| Deep venous thrombosis(DVT)refers to the abnormal coagulation of blood in the veins,complete or not completely blocking the blood vessels,and is a venous reflux disease.The pathogenesis of DVT is complicated,including intravenous endothelial cell,coagulation factor,fibrinolytic system,leukocyte,platelet and so on.In recent years,it is believed that reactive oxygen(ROS)produced by oxidative stress can lead to oxidative damage and even apoptosis of vascular endothelial cells,thereby promoting the formation of DVT.Numerous studies have found that in cardiovascular disease Nrf2 signaling pathways/HO-1 has strong antioxidant effect,can protect vascular endothelial cells from or reduce oxidative damage and the pathway is considered to be the strongest antioxidant effect.However,whether the signaling pathway plays a role in the formation of DVT has not been reported.Therefore,this study mainly discusses the role of Nrf2/ho-1 signaling pathway in the formation of mouse DVT.The DVT model of C57 mice was constructed by using the vena caval stenosis method to observe the effect of Nrf2 agonist and ho-1 inhibitor on the mice.The mRNA expression of Nrf2 and ho-1 gene in C57 mice were detected.The aim is to provide a new theoretical basis for finding the key targets of DVT.Objective1.The DVT model of C57 mice was constructed by inhaling anesthesia(isoflurane)and inferior vena cava stenosis.Use of Nrf2 agonists(TBHQ)inhibitors and HO-1(SnPP)intervention in C57BL/6 mice model of DVT,24 hours after building observed between groups in mouse models of DVT,ChengShuan mortality rate,the change of the inferior vena cava thrombus length,wet weight difference.Explore the relationship between Nrf2/ho-1 signaling pathway and DVT formation.2.For each group of inferior vena cava wall in C57BL/6 mice,using real-time fluorescent quantitative PCR(real-time-PCR)to detect each vein wall in C57BL/6 mice tissues Nrf2 and HO-1 gene mRNA expression change,explore the Nrf2 signaling pathways/HO-1 the role in the formation of DVT.Materials and MethodsThe first part:the C57 mouse DVT model was constructed to observe the effect of Nrf2 agonist and ho-1 inhibitor on the mouse suppository.1.Establishment of experimental group and DVT model:90 C57 mice,weight 25-30g,female.10 only according to the principle of random grouping is divided into blank control group(group A,n = 10),20 DVT model group(group B,n = 20),only Nrf2 agonist(TBHQ)DVT model group(group C,n = 20),Nrf2 agonist(TBHQ),HO-1 inhibitors(SnPP)DVT model group(D group,n = 20),HO-1 inhibitors(SnPP)DVT model group(D group,n = 20).In the 3 days before the experiment,group C mice were injected with TBHQ(16.7mg/kg)every 8 hours for 3 days.The mice in group D were given SnPP(5mg/kg)a day prior to the formation of the drug in group C.The mice of group D were given SnPP(5mg/kg)a day before the mold making.B,C,D and E were all used to construct the DVT model using isoflurane inhalation anesthesia and vena cava stenosis.2.The materials and test:the experiment building after 24 hours,choice method of cervical dislocation executed experimental mice,access to inferior vena cava in C57BL/6 mice group(from the parts to the common iliac vein ligation of joint inferior vena cava and its content),then the separation of the venous wall and its inner blood clots,measuring the mice after thrombus length,wet weight,packing-80 o saved for later use.3.Statistical analysis:statistical analysis was performed on the mortality rate,thrombosis rate,thrombus length and wet weight of each group of mice.SPSS 19.0 was used in statistical software,and the mean value of the measurement data was used.The comparison between the two groups was statistically significant using the single factor variance analysis,the least square method(LSD),and*p<0.05.The secend part:mRNA expression changes of Nrf2 and ho-1 in C57 mice.1.Trizol method was used to extract the total RNA in the inferior vena vena tissue of each group of mice,and the reverse transcription PCR(rt-pcr)was used to reverse transcription of RNA into cDNA in each group of mice.The mRNA expression of Nrf2 and ho-1 was detected by real-time fluorescence quantitative PCR assay in GAPDH.2.Statistical analysis:statistical software selected SPSS19.0.The mean value of the measurement data is plus or minus standard deviation(plus or minus s).In comparison,one-way anova,LSD(least square),and*p<0.05 were considered statistically significant.ResultsExperiment 1:the DVT model of C57 mice was constructed by vena caval stenosis to observe the effect of Nrf2 agonist and ho-1 inhibitor on the mice.1.Anesthetic effect:the experiment began to use 4%isoflurane for rapid induction,and 1.0%concentration isoflurane was used in the operation,and the anesthetic effect was ideal.After the operation,the time of post-operative anesthesia was faster,about 2min,and the respiratory depression in the individual mice during the anesthesia was reduced.After the anesthesia was closed and the air flow increased,the respiratory depression was rapidly alleviated.2.Each group mice death:A,B and C group mice mortality was 0%,D,E group mice death rates were 10%,5%,between group B and group C A,statistical analysis(*(p>0.05),there was no statistically significant difference.3.The type ChengShuan rate:A group of mice ChengShuan rate was 0%,B,C,D,E group assembly bolts rate were 68.4%,55.6%,63.2%,55.6%,between group C and group B and D to do statistical analysis,single factor analysis of variance)(*p<0.05),the difference was statistically significant.4.The thrombus length of the mice in each group:group A was not embolised,and the thrombus length of group B,C,D and E was 5.47 + 0.32mm,4.13 + 0.27mm,5.46 + 0.28mm,5.42 + 0.31m.Statistical analysis was performed between group C and group B and D(single factor variance analysis)(*p<0.05),and the difference was statistically significant.5.The wet weight of thrombus in each group was:group A did not have A thrombus,B,C,D and E were respectively:12.6 + + 3.lmg,8.4 + or 3.2mg,11.5 + + 3.5mg,12.3 + 3.3mg.Statistical analysis was performed between group C and group B and D(single factor variance analysis)(*p<0.05),and the difference was statistically significant.Experiment 2:mRNA expression changes of Nrf2 and ho-1 in C57 mice in each group.Real-time quantitative PCR was used to detect the relative expression of mRNA of Nrf2 and ho-1 genes in mice.The results of statistical analysis showed that the mRNA expression levels of Nrf2 and ho-1 genes in mice in groups A,B and C were statistically significant(*p<0.05)in 24 hours after DVT was formed.The mRNA expression of Nrf2 and ho-1 in group C mice was higher than that in group A and B(*p<0.05),and the mRNA expression of Nrf2 and ho-1 in group B mice was higher than that in group A(*p<0.05).The mRNA expression of ho-1 in the model group D and E was significantly different(*p<0.05),and the mRNA expression of ho-1 in the C model group was higher than that in group D(*p<0.05).Conclusion1.Activation of Nrf2 could inhibit the formation of DVT in C57 mice;2.Activation of Nrf2 may affect ho-1 and inhibit the formation of venous thrombosis in C57 mice;3.Nrf2/ho-1 signaling pathway may be a new target to prevent DVT formation. |
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