Study On Mechanisms Of Hypertension In Rat Adult Offspring Following Prenatal Exposure To Immuno-Inflammatory Stimulant: Screening For Candidate Genes Associated With Disease

Essential hypertension(EH) is one of the most common cardiovascular disease.And the complication caused by EH is one the main cause of human fatility.So it is important that investigation of the mechanism of EH for prevention and cure EH.Recently significant progress has been made in understanding the effect of inflammation on cardiovascular disease.More and more data from clinic practice and research tells us that hypertension is related with inflammation in certain ways.In the past research of our own team,we found that prenatal exposure to LPS or zymosan result in increases in blood pressure and body weight in rats offspring.This also is accordance with the modern developmental biology proposes "Fetal-Oringin Theory for Adult Chronic Diseases".The theory suggest that developmental plasticity of embryo and infant cause some adult chronicle diseases such as coronary heart disease,type2 diabetes,hypertension.That is to say that exterior stimulation during preganency may play a key role in chronicle adult disease.Together with our data support the possibility of the existence of this relationship.However,we want to know more fundamental answer:what factors and channels affect the growth of fetius after those exterior stimulation.Based on our research team,this essay intends to study how immuno-inflammatory stimulation affect the development of embryo.Through Affymetrix's GeneChip Rat Genome 230 Arrays,the gene expression profiles between immuno-inflammatory group and control group is studied.Methods1.Sprague-Dawley(SD) rats,dams in each group received i.p injections of 0.79 mg/kg LPS,8 mg/kg zymosan or sterile saline respectively on their gestational days 8,10,and 12.2.The serums were collected in tail nick at 2 h after the last injection,and the amniotic fluid was mixed at 2 h,12 h,24 h,48 h after the last injection.TNF-αand IL-6 levels of serum and amniotic fluid were measured by RIA method. 3.TNF-αand IL-6 mRNA levels were quantitated in amnion,placenta,amniotic fluid, Embryo and macrophage by real-time fluorescent quantitative-PCR.4.Gene expression profiles of each group was observed by Affymetrix's GeneChip Rat Genome 230 Arrays.5.The data of GeneChip were analyzed with Gene Ontology and GenMAPP for the biology function and the signal pathway.6.Twenty differentially expressed genes were selected to confirm the GeneChip data by real-time fluorescent quantitative-PCR.And these genes were studied in each group embryo by real-time fluorescent quantitative-PCR,Results and Conclusion1.The serum level of TNF-αand IL-6 in LPS group and zymosan group was higher than that in control group(P＜0.01).It showed that there was immuno-imflammatory response after LPS or zymosan injection in rats.The mRNA levels of TNF-αand IL-6 was very higher in macrophage than in other organization.2.In embryo,the mRNA level of IL-6 was more than other organization,but the mRNA level of TNF-αwas lower than other organization.However,the IL-6 mRNA levelof LPS group and zymosan group was higher several dozens times than control group on 24h and 48h.It suggested that IL-6 was important in the model that prenatalexposure to immuno-inflammatory stimulant results in increases of blood pressure and body weight in rats.3.Among the total 28000 genes on the GeneChip,in LPS group,183 genes were found to be higher expression(Ratio≥2 ) and 270 genes were lower expression(Ratio≤0.5 );in Zymosan group,144 genes were found to be higher expression(Ratio≥2 ) and 417 genes were lower expression(Ratio≤0.5 ).However,both in two group,50 genes were found to be higher expression(Ratio≥2 ) and 173 genes were lower expression(Ratio≤0.5 ).4.The data of genechip was analyzed with Gene Ontology for biology function.It is showed that the differentially expressed genes were sorted into binding,catalytic activity, transporter activity,signal transducer activity or transcription regulator on GO Molecular Function.5.The data of genechip was analyzed by GenMAPP software for signal pathway.It showed that in DNA replication,mRNA processing,translation factors,the differentially expressed genes were up-regulation in the LPS group and Zymosan group.And in calcium regulation in cardiac cell,smooth muscle contraction pathways,inflammatory response pathways,the down-regulation expressed genes are more than up-regulaion.6.We detected the mRNA level of 20 genes by real-time PCR,and found that 13 genes was consistent with the genechip data,but 4 genes was inconsistent.And the zymosan group of RGD1307150,Bcl11a,Tgfb2 were consistent with the genechip data.7.In the embryos of rats with immunolgy inflammation,real-time PCR show that the FgfS,Orc11 increased at 2 h,12 h point and down to normal level at 24 h and 48 h compared with control group.However,the profile of Bcl11a,Dusp9 mRNA appeared reverse.The level of Nfib mRNA decrease in every time point.The Egfr,Gap43,Penk1 remain normal at 2 h and just decreased at 12 h to 48 h.The Agtr2 was lower than control at 12 h.The Tgfb2 in zymosan group decreased at all point,but no change in the LPS group. With the genes function,we presumed that Fgf8,Orcll,Bcl11a might be correlated with acute regulation of immuno-inflammatory stimulus.Nfia,Nfib,Gap43 might be correlated with efforts of immuno-inflammatory stimulus on embryo's brain development,and Agtr2 might be on kidney development.Dusp9,Egfr,Ripx,Tgfb2 might be correlated with efforts of immuno-inflammatory stimulus on the proliferation and differentiation of embryo cell. However,the relationship between these efforts and prenatal exposure to immunoinflammatory stimulus results in increases of blood pressure was for further research.