Study On The Relationship Between EPHX2 Rs751141 Gene Polymorphism And Diabetic Vascular Complications And Its Mechanism

Background:Multiple epidemiological surveys show that type 2 diabetes mellitus(DM)is a complex metabolic disorder,and long-term high blood sugar will lead to multiorgan damage with serious vascular complications.At present,it is believed that the DM vascular complications in diabetic kidney disease(DKD)and ischemic stroke are closely related to genetic factors.In recent years,soluble epoxide hydrolase(sEH)in kidney disease especially the DKD and cerebral ischemic stroke has been paid attention,the study of sEH inhibitors and the EPHX2 knockout may become a new target for DKD and ischemic stroke.The rs751141 polymorphism(R287Q)of EPHX2 gene encoding sEH affects the activity of sEH and is associated with a variety of clinical diseases,and the relationship between the rs751141 polymorphism of EPHX2 gene and DKD,ischemic stroke diseases remains to be further studied.Objectives:1.This study aimed to verify whether the A allele of EPHX2 rs751141 is a protective factor of for DM vascular complications DKD and ischemic stroke.2.To study the relationship between the A allele of rs751141 polymorphism of EPHX2 and the activity of sEH hydrolase and the molecular mechanism of DKD.Methods:1.EPHX2 rs751141 gene polymorphism and diabetic vascular complications:1)Association of EPHX2 rs751141 polymorphism with DKD in type 2 DM patients:A total of 870 type 2 DM patients were hospitalized in China-Japan Friendship Hospital from February 2015 to June 2016.There were 464 non-diabetic kidney disease(non-DKD)patients(271male and 193female)and 406 DKD patients(258 male and 148 female).DNA was extracted from peripheral blood and SNP was detected by fluorescence probe method.The genotype frequencies,allele frequencies and Hardy-Weinberg balance were analyzed by chi-square test.Statistical analysis using Quanto software version 1.2.4.Data were analyzed with the SPSS 17.0 software.p<0.05 were considered significant.2)EPHX2 rs751141 gene polymorphism and type 2 DM associated with ischemic stroke:626 patients with type 2 DM were hospitalized in China-Japan Friendship Hospital from February 2015 to June 2016.There were 390 type 2 DM patients without ischemic stroke(241 male and 149 female)and 236 type 2 DM patients with ischemic stroke(127 male and 109 female).Detection method,data processing and statistical analysis is similar as 1.2.Study on the hydrolytic activity of EPHX2 recombinant plasmids and the molecular mechanism of rs751141 gene polymorphism in DKD:1)Construction of EPHX2 recombinant plasmids and the sEH hydrolase activity:Human cDNA as template,according to the GeneBank EPHX2 mRNA CDS primers.Using p-UCT DNA connection kit,the amplified cDNA fragment was cloned into p-UCT vector,and induced point mutations in the wild type,and a total of 4 single point mutations and 3 double point mutations were constructed,then the correct fragment was subcloned into eukaryotic expression vector pcDNA3.1/V5-His-A.EPHX2/pcDNA3.1/V5-His-A(WT),EPHX2/R287Q/pcDNA3.1/V5-His(R287Q),EPHX2/R287Q/R103C/pcDNA3.1/V5-His(R287Q+R103C),EPHX2/C154T/pcDN A3.1/V5-His(C154T),EPHX2/K55R/R103C/pcDNA3.1/V5-His(K55R+R103C),EPHX2/R103C/pcDNA3.1/V5-His(R103C),EPHX2/K55R/C154T/pcDNA3.1/V5-His(K55R+C154T),EPHX2/K55R/pcDNA3.1/V5-His(K55R)recombinant plasmids and pcDNA3.1/V5-His-A empty vector were transfected into HK2 cells by Lipofectamine2000,and green fluorescent protein(GFP)was transfected into the cells.The transfection efficiency was observed using fluorescence microscope.Western Blot was used to detect the expression of fusion protein.Eight different types of recombinant plasmids were transfected into HK2 cells with empty vector as control group.They were divided into two groups,one group of cell culture medium supplemented with sufficient 11,12-EET,and the other group was added with sEH inhibitor TPPU while adding 11,12-EET.11,12-DHET concentration in culture medium were assayed by ELISA method to evaluate the change of the hydrolysis products of 11,12-EET,in order to compare the differences among the recombinant plasmid in the hydrolysis activity of sEH.2)R287Q recombinant plasmid on the mechanism of DKD:WT,R287Q and NC recombinant plasmids were transfected into cultured normal HK2 cells.They were divided into two groups,one group was transfected with 48h,and the other group was given 11,12-EET cocultured with 24h.The supernatant,RNA and protein were detected by flow cytometry,RT-PCR and Western blot methods to detect the changes of the related indicators.Results:1.EPHX2 rs751141 gene polymorphism and DM vascular complications:1)The A allele frequency of rs751141 was significantly lower in DKD patients compared with Non-DKD controls(p<0.01),and the A allele of rs751141 was associated with a significantly lower risk of DKD after adjustment for multiple covariates in the additive and recessive genetic model(OR=0.68,95%CI=0.52-0.88,p<0.01;OR=0.28,95%CI=0.14-0.60,p<0.01).Significant association between rs751141 and homocysteine(Hcy)level on the risk of DKD was observed,indicating that in patients with the highest Hcy levels,the A allele showed marked association with lower risk of DKD in all three genetic models(p<0.01,p<0.05 and p<0.05).2)The correlation between EPHX2 rs751141 gene polymorphism and ischemic stroke showed that the A allele of EPHX2 rs751141 in type 2 DM patients without ischemic stroke group was significantly higher than that in type 2 DM patients with ischemic stroke(p<0.05).After adjusting for relevant risk factors,A allele carriers had a lower risk of ischemic stroke in both additive and dominant genetic models(OR=0.72,95%CI=0.55-0.97,p<0.05;OR=0.67,95%CI=0.48-0.95,p<0.05).2.Study on the relationship between the A allele of EPHX2 rs751141 polymorphism and sEH hydrolase activity and the molecular mechanism of DKD:1)The relationship between sEH recombinant plasmid and hydrolase activity:The successful construction of the one kind of WT and 7 kinds of mutant recombinant plasmids(R287Q,R287Q+R103C,C154T,K55R,R103C,K55R+R103C,K55R+C154T),and consruct HK-2 high expression system.When adding excessive 11,12-EET to cell culture medium,R287Q was significantly lower than that of WT(p<0.01);C154T was significantly higher than that of the WT(p<0.05).The addition of 11,12-EET to the cell culture medium and the addition of TPPU could significantly lower the 11,12-DHET content to the basal level.2)Inflammatory cytokines and signal pathway:The NO,IL-17A and IL-1 levels in supernatant of R287Q group were significantly lower than that of WT group(p<0.05).Compared with the WT group,the level of inflammatory cytokines in WT group was lower than that of 11,12-EET group,but was higher than that of mutant R287Q group with 11,12-EET.The RT-PCR results are also found in the R287Q group in eNOS,IL-17A,IL-6 and IL-1? mRNA expression level was lower than that of WT group.When cultured with 11,12-EET,the expression level of WT group of inflammatory cytokines in mRNA decreased,but still higher than R287Q group with 11,12-EET.Western blot method was used to detect NC group,WT group and R287Q group.The results showedthat R287Q group could reduce DKD inflammation by regulating PI3K/AKT signaling pathway.Conclusion:1.The A allele of EPHX2 rs751141 polymorphism has protective effects on DM vascular complications DKD and ischemic stroke.It is suggested that this site may be involved in the development of DM vascular complications.2.In vitro cell test,the mutant R287Q recombinant plasmid has lower 11,12-EET hydrolysis ability compared with WT.Compared with wild-type WT,mutant R287Q has a lower level of inflammation,exogenous 11,12-EET can reduce the level of inflammation in group WT.A allele may be involved in the molecular mechanism of DKD through PI3K/AKT signaling pathway.