Objective:Pulmonary hypertension(PH)is a severe pulmonary vascular disease that eventually leads to right ventricular failure and death.The purpose of this study was to investigate the mechanism by which pachymic acid...Objective:Pulmonary hypertension(PH)is a severe pulmonary vascular disease that eventually leads to right ventricular failure and death.The purpose of this study was to investigate the mechanism by which pachymic acid(PA)pretreatment affects PH and pulmonary vascular remodeling in rats.Methods:PH was induced via hypoxia exposure and administration of PA(5 mg/kg per day)in male Sprague-Dawley rats.Hemodynamic parameters were measured using a right ventricular floating catheter and pulmonary vascular morphometry was measured by hematoxylin-eosin(HE),a-SMA and Masson staining.MTT assays and EdU staining were used to detect cell proliferation,and apoptosis was analyzed by TUNEL staining.Western blotting and immunohistochemistry were used to detect the expression of proteins related to the Nrf2-Keapl-ARE pathway.展开更多
Acute lung injury(ALI)is a severe inflammatory condition with a high mortality rate,often precipitated by sepsis.The pathophysiology of ALI involves complex mechanisms,including inflammation,oxidative stress,and ferro...Acute lung injury(ALI)is a severe inflammatory condition with a high mortality rate,often precipitated by sepsis.The pathophysiology of ALI involves complex mechanisms,including inflammation,oxidative stress,and ferroptosis,a novel form of regulated cell death.This study explores the therapeutic potential of andrographolide(AG),a bioactive compound derived from Andrographis,in mitigating Lipopolysaccharide(LPS)-induced inflammation and ferroptosis.Our research employed in vitro experiments with RAW264.7 macrophage cells and in vivo studies using a murine model of LPS-induced ALI.The results indicate that AG significantly suppresses the production of pro-inflammatory cytokines and inhibits ferroptosis in LPS-stimulated RAW264.7 cells.In vivo,AG treatment markedly reduces lung edema,decreases inflammatory cell infiltration,and mitigates ferroptosis in lung tissues of LPS-induced ALI mice.These protective effects are mediated via the modulation of the Toll-like receptor 4(TLR4)/Kelch-like ECH-associated protein 1(Keap1)/Nuclear factor erythroid 2-related factor 2(Nrf2)signaling pathway.Molecular docking simulations identified the binding sites of AG on the TLR4 protein(Kd value:-33.5 kcal·mol^(-1)),and these interactions were further corroborated by Cellular Thermal Shift Assay(CETSA)and SPR assays.Collectively,our findings demonstrate that AG exerts potent anti-inflammatory and anti-ferroptosis effects in LPS-induced ALI by targeting TLR4 and modulating the Keap1/Nrf2 pathway.This study underscores AG's potential as a therapeutic agent for ALI and provides new insights into its underlying mechanisms of action.展开更多
The Keap1–Nrf2–ARE pathway is an important antioxidant defense mechanism that protects cells from oxidative stress and the Keap1–Nrf2 protein–protein interaction(PPI) has become an important drug target to upregul...The Keap1–Nrf2–ARE pathway is an important antioxidant defense mechanism that protects cells from oxidative stress and the Keap1–Nrf2 protein–protein interaction(PPI) has become an important drug target to upregulate the expression of ARE-controlled cytoprotective oxidative stress response enzymes in the development of therapeutic and preventive agents for a number of diseases and conditions. However, most known Nrf2 activators/ARE inducers are indirect inhibitors of Keap1–Nrf2PPI and they are electrophilic species that act by modifying the sulfhydryl groups of Keap1's cysteine residues. The electrophilicity of these indirect inhibitors may cause "off-target" side effects by reacting with cysteine residues of other important cellular proteins. Efforts have recently been focused on the development of direct inhibitors of Keap1–Nrf2 PPI. This article reviews these recent research efforts including the development of high throughput screening assays, the discovery of peptide and small molecule direct inhibitors, and the biophysical characterization of the binding of these inhibitors to the target Keap1 Kelch domain protein. These non-covalent direct inhibitors of Keap1–Nrf2 PPI could potentially be developed into effective therapeutic or preventive agents for a variety of diseases and conditions.展开更多
We performed a bioinformatics analysis with validation by multiple databases,aiming to evaluate the diagnostic and prognostic value of Kelch-like ECH-associated protein 1(Keapl)mRNA for lung cancer,and to explore poss...We performed a bioinformatics analysis with validation by multiple databases,aiming to evaluate the diagnostic and prognostic value of Kelch-like ECH-associated protein 1(Keapl)mRNA for lung cancer,and to explore possible mechanisms.Diagnostic performance of Keapl mRNA was determined by receiver operating characteristic(ROC)curve analysis.Prognostic implication of Keapl mRNA was estimated by Kaplan-Meier survival analysis.Co-expressed genes with both Keapl and Nfe2L2 were identified by LinkedOmics.Mechanisms of Keapl-Nfe2L2-co-expressed genes underlying the pathogenesis of lung cancer were explored by function enrichment and pathway analysis.The ROC curve analysis determined a good diagnostic performance of Keapl mRNA for lung squamous cell carcinoma(LUSC),with an area under the ROC curve(AUC)of 0.833,sensitivity of 72.7%,and specificity of 90.6%(P<0.001).Multivariate Cox regression recognized high Keapl mRNA to be an independent risk factor of mortality for overall lung cancer[hazard ratio(HR):11.034,P=0.044],but an independent antagonistic factor for lung adenocarcinoma(LUAD)(HR:0.404,P<0.001).Validation by UALCAN and GEPIA supported Oncomine findings regarding the diagnostic value of Keapl mRNA for LUSC,but denied its prognostic value.After screening,we identified 17 co-expressed genes with both Keapl and Nfe2L2 for LUAD,and 22 for LUSC,mainly enriched in signaling pathway of oxidative stress-induced gene expression via Nrf2.In conclusion,Keapl mRNA has a good diagnostic performance,but controversial prognostic efficacy for LUSC.The pathogenesis of lung cancer is associated with Keapl-Nfe2L2-co-expressed genes by signaling pathway of oxidative stress-induced gene expression via Nrf2.展开更多
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 pepti...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,5×10^(10)CFU(CFU:colony forming units)WB800,and 5×10^(10)CFU WB800-KR32,respectively,on Days 1-14 and all infused with ETEC K881×10^(10)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.展开更多
The treatment of hepatocellular carcinoma(HCC)has been dominated by multikinase inhibitors for more than a decade.However,drug resistance can severely restrict the efficacy of these drugs.Using CRISPR/CAS9 genome libr...The treatment of hepatocellular carcinoma(HCC)has been dominated by multikinase inhibitors for more than a decade.However,drug resistance can severely restrict the efficacy of these drugs.Using CRISPR/CAS9 genome library screening,we evaluated Kelch-like ECH-associated protein 1(KEAP1)as a key regulator of sorafenib’s susceptibility in HCC.We also investigated whether KEAP1’s knockdown can stabilize nuclear factor(erythroid-derived 2)-like 2(NRF2)protein levels that led to sorafenib’s resistance,including an NRF2 inhibitor that can synergize with sorafenib to abolish HCC’s growth in vitro and in vivo.Furthermore,we clarified that fibroblast growth factor 21(FGF21)is an important downstream regulator of NRF2 in HCC.Intriguingly,we observed that FGF21 bound to NRF2 through the C-terminus of FGF21,thereby stabilizing NRF2 by reducing its ubiquitination and generating a positive feedback loop in sorafenib-resistant HCC.These findings,therefore,propose that targeting FGF21 is a promising strategy to combat HCC sorafenib’s resistance.展开更多
基金This project was supported by the Natural Science Foundation of Hubei Province(No.2017CFB769).
文摘Objective:Pulmonary hypertension(PH)is a severe pulmonary vascular disease that eventually leads to right ventricular failure and death.The purpose of this study was to investigate the mechanism by which pachymic acid(PA)pretreatment affects PH and pulmonary vascular remodeling in rats.Methods:PH was induced via hypoxia exposure and administration of PA(5 mg/kg per day)in male Sprague-Dawley rats.Hemodynamic parameters were measured using a right ventricular floating catheter and pulmonary vascular morphometry was measured by hematoxylin-eosin(HE),a-SMA and Masson staining.MTT assays and EdU staining were used to detect cell proliferation,and apoptosis was analyzed by TUNEL staining.Western blotting and immunohistochemistry were used to detect the expression of proteins related to the Nrf2-Keapl-ARE pathway.
基金supported by China-ASEAN International Innovative Center for Health Industry of Traditional Chinese Medicine(No.AD20297142)Guangxi Collaborative Innovation Center for Scientific Achievements Transformation and Applicationon Traditional Chinese Medicine(No.05020058)。
文摘Acute lung injury(ALI)is a severe inflammatory condition with a high mortality rate,often precipitated by sepsis.The pathophysiology of ALI involves complex mechanisms,including inflammation,oxidative stress,and ferroptosis,a novel form of regulated cell death.This study explores the therapeutic potential of andrographolide(AG),a bioactive compound derived from Andrographis,in mitigating Lipopolysaccharide(LPS)-induced inflammation and ferroptosis.Our research employed in vitro experiments with RAW264.7 macrophage cells and in vivo studies using a murine model of LPS-induced ALI.The results indicate that AG significantly suppresses the production of pro-inflammatory cytokines and inhibits ferroptosis in LPS-stimulated RAW264.7 cells.In vivo,AG treatment markedly reduces lung edema,decreases inflammatory cell infiltration,and mitigates ferroptosis in lung tissues of LPS-induced ALI mice.These protective effects are mediated via the modulation of the Toll-like receptor 4(TLR4)/Kelch-like ECH-associated protein 1(Keap1)/Nuclear factor erythroid 2-related factor 2(Nrf2)signaling pathway.Molecular docking simulations identified the binding sites of AG on the TLR4 protein(Kd value:-33.5 kcal·mol^(-1)),and these interactions were further corroborated by Cellular Thermal Shift Assay(CETSA)and SPR assays.Collectively,our findings demonstrate that AG exerts potent anti-inflammatory and anti-ferroptosis effects in LPS-induced ALI by targeting TLR4 and modulating the Keap1/Nrf2 pathway.This study underscores AG's potential as a therapeutic agent for ALI and provides new insights into its underlying mechanisms of action.
基金the financial support of Grants CA133791, CA125868, and MH093197 from the National Institutes of Health, United States
文摘The Keap1–Nrf2–ARE pathway is an important antioxidant defense mechanism that protects cells from oxidative stress and the Keap1–Nrf2 protein–protein interaction(PPI) has become an important drug target to upregulate the expression of ARE-controlled cytoprotective oxidative stress response enzymes in the development of therapeutic and preventive agents for a number of diseases and conditions. However, most known Nrf2 activators/ARE inducers are indirect inhibitors of Keap1–Nrf2PPI and they are electrophilic species that act by modifying the sulfhydryl groups of Keap1's cysteine residues. The electrophilicity of these indirect inhibitors may cause "off-target" side effects by reacting with cysteine residues of other important cellular proteins. Efforts have recently been focused on the development of direct inhibitors of Keap1–Nrf2 PPI. This article reviews these recent research efforts including the development of high throughput screening assays, the discovery of peptide and small molecule direct inhibitors, and the biophysical characterization of the binding of these inhibitors to the target Keap1 Kelch domain protein. These non-covalent direct inhibitors of Keap1–Nrf2 PPI could potentially be developed into effective therapeutic or preventive agents for a variety of diseases and conditions.
文摘We performed a bioinformatics analysis with validation by multiple databases,aiming to evaluate the diagnostic and prognostic value of Kelch-like ECH-associated protein 1(Keapl)mRNA for lung cancer,and to explore possible mechanisms.Diagnostic performance of Keapl mRNA was determined by receiver operating characteristic(ROC)curve analysis.Prognostic implication of Keapl mRNA was estimated by Kaplan-Meier survival analysis.Co-expressed genes with both Keapl and Nfe2L2 were identified by LinkedOmics.Mechanisms of Keapl-Nfe2L2-co-expressed genes underlying the pathogenesis of lung cancer were explored by function enrichment and pathway analysis.The ROC curve analysis determined a good diagnostic performance of Keapl mRNA for lung squamous cell carcinoma(LUSC),with an area under the ROC curve(AUC)of 0.833,sensitivity of 72.7%,and specificity of 90.6%(P<0.001).Multivariate Cox regression recognized high Keapl mRNA to be an independent risk factor of mortality for overall lung cancer[hazard ratio(HR):11.034,P=0.044],but an independent antagonistic factor for lung adenocarcinoma(LUAD)(HR:0.404,P<0.001).Validation by UALCAN and GEPIA supported Oncomine findings regarding the diagnostic value of Keapl mRNA for LUSC,but denied its prognostic value.After screening,we identified 17 co-expressed genes with both Keapl and Nfe2L2 for LUAD,and 22 for LUSC,mainly enriched in signaling pathway of oxidative stress-induced gene expression via Nrf2.In conclusion,Keapl mRNA has a good diagnostic performance,but controversial prognostic efficacy for LUSC.The pathogenesis of lung cancer is associated with Keapl-Nfe2L2-co-expressed genes by signaling pathway of oxidative stress-induced gene expression via Nrf2.
基金supported by the Zhejiang Provincial Key R&D Program of China(No.2021C02008)the China Agriculture Research System of MOF and MARA(No.CARS-35)+2 种基金the National Natural Science Foundation of China(No.32022079)the Fundamental Research Funds for the Central Universities(No.2022QZJH46)the Taishan Industrial Leading Talents Project.
文摘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,5×10^(10)CFU(CFU:colony forming units)WB800,and 5×10^(10)CFU WB800-KR32,respectively,on Days 1-14 and all infused with ETEC K881×10^(10)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.
基金supported by the National Natural Science Foundation of China(81702981,81827804,81902367,81772546and LQ18H160010)Zhejiang Provincial Natural Science Foundation of China(LY20H160021 and Y15H160052)+1 种基金China Postdoctoral Science Foundation(2020T130584 and 2020M671755)Health Innovation Talent Support Project of Zhejiang Medical and Health Science and Technology Plan(2021447581)。
文摘The treatment of hepatocellular carcinoma(HCC)has been dominated by multikinase inhibitors for more than a decade.However,drug resistance can severely restrict the efficacy of these drugs.Using CRISPR/CAS9 genome library screening,we evaluated Kelch-like ECH-associated protein 1(KEAP1)as a key regulator of sorafenib’s susceptibility in HCC.We also investigated whether KEAP1’s knockdown can stabilize nuclear factor(erythroid-derived 2)-like 2(NRF2)protein levels that led to sorafenib’s resistance,including an NRF2 inhibitor that can synergize with sorafenib to abolish HCC’s growth in vitro and in vivo.Furthermore,we clarified that fibroblast growth factor 21(FGF21)is an important downstream regulator of NRF2 in HCC.Intriguingly,we observed that FGF21 bound to NRF2 through the C-terminus of FGF21,thereby stabilizing NRF2 by reducing its ubiquitination and generating a positive feedback loop in sorafenib-resistant HCC.These findings,therefore,propose that targeting FGF21 is a promising strategy to combat HCC sorafenib’s resistance.
