Effect And Mechanism Of Rac1 In Prenatal LPS Exposure Induced Transgenerational Hypertension In Offspring Rats

Background:Hypertension is one of the most crucial risk indicators of cardio-cerebrovascular diseases.Number of hypertensive patients around world has exceeded 1.1 billion,among which number in China will reach 300 million.With the fact of relatively low awareness and control rate of hypertension,prevention-control of hypertension in China is very austere.Therefore,it is important and urgent to further clarify pathogenesis of hypertension and to seek effective treatment strategy.Hypertension is considered to be result of interaction between gene and environment.Numerous studies suggest that there is a crucial link between the prenatal adverse stimulus and occurrence of hypertension.A British scholar named David Barker,proposed a theory of"developmental origin of health and disease",suggesting that cause of hypertension could be traced to embryonic development.Biological experiments also confirm that:prenatal adverse stimulus（malnutrition,excessive immune activation,etc.）can lead to increased incidence of cardiovascular diseases,such as hypertension,in offspring.Prenatal SD rats with intraperitoneal injection of lipopolysaccharide（LPS）can well imitate prenatal infection.Prenatal LPS exposure results in parent-placenta-fetus crosstalk and increased inflammatory effect with elevated production of IL-1/6,TNF-α,etc.Excessive inflammation results in impaired fetal development in utero,abnormal genetic modifications,and eventually leads to occurrence of hypertension and other pathological phenotype in offspring,but mechanism remains far from fully elucidated.Besides,more than 60%of hypertensive patients have clear family history,suggesting important role of genetic factors in the process of hypertension.Then,can the hypertension induced by prenatal LPS exposure be inherited,especially transgenerational inherited?And,what is the underlying mechanism?Results of large-scale GWAS,aiming at finding the key susceptible loci suggest that role of genetic factors in hypertension is far less than expected.Previously,studies on mechanism of transgenerational diseases mainly focus on reproductive and metabolic diseases.Anne C.Ferguson-Smith et al.successively confirmed the role of overall methylation level of sperm DNA and the inheritance of sperm ts RNA on the phenotype of abnormal metabolism in offspring.However,current studies in the field of cardiovascular disease are limited to the role of epigenetic modification in the contemporary pathogenesis of cardiovascular disease.Whether and how do cardiovascular diseases realize epigenetic inheritance,especially trans-generational action?Take hypertension as an example.Previously,relevant studies concerning inheritance of hypertension mainly focus on searching for gene mutations,but with little effect.As to complex multi-gene diseases,like hypertension,it is not effective to find the inherited mechanism,especially the trans-generational mechanism,through traditional methods.As an emerging field,epigenetics provide the possibility to explain the pathogenesis of inherited hypertension from the perspective of epigenetic regulation.Cells and organisms can respond differently to environment through genomic reprogramming.So how does DNA respond flexibly to environment changes?Epigenetic modifications,which control gene expression without changing DNA sequence,could achieve response to external stimuli in a relatively fast manner through chromatin remodeling,histone modification,DNA methylation,and micro RNA regulation.Currently,it has been confirmed that adverse external stimuli such as malnutrition and inflammatory stress can lead to excessive oxidative stress,and then induce abnormal epigenetic modifications.Long-term exposure of exogenous stimulation leads to the imbalance of oxidative-antioxidant system,resulting in excessive ROS level and substream abnormal epigenetic modification.Epigenetic modifications can be fixed and stable,providing a molecular environment adapted to development of relevent disease.Therefore,in the model of prenatal LPS exposure,it is certain in increase of oxidative stress.Whether or not the excessive oxidative stress leads to dysfunction of epigenetic modification on targeted molecule and then transgenerational action of hypertension?Thus,we propose following hypotheses:1.Prenatal LPS exposure can lead to natriuresis impairment,elevated blood pressure and trans-generational inheritance of hypertension in offspring rats;2.Transgenerational inheritance of prenatal LPS exposure induced hypertension is mediated by abnormal epigenetic modification.3.Excessive increase of oxidative stress level is the trigger factor of abnormal epigenetic modification.Therefore,our study will focus on exploring the molecular pathogenesis of prenatal LPS exposure induced hypertension,the epigenetic mechanism of trans-generational inheritance of hypertension and the upstream triggering factors of abnormal epigenetic modification.Methods:1.To observe differences in blood pressure and natriuresis between control group and LPS group in offspringsThe blood pressure was monitored continuously by tail-cuff method and telemetre method for 24 hours.Natriuresis was measured by metabolic cage.2.To screen out a target molecules which play crucial roles in prenatal LPS exposure induced hypertensionExecute m RNA-seq on renal tissue.Target molecules are deteceted by quantificational real-time polymerase chain reaction（qRT-PCR）and Western blot（WB）.Inhibitors of target molecules were applied to confirm their effects on blood pressure regulation.3.To detect exon mutation and promoter methylation of target molecule and to confirm effect of epigenetic modification on target moleculeThe exon region of target gene was sequenced to determine whether there were mutations.Methylation sequencing of target gene promoter was performed to determine whether there were differences in methylation levels between groups.Histone modifications were screened to determine whether there were differences in the expression,and chromatin immunoprecipitation was used to determine the deposition level of histone modifications on the target gene-promoter region.4.To explore role of oxidative stress in epigenetic modificationThe MDA,SOD level between groups and downstream molecular in prenatal LPS exposure and LPS+TEMPOL group were detected to uncover the possible triggering factors of abnormal epigenetic modifications.Result:1.Prenatal LPS exposure can lead to elevated blood pressure and natriuresis impairment in offspring rats.Furthermore,it was found that the prenatal LPS exposure induced hypertension was continuously transmitted to the F₃ generation.In F₄,although there was no significant difference in blood pressure between the groups at the basal state,there were significant differences in salt sensitivity between the groups.The elevated blood pressure and impaired urinary sodium excretion in F₄ of LPS group were greater than those in control group after the 2 month-induction of high salt（8%）diet.2.The m RNA and protein expression of Rac1 in LPS offspring were significantly higher than those in control group.There was a significant increase in renal nuclear translocation of MR and downstream ENa Cs expression level,along with no significant difference in serum aldosterone content between groups.After application of Rac1-specific inhibitor NSC23766,the increase of blood pressure,abnormality in natriuresis were significantly relieved and levels of MR nucleus accumulation and downstream ENa Cs were also decreased in LPS+NSC23766 group.3.No difference was found in exon mutation of Rac1 and in methylation level of Rac1promoter between groups.However,it was found that the expression of H3K9me2 and its deposition level on Rac1 promoter region were significantly lower in prenatal LPS exposure group.4.Oxidative stress levels of pregnant rats and F₁ offspring were significantly increased after prenatal LPS exposure.The demethyltransferase KDM3b of H3K9me2 was significantly increased in the ovary tissues and oocytes of F₁ in LPS group.After the application of Tempol,the changes in abnormal H3K9me2-Rac1 signaling and hypertension in F₁ and F₂ were significantly ameliorated.Conclusion:Prenatal LPS exposure results in significantly decreased H3K9me2 expression and deposition levels on Rac1 promoter,which leads to excessive increased Rac1 expression,elecvated MR nuclear accumulation and e Na Cs expression in offsprings.And finally,it results in natriuresis impairment and transgenerational hypertension.Increased oxidative stress level in pregnat rats and F₁ are the triggering factor of abnormal KDM3b function and H3K9me2modification in ovary and oocytes.And the oocytes with abnormal H3K9me2 modification are the inherited carrier of transgeneration hypertension.