Objective:Soluble epoxide hydrolase(sEH)emerges as a target of interest for inflammatory diseases.Piperine is a natural amide alkaloid from Piper nigrum and displays an inhibitory effect toward sEH,its chemical struct...Objective:Soluble epoxide hydrolase(sEH)emerges as a target of interest for inflammatory diseases.Piperine is a natural amide alkaloid from Piper nigrum and displays an inhibitory effect toward sEH,its chemical structural transformation was carried out in order to obtain a library of sEH inhibitors based on its skeleton.Methods:Structural transformation of piperine was carried out by chemical methods,and piperine derivatives were assayed for their sEH potentials.A mouse acute lung injury model was constructed by lipopolysaccharide(LPS).Hematoxylin and eosin(H&E)staining,immunofluorescence staining,Western Blot,and enzyme-linked immunosorbent assay were used for investigating the protective potential of sEH inhibitor 11h.Results:Piperine derivatives 11e,11h,11j,and 11o showed inhibitory potentials toward sEH with values of half maximal inhibitory concentration(IC50)from 20 to 70 nM.Compound 11h attenuated the pathological course of LPS-mediated acute lung injury(ALI)in vivo.Furthermore,levels of cytokines tumor necrosis factor alpha(TNF-α),interleukin 6(IL-6),myeloperoxidase(MPO),and lactate dehydrogenase(LDH)were decreased after administration of 11h.The LPS-mediated inflammation and redox unbalance,including expressions of cyclooxygenase-2(COX-2),heme oxygenase-1(HO-1),intercellular cell adhesion molecule-1(ICAM-1),vascular cell adhesion molecule-1(VCAM-1),p-p65/p65,glutamate-cysteine ligase modifier subunit(GCLM),and nuclear factor erythroid-2-related factor 2(Nrf2),were ameliorated through nuclear factor kappa B(NF-κB)and Nrf2 pathways via enhancing levels of epoxyeicosatrienoic acids(EETs)in LPS-exposed ALI mice after compound 11h treatment.Molecular docking demonstrated that the aromatic unsaturated group of 11h occupied a hydrophobic pocket and its urea group formed three hydrogen bonds with Asp333,Tyr381,and Tyr465,which stabilized the active conformation of the ligand.Conclusions:These findings demonstrated that compound 11h may serve as a lead compound for developing sEH inhibitors and treating inflammation related to diseases,such as ALI.展开更多
Dear Editor, Nicotine is a psychoactive alkaloid that is thought to play a key role in addiction to commercial tobacco products [1] and cotinine is its primary metabolite [2]. Pharmacological treatment, such as nicoti...Dear Editor, Nicotine is a psychoactive alkaloid that is thought to play a key role in addiction to commercial tobacco products [1] and cotinine is its primary metabolite [2]. Pharmacological treatment, such as nicotine replacement therapy （NRT）, is a valid solution to this problem. Tobacco smoke contains many carcinogens such as nitrosamines .展开更多
A two-layer method based on support vector machines (SVMs) has been developed to distinguish epoxide hydrolases (EHs) from other enzymes and to classify its subfamilies using its primary protein sequences. SVM classif...A two-layer method based on support vector machines (SVMs) has been developed to distinguish epoxide hydrolases (EHs) from other enzymes and to classify its subfamilies using its primary protein sequences. SVM classifiers were built using three different feature vectors extracted from the primary sequence of EHs: the amino acid composition (AAC), the dipeptide composition (DPC), and the pseudo-amino acid composition (PAAC). Validated by 5-fold cross tests, the first layer SVM clas- sifier can differentiate EHs and non-EHs with an accuracy of 94.2% and has a Matthew’s correlation coefficient (MCC) of 0.84. Using 2-fold cross validation, PAAC-based second layer SVM can further classify EH subfamilies with an overall accuracy of 90.7% and MCC of 0.87 as compared to AAC (80.0%) and DPC (84.9%). A program called EHPred has also been developed to assist readers to recognize EHs and to classify their subfamilies using primary protein sequences with greater accuracy.展开更多
In order to investigate the influence of silencing soluble epoxide hydrolase（sEH） with double-stranded small interfering RNA（siRNA） on cardiomyocytes apoptosis induced by doxorubicin（DOX）,two plasmids containing...In order to investigate the influence of silencing soluble epoxide hydrolase（sEH） with double-stranded small interfering RNA（siRNA） on cardiomyocytes apoptosis induced by doxorubicin（DOX）,two plasmids containing siRNA sequences specific to sEH were constructed and transfected into the primary cultured cardiomyocytes by using FuGENE HD transfection agents.The mRNA and protein expression levels of sEH were detected by semiquantitative RT-PCR and Western blotting respectively,and the plasmids that silenced sEH most significantly were selected,and renamed EH-R.The plasmids carrying a nonspecific siRNA coding sequence（PCN） served as the negative control.Cardiomyocytes were divided into four groups：control group,DOX group,PCN＋DOX group,and EH-R＋DOX group.Apoptosis of cardiomyocytes was induced by DOX at a concentration of 1 μmol/L.Apoptosis rate of cardiomyocytes was determined by flow cytometery.The protein expression levels of Bcl-2 and Bax were detected by Western blotting.The results showed that the expression of sEH was down-regulated by EH-R plasmid.The expression levels of sEH mRNA and protein in the EH-R＋DOX group were significantly decreased as compared with other groups（P0.01）.As compared with the control group,the apoptosis rate of cardiomyocytes in three DOX-treated groups was obviously increased,the expression levels of Bax increased,and those of Bcl-2 decreased（P0.01）.However,the expression levels of Bax were decreased,those of Bcl-2 increased and the apoptosis rate of cardiomyocytes obvi-ously decreased in EH-R＋DOX group when compared with those in the DOX group and the PCN＋DOX group（P0.01 for each）.It was concluded that the recombinant plasmids could be successfully constructed,and transfected into the primary cultured cardiomyocytes.They could ameliorate the DOX-induced cardiomyocytes apoptosis by selectively inhibiting the expression of sEH with RNAi and increasing the expression of Bcl-2.展开更多
To ensure proper dosage of a drug,analytical quantification of it in biofluid is necessary.Liquid chromatography mass spectrometry(LC-MS)is the conventional method of choice as it permits accurate identification and q...To ensure proper dosage of a drug,analytical quantification of it in biofluid is necessary.Liquid chromatography mass spectrometry(LC-MS)is the conventional method of choice as it permits accurate identification and quantification.However,it requires expensive instrumentation and is not appropriate for bedside use.Using soluble epoxide hydrolase(sEH)inhibitors(EC5026 and TPPU)as examples,we report development of a nanobody-based enzyme-linked immunosorbent assay(ELISA)for such small molecules and its use to accurately quantify the drug chemicals in human samples.Under optimized conditions,two nanobody-based ELISAs were successfully established for EC5026 and TPPU with low limits of detection of 0.085 ng/mL and 0.31 ng/mL,respectively,and two order of magnitude linear ranges with high precision and accuracy.The assay was designed to detect parent and two biologically active metabolites in the investigation of a new drug candidate EC5026.In addition,the ELISAs displayed excellent correlation with LC-MS analysis and evaluation of inhibitory potency.The results indicate that nanobody-based ELISA methods can efficiently analyze drug like compounds.These methods could be easily implemented by the bedside,in the field in remote areas or in veterinary practice.This work illustrates that nanobody based assays offer alternative and supplementary analytical tools to mass spectrometry for monitoring small molecule medicines during clinical development and therapy.Attributes of nanobody based pharmaceutical assays are discussed.展开更多
Epoxide hydrolase-catalyzed resolution of ethyl 3-phenylglydidate was investigated using resting cells of Pseudomonas sp. BZS21. Under the present conditions 26.2 % of (2R, 3S)- ethyl 3-phenylglycidate with ee value o...Epoxide hydrolase-catalyzed resolution of ethyl 3-phenylglydidate was investigated using resting cells of Pseudomonas sp. BZS21. Under the present conditions 26.2 % of (2R, 3S)- ethyl 3-phenylglycidate with ee value of 94.6 % was obtained from the racemic mixture.展开更多
The epoxide hydrolase gene(SpEH) from Sphingomonas sp. HXN-200 was synthesized and expressed in robust Escherichia coli cells that had a dual protection system. The enantioselectivity(E-value) of the recombinant SpEH ...The epoxide hydrolase gene(SpEH) from Sphingomonas sp. HXN-200 was synthesized and expressed in robust Escherichia coli cells that had a dual protection system. The enantioselectivity(E-value) of the recombinant SpEH was 7.7 and the yield of the remaining(R)-PGE was 24.3% for the hydrolysis of racemic phenyl glycidyl ether(rac-PGE). To improve the catalytic properties of SpEH, the site-directed mutagenesis was carried out based on homology modeling, sequence alignment and molecular docking. Six residues(V195, V196, F218,N226, Q312, and M332) near the active site were mutated to hydrophobic amino acids and the positive mutations were selected for combinatorial mutation. The optimal mutant SpEH^(V196A/N226A/M332A) had an enhanced E-value of 21.2 and a specific activity of 4.57 U·mg^-1-wet cells, which were 2.8-, and 2.3-fold higher than those of wild-type SpEH. The optimal temperature and p H for purified Sp EHV196 A/N226 A/M332 Ato catalyze the hydrolysis of rac-PGE were 25 ℃ and 7.0 with 200 U·mg^-1. The enantioselectivity and yield of the remaining(R)-PGE of E. coliSpEH^(V196A/N226A/M332A)increased from 7.7 to 21.2 and 24.3% to 40.9%, respectively. The molecular docking and kinetic parameter analyses showed that SpEH^(V196A/N226A/M332A) has a greater affinity toward(S)-PGE than(R)-PGE, and that it was more difficult for the O-atom of ASP170 to achieve the nucleophilic attack on the Cα of(R)-PGE, resulting in its improved enantioselectivity.展开更多
Background:Epoxyeicosatrienoic acids(EETs),which are metabolites of arachidonic acid catalyzed by cytochrome P450 epoxygenase,are degraded into inactive dihydroxyeicosatrienoic acids by soluble epoxide hydrolase(sEH)....Background:Epoxyeicosatrienoic acids(EETs),which are metabolites of arachidonic acid catalyzed by cytochrome P450 epoxygenase,are degraded into inactive dihydroxyeicosatrienoic acids by soluble epoxide hydrolase(sEH).Many studies have revealed that sEH gene deletion exerts protective effects against diabetes.Vascular calcification is a common complication of diabetes,but the potential effects of sEH on diabetic vascular calcification are still unknown.Methods:The level of aortic calcification in wild-type and Ephx2^(−/−)C57BL/6 diabetic mice induced with streptozotocin was evaluated by measuring the aortic calcium content through alizarin red staining,immunohistochemistry staining,and immunofluorescence staining.Mouse vascular smooth muscle cell lines(MOVAS cells)treated with β-glycerol phosphate(0.01 mol/L)plus advanced glycation end products(50 mg/L)were used to investigate the effects of sEH inhibitors or sEH knockdown and EETs on the calcification of vascular smooth muscle cells,which was detected by Western blotting,alizarin red staining,and Von Kossa staining.Results:sEH gene deletion significantly inhibited diabetic vascular calcification by increasing levels of EETs in the aortas of mice.EETs(especially 11,12-EET and 14,15-EET)efficiently prevented the osteogenic transdifferentiation of MOVAS cells by decreasing nidogen-2(NID2)expression.Interestingly,suppressing sEH activity by small interfering ribonucleic acid or specific inhibitors did not block osteogenic transdifferentiation of MOVAS cells induced by β-glycerol phosphate and advanced glycation end products.NID2 overexpression significantly abolished the inhibitory effect of sEH gene deletion on diabetic vascular calcification.Moreover,NID2 overexpression mediated by adeno-associated virus 9 vectors markedly increased insulin-like growth factor 2(IGF2)and phospho-ERK1/2 expression in MOVAS cells.Overall,sEH gene knockout inhibited diabetic vascular calcification by decreasing aortic NID2 expression and,then,inactivating the downstream IGF2-ERK1/2 signaling pathway.Conclusions:sEH gene deletion markedly inhibited diabetic vascular calcification through repressed osteogenic transdifferentiation of vascular smooth muscle cells mediated by increased aortic EET levels,which was associated with decreased NID2 expression and inactivation of the downstream IGF2-ERK1/2 signaling pathway.展开更多
Background Cigarette smoking is the major risk factor for chronic obstructive pulmonary disease (COPD) However, only 10%-20% of chronic heavy cigarette smokers develop symptomatic disease COPD is most likely th...Background Cigarette smoking is the major risk factor for chronic obstructive pulmonary disease (COPD) However, only 10%-20% of chronic heavy cigarette smokers develop symptomatic disease COPD is most likely the result of complex interactions between environmental and genetic factors Genetic susceptibility to COPD might depend on the variations in enzyme activities that detoxify cigarette smoke products, such as microsomal epoxide hydrolase (mEH) and glutathione S-transferase (GST) In this study, we investigated the relationship between polymorphisms in the genes encoding mEH and glutathione S-transferase P1 (GSTP1) and COPD in a Chinese population Methods Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to find mEH polymorphism in exon 3 (Tyr113→His), exon 4 (His139→Arg) and GSTP1 polymorphism in exon 5 (Ile105→Val) in 100 COPD patients and 100 age- and sex-matched healthy controls Results The proportion of mEH exon 3 heterozygotes was significantly higher in patients with COPD than that in the control subjects (42% vs 32%) The odds ratio ( OR ) adjusted by age, sex, body mass index (BMI) and cigarette years was 2 96 (95% CI 1 24-7 09) There was no marked difference in very slow activity genotype versus other genotypes between COPD patients and the controls When COPD patients were non-smokers, the OR of very slow activity genotype versus other genotypes was more than 1 00; and when COPD patients were smokers (current smokers and ex-smokers), the OR was less than 1 00 There was no significant difference in GSTP1 polymorphism adjusted by age, sex, BMI and smoking between COPD patients and the controls Conclusions mEH exon 3 heterozygotes might be associated with susceptibility to COPD in China The interaction might exist between mEH genotype and smoke The gene polymorphism for GSTP1 might not be associated with susceptibility to COPD in the Chinese population展开更多
Epoxyeicosatrienoic acids(EETs)have pleiotropic endogenous cardiovascular protective effects and can be hydrolyzed to the corresponding dihydroxyeicosatrienoic acids by soluble epoxide hydrolase(sEH).Heart failure wit...Epoxyeicosatrienoic acids(EETs)have pleiotropic endogenous cardiovascular protective effects and can be hydrolyzed to the corresponding dihydroxyeicosatrienoic acids by soluble epoxide hydrolase(sEH).Heart failure with preserved ejection fraction(HFpEF)has shown an increased prevalence and worse prognosis over the decades.However,the role of sEH activ-ity in HFpEF remains unclear.We enrolled 500 patients with HFpEF and 500 healthy controls between February 2010 and March 2016.Eight types of sEH-related eicosanoids were measured according to target metabolomics,and their correlation with clinical endpoints was also analyzed.The primary endpoint was cardiac mortality,and the secondary endpoint was a composite of cardiac events,including heart failure(HF)readmission,cardiogenic hospitalization,and all-cause mortal-ity.Furthermore,the effect of sEH inhibitors on cardiac diastolic function in HFpEF was investigated in vivo and in vitro.Patients with HFpEF showed significantly enhanced EET degradation by the sEH enzyme compared with healthy controls.More importantly,sEH activity was positively correlated with cardiac mortality in patients with HFpEF,especially in older patients with arrhythmia.A consistent result was obtained in the multiple adjusted models.Decreased sEH activity by the sEH inhibitor showed a significant effective effect on the improvement of cardiac diastolic function by ameliorating lipid disorders in cardiomyocytes of HFpEF mouse model.This study demonstrated that increased sEH activity was associated with cardiac mortality in patients with HFpEF and suggested that sEH inhibition could be a promising therapeutic strategy to improve diastolic cardiac function.Clinical trial identifier:NCT03461107(https://clini caltr ials.gov).展开更多
Soluble epoxide hydrolase(sEH) is related to arachidonic acid cascade and is over-expressed in a variety of diseases, making sEH an attractive target for the treatment of pain as well as inflammatory-related diseases....Soluble epoxide hydrolase(sEH) is related to arachidonic acid cascade and is over-expressed in a variety of diseases, making sEH an attractive target for the treatment of pain as well as inflammatory-related diseases. A new series of memantyl urea derivatives as potent sEH inhibitors was obtained using our previous reported compound 4 as lead compound. A preferential modification of piperidinyl to 3-carbamoyl piperidinyl was identified for this series via structure-based rational drug design. Compound A20 exhibited moderate percentage plasma protein binding(88.6%) and better metabolic stability in vitro. After oral administration, the bioavailability of A20 was 28.6%. Acute toxicity test showed that A20 was well tolerated and there was no adverse event encountered at dose of 6.0 g/kg. Inhibitor A20 also displayed robust analgesic effect in vivo and dose-dependently attenuated neuropathic pain in rat model induced by spared nerve injury, which was better than gabapentin and sEH inhibitor(±)-EC-5026. In one word, the oral administration of A20 significantly alleviated pain and improved the health status of the rats, demonstrating that A20 was a promising candidate to be further evaluated for the treatment of neuropathic pain.展开更多
AIM To investigate the contribution of polymorphisms in the CYP1A1, CYP2E1 and EPHX1 genes on sporadic colorectal cancer(SCRC) risk. METHODS Six hundred forty-one individuals(227 patients with SCRC and 400 controls) w...AIM To investigate the contribution of polymorphisms in the CYP1A1, CYP2E1 and EPHX1 genes on sporadic colorectal cancer(SCRC) risk. METHODS Six hundred forty-one individuals(227 patients with SCRC and 400 controls) were enrolled in the study. The variables analyzed were age, gender, tobacco and alcohol consumption, and clinical and histopathological tumor parameters. The CYP1A1 *2A, CYP1A1 *2C CYP2E1 *5B and CYP2E1 *6 polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP). The EPHX1 Tyr113 His, EPHX1 His139 Arg and CYP1A1 *2C polymorphisms were detected by real-time PCR. Chisquared test and binary logistic regression were used in the statistical analysis. Haplotype analysis was conducted using the Haploview program, version 2.05.RESULTS Age over 6 2 years was a risk factor for SCRC development(OR = 7.54, 95%CI: 4.94-11.50, P < 0.01). Male individuals were less susceptible to SCRC(OR = 0.55, 95%CI: 0.35-0.85, P < 0.01). The CYP2E1*5B polymorphism was associated with SCRC in the codominant(heterozygous genotype: OR = 2.66, 95%CI: 1.64-4.32, P < 0.01), dominant(OR = 2.82, 95%CI: 1.74-4.55, P < 0.01), overdominant(OR = 2.58, 95%CI: 1.59-4.19, P < 0.01), and log-additive models(OR = 2.84, 95%CI: 1.78-4.52, P < 0.01). The CYP2E1*6 polymorphism was associated with an increased SCRC risk in codominant(heterozygous genotype: OR = 2.81, 95%CI: 1.84-4.28, P < 0.01; homozygous polymorphic : OR = 7. 3 2, 9 5 % C I : 1.85-28.96, P < 0.01), dominant(OR = 2.97, 95%CI: 1.97-4.50, P < 0.01), recessive(OR = 5.26, 95%CI: 1.35-20.50, P = 0.016), overdominant(OR = 2.64, 95%CI: 1.74-4.01, P < 0.01), and log-additive models(OR = 2.78, 95%CI: 1.91-4.06, P < 0.01). The haplotype formed by the minor alleles of the CYP2E1*5B(C) and CYP2E1*6(A) polymorphisms was associated with SCRC(P = 0.002). However, the CYP1A1 *2A, CYP1A1 *2C, EPHX1 Tyr113 His and EPHX1 His139 Arg polymorphisms were not associated with SCRC.CONCLUSION In conclusion, the results demonstrated that CYP2E1*5B and CYP2E1*6 minor alleles play a role in the development of SCRC.展开更多
The arachidonic acid(AA)metabolic pathway participates in various physiological processes as well as in the development of malignancies.We analyzed genomic alterations in AA metabolic enzymes in the Cancer Genome Atla...The arachidonic acid(AA)metabolic pathway participates in various physiological processes as well as in the development of malignancies.We analyzed genomic alterations in AA metabolic enzymes in the Cancer Genome Atlas(TCGA)prostate cancer(PCa)dataset and found that the gene encoding soluble epoxide hydrolase(EPHX2)is frequently deleted in PCa.EPHX2 mRNA and protein expression in PCa was examined in multiple datasets by differential gene expression analysis and in a tissue microarray by immunohistochemistry.The expression data were analyzed in conjunction with clinicopathological variables.Both the mRNA and protein expression levels of EPHX2 were significantly decreased in tumors compared with normal prostate tissues and were inversely correlated with the Gleason grade and disease-free survival time.Furthermore,EPHX2 mRNA expression was significantly decreased in metastatic and recurrent PCa compared with localized and primary PCa,respectively.In addition,EPHX2 protein expression correlated negatively with Ki67 expression.In conclusion,EPHX2 deregulation is significantly correlated with the clinical characteristics of PCa progression and may serve as a prognostic marker for PCa.展开更多
The aim of this study was to explore the association of the genetic polymorphism of EPHX1 and EPHX2 with the susceptibility to chronic benzene poisoning(CBP).A case-control study of 268 patients with CBP and 268 healt...The aim of this study was to explore the association of the genetic polymorphism of EPHX1 and EPHX2 with the susceptibility to chronic benzene poisoning(CBP).A case-control study of 268 patients with CBP and 268 healthy workers matched by age and sex,all of whom were occupationally exposed to benzene,was conducted.The single nucleotide polymorphisms(SNPs,rs2854451,rs3738047,rs2234922 and rs1051741)of EPHX1 gene and the SNP(rs751141)of EPHX2 gene were tested by the TaqMan PCR method.In the subjects carrying the genotype of EPHX1 rs3738047 GG,the risk of CBP was decreased in the individuals simultaneously carrying EPHX1 rs2234922 G(P=0.02).Alternatively,in the subjects carrying the genotype of EPHX1 rs2234922 AA,the risk of CBP was increased in the individuals simultaneously carrying the allele of EPHX2 rs751141A(P=0.03).It was also found that there were potential interactions between alcohol consumption and the polymorphism of EPHX1 rs1051741(x_(H)^(2)=5.28,P=0.02)or rs2234922(x_(H)^(2)=6.71,P=0.01).Compared to individuals with EPHX1 rs1051741 CC or rs2234922 AA genotype in the drinkers,the risk of CBP in those carrying genotypes of EPHX1 rs1051741 CT+TT or rs2234922 AG+GG was decreased,respectively(P=0.04,P<0.01).Haplotype analysis of polymorphisms in EPHX1 showed that the risk of CBP was increased in the subjects with haplotype 2(rs2854451-A,rs3738047-G,rs2234922-A,rs1051741-C)or haplotype 4(rs2854451-G,rs3738047-A,rs2234922-G,rs1051741-T),but decreased in those with haplotype 6(rs2854451-G,rs3738047-G,rs2234922-G,rs1051741-T)or haplotype 10(rs2854451-A,rs3738047-A,rs2234922-G,rs1051741-T),respectively.Logistic regression analysis revealed that smoking might play a role in modifying the risk of CBP(OR=0.313,95%CI:0.123–0.794,P=0.015).The genetic polymorphism in EPHX1 may be associated with the risk of CBP in the Chinese occupational population and further research is needed for the association between the genetic polymorphism in EPHX2 and the susceptibility to CBP.展开更多
基金supported by the National Natural Science Foundation of China(82274069 and 82003580)Shenzhen science and technology research and development funds(JCYJ20190808171803553 and 2022071718149001)+4 种基金Young Scientific and Technological Talents(Level Two)in Tianjin(QN20230212)Tianjin Education Commission Research Program Project(2024KJ004)Young Elite Scientists Sponsorship Program by China Association of Chinese Medicine(2022-QNRC2-B09)“1+X”Research Project of the Second Hospital of Dalian Medical University(2024JJ11PT005)Eaglet Plan Project of Tianjin University of Traditional Chinese Medicine(XJS2024101)。
文摘Objective:Soluble epoxide hydrolase(sEH)emerges as a target of interest for inflammatory diseases.Piperine is a natural amide alkaloid from Piper nigrum and displays an inhibitory effect toward sEH,its chemical structural transformation was carried out in order to obtain a library of sEH inhibitors based on its skeleton.Methods:Structural transformation of piperine was carried out by chemical methods,and piperine derivatives were assayed for their sEH potentials.A mouse acute lung injury model was constructed by lipopolysaccharide(LPS).Hematoxylin and eosin(H&E)staining,immunofluorescence staining,Western Blot,and enzyme-linked immunosorbent assay were used for investigating the protective potential of sEH inhibitor 11h.Results:Piperine derivatives 11e,11h,11j,and 11o showed inhibitory potentials toward sEH with values of half maximal inhibitory concentration(IC50)from 20 to 70 nM.Compound 11h attenuated the pathological course of LPS-mediated acute lung injury(ALI)in vivo.Furthermore,levels of cytokines tumor necrosis factor alpha(TNF-α),interleukin 6(IL-6),myeloperoxidase(MPO),and lactate dehydrogenase(LDH)were decreased after administration of 11h.The LPS-mediated inflammation and redox unbalance,including expressions of cyclooxygenase-2(COX-2),heme oxygenase-1(HO-1),intercellular cell adhesion molecule-1(ICAM-1),vascular cell adhesion molecule-1(VCAM-1),p-p65/p65,glutamate-cysteine ligase modifier subunit(GCLM),and nuclear factor erythroid-2-related factor 2(Nrf2),were ameliorated through nuclear factor kappa B(NF-κB)and Nrf2 pathways via enhancing levels of epoxyeicosatrienoic acids(EETs)in LPS-exposed ALI mice after compound 11h treatment.Molecular docking demonstrated that the aromatic unsaturated group of 11h occupied a hydrophobic pocket and its urea group formed three hydrogen bonds with Asp333,Tyr381,and Tyr465,which stabilized the active conformation of the ligand.Conclusions:These findings demonstrated that compound 11h may serve as a lead compound for developing sEH inhibitors and treating inflammation related to diseases,such as ALI.
基金partly supported by the National Natural Science Foundation of China(81271475 and 81571297)
文摘Dear Editor, Nicotine is a psychoactive alkaloid that is thought to play a key role in addiction to commercial tobacco products [1] and cotinine is its primary metabolite [2]. Pharmacological treatment, such as nicotine replacement therapy （NRT）, is a valid solution to this problem. Tobacco smoke contains many carcinogens such as nitrosamines .
基金Project (No. 20542006) supported by the National Natural ScienceFoundation of China
文摘A two-layer method based on support vector machines (SVMs) has been developed to distinguish epoxide hydrolases (EHs) from other enzymes and to classify its subfamilies using its primary protein sequences. SVM classifiers were built using three different feature vectors extracted from the primary sequence of EHs: the amino acid composition (AAC), the dipeptide composition (DPC), and the pseudo-amino acid composition (PAAC). Validated by 5-fold cross tests, the first layer SVM clas- sifier can differentiate EHs and non-EHs with an accuracy of 94.2% and has a Matthew’s correlation coefficient (MCC) of 0.84. Using 2-fold cross validation, PAAC-based second layer SVM can further classify EH subfamilies with an overall accuracy of 90.7% and MCC of 0.87 as compared to AAC (80.0%) and DPC (84.9%). A program called EHPred has also been developed to assist readers to recognize EHs and to classify their subfamilies using primary protein sequences with greater accuracy.
文摘In order to investigate the influence of silencing soluble epoxide hydrolase（sEH） with double-stranded small interfering RNA（siRNA） on cardiomyocytes apoptosis induced by doxorubicin（DOX）,two plasmids containing siRNA sequences specific to sEH were constructed and transfected into the primary cultured cardiomyocytes by using FuGENE HD transfection agents.The mRNA and protein expression levels of sEH were detected by semiquantitative RT-PCR and Western blotting respectively,and the plasmids that silenced sEH most significantly were selected,and renamed EH-R.The plasmids carrying a nonspecific siRNA coding sequence（PCN） served as the negative control.Cardiomyocytes were divided into four groups：control group,DOX group,PCN＋DOX group,and EH-R＋DOX group.Apoptosis of cardiomyocytes was induced by DOX at a concentration of 1 μmol/L.Apoptosis rate of cardiomyocytes was determined by flow cytometery.The protein expression levels of Bcl-2 and Bax were detected by Western blotting.The results showed that the expression of sEH was down-regulated by EH-R plasmid.The expression levels of sEH mRNA and protein in the EH-R＋DOX group were significantly decreased as compared with other groups（P0.01）.As compared with the control group,the apoptosis rate of cardiomyocytes in three DOX-treated groups was obviously increased,the expression levels of Bax increased,and those of Bcl-2 decreased（P0.01）.However,the expression levels of Bax were decreased,those of Bcl-2 increased and the apoptosis rate of cardiomyocytes obvi-ously decreased in EH-R＋DOX group when compared with those in the DOX group and the PCN＋DOX group（P0.01 for each）.It was concluded that the recombinant plasmids could be successfully constructed,and transfected into the primary cultured cardiomyocytes.They could ameliorate the DOX-induced cardiomyocytes apoptosis by selectively inhibiting the expression of sEH with RNAi and increasing the expression of Bcl-2.
基金supported by NIEHS(RIVER Award,R35 ES030443)NIEHS(Superfund Award,P42 ES004699)+6 种基金NINDS(Counter ActProgram U54 NS127758)Juvenile Diabetes Research Foundation(2-SRA-2022-1210-S-B)Guangzhou Science and Technology Foundation(Grant No.:201903010034)Natural Resources Science Foundation of Guangdong Province(Grant No.:2018A030313926)Science and Technology Foundation Key R&D Program of Guangdong Province(Grant Nos.:2019B020209009 and 2019B020218009)R&D Program of Guangdong Province Drug Administration(Grant Nos.:2021TDZ09 and 2021YDZ06)supported by China Scholarship Council(CSC)(202108440382).
文摘To ensure proper dosage of a drug,analytical quantification of it in biofluid is necessary.Liquid chromatography mass spectrometry(LC-MS)is the conventional method of choice as it permits accurate identification and quantification.However,it requires expensive instrumentation and is not appropriate for bedside use.Using soluble epoxide hydrolase(sEH)inhibitors(EC5026 and TPPU)as examples,we report development of a nanobody-based enzyme-linked immunosorbent assay(ELISA)for such small molecules and its use to accurately quantify the drug chemicals in human samples.Under optimized conditions,two nanobody-based ELISAs were successfully established for EC5026 and TPPU with low limits of detection of 0.085 ng/mL and 0.31 ng/mL,respectively,and two order of magnitude linear ranges with high precision and accuracy.The assay was designed to detect parent and two biologically active metabolites in the investigation of a new drug candidate EC5026.In addition,the ELISAs displayed excellent correlation with LC-MS analysis and evaluation of inhibitory potency.The results indicate that nanobody-based ELISA methods can efficiently analyze drug like compounds.These methods could be easily implemented by the bedside,in the field in remote areas or in veterinary practice.This work illustrates that nanobody based assays offer alternative and supplementary analytical tools to mass spectrometry for monitoring small molecule medicines during clinical development and therapy.Attributes of nanobody based pharmaceutical assays are discussed.
基金supported by the Natural Science Foundation of Shandong Province(No.Y2000C21)the National Natural Science Foundation of China(No.30270047).
文摘Epoxide hydrolase-catalyzed resolution of ethyl 3-phenylglydidate was investigated using resting cells of Pseudomonas sp. BZS21. Under the present conditions 26.2 % of (2R, 3S)- ethyl 3-phenylglycidate with ee value of 94.6 % was obtained from the racemic mixture.
基金the National Natural Science Foundation of China(21676104,21878105,21908070)the National Key Research and Development Program of China(2018YFC1603400,2018YFC1602100)+3 种基金the Key Research and Development Program of Guangdong Province(2019B020213001)the Science and Technology Program of Guangzhou(201904010360)the Fundamental Research Funds for the Central Universities(2019PY15,2019MS100)the China Postdoctoral Science Foundation(BX20180102)for partially funding this work。
文摘The epoxide hydrolase gene(SpEH) from Sphingomonas sp. HXN-200 was synthesized and expressed in robust Escherichia coli cells that had a dual protection system. The enantioselectivity(E-value) of the recombinant SpEH was 7.7 and the yield of the remaining(R)-PGE was 24.3% for the hydrolysis of racemic phenyl glycidyl ether(rac-PGE). To improve the catalytic properties of SpEH, the site-directed mutagenesis was carried out based on homology modeling, sequence alignment and molecular docking. Six residues(V195, V196, F218,N226, Q312, and M332) near the active site were mutated to hydrophobic amino acids and the positive mutations were selected for combinatorial mutation. The optimal mutant SpEH^(V196A/N226A/M332A) had an enhanced E-value of 21.2 and a specific activity of 4.57 U·mg^-1-wet cells, which were 2.8-, and 2.3-fold higher than those of wild-type SpEH. The optimal temperature and p H for purified Sp EHV196 A/N226 A/M332 Ato catalyze the hydrolysis of rac-PGE were 25 ℃ and 7.0 with 200 U·mg^-1. The enantioselectivity and yield of the remaining(R)-PGE of E. coliSpEH^(V196A/N226A/M332A)increased from 7.7 to 21.2 and 24.3% to 40.9%, respectively. The molecular docking and kinetic parameter analyses showed that SpEH^(V196A/N226A/M332A) has a greater affinity toward(S)-PGE than(R)-PGE, and that it was more difficult for the O-atom of ASP170 to achieve the nucleophilic attack on the Cα of(R)-PGE, resulting in its improved enantioselectivity.
基金supported by the National Natural Science Foundation of China(Nos.82070825,81873512,and 82004254).
文摘Background:Epoxyeicosatrienoic acids(EETs),which are metabolites of arachidonic acid catalyzed by cytochrome P450 epoxygenase,are degraded into inactive dihydroxyeicosatrienoic acids by soluble epoxide hydrolase(sEH).Many studies have revealed that sEH gene deletion exerts protective effects against diabetes.Vascular calcification is a common complication of diabetes,but the potential effects of sEH on diabetic vascular calcification are still unknown.Methods:The level of aortic calcification in wild-type and Ephx2^(−/−)C57BL/6 diabetic mice induced with streptozotocin was evaluated by measuring the aortic calcium content through alizarin red staining,immunohistochemistry staining,and immunofluorescence staining.Mouse vascular smooth muscle cell lines(MOVAS cells)treated with β-glycerol phosphate(0.01 mol/L)plus advanced glycation end products(50 mg/L)were used to investigate the effects of sEH inhibitors or sEH knockdown and EETs on the calcification of vascular smooth muscle cells,which was detected by Western blotting,alizarin red staining,and Von Kossa staining.Results:sEH gene deletion significantly inhibited diabetic vascular calcification by increasing levels of EETs in the aortas of mice.EETs(especially 11,12-EET and 14,15-EET)efficiently prevented the osteogenic transdifferentiation of MOVAS cells by decreasing nidogen-2(NID2)expression.Interestingly,suppressing sEH activity by small interfering ribonucleic acid or specific inhibitors did not block osteogenic transdifferentiation of MOVAS cells induced by β-glycerol phosphate and advanced glycation end products.NID2 overexpression significantly abolished the inhibitory effect of sEH gene deletion on diabetic vascular calcification.Moreover,NID2 overexpression mediated by adeno-associated virus 9 vectors markedly increased insulin-like growth factor 2(IGF2)and phospho-ERK1/2 expression in MOVAS cells.Overall,sEH gene knockout inhibited diabetic vascular calcification by decreasing aortic NID2 expression and,then,inactivating the downstream IGF2-ERK1/2 signaling pathway.Conclusions:sEH gene deletion markedly inhibited diabetic vascular calcification through repressed osteogenic transdifferentiation of vascular smooth muscle cells mediated by increased aortic EET levels,which was associated with decreased NID2 expression and inactivation of the downstream IGF2-ERK1/2 signaling pathway.
基金ThisstudywassupportedbytheNationalScienceandTechnologyProject[No 2 001BA703B03(A)]andtheBeijingNaturalScienceFundImbursementProject (No 70 2 2 0 14 )
文摘Background Cigarette smoking is the major risk factor for chronic obstructive pulmonary disease (COPD) However, only 10%-20% of chronic heavy cigarette smokers develop symptomatic disease COPD is most likely the result of complex interactions between environmental and genetic factors Genetic susceptibility to COPD might depend on the variations in enzyme activities that detoxify cigarette smoke products, such as microsomal epoxide hydrolase (mEH) and glutathione S-transferase (GST) In this study, we investigated the relationship between polymorphisms in the genes encoding mEH and glutathione S-transferase P1 (GSTP1) and COPD in a Chinese population Methods Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to find mEH polymorphism in exon 3 (Tyr113→His), exon 4 (His139→Arg) and GSTP1 polymorphism in exon 5 (Ile105→Val) in 100 COPD patients and 100 age- and sex-matched healthy controls Results The proportion of mEH exon 3 heterozygotes was significantly higher in patients with COPD than that in the control subjects (42% vs 32%) The odds ratio ( OR ) adjusted by age, sex, body mass index (BMI) and cigarette years was 2 96 (95% CI 1 24-7 09) There was no marked difference in very slow activity genotype versus other genotypes between COPD patients and the controls When COPD patients were non-smokers, the OR of very slow activity genotype versus other genotypes was more than 1 00; and when COPD patients were smokers (current smokers and ex-smokers), the OR was less than 1 00 There was no significant difference in GSTP1 polymorphism adjusted by age, sex, BMI and smoking between COPD patients and the controls Conclusions mEH exon 3 heterozygotes might be associated with susceptibility to COPD in China The interaction might exist between mEH genotype and smoke The gene polymorphism for GSTP1 might not be associated with susceptibility to COPD in the Chinese population
基金supported by grants from the National Natural Science Foundation of China(81790624[to D.W.W.],81900342[to L.P.]and 81790621[to Y.Z.]).
文摘Epoxyeicosatrienoic acids(EETs)have pleiotropic endogenous cardiovascular protective effects and can be hydrolyzed to the corresponding dihydroxyeicosatrienoic acids by soluble epoxide hydrolase(sEH).Heart failure with preserved ejection fraction(HFpEF)has shown an increased prevalence and worse prognosis over the decades.However,the role of sEH activ-ity in HFpEF remains unclear.We enrolled 500 patients with HFpEF and 500 healthy controls between February 2010 and March 2016.Eight types of sEH-related eicosanoids were measured according to target metabolomics,and their correlation with clinical endpoints was also analyzed.The primary endpoint was cardiac mortality,and the secondary endpoint was a composite of cardiac events,including heart failure(HF)readmission,cardiogenic hospitalization,and all-cause mortal-ity.Furthermore,the effect of sEH inhibitors on cardiac diastolic function in HFpEF was investigated in vivo and in vitro.Patients with HFpEF showed significantly enhanced EET degradation by the sEH enzyme compared with healthy controls.More importantly,sEH activity was positively correlated with cardiac mortality in patients with HFpEF,especially in older patients with arrhythmia.A consistent result was obtained in the multiple adjusted models.Decreased sEH activity by the sEH inhibitor showed a significant effective effect on the improvement of cardiac diastolic function by ameliorating lipid disorders in cardiomyocytes of HFpEF mouse model.This study demonstrated that increased sEH activity was associated with cardiac mortality in patients with HFpEF and suggested that sEH inhibition could be a promising therapeutic strategy to improve diastolic cardiac function.Clinical trial identifier:NCT03461107(https://clini caltr ials.gov).
基金funded by the Liaoning Revitalization Talents Program(XLYC1908031,China)Basic Research Project of Department of Education of Liaoning Province-natural sciences(2020LJC02,China)+2 种基金Major Basic Research Project of Natural Science Foundation of Shandong Province(ZR2018ZC1056,China)partial support was provided by the NIH-NIEHS RIVER Award(R35 ES030443-01,USA)the NIEHS Superfund Research Program(P42 ES004699,USA)。
文摘Soluble epoxide hydrolase(sEH) is related to arachidonic acid cascade and is over-expressed in a variety of diseases, making sEH an attractive target for the treatment of pain as well as inflammatory-related diseases. A new series of memantyl urea derivatives as potent sEH inhibitors was obtained using our previous reported compound 4 as lead compound. A preferential modification of piperidinyl to 3-carbamoyl piperidinyl was identified for this series via structure-based rational drug design. Compound A20 exhibited moderate percentage plasma protein binding(88.6%) and better metabolic stability in vitro. After oral administration, the bioavailability of A20 was 28.6%. Acute toxicity test showed that A20 was well tolerated and there was no adverse event encountered at dose of 6.0 g/kg. Inhibitor A20 also displayed robust analgesic effect in vivo and dose-dependently attenuated neuropathic pain in rat model induced by spared nerve injury, which was better than gabapentin and sEH inhibitor(±)-EC-5026. In one word, the oral administration of A20 significantly alleviated pain and improved the health status of the rats, demonstrating that A20 was a promising candidate to be further evaluated for the treatment of neuropathic pain.
基金Supported by Sao Paulo Research Foundation(FAPESP),No.2011/23969-1 and No.2012/02473-0Coordination for the Improvement of Higher Education Personnel(CAPES)(Master grant)and National Council of Technological and Scientific Development(CNPq),No.310582/2014-8
文摘AIM To investigate the contribution of polymorphisms in the CYP1A1, CYP2E1 and EPHX1 genes on sporadic colorectal cancer(SCRC) risk. METHODS Six hundred forty-one individuals(227 patients with SCRC and 400 controls) were enrolled in the study. The variables analyzed were age, gender, tobacco and alcohol consumption, and clinical and histopathological tumor parameters. The CYP1A1 *2A, CYP1A1 *2C CYP2E1 *5B and CYP2E1 *6 polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP). The EPHX1 Tyr113 His, EPHX1 His139 Arg and CYP1A1 *2C polymorphisms were detected by real-time PCR. Chisquared test and binary logistic regression were used in the statistical analysis. Haplotype analysis was conducted using the Haploview program, version 2.05.RESULTS Age over 6 2 years was a risk factor for SCRC development(OR = 7.54, 95%CI: 4.94-11.50, P < 0.01). Male individuals were less susceptible to SCRC(OR = 0.55, 95%CI: 0.35-0.85, P < 0.01). The CYP2E1*5B polymorphism was associated with SCRC in the codominant(heterozygous genotype: OR = 2.66, 95%CI: 1.64-4.32, P < 0.01), dominant(OR = 2.82, 95%CI: 1.74-4.55, P < 0.01), overdominant(OR = 2.58, 95%CI: 1.59-4.19, P < 0.01), and log-additive models(OR = 2.84, 95%CI: 1.78-4.52, P < 0.01). The CYP2E1*6 polymorphism was associated with an increased SCRC risk in codominant(heterozygous genotype: OR = 2.81, 95%CI: 1.84-4.28, P < 0.01; homozygous polymorphic : OR = 7. 3 2, 9 5 % C I : 1.85-28.96, P < 0.01), dominant(OR = 2.97, 95%CI: 1.97-4.50, P < 0.01), recessive(OR = 5.26, 95%CI: 1.35-20.50, P = 0.016), overdominant(OR = 2.64, 95%CI: 1.74-4.01, P < 0.01), and log-additive models(OR = 2.78, 95%CI: 1.91-4.06, P < 0.01). The haplotype formed by the minor alleles of the CYP2E1*5B(C) and CYP2E1*6(A) polymorphisms was associated with SCRC(P = 0.002). However, the CYP1A1 *2A, CYP1A1 *2C, EPHX1 Tyr113 His and EPHX1 His139 Arg polymorphisms were not associated with SCRC.CONCLUSION In conclusion, the results demonstrated that CYP2E1*5B and CYP2E1*6 minor alleles play a role in the development of SCRC.
基金Financial support for this study was supported by Yunnan Fundamental Research Projects(No.2019FE001[-277]and 2019FE001[-005])the Project for Innovation Team of Yunnan Provincial Science and Technology Department,China(No.2018HC005)Qujing Affiliated Hospital of Kunming Medical University(2019YJKT11 and 2020YJKT03).
文摘The arachidonic acid(AA)metabolic pathway participates in various physiological processes as well as in the development of malignancies.We analyzed genomic alterations in AA metabolic enzymes in the Cancer Genome Atlas(TCGA)prostate cancer(PCa)dataset and found that the gene encoding soluble epoxide hydrolase(EPHX2)is frequently deleted in PCa.EPHX2 mRNA and protein expression in PCa was examined in multiple datasets by differential gene expression analysis and in a tissue microarray by immunohistochemistry.The expression data were analyzed in conjunction with clinicopathological variables.Both the mRNA and protein expression levels of EPHX2 were significantly decreased in tumors compared with normal prostate tissues and were inversely correlated with the Gleason grade and disease-free survival time.Furthermore,EPHX2 mRNA expression was significantly decreased in metastatic and recurrent PCa compared with localized and primary PCa,respectively.In addition,EPHX2 protein expression correlated negatively with Ki67 expression.In conclusion,EPHX2 deregulation is significantly correlated with the clinical characteristics of PCa progression and may serve as a prognostic marker for PCa.
基金This work was funded by the Chinese National Key Basic Research and Development Program grant(No.2002CB512902)National Natural Science Foundation of China(Grant No.30271113).
文摘The aim of this study was to explore the association of the genetic polymorphism of EPHX1 and EPHX2 with the susceptibility to chronic benzene poisoning(CBP).A case-control study of 268 patients with CBP and 268 healthy workers matched by age and sex,all of whom were occupationally exposed to benzene,was conducted.The single nucleotide polymorphisms(SNPs,rs2854451,rs3738047,rs2234922 and rs1051741)of EPHX1 gene and the SNP(rs751141)of EPHX2 gene were tested by the TaqMan PCR method.In the subjects carrying the genotype of EPHX1 rs3738047 GG,the risk of CBP was decreased in the individuals simultaneously carrying EPHX1 rs2234922 G(P=0.02).Alternatively,in the subjects carrying the genotype of EPHX1 rs2234922 AA,the risk of CBP was increased in the individuals simultaneously carrying the allele of EPHX2 rs751141A(P=0.03).It was also found that there were potential interactions between alcohol consumption and the polymorphism of EPHX1 rs1051741(x_(H)^(2)=5.28,P=0.02)or rs2234922(x_(H)^(2)=6.71,P=0.01).Compared to individuals with EPHX1 rs1051741 CC or rs2234922 AA genotype in the drinkers,the risk of CBP in those carrying genotypes of EPHX1 rs1051741 CT+TT or rs2234922 AG+GG was decreased,respectively(P=0.04,P<0.01).Haplotype analysis of polymorphisms in EPHX1 showed that the risk of CBP was increased in the subjects with haplotype 2(rs2854451-A,rs3738047-G,rs2234922-A,rs1051741-C)or haplotype 4(rs2854451-G,rs3738047-A,rs2234922-G,rs1051741-T),but decreased in those with haplotype 6(rs2854451-G,rs3738047-G,rs2234922-G,rs1051741-T)or haplotype 10(rs2854451-A,rs3738047-A,rs2234922-G,rs1051741-T),respectively.Logistic regression analysis revealed that smoking might play a role in modifying the risk of CBP(OR=0.313,95%CI:0.123–0.794,P=0.015).The genetic polymorphism in EPHX1 may be associated with the risk of CBP in the Chinese occupational population and further research is needed for the association between the genetic polymorphism in EPHX2 and the susceptibility to CBP.
