健脾祛痰化瘀法对脾虚痰浊AS巴马小型猪心肌线粒体作用及机制研究
发布时间：2018-08-14 15:38
【摘要】：目的:通过观察健脾祛痰化瘀法对脾虚痰浊动脉粥样硬化巴马小猪心肌细胞线粒体的能量代谢的影响,以氧化应激为切入点,探讨健脾祛痰化瘀法改善脾虚痰浊动脉粥样硬化的巴马小型猪心肌损伤的作用机制。材料与方法:健康巴马小型猪15只,月龄6-8个月,随机分为正常组、模型组、健脾祛痰化瘀组3组,每组5只。造模:正常对照组予基础饲料喂饲,模型组、健脾祛痰化瘀组采用高脂高热量喂养,喂养第3周,模型组与健脾祛痰化瘀组予冠状动脉球囊拉伤手术干预建立动脉粥样硬化模型,第6周加入跑步机跑步过劳,建立脾虚模型[1],共喂养48周。药物干预:高脂喂饲24周后健脾祛痰化瘀组予健脾祛痰化瘀方药搅拌于饲料中喂食。第48周末取心肌组织。油红O染色观察模型组心肌脂肪形成情况,HE染色观察巴马小型猪心肌细胞的变化,电镜观察心肌细胞线粒体的变化;比色法观察心肌细胞线粒体呼吸链复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ和ATP的含量,PCR array的方法观察各组巴马小型猪心肌线粒体能量代谢通路相关基因m RNA表达,Na+-K+-ATP酶和ATP5F1酶的活性观察心肌线粒体的能量变化;检测各组巴马小型猪的心肌线粒体ROS、CAT、MDA、GSH-PX的含量;检测各组巴马小型猪心肌细胞Bax、Bcl-2、细胞色素C、Caspase3的含量。结果:1健脾祛痰化瘀法对脾虚痰浊动脉粥样硬化巴马小型猪心肌形态的影响1.1油红O染色后脂肪成红色,细胞核成蓝色,间质无色。心肌油红O显色表示:正常组心肌细胞排列有序,无橘红色的脂肪细胞浸润。与正常组比较,模型组心肌细胞内呈现油红着色,脂滴多。与模型组比较,中药治疗组心肌脂粒明显减少。1.2在光镜下,正常组巴马小型猪心肌细胞为短圆柱型并有分支,细胞核位于中央,成椭圆形,心肌细胞轮廓清晰,胞浆可见肌原纤维平行排列心肌细胞无炎性浸润。模型组巴马小猪心肌细胞出现炎性浸润,心肌纤维肿胀,甚至出现坏死区。与模型组比较,中药治疗组心肌细胞炎性浸润,心肌纤维肿胀,坏死明显减少。1.3电镜下,正常组心肌纤维排列整齐,线粒体数量和形体正常,可见完整的细胞核、线粒体。Z线清楚,肌纤维间线粒体较多,肌节清晰、核仁明显。线粒体无融合、肿胀、变形等。模型组心肌纤维排列较紊乱,心肌细胞核形极不规则,闰盘较为紊乱。心肌细胞结构模糊,肌纤维部分溶解断裂,Z线清楚,心肌纤维间线粒体部分嵴减少或外膜局部破损,线粒体肿胀内质网空泡变性。肌纤维部分变性、断裂,甚至溶解,提示模型制备成功。中药治疗组心肌纤维排列整齐,Z线清楚,肌纤维间线粒体较多,但线粒体部分外膜局部破损,可见心肌闰盘。2健脾祛痰化瘀法对脾虚痰浊动脉粥样硬化巴马小型猪心肌线粒体能量代谢的影响2.1巴马小型猪心肌线粒体呼吸链复合物Ⅰ活性的检测:各组巴马小型猪心肌线粒体呼吸链复合物Ⅰ活性比较,与正常组相比,模型组巴马小型猪心肌线粒体呼吸链复合物Ⅰ活性下降,但无统计学差异(P0.05),药物干预之后与模型组比较,中药治疗组显著上调,但无明显统计学差异(P0.05)。2.2巴马小型猪心肌线粒体呼吸链复合物Ⅱ活性的检测:各组心肌线粒体呼吸链复合物Ⅱ活性比较,与正常组相比,模型组心肌线粒体呼吸链复合物Ⅱ活性明显下降(P0.01),药物干预之后与模型组比较,中药治疗组显著上调(P0.05)。2.3巴马小型猪心肌线粒体呼吸链复合物Ⅲ活性的检测:各组心肌线粒体呼吸链复合物Ⅲ活性比较,与正常组相比,模型组心肌线粒体呼吸链复合物Ⅲ活性明显下降(P0.01),药物干预之后与模型组比较,中药治疗组显著上调(P0.05)。2.4巴马小型猪心肌线粒体呼吸链复合物Ⅳ活性的检测:各组心肌线粒体呼吸链复合物Ⅳ活性比较,与正常组相比,模型组心肌线粒体呼吸链复合物Ⅳ活性明显下降(P0.01),药物干预之后与模型组比较,中药治疗组显著上调(P0.05)。2.5巴马小型猪心肌线粒体呼吸链复合物Ⅴ活性的检测:各组心肌线粒体呼吸链复合物Ⅴ活性比较,与正常组相比,模型组心肌线粒体呼吸链复合物Ⅴ活性明显下降(P0.01),药物干预之后与模型组比较,中药治疗组显著上调(P0.01)。2.6巴马小型猪心肌线粒体ATP活性的检测:各组心肌线粒体ATP活性比较,与正常组相比,模型组心肌线粒体ATP活性明显下降(P0.01),药物干预之后与模型组比较,总要治疗组显著上调(P0.01)。2.7 PCR array法检测线粒体能量代谢相关基因m RNA的表达:与正常组相比,模型组线粒体能量代谢相关基因m RNA的表达有生物学意义的变化的基因上调≥4倍的基因有9个,分别是:DNAJB1、OXA1L、NDUFA6、NDUFB8、NDUFB9、NDUFS2、NDUFS8、PPA2、NNT,占总基因数的10%,下调≥4倍的基因有17个,分别是:ATP5A1、ATP5L、ATP5F1、ATP4A、ATP6V1E2、ATP6V0A2、ATP6V1C2、ATP6V1G3、ATP6V0D2、ATP12A、BCS1L、SDHA、SDHB、LHPP、NDUFS1、SDHD、SLC25A15,占总基因数的20%。中药治疗后与模型组相比,下调的≥4倍基因有2个,分别是:NDUFB3、NDUFC2,占总基因数的2%,上调的≥4倍基因有7个,分别是:ATP5A1、ATP5F1、ATP6V1E2、ATP5E、ATP6V0A2、SDHC、SDHD占基因综述的8%。其中、ATP5F1、ATP5E两个基因变化的最明显。2.8巴马小型猪心肌Na+-K+-ATP酶表达的检测:模型组Na+-K+-ATP酶表达较正常组明显减少(P0.01),中药治疗组Na+-K+-ATP酶的活性与模型组比较明显升高(P0.01)。2.9巴马小型猪心肌ATP5F1蛋白表达的检测:模型组ATP5F1蛋白表达较正常组明显减少(P0.01),中药治疗组ATP5F1的表达与模型组比较明显升高(P0.01)。3健脾祛痰化瘀法通过影响线粒体氧化应激改善脾虚痰浊动脉粥样硬化巴马小型猪心肌细胞凋亡。3.1巴马小型猪心肌ROS含量的检测:各组心肌ROS含量比较,与正常组相比,模型组心肌线粒体ROS含量明显上调(P0.05),药物干预之后与模型组比较,中药治疗组显著下调(P0.01)。3.2巴马小型猪心肌MDA含量的检测:各组心肌MDA含量比较,与正常组相比,模型组心肌MDA含量明显上升(P0.01),药物干预之后与模型组比较,中药治疗组显著下调(P0.01)。3.3巴马小型猪心肌GSH-PX含量的检测:各组心肌GSH-PX含量比较,与正常组相比,模型组心肌GSH-PX含量明显下调(P0.01),药物干预之后与模型组比较,中药治疗组显著上调(P0.05)。3.4巴马小型猪心肌CAT含量的检测:各组心肌CAT含量比较,与正常组相比,模型组心肌CAT含量明显下调(P0.01),药物干预之后与模型组比较,中药治疗组显著上调(P0.01)。3.5巴马小型猪心肌Bax蛋白表达的检测:各组心肌Bax蛋白表达比较,与正常组相比,模型组心肌Bax含量明显上调(P0.01),药物干预之后与模型组比较,中药治疗组显著下调(P0.05)。3.6巴马小型猪心肌Bcl-2蛋白表达的检测:各组心肌Bcl-2蛋白表达比较,与正常组相比,模型组心肌Bcl-2明显下调(P0.01),药物干预之后与模型组比较,中药治疗组显著上调Bcl-2含量(P0.01)。3.7巴马小型猪心肌Cyto C蛋白表达的检测:各组心肌Cyto C蛋白表达比较,与正常组相比,模型组心肌Cyto C蛋白含量明显上调(P0.01),药物干预之后与模型组比较,中药治疗组显著下调(P0.01)。3.8巴马小型猪心肌Caspase3蛋白表达的检测:各组心肌Caspase3含量比较,与正常组相比,模型组心肌Caspase3含量明显上调(P0.01),药物干预之后与模型组比较,中药治疗组显著下调(P0.01)。结论:1.同课题组的研究已经完成了脾虚痰浊动脉粥样硬化巴马小型猪模型的建立和评价。健脾祛痰化瘀法减少了脾虚痰浊动脉粥样硬化所致的巴马小型猪心肌线粒体的损伤。2.健脾祛痰化瘀法可以增加线粒体电子传递链上复合物的活性,增加ATP合成,改善线粒体能量代谢来减轻脾虚痰浊动脉粥样硬化对心肌线粒体的损伤从而实现对线粒体功能和结构的保护作用。3健脾祛痰化瘀法可以减少脾虚痰浊动脉粥样硬化巴马小型猪心肌氧自由基、MDA的生成,增加CAT、GSH-PX的表达,通过其抗氧化的功能,减少了线粒体的氧化应激从而减少巴马小型猪心肌细胞Bax、Cyto C,Caspase3表达,增加Bcl-2的表达,减少心肌细胞损伤。
[Abstract]:Objective: To observe the effect of invigorating spleen and expelling phlegm and removing blood stasis on mitochondrial energy metabolism in Bama piglets with atherosclerosis due to spleen deficiency and phlegm turbidity. * 15 pigs, 6-8 months old, were randomly divided into 3 groups: normal group, model group, Jianpi expectorant Huayu group, 5 rats in each group. Model: normal control group was fed with basal diet, model group, spleen strengthening, expectorant and dissipating stasis group were fed with high fat and high calorie for third weeks. The model of atherosclerosis was established by adding treadmill running overwork in the 6th week and feeding spleen deficiency model for 48 weeks.Medication intervention: After 24 weeks of high-fat feeding, the group of invigorating spleen and removing phlegm and removing blood stasis was stirred into the feed and fed.At the end of the 48th week, the myocardial tissue was taken.The formation of myocardial fat in the model group was observed by oil red O staining, and the Bama was observed by HE staining. * the changes of myocardial cells in minipigs, the changes of mitochondria in cardiac muscle cells were observed under electron microscope. The contents of mitochondrial respiratory chain complexes I, II, III, IV, V and ATP were observed by colorimetry. The expression of M RNA, Na+-K+-ATP * and ATP5F1 enzymes in myocardial mitochondrial energy metabolism pathway in Bama miniature pigs were observed by PCR array. Observe the myocardial mitochondrial energy changes; detect the contents of ROS * *, CAT, MDA and GSH-PX in myocardial mitochondria of Bama minipigs in each group; detect the contents of Bax, Bcl-2, cytochrome C and Caspase3 in the myocardial cells of Bama miniature pigs. Results: 1 * the influence of spleen strengthening, expectorant and stasis removing methods on the myocardial morphology of Bama miniature pigs with spleen deficiency, phlegm and turbid, 1.1 oil red O After staining, the fat was red, the nucleus was blue, and the stroma was colorless. Myocardial oil red O staining showed that the myocardial cells in the normal group were arranged orderly without the infiltration of orange-red adipocytes. Compared with the normal group, the myocardial cells in the model group showed oil red staining and more lipid droplets. The normal group of Bama miniature pigs * cardiomyocytes were short cylindrical and branched, the nuclei were located in the center, oval shape, the myocardial cell contour was clear, the myofibrils were arranged in parallel with the cardiomyocytes * without inflammatory infiltration. In the model group, inflammatory cells infiltrated, cardiac muscle fibers swelled and even necrotic areas appeared in the Bama minipig. Under electron microscope, the myocardial fibers in the normal group were arranged orderly, the number and shape of mitochondria were normal, complete nuclei, mitochondria were visible, Z mitochondria were clear, there were more mitochondria between myofibers, sarcomeres were clear, nucleoli were obvious, mitochondria were not fused, swollen, deformed and so on. The myocardial fibers were arranged disorderly, the nuclei of myocardial cells were irregular and intercalated discs were disorderly. The structure of myocardial cells was blurred, the myocardial fibers were partially dissolved and broken, the Z-line was clear, the mitochondrial ridge between myocardial fibers was reduced or the adventitia was damaged, the mitochondria was swollen and endoplasmic reticulum was vacuolated. In the Chinese medicine treatment group, the cardiac muscle fibers were arranged orderly, the Z line was clear, the mitochondria of the muscle fibers were more, but the mitochondria were partially damaged. The effect of the intercalated disc.2 * the method of invigorating the spleen, eliminating phlegm and removing blood stasis * on the energy metabolism of the myocardial mitochondria in Bama miniature pigs with spleen deficiency, phlegm and turbidity was observed. 2.1 the myocardial mitochondrial respiratory chain of Bama miniature pigs was recovered. The activity of compound I *: compared with the normal group, the activity of myocardial mitochondrial respiratory chain complex I * in Bama miniature pigs decreased, but there was no statistical difference (P0.05). P0.05 (.2.2) * detection of myocardial mitochondrial respiratory chain complex II activity in.2.2 Bama miniature pigs: compared with the normal group, the activity of myocardial mitochondrial respiratory chain complex II decreased significantly (P0.01). .05).2.3 * Bama miniature pig myocardial mitochondrial respiratory chain complex III activity: compared with the normal group, the respiratory chain complex III activity of myocardial mitochondria decreased significantly compared with the normal group (P0.01). After intervention, the Chinese medicine treatment group significantly increased (P0.05).2.4 bar compared with the model group. * detection of myocardial mitochondrial respiratory chain complex IV activity in horses miniature pigs: compared with the normal group, the activity of mitochondrial respiratory chain complex IV in each group was significantly lower than that in the normal group (P0.01). After drug intervention, compared with the model group, the Chinese medicine treatment group significantly increased (P0.05).2.5 Bama miniature pig * Detection of myocardial mitochondrial respiratory chain complex V activity: compared with the normal group, the activity of myocardial mitochondrial respiratory chain complex V significantly decreased in all groups (P0.01). Compared with the model group, the myocardial mitochondrial respiratory chain complex V activity in the model group was significantly increased (P0.01).2.6 Bama miniature pig myocardial mitochondria * Detection of mitochondrial ATP activity: Compared with the normal group, the activity of myocardial mitochondrial ATP in the model group decreased significantly (P 0.01). After drug intervention, compared with the model group, the total treatment group significantly increased (P 0.01). 2.7 PCR array method was used to detect the expression of mitochondrial energy metabolism-related gene m RNA: Compared with the normal group, the model group was significantly increased (P 0.01). The expression of M RNA related to mitochondrial energy metabolism in group A was up-regulated by more than four times in 9 genes, which were DNA JB1, OXA1L, NDUFA6, NDUFB8, NDUFB9, NDUFS2, NDUFS8, PPA2, NNT, accounting for 10% of the total gene number, and down-regulated by more than four times in 17 genes: ATP5A1, ATP5L, ATP5F1, ATP4A, ATP6V1E2, ATP6V0A2, ATP6V1C2, ATP6V1C2, ATP6V1C2. ATP6V1G3, ATP6V1G3, ATP6V0D2, ATP6V0D2, ATP12A, ATP12A, BCS1L, SDHA, SDHA, SDHB, LHPP, NDUFS1, SDHD, SDHD, SLC25A15 15, accounted for 20% of the total gene. Compared with the model group, there were 2 down-regulated (>4-fold) genes, respectively: NDUFB3, NDUFC2, 2, 2% of the total gene number of NDUFB3, NDUFC2, 7 up-regulated (>4-fold) genes, respectively: ATP5A1, ATP5F11 F1F1F1F1F1F11 F12, ATP6V1E2, ATP5E 2, ATP5E 5E 5 E, ATP5E In the meantime, it is necessary to study the relationship between the two. Among the 8%., ATP5F1 and ATP5E were the two most significant changes in the expression of Na+-K+-ATP * enzymes in the.2.8 Bama miniature pigs: the expression of Na+-K+-ATP enzymes in the model group was significantly lower than that in the normal group (P0.01), and the activity of Na+-K+-ATP * in the Chinese medicine treatment group was significantly higher than that in the model group (P0.01). The expression of ATP5F1 protein in the model group was significantly lower than that in the normal group (P0.01). The expression of ATP5F1 in the Chinese medicine treatment group was significantly higher than that in the model group (P0.01).3 * * the method of invigorating the spleen, eliminating phlegm and removing blood stasis could improve the myocardial apoptosis of Bama miniature pigs by affecting mitochondrial oxidative stress, and the detection of myocardial ROS content in Bama miniature pigs. Compared with the normal group, the myocardial mitochondrial ROS content in the model group was significantly higher than that in the normal group (P0.05). After drug intervention, compared with the model group, the Chinese medicine treatment group * significantly reduced (P0.01).3.2 MDA myocardial content in Bama minipigs compared with the model group: the myocardial MDA content of each group was significantly higher than that of the normal group, and the MDA content of the model group increased significantly compared with the normal group (P0.) (P0.) ROS 01) after drug intervention, compared with the model group, the Chinese medicine treatment group significantly reduced (P0.01).3.3 * Bama miniature pig myocardial GSH-PX content detection: compared with the normal group, the myocardial GSH-PX content of the model group was significantly lower than that of the normal group (P0.01). After the intervention, compared with the model group, the Chinese medicine treatment group significantly increased (P0.05).3.4 bar (GSH-PX). * the detection of myocardial CAT content in equine miniature pigs: compared with the normal group, the myocardial CAT content in each group was significantly lower than that in the normal group (P0.01). Compared with the model group, the myocardial * CAT content of the model group was significantly lower than that of the model group (CAT). Compared with the model group, the Bax content in the myocardium of the model group was significantly increased (P0.01). After intervention * compared with the model group, the Chinese medicine treatment group significantly reduced (P0.05).3.6 Bcl-2 protein expression in Bama minipigs: compared with the normal group, the expression of Bcl-2 protein in the myocardium of the two groups was significantly lower than that in the normal group (P0.01). Compared with the traditional Chinese medicine treatment group, the Bcl-2 content (P0.01) was significantly increased * the expression of Cyto C protein was detected in.3.7 Bama miniature pigs. Compared with the normal group, the expression of Cyto C protein in the model group was significantly higher than that in the normal group (P0.01). * the detection of Caspase3 protein expression in porcine myocardium: compared with the normal group, the content of Caspase3 in the myocardium of each group was significantly higher than that in the normal group (P0.01). After intervention, the Chinese medicine treatment group was significantly lower than that in the model group (P0.01). Conclusion: 1. the study of the same subject group has completed the spleen deficiency, phlegm turbidity, atherosclerosis, Bama minor, and Caspase3. * the establishment and evaluation of the pig model. Jianpi expectorant * Huayu method reduced the myocardial mitochondrial injury of Bama miniature pigs caused by spleen deficiency, phlegm and turbid atherosclerosis..2., invigorating the spleen, eliminating phlegm and removing blood stasis can increase the activity of complexes on the electron transport chain of mitochondria, increase ATP synthesis, and improve mitochondrial energy metabolism to relieve atherosclerosis of the spleen asthenia, phlegm and turbid arteries. The method of invigorating the spleen and expelling phlegm and removing blood stasis can reduce the production of oxygen free radicals and MDA, increase the expression of CAT and GSH-PX in the myocardium of Bama miniature pigs with atherosclerosis due to spleen deficiency and phlegm turbidity, and reduce the oxidative stress of the mitochondria through its antioxidant function. * the expression of Bax, Cyto C and Caspase3 in myocardial cells of Ma mini pigs increased Bcl-2 expression and reduced myocardial cell injury.
【学位授予单位】：辽宁中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R259
本文编号：2183335
[Abstract]:Objective: To observe the effect of invigorating spleen and expelling phlegm and removing blood stasis on mitochondrial energy metabolism in Bama piglets with atherosclerosis due to spleen deficiency and phlegm turbidity. * 15 pigs, 6-8 months old, were randomly divided into 3 groups: normal group, model group, Jianpi expectorant Huayu group, 5 rats in each group. Model: normal control group was fed with basal diet, model group, spleen strengthening, expectorant and dissipating stasis group were fed with high fat and high calorie for third weeks. The model of atherosclerosis was established by adding treadmill running overwork in the 6th week and feeding spleen deficiency model for 48 weeks.Medication intervention: After 24 weeks of high-fat feeding, the group of invigorating spleen and removing phlegm and removing blood stasis was stirred into the feed and fed.At the end of the 48th week, the myocardial tissue was taken.The formation of myocardial fat in the model group was observed by oil red O staining, and the Bama was observed by HE staining. * the changes of myocardial cells in minipigs, the changes of mitochondria in cardiac muscle cells were observed under electron microscope. The contents of mitochondrial respiratory chain complexes I, II, III, IV, V and ATP were observed by colorimetry. The expression of M RNA, Na+-K+-ATP * and ATP5F1 enzymes in myocardial mitochondrial energy metabolism pathway in Bama miniature pigs were observed by PCR array. Observe the myocardial mitochondrial energy changes; detect the contents of ROS * *, CAT, MDA and GSH-PX in myocardial mitochondria of Bama minipigs in each group; detect the contents of Bax, Bcl-2, cytochrome C and Caspase3 in the myocardial cells of Bama miniature pigs. Results: 1 * the influence of spleen strengthening, expectorant and stasis removing methods on the myocardial morphology of Bama miniature pigs with spleen deficiency, phlegm and turbid, 1.1 oil red O After staining, the fat was red, the nucleus was blue, and the stroma was colorless. Myocardial oil red O staining showed that the myocardial cells in the normal group were arranged orderly without the infiltration of orange-red adipocytes. Compared with the normal group, the myocardial cells in the model group showed oil red staining and more lipid droplets. The normal group of Bama miniature pigs * cardiomyocytes were short cylindrical and branched, the nuclei were located in the center, oval shape, the myocardial cell contour was clear, the myofibrils were arranged in parallel with the cardiomyocytes * without inflammatory infiltration. In the model group, inflammatory cells infiltrated, cardiac muscle fibers swelled and even necrotic areas appeared in the Bama minipig. Under electron microscope, the myocardial fibers in the normal group were arranged orderly, the number and shape of mitochondria were normal, complete nuclei, mitochondria were visible, Z mitochondria were clear, there were more mitochondria between myofibers, sarcomeres were clear, nucleoli were obvious, mitochondria were not fused, swollen, deformed and so on. The myocardial fibers were arranged disorderly, the nuclei of myocardial cells were irregular and intercalated discs were disorderly. The structure of myocardial cells was blurred, the myocardial fibers were partially dissolved and broken, the Z-line was clear, the mitochondrial ridge between myocardial fibers was reduced or the adventitia was damaged, the mitochondria was swollen and endoplasmic reticulum was vacuolated. In the Chinese medicine treatment group, the cardiac muscle fibers were arranged orderly, the Z line was clear, the mitochondria of the muscle fibers were more, but the mitochondria were partially damaged. The effect of the intercalated disc.2 * the method of invigorating the spleen, eliminating phlegm and removing blood stasis * on the energy metabolism of the myocardial mitochondria in Bama miniature pigs with spleen deficiency, phlegm and turbidity was observed. 2.1 the myocardial mitochondrial respiratory chain of Bama miniature pigs was recovered. The activity of compound I *: compared with the normal group, the activity of myocardial mitochondrial respiratory chain complex I * in Bama miniature pigs decreased, but there was no statistical difference (P0.05). P0.05 (.2.2) * detection of myocardial mitochondrial respiratory chain complex II activity in.2.2 Bama miniature pigs: compared with the normal group, the activity of myocardial mitochondrial respiratory chain complex II decreased significantly (P0.01). .05).2.3 * Bama miniature pig myocardial mitochondrial respiratory chain complex III activity: compared with the normal group, the respiratory chain complex III activity of myocardial mitochondria decreased significantly compared with the normal group (P0.01). After intervention, the Chinese medicine treatment group significantly increased (P0.05).2.4 bar compared with the model group. * detection of myocardial mitochondrial respiratory chain complex IV activity in horses miniature pigs: compared with the normal group, the activity of mitochondrial respiratory chain complex IV in each group was significantly lower than that in the normal group (P0.01). After drug intervention, compared with the model group, the Chinese medicine treatment group significantly increased (P0.05).2.5 Bama miniature pig * Detection of myocardial mitochondrial respiratory chain complex V activity: compared with the normal group, the activity of myocardial mitochondrial respiratory chain complex V significantly decreased in all groups (P0.01). Compared with the model group, the myocardial mitochondrial respiratory chain complex V activity in the model group was significantly increased (P0.01).2.6 Bama miniature pig myocardial mitochondria * Detection of mitochondrial ATP activity: Compared with the normal group, the activity of myocardial mitochondrial ATP in the model group decreased significantly (P 0.01). After drug intervention, compared with the model group, the total treatment group significantly increased (P 0.01). 2.7 PCR array method was used to detect the expression of mitochondrial energy metabolism-related gene m RNA: Compared with the normal group, the model group was significantly increased (P 0.01). The expression of M RNA related to mitochondrial energy metabolism in group A was up-regulated by more than four times in 9 genes, which were DNA JB1, OXA1L, NDUFA6, NDUFB8, NDUFB9, NDUFS2, NDUFS8, PPA2, NNT, accounting for 10% of the total gene number, and down-regulated by more than four times in 17 genes: ATP5A1, ATP5L, ATP5F1, ATP4A, ATP6V1E2, ATP6V0A2, ATP6V1C2, ATP6V1C2, ATP6V1C2. ATP6V1G3, ATP6V1G3, ATP6V0D2, ATP6V0D2, ATP12A, ATP12A, BCS1L, SDHA, SDHA, SDHB, LHPP, NDUFS1, SDHD, SDHD, SLC25A15 15, accounted for 20% of the total gene. Compared with the model group, there were 2 down-regulated (>4-fold) genes, respectively: NDUFB3, NDUFC2, 2, 2% of the total gene number of NDUFB3, NDUFC2, 7 up-regulated (>4-fold) genes, respectively: ATP5A1, ATP5F11 F1F1F1F1F1F11 F12, ATP6V1E2, ATP5E 2, ATP5E 5E 5 E, ATP5E In the meantime, it is necessary to study the relationship between the two. Among the 8%., ATP5F1 and ATP5E were the two most significant changes in the expression of Na+-K+-ATP * enzymes in the.2.8 Bama miniature pigs: the expression of Na+-K+-ATP enzymes in the model group was significantly lower than that in the normal group (P0.01), and the activity of Na+-K+-ATP * in the Chinese medicine treatment group was significantly higher than that in the model group (P0.01). The expression of ATP5F1 protein in the model group was significantly lower than that in the normal group (P0.01). The expression of ATP5F1 in the Chinese medicine treatment group was significantly higher than that in the model group (P0.01).3 * * the method of invigorating the spleen, eliminating phlegm and removing blood stasis could improve the myocardial apoptosis of Bama miniature pigs by affecting mitochondrial oxidative stress, and the detection of myocardial ROS content in Bama miniature pigs. Compared with the normal group, the myocardial mitochondrial ROS content in the model group was significantly higher than that in the normal group (P0.05). After drug intervention, compared with the model group, the Chinese medicine treatment group * significantly reduced (P0.01).3.2 MDA myocardial content in Bama minipigs compared with the model group: the myocardial MDA content of each group was significantly higher than that of the normal group, and the MDA content of the model group increased significantly compared with the normal group (P0.) (P0.) ROS 01) after drug intervention, compared with the model group, the Chinese medicine treatment group significantly reduced (P0.01).3.3 * Bama miniature pig myocardial GSH-PX content detection: compared with the normal group, the myocardial GSH-PX content of the model group was significantly lower than that of the normal group (P0.01). After the intervention, compared with the model group, the Chinese medicine treatment group significantly increased (P0.05).3.4 bar (GSH-PX). * the detection of myocardial CAT content in equine miniature pigs: compared with the normal group, the myocardial CAT content in each group was significantly lower than that in the normal group (P0.01). Compared with the model group, the myocardial * CAT content of the model group was significantly lower than that of the model group (CAT). Compared with the model group, the Bax content in the myocardium of the model group was significantly increased (P0.01). After intervention * compared with the model group, the Chinese medicine treatment group significantly reduced (P0.05).3.6 Bcl-2 protein expression in Bama minipigs: compared with the normal group, the expression of Bcl-2 protein in the myocardium of the two groups was significantly lower than that in the normal group (P0.01). Compared with the traditional Chinese medicine treatment group, the Bcl-2 content (P0.01) was significantly increased * the expression of Cyto C protein was detected in.3.7 Bama miniature pigs. Compared with the normal group, the expression of Cyto C protein in the model group was significantly higher than that in the normal group (P0.01). * the detection of Caspase3 protein expression in porcine myocardium: compared with the normal group, the content of Caspase3 in the myocardium of each group was significantly higher than that in the normal group (P0.01). After intervention, the Chinese medicine treatment group was significantly lower than that in the model group (P0.01). Conclusion: 1. the study of the same subject group has completed the spleen deficiency, phlegm turbidity, atherosclerosis, Bama minor, and Caspase3. * the establishment and evaluation of the pig model. Jianpi expectorant * Huayu method reduced the myocardial mitochondrial injury of Bama miniature pigs caused by spleen deficiency, phlegm and turbid atherosclerosis..2., invigorating the spleen, eliminating phlegm and removing blood stasis can increase the activity of complexes on the electron transport chain of mitochondria, increase ATP synthesis, and improve mitochondrial energy metabolism to relieve atherosclerosis of the spleen asthenia, phlegm and turbid arteries. The method of invigorating the spleen and expelling phlegm and removing blood stasis can reduce the production of oxygen free radicals and MDA, increase the expression of CAT and GSH-PX in the myocardium of Bama miniature pigs with atherosclerosis due to spleen deficiency and phlegm turbidity, and reduce the oxidative stress of the mitochondria through its antioxidant function. * the expression of Bax, Cyto C and Caspase3 in myocardial cells of Ma mini pigs increased Bcl-2 expression and reduced myocardial cell injury.
【学位授予单位】：辽宁中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R259
【参考文献】
相关期刊论文 前9条
1 杜莹;贾连群;杨关林;袁秀敏;王英;宋囡;何文智;王俊岩;;健脾降脂中药对脾失健运膏脂转输障碍大鼠肝脏胆固醇代谢相关基因表达的影响[J];辽宁中医杂志;2014年08期
2 袁东超;李艳娟;张哲;张会永;肖蕾;齐文诚;吴瑾;孔德昭;杨关林;;基于logistic分析的心脑合病痰瘀互结证诊断探索[J];中国卫生统计;2014年02期
3 卢文彪;李丕彦;楚英兰;;健身舞蹈对中老年女性血脂、氧自由基及抗氧化酶的影响[J];中国老年学杂志;2012年14期
4 王志强;李炳超;;半夏药理作用研究进展[J];山西医药杂志(下半月刊);2009年01期
5 马中建;刘红丽;;健脾调脂饮治疗高脂血症的临床观察[J];中医药导报;2008年05期
6 龙全江,徐雪琴,胡昀;白术的化学、药理与炮制研究进展[J];中国中医药信息杂志;2004年11期
7 杨利;邓铁涛教授“冠心三论”[J];湖南中医药导报;2004年06期
8 林来胜;中医对高脂血症的认识及治疗探讨[J];陕西中医;2001年02期
9 郑雪君;中药降脂机理及祛脂途径初探[J];江西中医药;1996年03期
，本文编号：2183335
本文链接：https://www.wllwen.com/zhongyixuelunwen/2183335.html
最近更新
教材专著
