组肝组织6、12、24 h Nrf2 mRNA(1.59±0.13,3.65±0.11,2.35±0.11 vs.1.04±1.01,P<0.01)和HO-1 mRNA(2.44±0.19,4.77±0.18,3.82±0.17 vs.1.12±0.06,P<0.01)水平高于对照组,差异均有统计学意义。ALF组肝组织MPO(4.10±0.70,9.77±1.15,7.23±0.40 vs.2.07±0.42,P<0.01)、Nrf2(4.03±0.80,9.03±0.50,6.07±0.47 vs.2.63±0.38,P<0.01)和HO-1(1.73±0.21,5.17±0.51,3.03±0.32 vs.0.97±0.21,P<0.01)蛋白表达于12 h达最高值,ALF组各时间点与对照组比较均差异有统计学意义。结论 MPO可能通过氧化应激和炎症反应影响Nrf2/HO-1信号通路,在ALF中发挥重要作用。