| Objective:The development of hypertensive end organ damage(EOD)plays a pivotal role in the occurrence of hypertensive complications. It is therefore essential to reverse, reduce or prevent EOD from happening in the treatment of hypertension. Renin-angiotensin system may also play an important role in hypertensive EOD. Recently, an emerging concept contends that inflammation plays a predominant role in the progression of hypertension and is also involved in the triggering of hypertension-associated cardiovascular complications. Female sex hormones have the ability to affect blood pressure levels. Accumulating evidence from experimental studies showed that estrogens have a cardioprotective influence in women by improving endothelial function, and regulating the response to inflammation. Therefore, the present studyies were designed three parts to evealuatd the influence of estrogens on local renin-angiotensin system and inflammatory factors in hypertensive rats.Methods and Results:1. Local RAS and inflammatory factors are involved in cardiovascular hypertrophy in spontaneously hypertensive ratsSHR and WKY rats were used in this study. The blood pressure and baroreflex sensitivity were measured in conscious state. After hemodynamic monitoring and BRS studies, the animal was weighed, anesthetized, and killed by decapitation. The aorta and heart were immediately excised and rinsed in cold physiological saline for histopath-ological examination. The mRNA levels of angiotensinogen, renin, ACE, ACE2, AT1, AT2 in local tissue were determined using quantitative real-time polym-erase chain reaction. The plasma was collected for the determination of angiotensin II concentration using the radioimmunoassay kit. The main findings of this study are as following: 1.1 Hemodynamic parameter, plasma AngⅡ, serum inflammatory factors and EOD in SHRCompared with WKY rats, BP, BPV, plasma AngⅡconcentration, serum IL-1βand IL-6 concentration were significantly higher, while HP, HPV and BRS were significantly lower in SHR. SHR present with obvious cardiovascular hypertrophy characterized by increased LVW/BW and AW/length.1.2 mRNA expression of RAS and inflammatory factors in the heart and aorta of SHRCompared with WKY rats, mRNA levels of angiotensinogen, renin, ACE2, AT1 and AT2 in the heart of SHR increased by 314%,212%,119%,215% and 250% respectively. There was no difference in ACE mRNA in the heart between SHR and WKY rats. mRNA levels of ACE, ACE2, AT1 and AT2 in the aorta of SHR increased by 116%,86%, 107% and 110% respectively when compared with those of WKY rats. There was no significant difference in angiotensinogen and renin mRNA in the aorta between SHR and WKY rats.In addition, mRNA levels of TNFα, IL-1βand IL-6 in the heart of SHR increased by 213%,132% and 91% respectively, and mRNA levels of TNFa and IL-6 in the aorta of SHR increased by 220% and 458% respectively, when compared with WKY rats. There was no significant difference in IL-1βmRNA in the aorta between SHR and WKY rats.1.3 Correlations between hemodynamics, plasma AngⅡconcentration, serum inflammatory factors and cardiovascular hypertrophy in SHRBP and BPV were found to be positively correlated with LVW/BW and AW/length, while BRS was negatively correlated with cardiovascular hypertrophy. Plasma AngⅡwas insignificantly correlated with any cardiovascular damage parameter.1.4 Correlations between local mRNA expression of RAS and inflammatory factors and cardiovascular damage in SHRACE, ACE2 and TNF-αmRNA expression in the heart was positively correlated with LVW/BW, while renin, ACE2, TNF-α, IL-1βand IL-6 mRNA expression in the aorta was also positively concerned with AW/length.1.5 Relationships between local tissue RAS mRNA and inflammatory factor mRNA in SHRRenin, AT1 and AT2 mRNA expression in the heart was positively correlated with TNF-αmRNA. ACE2 and AT2 mRNA expression in the aorta was also positively correlated with TNF-αmRNA. Angiotensinogen, renin, ACE, ACE2 and AT2 mRNA expression in the heart was positively correlated with IL-6 mRNA. Angiotensinogen, renin, ACE2, AT1 and AT2 mRNA expression in the aorta was positively correlated with IL-6 mRNA1.6 Stepwise multivariate regression analysisThe regression equations indicated that LV weight/BW was independently correlated with DBP(β=0.493, P< 0.01; whereβis the standardized partial regressive coefficient)and ACE gene expression(β=0.449, P<0.01). Aortic weight/length was independently correlated with BRS(β=-0.810, P<0.01). The determinant coefficient (R2) for cardiovascular damage averaged 0.596. This value means that the independent hemodynamic factors, DBP, BRS and ACE mRNA expression level explain 59.6% of the variation in the hypertensive cardiovascular hypertrophy.2. Gender related influence on inflammatory factors in spontaneously hypertensive ratsBoth male and female SHR rats were used in this study. The blood pressure was measured in conscious state by using the tail-cuff plethysmography method. The rats was anesthetized, and a polyether cannula (PE-50) was inserted into the carotid artery to collecting blood sample, the kidneys were collected and immediately frozen in liquid nitrogen. The aorta and heart were immediately excised and rinsed in cold physiological saline for histopathological examination. The mRNA levels of TNF-a, IL-1βand IL-6 in local tissue were determined using quantitative real-time polymerase chain reaction. The plasma was determined of angiotensinⅡ, ET-1 concentration, the serum of TNF-a, IL-1βand IL-6 concentration was determined by using the radioimmunoassay kit. The main finding of this study are as following:2.1 Hemodynamic parameter, plasma AngⅡ, serum inflammatory factors and EOD in SHRCompared with SHR-12m group, SBP and body weight(BW)were no change in SHR-6m group. SBP, BW, LVW/RVW and AW/length were significantly higher in the same age male SHR when compared with female SHR, and VW/BW, LVW/BW, RVW/BW were significantly lower in the same age male SHR. Compared with SHR-6m-female, VW/BW, LVW/RVW, RKW/BW and AW/length were significantly higher in the SHR-12m-female group. Compared with SHR-6m-male group, RVW/BW, LVW/RVW, RKW/BW and AW/length were significantly higher in the SHR-12m-male group.2.2 plasma Ang II, ET-1 and serum inflammatory factors in SHR Compared with female SHR, plasma AngⅡwas significantly higher in the male SHR, plasma ET-1 was significantly lower in the male SHR. The serum IL-1βwas significantly higher in the SHR-6m-female group than those of SHR-6m-male group, compared with SHR-12m-male group, serum IL-1βwas significantly lower in the SHR-12m-female group. Compared with SHR-male, serum IL-6 was significantly higher in the same age SHR-female, compared with SHR-6m group, serum IL-6 was significantly higher in the same sex SHR-12m group. serum TNF-αwas no significantly change in the SHR-6m group, compared with SHR-12m-male group, serum TNF-a was significantly lower in the SHR-12m-female group.2.3 Correlations between plasma AngⅡ, ET-1 and serum inflammatory factors in SHRPlasma Ang II was found to be positively correlated with serum IL-1βand IL-6. Plasma Ang II was positively correlated with serum E2. Plasma ET-1 was not any correlated with serum inflammatory factors in SHR.2.4 mRNA expression of inflammatory factors in the kidney of SHRgroup Compared with SHR-6m-female group, mRNA levels of IL-6 in the kidney of SHR-6m-male group increased by 53%, Compared with SHR-12m-female group, mRNA levels of IL-6 in the kidney of SHR-12m-male group increased by 272%. Compared with SHR-6m-male group, mRNA levels of IL-1βin the kidney of SHR-12 m-male group by 33%. Compared with SHR-6m-female group, mRNA levels of TNFa in the kidney of SHR-6m-male group increased by 54%, Compared with SHR-12m-female group, mRNA levels of TNFa in the kidney of SHR-12m-male group increased by 50%. The mRNA levels of TNFa in the kidney of same age SHR(6m,12m)did not change significantly.3. Gender differences in development of renovascular hypertension10-week-old SD rats were prepared by the narrow side of the renal artery to development renovascular hypertension(2K1C). After removal of both ovaries of a group of female rats(VOX),2K1C operation was performed in the same female rats. After 6 months of regular feeding, rats were instrumented to determine blood pressure(BP) in anesthesia state. After BP recording, a polyethy cannula(PE-50) was inserted into the carotid artery to collecting blood for determination of AngⅡ, ET-1 and inflammatory cytokines by using ELISA kit. Rats were killed and the thoracic aorta, heart, kidney were collected and frozen. Observation of target organ damage. Western Blot method was applicated to detect the protein expression of ACE, ACE2, AT1 and AT2 in the thoracic aorta, heart and kidney tissue. The main findings of this study are as following: 3.1 Hemodynamic parameter in 2K1C ratsCompared with 2K1C-sham-male rats, SBP.DBP were significantly increased in all 2K1C rats, and HP was significantly lower in 2K1C-female rats. SBP,DBP were significantly higher in 2K1C-female rats and similar in 2K1C-OVX rats when compared with 2K1C-male rats.3.2 EOD parameter in 2K1C ratsVW/BW, LVW/BW, LVW/BW and RKW/BW were significantly higher in 2K1C rats. VW/BW, LVW/BW, LVW/BW and RKW/BW were significantly higher in 2K1C-female rats when compared with 2K1C-male rats and 2K1C-OVX rats. Compared with 2K1C-sham-male rats, AW/length were significantly higher in 2K1C-male, and was similar in 2K1C-OVX rats and 2K1C-female rats.3.3 plasma AngⅡ, ET-1 and serum inflammatory factors in 2K1C ratsCompared with 2K1C-sham-male rats, plasma AngⅡwere significantly higher in 2K1C-male, and similar in 2K1C-OVX rats and 2K1C-female rats, plasma ET-1 were significantly higher in all 2K1C rats.plasma ET-1 were significantly higher in 2K1C-female rats when compared with 2K1C-male rats and 2K1C-OVX rats. Compared with 2K1C-sham-male rats, serum IL-1βwere significantly higher in 2K1C-OVX rats and were significantly lower in 2K1C-female rats, serum IL-6 were significantly lower in 2K1C-OVX rats and another groups did not change. Compared with 2K1C-sham-male rats, serum TNF-a were significantly higher in all 2K1C rats.3.4 Protein levels of ACE, ACE2, AT1 and AT2 in 2K1C ratsThoracic aorta tissue:the protein levels of ACE were significantly higher in 2K1C-female rats and 2K1C-male rats, were similar in 2K1C-OVX rats when compared with 2K1C-sham-male rats.the protein levels of ACE were significantly higher in 2K1C-female rats when compared with 2KIC-male rats. The protein levels of ACE2 were significantly higher in 2K1C-OVX rats and in 2K1C-female rats, on change in 2K1C-male rats when compared with 2K1C-male rats. The protein levels of AT1 receptor were on significantly change in all 2K1C rats when compared with 2K1C-sham-male rats. Compared with 2KlC-male rats, the protein levels of AT1 receptor were significantly lower in 2K1C-OVX rats and in 2K1C-female rats. Compared with 2K1C-sham-male rats the protein levels of AT2 receptor were significantly higher in all 2K1C rats.Kidney tissue:Compared with 2K1C-sham-male rats, the protein levels of ACE were significantly higher in 2K1C-female rats, the protein levels of ACE2 were no significantly change in all 2K1C rats, the protein levels of AT1 receptor were significantly lower in 2K1C-female rats, and the protein levels of AT2 receptor were also significantly lower in 2K1C-female rats.Heart tissue:Compared with 2K1C-sham-male rats, the protein levels of ACE were significantly higher in 2K1C-female rats, the protein levels of ACE2 were significantly lower in 2K1C-female rats and in 2K1C-male rats, the protein levels of AT1 receptor were significantly higher in 2K1C-female rats and in 2K1C-male rats. Compared with 2K1C-male rats, AT1 receptor were significantly higher in 2K1C-female rats. Compared with 2K1C-sham-male rats, the protein levels of AT2 receptor were significantly lower in 2K1C-OVX rats.Conclusions:1 Gene expression of all the components of RAS, including ACE2 and AT2 receptor in the heart and the aorta, was increased in SHR when compared with WKY rats; Hypertensive cardiovascular hypertrophy was significantly related to mRNA expression of local RAS and inflammatory factors, but not to plasma AngⅡin SHR; Stepwise multiple-regression analysis showed that left ventricular hypertrophy was mainly determined by increased DBP and cardiac ACE gene expression; and aortic hypertrophy was mainly correlated with decreased baroreflex sensitivity.2. Effects of estrogen on the adult(6 months)blood pressure in spontaneously hypertensive rats and target organ damage have a certain effect of mitigation; the plasma concentrations of AngⅡwas significantly higher in the male SHR, the plasma concentrations of ET-1 was significantly lower in the male SHR. Female adult spontaneously hypertensive rats serum concentrations of IL-6 and renal tissue mRNA expression was significantly lower than the male same age group, and increased with age; the serum concentrations of TNFa in the elderly female groups was significantly lower than the male same age group, the TNFa mRNA expression levels of female adult and elderly groups in spontaneously hypertensive rat was significantly lower than the male same age group. The serum concentrations of IL-1β,IL-6 and estradiol was significantly related to the plasma concentrations of Ang II.3.10-week-old SD rats were prepared by the narrow side of the renal artery to development renovascular hypertension(2K1C). After removal of both ovaries of a group of female rats(VOX)to development low levels of estrogen,2K1C operation was performed in one group female rats. Compared with 2K1C-sham-male rats, SBP,DBP were significantly higher in all 2K1C rats. SBP,DBP were significantly higher in 2K1C-female rats and similar in 2K1C-OVX rats when compared with 2K1C-male rats. It is likely that the effect of estrogen on renovascular hypertension was depending on promoting promote the role of AngⅡby increasing the release of ET-1, increasing the protein expression levels of ACE in heart, thoracic aorta and kidney tissue, and increasing the protein expression level of AT1 receptors in heart tissue.
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