Effect And Mechanism Of Puerarin On Blood Pressure In Spontaneously Hypertensive Rats

Hypertension is one of the most important risk factors for cardiovascular and cerebrovascular diseases,and is also the cause of morbidity,disability and mortality in the world.Activation of the RAAS system,hyperactivity of the sympathetic nervous system,renal water and sodium retention have been recognized although pathogenesis of hypertension has not been fully elucidated.In recent years,more and more evidences show that oxidative stress and oxidative damage are the main causes of vascular injury,which may be related to the pathogenesis of hypertension.ENOS is mainly secreted from vascular endothelial cells and myocardial cells,and is a specific enzyme for NO.it played important roles in a variety of physiological and pathological process including oxidative injury,endothelial protection,vasomotor function,platelet adhesion and aggregation.Therefore,it is of great clinical significant values to study the role of eNOS and its chaperone in vascular protection for the prevention and treatment of hypertension.Radix Pueraria was found in the book of "Shen Nong's herbal classic" from Eastern Han Dynasty,which has been nearly 2000 years.On the basis of inheriting forerunners'experience,pueraria was classified as drugs for treating exterior syndromes in the book of modern chinese medicine.Puerarin is one of the main effective components of Traditional Chinese Medicine Radix Puerariae.Its chemical name is 8-?-D-glucopyranose-4',7-dihydroxyisoflavone,and relative molecular weight is about 416.38.Puerarin has effect on reducing myocardial oxygen consumption,expanding cardiovascular and cerebrovascular,improving myocardial contraction,promoting microcirculation.It plays an important role in the treatment of cardiovascular diseases such as coronary heart disease,myocardial ischemia reperfusion injury,ischemic stroke,hypertension and senile dementia.However,the clinical application of puerarin has been limited since the adverse reactions reported in the end of twentieth century,such as intravascular hemolysis.At present,the methods of reducing hemolysis include taurine compatibility,submicron encapsulation,polyethylene glycol polymer materials modification and so on.In our study,it was used to study the effects of intraperitoneal injection of puerarin on function of liver and kidney,and the hemolytic index was also observed.And results showed no significant changes in the puerarin group.So,how is the antihypertensive effect of puerarin,and what is the mechanism?With this question,antihypertensive mechanism of puerarin was studied with qPCR array,who owned comprehensive and precise advantages,in the first part of the experiment.The results suggested that mechanism of puerarin antihypertension closely related to the increase of eNOS activity and the regulation of eNOS/cGMP pathway.Based on this,in order to further study factors that affect the activity of eNOS,molecular chaperones related to eNOS was found firstly with the help of network pharmacology database.Then by qPCR array technology,effect and mechanism of Puerarin on oxidative damage of aorta under chronic hypertension was studied.This study contains two parts:literature review and experimental research.1 Literature review:including three reviews:the first one is "Research Progress of Radix Pueraria from ancient times to present",and second one is "Clinical and Basic Research Process of Puerarin Preventing Hypertension",the other is "Oxidative Stress and Hypertension".2.Experimental research:including the following two parts:Studyl Effect and Mechanism of Puerarin on Blood Pressure in SHR Objective:To explore the effect and mechanism of puerarin on blood pressure in spontaneously hypertensive rats(SHR).Methods:Wistar Kyoto rats(WKY)were set up for control and sixty 10 week old SHR were divided into four groups randomly including model group,losartan group(32.5mg·Kg-1,intragastric administration),normal dose puerarin and high dose puerarin group(40mg kg-1 and 80mg·kg-1,intraperitoneal injection,respectively).In control and model groups,the same volume of saline was given intraperitoneally.Drug treatment lasted 10 weeks.Systolic pressure,diastolic pressure and heart rate were tested weekly.Ultrasonic cardiogram technology was used for heart functions and aortic diameter.Aortic tissue pathomorphology was observed by Masson staining and Homogenate preparation.Renin-angiotensin-aldosterone levels of plasma were measured with biochemical assay.84 genes related to hypertension were tested by qPCR array,and differentially expressed genes were analysed depended on databases of String and KEGG Pathway.Lastly,some interesting molecules were tested by Western blot.Results:Systolic blood pressure(SBP)and diastolic blood pressure(DBP)of model group were obviously increased than control group(P<0.001),SBP and DBP of high dose puerarin group and losartan group were decreased significantly(P<0.05 or P<0.01)compared with model group.Heart rate(HR)of model group was apparently improved than control group(P<0.05),and HR of high and normal dose puerarin were both down regulated effectively compared with model group(P<0.01),while there was no statistical significance between losartan and model group(P>0.05).Aortic diameter of model group increased significantly than control group(P<0.001).Losartan group decreased effectively(P<0.05)than model group and puerarin group did not.Value of EF and FS in model group significantly decreased(P<0.01).And compared with model group,value of EF and FS of normal and high dose puerarin group were up regulated apparently(P<0.001),while losartan group was of no statistical significance(P>0.05).Aortic intima-media thickness of model group increased evidently compared with control group(P<0.001)and there were considerably differences in aortic intima-media thickness between all drug groups and model group(P<0.001).Plasma level of angiotensin ?,renin and aldosterone in model group had no statistical difference than control group(P>0.05).Level of angiotensin ? of normal and high dose puerarin decreased significantly compared with both model and losartan group(P<0.01).There were considerably differences in renin level between all drug groups and model group(P<0.01).The aldosterone levels in different administration groups had no statistically significant difference(P>0.05).The results of network analysis suggested eNOS was related to Cav1 and Agtr1b,and Adrb1,Agtr1b,Gucy1a3,Gucy1b3,Kcnmal,Mylk,Mylk3,eNOS,Pde3a and Pde5a participated in the activation of cGMP-PKG pathway,Agtr1b,Gucy1a3,Gucy1b3,Kcnmal,Mylk,Mylk3 and Ptgir involved in contraction of vascular smooth muscles,while Gucyla3,Gucy1b3,Mylk,Mylk3,eNOS and Ptgir related to platelet activation.Serum level of cGMP and PGI2 in model group significantly decreased(P<0.05).And compared with model group,cGMP and PGI2 of losartan group,normal and high dose puerarin group were up regulated apparently(P<0.01 or P<0.001).Protein expression of p-eNOS in model group was significantly decreased(P<0.01).Compared with model group,p-eNOS expression of high dose puerarin group increased obviously(P<0.05)and losartan group increased not significantly(P>0.05).Protein expression of eNOS in all groups had no statistically significant difference(P>0.05).Agtrl protein expression in model group was significantly higher(P<0.01),and that of losartan and high dose puerarin group decreased effectively(P<0.01).Protein expression of Agtr2 in all groups had no statistically significant difference(P>0.05).Conclusion:Puerarin has an antihypertensive effect,and its mechanism may be related to regulation of RAAS levels,the enhancement of eNOS regulated by Cavl and Agtrl,and the balance of eNOS/cGMP pathway.Study2 To Futher Stdudy the Mechanism of Puerarin on Blood Pressure in SHR Based on on eNOS Related GenesObjective:To futher stdudy the mechanism of puerarin on blood pressure in shr based on on enos related genes.Methods:Grouping methods and medications of Wistar Kyoto rats(WKY)and pontaneously hypertensive rats(SHR)were the same as part 1.The level of NO,O2-·,SOD and MDA in serum were detected by biochemical Method.Serum eNOS level was measured with Elisa assay.84 genes related to oxidative stress were tested by qPCR array,and differentially expressed genes were analysed depended on databases of String and KEGG Pathway.Lastly,some interesting molecules were validated d by Western blot.Results:Serum levels of NO in model group decreased significantly than control group(P<0.001).NO both in puerarin and losartan group increased effectively compared with model group(P<0.01 or P<0.001).MDA level of model group increased significantly than control group(P<0.001).And MDA in puerarin and losartan group decreased obviously(P<0.01 or P<0.001).O2-·level of model group was down regulated markedly than control group(P<0.001).There were no significant differences in the level of O2-· among administration groups(P>0.05).No difference statistically among all groups was observed in SOD level.Level of eNOS in model group decreased significantly than control group(P<0.001).And compared with model group,eNOS in all drug groups were improved effectively(P<0.001 or P<0.01).Network analysis of 12 differentially expressed genes suggested eNOS closely related to Hsp90?1 and Cavl.While,Hsp90?1,eNOS,Pik3cg and Pten participated in PI3K-Akt pathway,Hsp90?1,Hspal,eNOS and Pik3cg were involved in the regulation of estrogen pathway and Cavl,Pik3cg and Pten were related to focal adhesion.Protein expression of Hsp90 in model group was significantly increased(P<0.05).Compared with model group,Hsp90 expression of high dose puerarin and losartan group decreased obviously(P<0.05 or P<0.001).There was no difference for PI3K between model and control group(P>0.05).Compared with model group,PI3K expression in high dose puerarin and losartan group increased effectively(P<0.001 or P<0.01).Akt and p-Akt expression in all groups had no statistical difference(P>0.05).Conclusion:Puerarin has an effect on anti-oxidative stress in SHR,and its mechanism may be related to down regulation of serum MDA level and up regulation of NO and eNOS level.And it also has connection with enhancement of eNOS activity regulated by Hsp90,Hsp70 and Nox.Anti-oxidative enzyme system of played important role in the protection of aorta.PI3K/Akt pathway was also involved in the mechanism of puerarin suppressing oxidative stress resulted by hypertension.