Effects Of Different Level Of Estrogen And (or) Progesterone In The Reproductive Phase On The Blood Pressure During The Perimenopausal And Postmenopausal Phase In Rats

BackgroundEssential hypertension（referred to as hypertension）is the most common chronic non-communicable diseases,which is the primary risk factor of cardiovascular and cerebrovascular disease.Hypertension have a series of epidemiological characteristics including high incidence,wide geographical distribution.gender differences,age trends and so on.Results of studies showed that the blood pressure has no difference between boy and girl before the adolescence:the incidence was significantly lower than the same age men in childbearing-age women before perimenopause;the prevalence of hypertension is more common compared with their husband in the middle-aged or postmenopausal women.This gender difference in hypertension among physiological stages was highly concerned.In women.the incidence of hypertensive disease increases with age.The prevalence of hypertension was 26.7%in 50-59 years old women during perimenopausal phase.while it was almost 2 times of the prevalence of perimenopausal women in 70-79 years old.In addition,after adjusting for age,the risk of hypertensive disorder was significantly higher in postmenopausal women than childbearing age women,and the prevalence of hypertension was higher among young women with premature ovarian failure compared with healthy women in childbearing age.It was suggesting that female reproductive hormones’ deficiency may be associated with the higher risk of hypertension.The female reproductive hormones in women include follicle stimulating hormone,luteinizing hormone,estrogen,progesterone,prolactin and so on.The estradiol（E₂）and progesterone（P）,one of the steroid hormone,plays a vital role in formation of menstrual cycle,the growth and development of secondary sexual characteristics,the improvement of reproductive function and the health of multiple systems.In normal menstrual cycles,the blood pressure showed a corresponding change with the fluctuation of the reproductive hormone level,which reflected the regulation of reproductive hormones on blood pressure.A series of physiological changes of reproductive hormones in the perimenopausal period affect the pathogenesis of many diseases.For example,the estrogen concentration in postmenopausal women reduced by 60%resulted in the proportion of androgen increased,such lacking "female advantage" changes may lead to an increase in blood pressure.Therefore.it can be speculated that the low incidence of hypertension in women of childbearing age may be related to higher levels of-E₂ and P in the body.Studies have found that higher levels of E₂ and P in reproductive phase were associated with better cognitive function and fewer mood disorders after menopause.However,the association between higher endogenous levels of E₂ and（or）P in reproductive phase and its perimenopausal or postmenopausal blood pressure have not been reported.Therefore,it deserves to explore the relationships between the high endogenous levels of E₂,and/or P in their own reproductive age and the vascular endothelial function after menopause.ObjectivesThis study focused on the relationship between endogenous E₂ and P exposure and long-term blood pressure regulation.The aim of this study was to investigate the effects and mechanisms of different level of endogenous E₂ and P on the perimenopause and postmenopausal blood pressure regulation through establishing an artificial estrous cycle,and further distinguish the seprate role of E₂,P alone.The results can provide a reference basis for the individualized assessment of postmenopausal hypertension risk in women of childbearing age and provide targeted recommendations for the prevention and control of postmenopausal women with hypertension.Methods1.Animals and groupingA total of 160 healthy female Wistar rats were provided by the Experimental Animal Center of Shandong University in two batches.The first batch of rats was used for Phase I experiment,and the second batch of rats was used for Phase Ⅱexperiment.Animals were housed in SPF（No Specific Pathogens,SPF）room with constant temperature（50 ± 60%）and constant temperature（22 ±0.5℃）,free to feed feed and tap water.2.Experimental designThe experimental study is divided into two stages.Stage I experiment:To explore the effects and changes of 17-β E₂ and P levels during reproductive phase on perimenopausal and postmenopausal blood pressure.Stage II experiment:To explore the separate effects of high dose 17-β E₂ or high dose P on the perimenopausal or postmenopausal blood pressure.The 100 rats in stage Ⅰ experiment were randomly divided into 5 groups:low dose of estrogen and progesterone（low dose 17-β E₂ plus low dose P.LELP）.normal dose of estrogen and progesterone（High dose 17-β E₂ plus normal dose P,NENP）.high dose of estrogen and progesterone(high dose 17-（3 E₂ plus high dose P.HEHP）.ovariectomized group and sham-ovariectomized group.In the stage Ⅱ experiment.60 rats were randomly divided into three groups:high-dose estrogen group(high dose 17-（3 E₂ plus low dose P.HELP）,high-dose progesterone group（high dose P plus low dose 17-beta E₂.HPLE）and ovariectomized group.Each group contained 20 rats.3.Establishment of artificial estrous cycle modelAnimals in stage Ⅰ and stage Ⅱ experiment were monitored（except sham operation group）through reproductive imitation phase,perimenopausal imitation phase and postmenopausal imitation phase after ovariectomy to observe the different level of estrogen and/or progesterone in reproductive on the blood pressure of perimenopausal and postmenopausal period.Reproductive imitation phase:The exogenous supplemental hormone levels in the artificial estrous cycle model to simulate the natrual estrous cycle were given as reference dose.The specific 17-β E₂ and P dosages in the Stage I experiment and Stage Ⅱ experiment were given in Table 2.The 17-β E₂ and P were dissolved in peanut oil daily on 16:00-18:00 through gavage administration,the volume was 0.1mL/100g.6 days for a complete artificial estrous cycle.The ovariectomized group and sham-ovariectomized group were given the same volume of peanut oil as the control group.Perimenopausal imitation phase:On the basis of the experimental dose of the reproductive imitation phase,the dosages were decreased half in each cycle on the next 5 artificial estrous cycle.From the beginning of the 9th week to the end of the 13th week.Postmenopausal imitation phase:6 consecutive cycles of hormones withdrawal phase were imitated of postmenopausal phase after decreasing dosages in perimenopausal imitation phase.The complete menopause simulation period begins at the 14th week to the ends of the 19th week.4.Observation indicators and sample collectionTail arterial indicators included systolic blood pressure（SBP）,diastolic blood pressure（DBP）.heart rate（HR）.Using noninvasive sphygmomanometer（BP-1998A,softron）to monitor arterial indicators in the diestrus phase of an estrous cycle.Then the body mass was recorded after measuring blood pressure.The samples were collected at 8 points,chronically as time before ovariectomy,the end of the reproductive imitation phase,each cycle of the perimenopausal imitation phase and the end of the postmenopause imitation phase.At each point,two rats were sacrificed,and the serum and abdominal aorta samples were collected and stored at-80℃.5.Detection of serum hormones and proteins and its mRNA expression levels in abdominal aortaThe levels of serum E₂ and P.as well as the estrogen receptor-β（ER-β）,angiotensin Ⅱ type 1 receptor（AT1R）and vascular endothelial growth factor（VEGF）proteins in abdominal aorta were measured by enzyme-linked immunosorbent assay（ELISA）.The expression of ER-βmRNA,VEGF mRNA and AT1R mRNA in the abdominal aorta were detected by semi-quantitative RT-PCR.6.Statistical analysisData were analyzed by SPSS 19.0.The repeated measurement design of variance analysis was used to compare the differences in vascular indicators and body weight among groups during imitation phases.Pearson correlation analysis was used to analyze the correlation between related proteins and its mRNA expression or serum hormone levels or blood pressures.The differences of hormone concentration.protein and mRNA expression in each point were compared by one-way analysis of variance or nonparametric rank sum test（Kruskal-Wallis H test）.P<0.05 was considered statistical significance.Results1.Effects of different levels of estrogen and progesterone in reproductive phase on the blood pressure of perimenopausal and postmenopausal phase（1）Arterial indexs in simulation phasesDuring the reproductive imitation phase,the effect of intergroup treatment factors significantly affected the DBP among groups.The SBP and DBPof HEHP group were significantly higher than those in the control group（PSBP = 0.027.PDBP ＝0.005）.During the perimenopausal imitation phase,the DBP of perimenopausal imitation phase was significantly affected by the intergroup treatment factors.The DBP of the NENP and the HEHP groups were significantly higher than that of the control group（ovariectomy group）（P=0.047;P = 0.013）.During the postmenopausal imitation phase,the intergroup treatment factors and the interaction between time and group treatment affected the SBP and DBP among groups.The DBP in the HEHP group was significantly higher than those in the control group（PDBP=0.037.PHR＝=0.025）.（2）Hormone levels during the imitation phasesThe serum E₂ concentration in the NENP group and HEHP group were significantly higher than that in the control group（P<0.001）.whereas the serum E₂ concentration in the LELP group was lower than that in the control group during imitation phases（P<0.05）.The serum P concentration in the NENP and the HEHP groups were significantly higher than that in the control group at the reproductive imitation phase and the perimenopausal imitation phase（P<0.001）.and the serum P concentration in the HEHP group was significantly higher than that in the control group during postmenopausal imitation phase（P<0.001）.2.Effects of high dose of estrogen or high dose of progesterone in reproductive phase on the blood pressure of perimenopausal and postmenopausal phase（1）Arterial indexs in simulation phasesDuring the reproductive imitation phase,the effect of intergroup treatment affected the SBP and DBP among groups.The SBP of HELP group and HPLE group were significantly higher than those in the control group（HELP:PSBP= 0.011,PHR＝0.030:HPLE:PSBP = 0.041,PHR = 0.061）.During the perimenopausal imitation phase.the changes of SBP and DBP in each group were affected by the intergroup treatment effect.The levels of SBP in HELP group and HPLE group were significantly higher than those in the control group,and the DBP of the HELP group was significantly higher than those in the control group（HELP group:PSBP = 0.002,PDB= 0.010;HPLE group:PSBP =0.009,PDBP= 0.674）.During the postmenopausal imitation phase,the effects of intergroup treatment significantly affected the SBP and DBP among groups.Compared with the control group,the SBP of HELP group and HPLE group were higher than that of the control group.and the DBP of the HELP group significantly increased（HELP group:PSBP =0.001.PDBP=0.004;HPLE group:PSBP = 0.0O01 PDBP= 0.272）.（2）Hormone levels during the imitation phasesThe serum E₂ concentration in the HELP group and the serum P concentration in the HPLE group was significantly higher than those in the control group during imitation phases（P<0.05）.3.Effects of different levels of estrogen and（or）progesterone during the reproductive phase on the expression level of ER-P related proteins and its mRNA during the perimenopausal and postmenopausal phase（1）Effects of different levels of estrogen and progesterone during the reproductive phase on the protein and mRNA expression of ER-β,VEGF,AT1RThe protein and mRNA expression levels of ER-β in the abdominal aorta of NENP group and HEHP group were significantly higher than those in the control group during reproductive and perimenopausal imitation phases（P<0.001）.The expression of ER-β protein in the abdominal aorta was positively correlated with the serum E₂ and P concentrations（rE₂=0.670.PE₂<.001:rP = 0.640.PP><0.001）.The expression of ER-β mRNA was related to the protein expression level（r = 0.864.P<0.0011）.The protein and mRNA expression levels of VEGF in abdominal aorta of the NENP group and HEHP group were significantly higher than those in the control group during the productive and perimenopausal imitation phases（P<0.001）.The expression of VEGF in the abdominal aorta was positively correlated with the serum E₂ and P（rE₂ = 0.824.PE₂<0.001;rP = 0.777.Pp<0.001）.The expression level of VEGFa mRNA was closely related to the expression of protein（r =0.827,P<0.001）.The protein and mRNA expression of AT1R in the abdominal aorta of the HEHP group was significantly higher than that in the control group（P<0.01）.The serum E₂ was positively correlated with the protein expression of AT1R in the abdominal aorta（r = 0.209,P =0.046）.The expression level of AT1R mRNA was closely related to its protein expression（r = 0.657,P<0.001）.（2）Effects of high dose of estrogen or high dose of progesterone during the reproductive phase on the protein and mRNA expression of ER-β,VEGF,AT1RThe protein and mRNA expression of ER-β in the abdominal aorta of the HELP group was significantly higher than that of the control group in the reproductive and perimenopausal imitation phases（P<0.001）.The mRNA expression of ER-β in the abdominal aorta of the HPLE group was significantly higher than that of the control group in all imitation phases（P<0.01）.The protein expression of ER-βmRNA was positively correlated with serum E₂（r =0.676,P<0.001）.and the expression of ER-βmRNA was positively correlated with its protein（r = 0.371.P<0.001）.The protein and mRNA expression of VEGF in the abdominal aorta of the HELP group was significantly higher than that in the control group during the reproductive and perimenopausal imitation phases（P<0.05）.The expression of VEGF in the abdominal aorta was positively correlated with the serum E₂ concentration（r = 0.624.P<0.001）.and was not statistically correlated with the SBP and DBP（rSBP =0.132.PSBP = 0.189:rDBP=0.030,PDBP= 0.767）.The expression of VEGFa mRNA was positively correlated with its protein（r = 0.241.P = 0.022）.The protein and mRNA expression of AT1R in the abdominal aorta of the HELP group was significantly higher than that in the control group during imitation phases（P<0.05）.The protein expression of AT1R was positively correlated with serum E₂ concentration and SBP among groups（rE₂ = 0.347.P E₂<0.001:rSBP=0.370.PSBP=0.008）,and there was no significant correlation with DBP（r=0.037,P=0.795）.The expression of AT1R protein was significantly correlated with its mRNA expression（r=0.259,P =0.004）.Conclusion1.Reproductive hormone levels in the reproductive phase of rats have an effect on the perimenopausal and postmenopausal blood pressure;2.High level of-endogenous E₂ and P in the reproductive phase of rats significantly increased its DBP and HR in the perimenopausal and postmenopausal phase;low levels of endogenous E₂ and P in the reproductive phase had no significant effect on postmenopausal DBP;3.Separate high dose of E₂ in the reproductive phase of rats increased its SBP and DBP of perimenopausal and postmenopausal phase;separate high dose of P in the reproductive phase increased its SBP of perimenopausal and postmenopausal phase,and enhancing the pulse pressure;4.The expression of VEGF in the abdominal aorta was correlated with the serum E₂,the AT1R protein was correlated with serum E₂ and SBP among groups.The elevation effects of blood pressure in high dose E₂ and/or P group were associated with the up-regulation of AT1R protein and mRNA expression in the abdominal aorta.While the increasing level of VEGF protein and its mRNA expression in the abdominal aorta can not reverse the rising trend in blood pressure.