参草通脉颗粒干预慢性心衰大鼠klotho蛋白及抗心室重构机制研究

发布时间：2018-05-05 05:22

本文选题：参草通脉颗粒 + klotho　；参考：《辽宁中医药大学》2017年硕士论文

【摘要】：目的:通过建立慢性心衰(CHF)大鼠模型,观察参草通脉颗粒对慢性心衰大鼠心肌及肾组织klotho的影响以及心肌组织结构的改变。围绕参草通脉颗粒对大鼠模型慢性心衰症状的作用机理和作用靶点,分别从基因、蛋白和组织形态学等渠道发现和探索将中医药防治作为治疗慢性心衰重要手段的科学依据。材料与方法:1.通过对实验大鼠采取结扎冠状动脉前降支造成局部心梗,再对大鼠进行游泳及减食等方法造成慢性心衰模型,后观察大鼠状态,且经多普勒超声心动图检测射血分数(EF)≤55%或CI≤180ml/min/kg,判定慢性心衰大鼠模型造模是否成功。2.将建立的慢性心衰大鼠模型分4组培养。首先,设立假手术组模型用于后期对比研究,该组模型不采取任何药物干预,仅用常规等容积蒸馏水灌胃。其次,保留一组心衰大鼠模型为模型组,该组采取同假手术组培养方式。第三,设立西药组采取灌服赖诺普利干预。最后,设立中药组模型,灌服参草通脉颗粒,中药组按照9.2g生药/Kg/d灌服参草通脉颗粒,赖诺普利西药组1.5mg生药/Kg/d。预计培养4周时间,随后经大鼠尾静脉采血后,麻醉处死全部大鼠,取其大鼠心脏心肌组织及肾组织。3.将所取各组样本分别通过ELISA法、蛋白免疫印迹技术和实时荧光定量PCR技术收集相关数据,主要对比不同组模型间BNP浓度,心肌及肾组织klotho的变化情况及电镜观察各组间心肌细胞组织形态学变化情况。结果:1.慢性心衰大鼠BNP浓度与假手术组比较,治疗组(中药组,西药组)及模型组均升高,p0.05。其中假手术组BNP浓度为450±37.03pg/ml,模型组BNP浓度1017.55±98.6pg/ml,中药组与西药组BNP浓度分别为681.08±75.31pg/ml,686.53±74.86pg/ml,p0.05。2.慢性心衰大鼠模型组心肌klotho mRNA表达明显降低,为0.0003±0.00004(与假手术组比较P0.01);西药组样本心肌klotho mRNA具体数值为0.0020±0.0009相较于中药组(具体数值为0.0012±0.0006)p0.05,组间比较治疗组与模型组P0.01与假手术组P0.01。3.慢性心衰大鼠模型组心肌klotho蛋白表达明显下降,为0.0291±0.0025(与假手术组比较P0.01);西药组样本心肌klotho蛋白具体数值为0.2475±0.1407,相较于中药组(具体数值为0.215±0.077)p0.05,组间比较治疗组与模型组P0.01与假手术组P0.01。4.慢性心衰模型组肾组织klotho mRNA表达明显降低,为0.0022±0.0007,(与假手术组比较P0.05);西药组样本肾klotho mRNA具体数值为0.0067±0.00209,相较于中药组(具体数值为0.0063±0.0022)p0.05,组间比较治疗组与模型组P0.01与假手术组P0.01。5.慢性心衰模型组肾组织klotho蛋白表达明显降低,为0.0639±0.0203(与假手术组比较P0.01);西药组样本肾klotho蛋白具体数值为0.2150±0.0303,相较于中药组(数值为0.2057±0.0457)p0.05,组间比较治疗组与模型组P0.01与假手术组P0.01。6.假手术组,可见完整的心肌细胞,肌纤维整齐有序,呈均匀清晰的Z线排列,肌节可见明显的周期性,心肌组织间质无水肿,大量椭圆形线粒体排列整齐于心肌肌纤维中。模型组,肌节结构不清或消失,即肌原纤维断裂、溶解,肌节紊乱,心肌组织间质见明显水肿,坏死区域面积大,坏死严重,线粒体形态不一,见肿胀、破裂或消失,空泡化严重。中药组,肌节较清晰,仍有局部肌纤维断裂、溶解,线粒体排列于肌丝之间,呈轻度水肿,尚有部分线粒体空化。西药组,肌节较清晰,部分肌纤维断裂、溶解,线粒体排列于肌丝之间,轻度肿胀,且空化线粒体较少。结论:1.通过对大鼠模型超声心动图检测,并且辅以对比健康大鼠行为,证明利用结扎冠状动脉前降支配合减食与力竭式游泳可成功造成大鼠心衰模型。2.通过4周参草通脉颗粒的治疗后利用ELISA法检测BNP浓度下降,通过实时荧光定量PCR、Western blot检测大鼠心肌组织,发现参草通脉颗粒对心肌组织klotho mRNA与蛋白有明显升高其表达的作用;并且通过电镜观察心肌组织证明参草通脉颗粒可起到抑制或逆转心室重构的作用。3.通过4周参草通脉颗粒的治疗后利用实时荧光定量PCR、Western blot检测大鼠肾组织,发现参草通脉颗粒对肾组织klothomRNA与蛋白有明显升高其表达的作用;参草通脉颗粒可起到抑制肾损伤的作用。
[Abstract]:Objective: by establishing a rat model of chronic heart failure (CHF), we observed the effect of Shen Cao Tongmai Granule on the Klotho of myocardium and kidney tissue in chronic heart failure rats and the changes of the structure of myocardium. The mechanism and target points of Shen Cao Tong Mai Granule on the symptoms of chronic heart failure in rat model were discussed from gene, protein and histomorphology. We found and explored the scientific basis of traditional Chinese medicine as an important means to treat chronic heart failure. Materials and methods: 1. by ligating the anterior descending branch of the coronary artery to cause local myocardial infarction in experimental rats, and then swimming and reducing food in rats, the model of chronic heart failure was created, and then the state of rats was observed, and the Doppler echocardiography was used to examine the rats. The score of ejection fraction (EF) was less than 55% or CI < 180ml/min/kg. To determine whether the model of chronic heart failure rat model was successful or not, the model of chronic heart failure in.2. would be divided into 4 groups. First, the model of sham operation group was set up for the later contrast study. The model group of heart failure rats was the model group, and the group adopted the same group as the sham operation group. Third, the western medicine group was set up to take the intervention of lisinopril. Finally, the Chinese medicine group was set up and filled with Shen Cao Tongmai granule. The Chinese medicine group was filled with Shen Cao Tong Mai Granule according to the 9.2g raw medicine /Kg/d, and the /Kg/d. of the 1.5mg medicine of lisinopixi drug group was expected to be cultivated for 4 weeks. After the rat tail vein was collected, all rats were killed, and the cardiac muscle tissue and.3. of the rats were taken to collect all the samples by ELISA, protein immunoblotting and real-time fluorescence quantitative PCR. The concentration of BNP and the changes of Klotho in the myocardium and kidney tissue were compared. Results: 1. the concentration of BNP in the chronic heart failure rats was higher than that in the sham group. The treatment group (Chinese medicine group, western medicine group) and the model group were all increased, and the concentration of BNP in the sham operation group was 450 + 37.03pg/ml, the concentration of BNP in the model group was 1017.55 + 98.6pg/ml, and the concentration of BNP in the Chinese medicine group and the western medicine group was respectively the concentration of BNP in the Chinese medicine group and the western medicine group. 681.08 + 75.31pg/ml, 686.53 + 74.86pg/ml, the expression of Klotho mRNA in the rat model group of p0.05.2. chronic heart failure was significantly reduced, 0.0003 + 0.00004 (compared with the sham operation group P0.01), and the specific value of Klotho mRNA in the western medicine group was 0.0020 + 0.0009 compared to the Chinese medicine group (with the body value of 0.0012 + 0.0006) P0.05, the comparison between the group and the model group was compared with the model group. The expression of Klotho protein in the myocardium of the model group P0.01 and the sham group P0.01.3. was 0.0291 + 0.0025 (compared with the sham operation group P0.01), and the specific value of Klotho protein in the western medicine group was 0.2475 + 0.1407, compared with the traditional Chinese medicine group (the specific value was 0.215 + 0.077) P0.05, and the comparative treatment group and the model group P0.01 were compared. The expression of Klotho mRNA in renal tissue of P0.01.4. chronic heart failure model group was significantly lower than that in sham group, which was 0.0022 + 0.0007 (compared with sham operation group P0.05), and the specific value of Klotho mRNA in the sample kidney of Western medicine group was 0.0067 + 0.00209, compared with the traditional Chinese Medicine group (the specific value was 0.0063 + 0.0022) P0.05, and the comparison group was compared with the model group P0.01 and the sham operation group P. In 0.01.5. chronic heart failure model group, the expression of Klotho protein in renal tissue decreased significantly, which was 0.0639 + 0.0203 (compared with sham operation group P0.01). The specific value of Klotho protein in the western medicine group was 0.2150 + 0.0303, compared with the traditional Chinese medicine group (0.2057 + 0.0457) P0.05, and the comparison treatment group and the model group P0.01 and the sham operation group P0.01.6. sham operation group, The complete myocardial cells were seen, the muscle fibers were orderly and orderly, the Z lines were arranged in a uniform and clear line, the myofibrils were obvious periodicity, the interstitial tissue of the myocardium was not edema, and a large number of oval mitochondria were arranged neatly in the muscle fibers. The model group, the myofibril structure was not clear or disappeared, that is, the myofibril rupture, dissolving, the myofibril disorder, and the interstitial tissue of the myocardium. Edema, necrotic area large area, necrosis serious, mitochondria form different, see swelling, rupture or disappearance, vacuolization serious. The Chinese medicine group, the myojoints are clear, there are still local muscle fiber fracture, dissolve, the mitochondria are arranged between the myes, with a mild edema, the western medicine group, the myojoints are clearer, some muscle fibers break and dissolve. The mitochondria were arranged between the muscle fibers, slightly swelling and the cavitation mitochondria were less. Conclusion: 1. through the echocardiographic examination of the rat model and the behavior of the healthy rats, it is proved that the model.2. of the rat heart failure model can be successfully made through the ligature of the anterior descending branch of the coronary artery and the exhaustive swimming of the rats through 4 weeks. After treatment, ELISA method was used to detect the decrease of BNP concentration. The myocardial tissue of rats was detected by real-time fluorescence quantitative PCR and Western blot. The effect of Shen Cao Tong Mai Granule on the expression of Klotho mRNA and protein was found in the myocardium, and the myocardial tissue was observed by electron microscope to prove that the ginseng Tongmai granule could inhibit or reverse the ventricular remodeling. The effect of.3. on renal tissue of rats was detected by real-time fluorescence quantitative PCR and Western blot after 4 weeks of ginseng Tongmai Granules. The effect of Shen Cao Tong Mai Granule on the expression of klothomRNA and protein in renal tissue was found, and the effect of Shen Cao Tongmai Granules could inhibit the renal injury.

【学位授予单位】：辽宁中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

【参考文献】