| Objective:Based on the evaluation methods of traditional pharmacology,network pharmacology and molecular biology,from many aspects of systolic blood pressure,heart rate,macroscopic characterization,behavioristics,micro-physicochemical index and pathological diagnosis,to evaluate the effect of Wuweijiangyasan comprehensively.Then in the basis of the determination of the antihypertensive effect of Wuweijiangyasan,using the exisent targets ACE and ACE2 as the breakthrough points which are closly related to the pathogenesis of hypertension.At the same time,combined with the drug targets AR and ER from the predicting network pharmacology.We apply RT-PCR and Western blot technologies to realize the preliminary exploration on the antihypertensive mechanism of Wuweijiangyasan.It will not only provide a scientific basis for resistant hypertension,but also achieve the purpose of providing the scientific ideas and methods for traditional Chinese medicine compound to resistant hypertension on the efficacy and mechanism research.Methods:1.Using the animal pharmacodynamic evaluation method:Then based on observation caliber like the blood pressure,heart rate,body weight,macroscopic characterization,behavioristics,micro-physicochemical index and pathological diagnosis:14 weeks old SHR rats were randomly divided into model group,Wuwei group,Sancao group,Niuhuang group,Valsartan group and WKY group,which used as the normal control group,they were administrated for 10 weeks.Furthermore,it is according to the initial blood pressure and weight to group.The blood pressure and heart rate were obtained by measuring the rats by BP-2010A.The blood pressure was observed twice a week for observing dynamically.Moreover the changes of the basic condition of SHR rats were observed closely and the body weight was measured once a week,as well as the degree of irritability and the rotation tolerance time test and so on.After the experiment finished,use the abdominal aorta method and then isolate or prepare serum for biochemical tests.This part is to study the effect of Wuweijiangyasan on blood physical and chemical indexes of rats.At the same time,conduct the pathologic examination of target organs on heart and kidney tissue,contains these steps of paraffin embedding,HE staining,neutral gum seal,microscopic observation and photography.Under the optical microscope,detecting the pathological changes of the tissue in the rats,and then explore the pathological changes of the heart and kidneys,which will contributed to the comprehensive evaluation of antihypertensive effective of Wuweijiangyasan.2.The antihypertensive mechanism of Wuweijiangyasan:Applying the method of network pharmacology and molecular biology to study then adopted the technique of real-time fluorescent quantitative PCR and Western blot method for testing:study the expression level of ACE andACE2 gene and protein in the heart and kidney tissues in the experimental rats.At the same time in the detection of heart and kidney AR,ER alpha protein expression level,to the purpose of examining the accuracy of pre-network pharmacological prediction,namely,whether Wuweijiangyasan decompression by adjusting the AR,ER alpha.Results:1.Pharmacodynamics of Wuweijiangyasan:(1)SBP influence:after 10 weeks of administration:the blood pressure of model group rising;while the Wuwei group in the onset to decrease significantly at the 2 weeks,moreover,its blood pressure come to maintain a stable low level after 4 weeks;the SBP of Sancao group was down in administration within 0-4 weeks,so as to the period after 8 weeks;but get a SBP recovery for 4-8 weeks.The Niuhuang group get the blood pressure decreased effectively after the administration during the 0-1 weeks and 4-8weeks;The Valsartan was continuous decompression.(2)Heart rate influence:there was statistical difference between the Wuwei group and the model group,the heart rate of the model group was significantly increased,while the heart rate of the Wuwei group was stable after administration.(3)Weight influence:the model group was the fastest growth rate of body weight,however,the weight growth of Wuwei is slow and the smallest.(4)Behavioral influence:?the degree of easy to provoke:it is the time for Wuwei after 5 weeks when the degree of irritability tends to stabilize;?Rotation tolerance time:the rotation tolerance time increased and has been stabilized of the Wuweijiangyasan group after 2 weeks of administration,which is also consistent with the change in blood pressure.(5)Effect on the general characterization:It is found that the rats in the Wuweijiangyasan group are get together to climb,screaming,bow,manic,fixed difficult,deep yellow urine,dry stool,stool less et al,these symptoms followed changes with the fluctuations in blood pressure;in other words,blood pressure increase,so that every symptoms aggravated,while blood pressure decreased,then the symptoms alleviated,such as their climbing,scratches reduced;the degree of screaming relieved,even without grabbing resistance,urine is clear,the stool volume gradually increased,stool thin and so on;at the other hand,the model group become more aggravated,the difference is obvious.(6)Blood fat influence:The comparison with the model group is that the CHOL,TG and LDL-C were all decreased,and the difference was statistically significantally(P<0.05).(7)Influence on the pathological changes of heart and kidney:the SHR rats in the model group with intravascular blood stasis,and small arterial muscle was thickening,cell size is uneven or irregular,cell vacuolar degeneration,blood cavity narrowing are easy to see.Additionally,their Kidney glomerular enlargement,blood stasis significantly,and part of the basement membrane mild thickening,capsule capsule exudation,curved tube swelling,a mesh-like changes,interstitial infiltration of inflammation.However,the heart and kidney lesions of the Wuwei group had some reduction in different degrees compared with the model group.2.The antihypertension mechanism of Wuweijiangyasan(1)Effects of the expression of ACE and ACE2 mRNA in rat heart and kidney:Compared with the model group,the expression of ACE mRNA in the heart of SHR rats was down-regulated,on the other hand,the expression of ACE2 mRNA was increased(P<0.01).ACE mRNA and ACE2 mRNA in the heart tissue of SHR rats were not significantly different from those in the normal group(P>0.05).There was no significant difference in the expression of ACE mRNA between the normal group,the model group,the Wuwei group and the valsartan group(P>0.05),indicating that there was no significant effect of Wuweijiangyasan on kidney ACE mRNA(P>0.05).Compared with the model group,the expression level of ACE2 mRNA in SHR rats was significantly lower than that in the normal group(P<0.05).However,the expression of ACE2 mRNA in SHR rats was significantly higher than that in the control group(P<0.05).(2)Effects of ACE,ACE2,ER alpha and AR protein expression in rat heart and kidney:?There was little difference between the normal group and the Wuwei group in the expression of heart ACE and ACE2 protein(P>0.05).Then compared with the model group rats,Wuwei group can significantly increase the content of heart ACE2 protein(P<0.05),which is consistented with its expression of ACE2mRNA.? At the same time,compared with the model group,the expression of heart and kidney AR?ERa protein showed no significant difference with Wuwei group(P>0.05).Conclusions:1.Wuweijiangyasan pharmacodynamics:(1)Reduction Smooth:The effect of Wuweijiangyasan lowering blood pressure is slower,but the advantage of the regulator is more obvious in the late period,and the latter is better than the Sancao and Niuhuang groups.It is believed that the blood pressure drop continuous,stable and small fluctuations for Wuweijiangyasan.However,the blood pressure of the model group continued to rise,and compared with the model group,the effect of Wuwei was statistically significant(P<0.05).(2)Stable heart rate:The heart rate of the model group was significantly increased,while Wuwei group was stable.Heart rate run rapid,then systolic blood pressure and diastolic blood pressure were increased,and the latter was higher than the former,heart rate slowed down,diastolic blood pressure decreased more than systolic blood pressure.And after 2 weeks'treatment,the heart rate and blood pressure were decreased and remained stable,and the change trend was consistent with Wuwei drugs.(3)Slow weight gain:The weight of the model group increased rapidly,and the weight gain of the Wuwei group was slow and the amplitude was the smallest.(4)Sedative effect:Compared with the model group,the Wuwei groups were low and stable in the degree of easy to provoke,and the volatility is small;as well as the rotarytolerance time increased,which has been stable and smaller than the model group.The two trends are consistent with the blood pressure downward trend.(5)Improved characterization:Wuweijiangyasan can improve SHR rats in blood pressure and hypertension associated symptoms,such as grasping resistance,screaming,fixing difficulties and stool,urine and so on with the decline in blood pressure,while the model group increased gradually with the increase of blood pressure.(6)Lipid-lowering Obviously:Wuweijiangyasan has obvious lipid-lowering effect.It can reduce the level of CHOL,TG and LDL-C.(7)Target organ Protection:Wuweijiangyasan can relieve the athological damage of heart and kidney,which can play a certain degree of protection of target organ function.2.The possible hypotensive mechanism of Wuweijiangyasan(1)Regulation of cardiac RAAS to hypotension:Wuweijiangyasan can reduce the level of ACEmRNA in the heart of SHR rats,so inhibit the activity of ACE in heart,and can increase the expression level of ACE2mRNA and protein in heart at the same time.(2)Wuweijiangyasan had no difference onAR?ERa protein of heart and kidney... |
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