Study On Jianpi Recipe Remodeling Of SHR Arterial Blood Pressure And Cardiac Muscle Qishen

Purpose：This study is to optimize the extraction of Jianpi Yiqi decoctio byusing Central Composite Design/Response Surface Methodology and observe theeffects of Qishen Jianpi decoction on anti-hypertension in the rats withspontaneous hypertension and explore the impacts on protein and mRNA expressionof AngⅡ，TGF-β1，Smad2and Smad3, MMP-1,MMP-2,MMP-9collagen I and collagenⅢ,by which we can value its antihypertensive effect and also investigate thefunction of Qishen Jianpi decoction in the mechanisms prevent the developmentof arterial blood pressure in SHR and inhibit accumulation of cardiac collagenfibers. At the same time, we will also discuss how the Qishen Jianpi decoctionregulates the expression of AngⅡ and influences the content of signalingmolecules which are in the TGF-β1/Smad thoroughfare. Besides, we will futherto research about how the Qishen Jianpi decoction influence the expression ofMMP-1,MMP-2,MMP-9collagenI and collagenⅢof cardiac collagen fibers. Meanwhile,we also explore the mechanisms of inhibiting accumulation of cardiac collagenfibers during the process of hypertension. All the experiments have offered thescientific basis and theories for the use of Qishen Jianpi decoction in theclinical treatment and also explain its molecule process of protecting theMyocardial tissue.Material and method：Theeffectsofthreeindependentvariables,Extractiontimes, ratio of material to solvent and extracting time were investigated withthe content of Astragaloside IV and Astragalus Polysaccharides. Responsesurfaces were delineated according to the mathematic models, and optimumformulations were selected.Using the randomized controlled method to divide the6024-week-old SHR intothe model group (giving water,SHR group), perindopril group [perindopril,0.4mg/(Kg·d)], perindopril associated with herbgroup[perindopril0.4mg/(Kg·d)+medium gose of herb], and another3groupsindividually high level, middle level and low level dose of herb(the dose isdecided according to the100g weight which are4g,2g,1g) and the normal groupis10mormal arterial pressure WKY at the same age and the same kind (givingpure water, WKY group). The rats are fed once a day. We will detect the systolicpressure’s change of their caudal artery with BP-98A intelligentsphygmomanometer before and2hours,2weeks,4weeks, and6weeks after givingthem the medicine, and the measuring time is2hours after the medicine giving.It will last6weeks.The rats will be fed only water for24hours, then these groups of rats willbe weighed after given10%chloral hydrate’s anesthetic. We will fix the ratson the desk after anesthetic and sterilize their belly skin with0.5%Iodophor,then we will cut off their belly skin and widen the thoracic cavity dlong theabdominal cavity to make the heart seeable, and take about50g tissue to putit in the EP canal combined with1ml Trizol in the refrigerator under thetemperature of-70℃, RT-PCR method will be used to detect the mRNA content ofAngⅡ, TGF-β1, MMP-1, MMP-2, MMP-9; Take another50g tissue to put it in theEP canal in the refrigerator under the temperature of-70℃, and the Westernblot method will be used to detect theexpression of AngⅡ, TGF-β1, Smad2, Smad3,MMP-1, MMP-2and MMP-9; The rest of tissue will be put into4%paraformal-dehydeto carry on the observation of pathology images and at the same time detect theexpression of CollagenI and CollagenⅢ.The data will be checked with one factor analysis of variance using theSPSS17.0software, and LSD method will be used to make comparison in the wayof (X±s), if P＜0.05, it will have significant difference.Results：1. Central Composite Design／Response Surface Methodology is successfully usedto optimize the extraction of Jianpi Yiqi decoctio. All the investigated response variables were found to be highly dependenton the formulation variables. The optimized values of extraction times, ratioof material to solvent and extracting time were2,9and1.5, respectively. Theobserved values agreed well with model predicted values.2. The influence of Qishen Jianpi decoction on the SHR about the caudal artery’ssystolic pressure.The statistical results explanation: before giving the medicine, thesystolic pressure of these groups of rats are all higher than the group WKY,P<0.01;4hours after giving them the medicine, there is no obvious change aboutthe systolic pressure; After2weeks, the pressure is obvious getting down forthe perindopril group and the perindopril associated with herb group, andcomparing with the model group, it has the statistical meaning, P<0.01; After4weeks and6weeks, the pressure is continuously getting down and it has thestatistical meaning comparing with the model group, P<0.01. while for the3leveldose of herb groups, the pressure is also getting down at some extent after2weeks of using the medicine, however, after4and6weeks, the pressure is notgetting down clearly, and comparing with the model group, it has no statisticalmeaning.3. The influence of Qishen Jianpi decoction on the cardiac collagen fibersmorphology and the expression of CollagenI and CollagenⅢ.The cardiac collagen fibers of WKY group are clear and arrange regularly,interstitial collagen fibers is little; The interstitial fibroblast and collagenfibers are clearly in SHR group. Compared with control group, the shape ofmitochondria is better, interstitial fibroblast and collagen fibers are lessin the perindopril group, the perindopril associated with herb group and thehigh dose of herb group. The protein expression of CollagenI and CollagenⅢ issignificantly decreased in perindopril group, perindopril associated with herbgroup and high level dose group compared with SHR group, P<0.01, P<0.05, andthe expression of CollagenⅢ was significantly decreased in middle level dosegroup,P<0.05. 4. The influence of Qishen Jianpi decoction on the dose of signaling moleculewhich are in the SHR TGF-β1/Smad thoyoughfare.Comparing with the SHR group, the mRNA content of TGF-β1and AngII of theperindopril group, the perindopril associated with herb group and the high doseof herb group is obviously getting down, P<0.05. Comparing with the SHR group,TGF-β1protein’s expression quantity of the perindopril group, the perindoprilassociated with herb group and the3dose of herb group is clearly decrease,P<0.01，P<0.05; The content of AngII and Smad3protein of the perindopril group,the perindopril associated with herb group and the high dose of herb group ismuch lower than the SHR group, P<0.01，P<0.05; The protein expression of Smad2is decreased in perindopril group， perindopril associated with herb group,P<0.01，P<0.055. The influence of Qishen Jianpi decoction on the expression of MMP-1, MMP-2,MMP-9and CollagenI, Ⅲ.The mRNA and the protein expression of MMP-1, MMP-2and MMP-9wassignificantly increased in perindopril group,perindopril associated with herbgroup and high dose group compared with SHR group, P<0.01，P<0.05, and theexpression of MMP-1was significantly increased in middle dose group, P<0.05;The protein expression of Collagen I and CollagenⅢ was significantly decreasedin perindopril group, perindopril associated with herb group and high dose groupcompared with SHR group, P<0.01, P<0.05, and the expression of CollagenⅢ wassignificantly decreased in middle dose group, P<0.05.Conclusion：1. Central Composite Design／Response Surface Methodology is successfully usedto optimize the extraction of Jianpi Yiqi decoctio.2. The middle level dose of the Qishen Jianpi decoction combined with perindoprilshows the better lowing pressure effect on the caudal artery systolic pressure,after4weeks treatment, it will reach the stable level.3. The Qishen Jianpi decoction combined with perindopril and the high level dose of Qishen Jianpi decoction can improve SHR cardiac pathology images conformation.By inhibiting the expression of Collagen I and CollagenⅢ so as to reverse theaccumulation of cardiac collagen fibers.4. By regulating the factors from TGF-β1/Smad signaling pathway and AngII,Qishen Jianpi decoction combined with perindopril could modify the functionobstacle of hearts in the development of arterial blood pressure in SHR.5. The Qishen Jianpi decoction combined with perindopril and the high level doseof Qishen Jianpi decoction can stimulate the expression of MMP-1, MMP-2and MMP-9to exert its function of lowing blood pressure and protect the hearts.