【摘要】：目的:通過(guò)觀察健脾祛痰化瘀法對(duì)脾虛痰濁動(dòng)脈粥樣硬化巴馬小豬心肌細(xì)胞線粒體的能量代謝的影響,以氧化應(yīng)激為切入點(diǎn),探討健脾祛痰化瘀法改善脾虛痰濁動(dòng)脈粥樣硬化的巴馬小型豬心肌損傷的作用機(jī)制。材料與方法:健康巴馬小型豬15只,月齡6-8個(gè)月,隨機(jī)分為正常組、模型組、健脾祛痰化瘀組3組,每組5只。造模:正常對(duì)照組予基礎(chǔ)飼料喂飼,模型組、健脾祛痰化瘀組采用高脂高熱量喂養(yǎng),喂養(yǎng)第3周,模型組與健脾祛痰化瘀組予冠狀動(dòng)脈球囊拉傷手術(shù)干預(yù)建立動(dòng)脈粥樣硬化模型,第6周加入跑步機(jī)跑步過(guò)勞,建立脾虛模型[1],共喂養(yǎng)48周。藥物干預(yù):高脂喂飼24周后健脾祛痰化瘀組予健脾祛痰化瘀方藥攪拌于飼料中喂食。第48周末取心肌組織。油紅O染色觀察模型組心肌脂肪形成情況,HE染色觀察巴馬小型豬心肌細(xì)胞的變化,電鏡觀察心肌細(xì)胞線粒體的變化;比色法觀察心肌細(xì)胞線粒體呼吸鏈復(fù)合物Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ和ATP的含量,PCR array的方法觀察各組巴馬小型豬心肌線粒體能量代謝通路相關(guān)基因m RNA表達(dá),Na+-K+-ATP酶和ATP5F1酶的活性觀察心肌線粒體的能量變化;檢測(cè)各組巴馬小型豬的心肌線粒體ROS、CAT、MDA、GSH-PX的含量;檢測(cè)各組巴馬小型豬心肌細(xì)胞Bax、Bcl-2、細(xì)胞色素C、Caspase3的含量。結(jié)果:1健脾祛痰化瘀法對(duì)脾虛痰濁動(dòng)脈粥樣硬化巴馬小型豬心肌形態(tài)的影響1.1油紅O染色后脂肪成紅色,細(xì)胞核成藍(lán)色,間質(zhì)無(wú)色。心肌油紅O顯色表示:正常組心肌細(xì)胞排列有序,無(wú)橘紅色的脂肪細(xì)胞浸潤(rùn)。與正常組比較,模型組心肌細(xì)胞內(nèi)呈現(xiàn)油紅著色,脂滴多。與模型組比較,中藥治療組心肌脂粒明顯減少。1.2在光鏡下,正常組巴馬小型豬心肌細(xì)胞為短圓柱型并有分支,細(xì)胞核位于中央,成橢圓形,心肌細(xì)胞輪廓清晰,胞漿可見(jiàn)肌原纖維平行排列心肌細(xì)胞無(wú)炎性浸潤(rùn)。模型組巴馬小豬心肌細(xì)胞出現(xiàn)炎性浸潤(rùn),心肌纖維腫脹,甚至出現(xiàn)壞死區(qū)。與模型組比較,中藥治療組心肌細(xì)胞炎性浸潤(rùn),心肌纖維腫脹,壞死明顯減少。1.3電鏡下,正常組心肌纖維排列整齊,線粒體數(shù)量和形體正常,可見(jiàn)完整的細(xì)胞核、線粒體。Z線清楚,肌纖維間線粒體較多,肌節(jié)清晰、核仁明顯。線粒體無(wú)融合、腫脹、變形等。模型組心肌纖維排列較紊亂,心肌細(xì)胞核形極不規(guī)則,閏盤(pán)較為紊亂。心肌細(xì)胞結(jié)構(gòu)模糊,肌纖維部分溶解斷裂,Z線清楚,心肌纖維間線粒體部分嵴減少或外膜局部破損,線粒體腫脹內(nèi)質(zhì)網(wǎng)空泡變性。肌纖維部分變性、斷裂,甚至溶解,提示模型制備成功。中藥治療組心肌纖維排列整齊,Z線清楚,肌纖維間線粒體較多,但線粒體部分外膜局部破損,可見(jiàn)心肌閏盤(pán)。2健脾祛痰化瘀法對(duì)脾虛痰濁動(dòng)脈粥樣硬化巴馬小型豬心肌線粒體能量代謝的影響2.1巴馬小型豬心肌線粒體呼吸鏈復(fù)合物Ⅰ活性的檢測(cè):各組巴馬小型豬心肌線粒體呼吸鏈復(fù)合物Ⅰ活性比較,與正常組相比,模型組巴馬小型豬心肌線粒體呼吸鏈復(fù)合物Ⅰ活性下降,但無(wú)統(tǒng)計(jì)學(xué)差異(P0.05),藥物干預(yù)之后與模型組比較,中藥治療組顯著上調(diào),但無(wú)明顯統(tǒng)計(jì)學(xué)差異(P0.05)。2.2巴馬小型豬心肌線粒體呼吸鏈復(fù)合物Ⅱ活性的檢測(cè):各組心肌線粒體呼吸鏈復(fù)合物Ⅱ活性比較,與正常組相比,模型組心肌線粒體呼吸鏈復(fù)合物Ⅱ活性明顯下降(P0.01),藥物干預(yù)之后與模型組比較,中藥治療組顯著上調(diào)(P0.05)。2.3巴馬小型豬心肌線粒體呼吸鏈復(fù)合物Ⅲ活性的檢測(cè):各組心肌線粒體呼吸鏈復(fù)合物Ⅲ活性比較,與正常組相比,模型組心肌線粒體呼吸鏈復(fù)合物Ⅲ活性明顯下降(P0.01),藥物干預(yù)之后與模型組比較,中藥治療組顯著上調(diào)(P0.05)。2.4巴馬小型豬心肌線粒體呼吸鏈復(fù)合物Ⅳ活性的檢測(cè):各組心肌線粒體呼吸鏈復(fù)合物Ⅳ活性比較,與正常組相比,模型組心肌線粒體呼吸鏈復(fù)合物Ⅳ活性明顯下降(P0.01),藥物干預(yù)之后與模型組比較,中藥治療組顯著上調(diào)(P0.05)。2.5巴馬小型豬心肌線粒體呼吸鏈復(fù)合物Ⅴ活性的檢測(cè):各組心肌線粒體呼吸鏈復(fù)合物Ⅴ活性比較,與正常組相比,模型組心肌線粒體呼吸鏈復(fù)合物Ⅴ活性明顯下降(P0.01),藥物干預(yù)之后與模型組比較,中藥治療組顯著上調(diào)(P0.01)。2.6巴馬小型豬心肌線粒體ATP活性的檢測(cè):各組心肌線粒體ATP活性比較,與正常組相比,模型組心肌線粒體ATP活性明顯下降(P0.01),藥物干預(yù)之后與模型組比較,總要治療組顯著上調(diào)(P0.01)。2.7 PCR array法檢測(cè)線粒體能量代謝相關(guān)基因m RNA的表達(dá):與正常組相比,模型組線粒體能量代謝相關(guān)基因m RNA的表達(dá)有生物學(xué)意義的變化的基因上調(diào)≥4倍的基因有9個(gè),分別是:DNAJB1、OXA1L、NDUFA6、NDUFB8、NDUFB9、NDUFS2、NDUFS8、PPA2、NNT,占總基因數(shù)的10%,下調(diào)≥4倍的基因有17個(gè),分別是:ATP5A1、ATP5L、ATP5F1、ATP4A、ATP6V1E2、ATP6V0A2、ATP6V1C2、ATP6V1G3、ATP6V0D2、ATP12A、BCS1L、SDHA、SDHB、LHPP、NDUFS1、SDHD、SLC25A15,占總基因數(shù)的20%。中藥治療后與模型組相比,下調(diào)的≥4倍基因有2個(gè),分別是:NDUFB3、NDUFC2,占總基因數(shù)的2%,上調(diào)的≥4倍基因有7個(gè),分別是:ATP5A1、ATP5F1、ATP6V1E2、ATP5E、ATP6V0A2、SDHC、SDHD占基因綜述的8%。其中、ATP5F1、ATP5E兩個(gè)基因變化的最明顯。2.8巴馬小型豬心肌Na+-K+-ATP酶表達(dá)的檢測(cè):模型組Na+-K+-ATP酶表達(dá)較正常組明顯減少(P0.01),中藥治療組Na+-K+-ATP酶的活性與模型組比較明顯升高(P0.01)。2.9巴馬小型豬心肌ATP5F1蛋白表達(dá)的檢測(cè):模型組ATP5F1蛋白表達(dá)較正常組明顯減少(P0.01),中藥治療組ATP5F1的表達(dá)與模型組比較明顯升高(P0.01)。3健脾祛痰化瘀法通過(guò)影響線粒體氧化應(yīng)激改善脾虛痰濁動(dòng)脈粥樣硬化巴馬小型豬心肌細(xì)胞凋亡。3.1巴馬小型豬心肌ROS含量的檢測(cè):各組心肌ROS含量比較,與正常組相比,模型組心肌線粒體ROS含量明顯上調(diào)(P0.05),藥物干預(yù)之后與模型組比較,中藥治療組顯著下調(diào)(P0.01)。3.2巴馬小型豬心肌MDA含量的檢測(cè):各組心肌MDA含量比較,與正常組相比,模型組心肌MDA含量明顯上升(P0.01),藥物干預(yù)之后與模型組比較,中藥治療組顯著下調(diào)(P0.01)。3.3巴馬小型豬心肌GSH-PX含量的檢測(cè):各組心肌GSH-PX含量比較,與正常組相比,模型組心肌GSH-PX含量明顯下調(diào)(P0.01),藥物干預(yù)之后與模型組比較,中藥治療組顯著上調(diào)(P0.05)。3.4巴馬小型豬心肌CAT含量的檢測(cè):各組心肌CAT含量比較,與正常組相比,模型組心肌CAT含量明顯下調(diào)(P0.01),藥物干預(yù)之后與模型組比較,中藥治療組顯著上調(diào)(P0.01)。3.5巴馬小型豬心肌Bax蛋白表達(dá)的檢測(cè):各組心肌Bax蛋白表達(dá)比較,與正常組相比,模型組心肌Bax含量明顯上調(diào)(P0.01),藥物干預(yù)之后與模型組比較,中藥治療組顯著下調(diào)(P0.05)。3.6巴馬小型豬心肌Bcl-2蛋白表達(dá)的檢測(cè):各組心肌Bcl-2蛋白表達(dá)比較,與正常組相比,模型組心肌Bcl-2明顯下調(diào)(P0.01),藥物干預(yù)之后與模型組比較,中藥治療組顯著上調(diào)Bcl-2含量(P0.01)。3.7巴馬小型豬心肌Cyto C蛋白表達(dá)的檢測(cè):各組心肌Cyto C蛋白表達(dá)比較,與正常組相比,模型組心肌Cyto C蛋白含量明顯上調(diào)(P0.01),藥物干預(yù)之后與模型組比較,中藥治療組顯著下調(diào)(P0.01)。3.8巴馬小型豬心肌Caspase3蛋白表達(dá)的檢測(cè):各組心肌Caspase3含量比較,與正常組相比,模型組心肌Caspase3含量明顯上調(diào)(P0.01),藥物干預(yù)之后與模型組比較,中藥治療組顯著下調(diào)(P0.01)。結(jié)論:1.同課題組的研究已經(jīng)完成了脾虛痰濁動(dòng)脈粥樣硬化巴馬小型豬模型的建立和評(píng)價(jià)。健脾祛痰化瘀法減少了脾虛痰濁動(dòng)脈粥樣硬化所致的巴馬小型豬心肌線粒體的損傷。2.健脾祛痰化瘀法可以增加線粒體電子傳遞鏈上復(fù)合物的活性,增加ATP合成,改善線粒體能量代謝來(lái)減輕脾虛痰濁動(dòng)脈粥樣硬化對(duì)心肌線粒體的損傷從而實(shí)現(xiàn)對(duì)線粒體功能和結(jié)構(gòu)的保護(hù)作用。3健脾祛痰化瘀法可以減少脾虛痰濁動(dòng)脈粥樣硬化巴馬小型豬心肌氧自由基、MDA的生成,增加CAT、GSH-PX的表達(dá),通過(guò)其抗氧化的功能,減少了線粒體的氧化應(yīng)激從而減少巴馬小型豬心肌細(xì)胞Bax、Cyto C,Caspase3表達(dá),增加Bcl-2的表達(dá),減少心肌細(xì)胞損傷。
[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.
【學(xué)位授予單位】：遼寧中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R259

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