芪参健脾方对SHR大鼠动脉血压及心肌重塑影响的研究

【作者】 谷丽艳；

【导师】 张立德；

【作者基本信息】 辽宁中医药大学 ， 中西医结合基础， 2013， 博士

【摘要】 目的：本实验通过优化芪参健脾方的提取工艺，以及观察芪参健脾方对自发性高血压大鼠（SHR）尾动脉收缩压以及心肌组织内血管紧张素Ⅱ（AngⅡ）、TGF-β1/Smad信号转导途径各因子、基质金属蛋白酶1、2、9（MMP-1，MMP-2，MMP-9）和I，Ⅲ型胶原蛋白（Collagen I，CollagenⅢ）表达的影响，评价其降压效果，并考察芪参健脾方对SHR心肌纤维化，高血压所致的心肌重塑的过程中各信号分子含量表达的影响，探索芪参健脾方调节血管紧张素Ⅱ、TGF-β1/Smad信号转导途径内各因子表达水平的机制；进一步研究芪参健脾方对SHR心肌组织质金属蛋白酶1、2、9和和I，Ⅲ型胶原蛋白表达的影响，探求高血压进程中心肌重塑的改善机制；为临床辨证治疗施以芪参健脾方提供科学的实验数据与理论基础，阐释其降压及心肌保护作用的分子机制。材料与方法：以水煎法提取补脾益气汤中的有效部位，以黄芪甲苷含量为指标，采用星点设计-效应面法考察提取次数、溶媒用量、提取时间3个因素对黄芪甲苷含量的影响，优选最佳提取工艺条件。采用随机对照法将60只24周龄的SHR分为模型组（给予蒸馏水，SHR组）、培哚普利组（培哚普利，0.44mg/kg/d）、培哚普利联合中药组（培哚普利，0.4mg/kg/d+中药中剂量，中加西组）、中药高、中、低剂量组（用药量按照每100g大鼠体质量分别折算为4g，2g，1g），每组10只，以同龄同种系正常血压的京都种大鼠（WKY）10只作为正常组（WKY组），大鼠每日一次灌胃。智能无创血压计测量大鼠初次给药前，首次给药后2h，第2周，4周和6周给药后2h大鼠尾动脉收缩压变化情况，连续治疗6周。大鼠禁食、自由饮水24h后，各组大鼠称重，10%水合氯醛腹腔麻醉（3mg/kg）。大鼠麻醉后固定于鼠台上，用0.5%碘伏消毒大鼠胸腹部皮肤，沿左侧胸骨旁斜形切口，上界为两前肢后缘线，下界为第五肋间，依次剪开皮肤、浅筋膜和深筋膜，在靠胸骨缘处用止血钳钝性分离第3、4肋间，用专用开胸器撑开3、4肋间暴露心脏，将棉纱放置在肺组织旁，防治损伤肺组织。活体取出心脏，迅速取材，取50mg心肌组织放入EP管中并加入1ml Trizol试剂，-70℃冰箱保存，PCR方法检测心肌AngⅡ、TGF-β1、MMP-1，MMP-2和MMP-9的mRNA表达；另取心肌组织50mg入EP管中，-70℃冰箱保存，western方法检测MMP-1，MMP-2和MMP-9蛋白的表达；剩余心肌组织用4%多聚甲醛固定，进行心肌病理形态学观察及免疫组化方法检测I，Ⅲ型胶原蛋白表达。采用SPSS17.0统计学软件对数据进行处理，实验数据以均数±标准差(X±s)表示，组间均数比较采用单因素方差分析（ANOVA），P＜0.05为差异有统计学意义。结果：1.补脾益气汤的最佳提取工艺采用星点设计-效应面法优选了补脾益气汤的最佳提取工艺，优选后的理论值和预测值相近，说明所建立的数学模型的预测性较好。2.芪参健脾方对SHR尾动脉收缩压的影响统计结果表明，用药前，各组大鼠血压均高于WKY组，P＜0.01；药后2h，各组大鼠收缩压无明显变化；用药2周后，中加西药组、西药组血压明显降低，与模型组相比有统计学意义，P＜0.01，用药4周及6周后，中加西药组、西药组血压持续下降，与模型组相比有统计学意义，P＜0.01；中药高、中、低剂量组血压用药2周后也有一定程度的下降，用药4周及6周后血压下降不明显，与模型组相比无统计学意义。3.芪参健脾方对SHR心肌组织形态学及Collagen I和CollagenⅢ的表达的影响WKY组大鼠心肌组织胶原纤维分布比较均匀、纤细，心肌间质无纤维化现象；与WKY组相比，SHR组心肌组织呈典型心肌肥厚改变，心肌细胞排列杂乱无章，被深入的纤维分隔紧裹，细胞间网络增粗变密，心肌纤维走向紊乱，可见溶解断裂，细胞间质纤维化增多。与SHR组相比，培哚普利组、培哚普利联合中药组和中药高剂量组心肌细胞直径明显减小，大小一致，排列整齐，心肌纤维无断裂，纤维增生减少。与SHR模型组比较，培哚普利组、培哚普利联合中药组和中药高剂量组中Collagen I和CollagenⅢ的表达显著下降（P<0.01，P<0.05），中剂量组中CollagenⅢ的表达也明显减少（P<0.05）。4.芪参健脾方对SHR心肌组织AngⅡ和TGF-β1/Smad信号转导途径中各信号分子含量的影响与模型组比较，培哚普利组、培哚普利联合中药组和中药高剂量组中TGF-β1、AngII mRNA的表达量显著下降（P<0.05）。与模型组比较，培哚普利组，培哚普利联合中药组，中药高、中、低各剂量组TGF-β1蛋白的表达显著降低（P<0.01，P<0.05），培哚普利组，培哚普利联合中药组，中药高剂量组AngⅡ蛋白和Smad3蛋白的表达量明显低于SHR组（P<0.01，P<0.05），培哚普利组，培哚普利联合中药组Smad2蛋白的表达量明显低于SHR组（P<0.01，P<0.05）。5.芪参健脾方对SHR心肌组织MMP-1，MMP-2和MMP-9表达的影响与SHR模型组比较，培哚普利组、培哚普利联合中药组和中药高剂量组中MMP-1，MMP-2和MMP-9的mRNA和蛋白的表达量都显著增加（P<0.01，P<0.05），中药中剂量组MMP-1蛋白的表达也量明显增加（P<0.05）；与正常组比较，SHR组心肌呈典型心肌纤维化改变；与SHR模型组比较，培哚普利联合中药组、培哚普利组和芪参健脾方高剂量组中Collagen I和CollagenⅢ的表达显著下降（P<0.01，P<0.05），中剂量组中CollagenⅢ的表达也明显减少（P<0.05）。结论：1.采用星点设计-效应面法可优化补脾益气汤的提取工艺。2.芪参健脾方联合培哚普利治疗对SHR尾动脉收缩压表现出较好的降压效果，治疗4周达到稳定期。3.芪参健脾方联合培哚普利及单方高剂量的应用能有效改善SHR心肌病理形态结构，同时显著下调了Collagen I，CollagenⅢ的表达，逆转高血压导致的心肌重塑。4.芪参健脾方联合培哚普利通过调节SHR心肌组织AngⅡ和TGF-β1/Smad信号转导途径中各信号分子的表达，改善高血压进程中心肌细胞的损伤和功能障碍。5.芪参健脾方联合培哚普利及单方高剂量的应用，明显促进了MMP-1，MMP-2和MMP-9的表达，从而抑制心肌纤维化，发挥降压及保护心肌组织的功能。更多还原

【Abstract】 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.更多还原