| Purpose: To observe the effects of energy metabolism of jianpi qutan huayu recipe on mitochondria of the myocardial in pixu tanzhuo atherosclerosis bama mini-pigs, explore the improve mechanism of the jianpi qutan huayu recipe of the injury myocardial in pixu tanzhuo atherosclerosis Bama mini-pigs, point to the oxidative stress.Methods: 15 health Bama mini-pigs, 6-8 months of age, randomly distributed all the bama mini-pigs into normal group, model group, jianpi qutan huayu 3 groups,5 pigs each group. Modeling: the normal control group were fed the basal diet for 48 weeks, the other groups were fed high fat and high calorie fodder for 48 weeks, in the third week the model group, jianpi qutan huyu groups received surgical intervention of coronary artery, establish atherosclerosis models. In the 6th week jianpi qutan huayu group was adding treadmill running intervention, establish pixu models.Stirring the Chinese medicine in the feed fed after the 24 th week in jianpi qutan huayu group. At the end of 48 th week collect the myocardial tissue of the pigs. Stain the Myocardial tissue by oil red O, observe pathological changes in myocardial mitochondria of cardiac cells by electron microscopy; To observe respiratory chain complexes I, II, III, IV, V and ATP content of myocardial mitochondrial and observe the expression which was detected by PCR array mitochondrial energy metabolism related gene m RNA in each group, the activity of Na+-K+-ATPase and ATP5F1 enzyme. To observe the content of ROS、CAT、MDA、GSH-Px of myocardial mitochondrial in the mini-pigs. To observe the content change of Bcl-2、 Bax、Cytochrome c、Caspase3 of myocardial mitochondrial in mini-pigs.Result: Dissertation 1 Effect of jianpi qutan huayu recipe on myocardial morphology in pixu tanzhuo atherosclerosis Bama mimi-pig 1.1 After the stain of oil red fad cells turn into red, the nucleus are blue, interstitial colorless.The Oil Red O of myocardial chromogenic showed: myocardial cells of the normal group were arranged in order, no orange fat cell infiltration, compared with the normal group, model group presented myocardial cells oil red coloring, more lipid droplets. Compared with the modle group, the myocardial lipid particle was significantly reduced in chinses medicine reatment group. 1.2 In the light microscope, the normal group Bama mini pig cardiomyocytes short cylindrical and has a branch, located in the central nucleus, into an oval, a clear outline of myocardial cells, cytoplasm of the muscle fibers are arranged in parallel cardiomyocytes no inflammatory infiltration. Bama pig model group myocardial cells showed inflammatory infiltration, myocardial fiber swelling, and even necrosis. ompared with the modle group, myocardial cells and mitochondria was reduced in chinses medicine reatment group. 1.3 The electron microscope showed: Normal cardiac muscle fibers arranged in neat rows, the number of mitochondria and normal physical, visible intact nuclei, mitochondria. Z clear line between the muscle fibers more mitochondria, sarcomere clear, prominent nucleoli. No mitochondrial fusion, swelling and deformation.model group: myocardial fibers arranged in a very irregular shaped nuclei myocardium, intercalated disk is more disorder. The structure of myocardial cells are fuzzy, partial dissolution of muscle fiber rupture, Z line is clear, myocardial fibers decreased mitochondrial outer membrane part ridge or partial damage, swelling of endoplasmic reticulum mitochondria degeneration. Partially denatured muscle fibers, fracture, or even dissolve, in Figure, Pormoted model was successful. Chinese medicine treatment of myocardial fibers arranged in neat rows, Z line was clear between the muscle fibers more mitochondria, the outer membrane of the mitochondrial was partially local damage, visible myocardial intercalated disc. Dissertation 2 The effects of energy metabolism of jianpi qutan huayu recipe on mitochondria of the myocardial in pixu tanzhuo atherosclerosis bama mini-pigs, 2.1 Comparison of the complexⅠactivity in mini-pigs’ myocardial mitochondrial in each group:compared with the normal control group,the activity of complexⅠin model group was not significantly increased(P>0.05).Compared with the modle group,the complexⅠin Chinese medicine treatment group was not significantly lower than the modle group(P>0.05); 2.2 Comparison of the complex Ⅱ activity in mini-pigs’ myocardial mitochondrial in each group:compared with the normal control group,the activity of complex Ⅱ in model group was was significantly lower than the normal group(P<0.01),compared with the modle group,the complexⅡin Chinese medicine treatment group was significantly increased than the modle group(P<0.05); 2.3 Comparison of the complex Ⅲ activity in mini-pigs’ myocardial mitochondrial in each group:compared with the normal control group,the activity of complex Ⅲ in model group was was significantly lower than the normal group(P<0.01),compared with the modle group,the complexⅢin Chinese medicine treatment group was significantly increased than the modle group(P<0.05); 2.4 Comparison of the complex Ⅳ activity in mini-pigs’ myocardial mitochondrial in each group:compared with the normal control group,the activity of complex Ⅳ in model group was was significantly lower than the normal group(P<0.01),compared with the modle group,the complex Ⅳ in Chinese medicine treatment group was significantly increased than the modle group(P<0.05); 2.5 Comparison of the complex Ⅴ activity in mini-pigs’ myocardial mitochondrial in each group:compared with the normal control group,the activity of complex Ⅴ in model group was was significantly lower than the normal group(P<0.01),compared with the modle group,the complex Ⅴ in Chinese medicine treatment group was significantly increased than the modle group(P<0.01); 2.6 Comparison of mini-pigs complex ATPase activity in each group:compared with the normal control group,the model group ATPase activity was significantly lower than the normal group(P<0.01),compared with the modle group,the ATPase activity of tne Chinese medicin treatment group was significantly increased than the modle group(P<0.01); 2.7 The expression was detected by PCR array mitochondrial energy metabolism related gene m RNA: Compared with normal group, There were 9 significant biologically mitochondrial energy metabolism related genes upregulated expression in model group.They are DNAJB1、OXA1L、NDUFA6、NDUFB8、NDUFB9、NDUFS2、NDUFS8、PPA2、NNT,10% of the total number of genes,17 down-regulated genes,they are:ATP5A1、ATP5L、ATP5F1、ATP4A、ATP6V1E2、ATP6V0A2、ATP6V1C2、ATP6V1G3、ATP6V0D2、ATP12A、BCS1L、SDHA、SDHB、LHPP、NDUFS1、SDHD、SLC25A15,20% of the total number of genes. Traditional Chinese medicine treatment group compared with the model, there are two up-regulated genes,they are:NDUFB3、 NDUFC2,2% of the total number of genes. 7 down-regulated genes,they are:ATP5A1、ATP5F1、ATP6V1E2、ATP5E、ATP6V0A2、SDHC、SDHD 8% of the total number of genes.ATP5F1、ATP5E change obviously。 2.8 Comparison of mini-pigs complex Na+-K+-ATPase activity in each group:compared with the normal control group,the activity of Na+-K+-ATPase in model group was significantly lower than the normal group(P <0.01),compared with the modle group,the activity of Na+-K+-ATPase in Chinese medicine treatment group was significantly increased than the modle group(P <0.01); 2.9 Comparison of mini-pigs ATP5F1 protein in each group:compared with the normal control group,the express of ATP5F1 protein in model group was significantly lower than the normal group(P <0.01),compared with the modle group,the express of ATP5F1 protein in Chinese medicine treatment group was significantly increased than the modle group(P <0.01); Dissertation 3 The effect on the apoptosis of myocardial in pixu tanzhuo atherosclerosis bama mini-pigs, by affecting mitochondrial oxidative stress. 3.1. Comparison of mini-pigs ROS content in each group:compared with the normal control group,the content of ROS in model group was significantly increased than the normal group(P <0.05),compared with the modle group,the content of ROS in Chinese medicine treatment group was significantly lower than the modle group(P <0.01); 3.2.Comparison of mini-pigs MDA content in each group:compared with the normal control group,the content of MDA in model group was significantly increased than the normal group(P <0.01),compared with the modle group,the content of MDA in Chinese medicine treatment group was significantly lower than the modle group(P <0.01); 3.3 Comparison of mini-pigs GSH-PX content in each group:compared with the normal control group,the content of GSH-PX in model group was significantly lower than the normalgroup(P <0.01),compared with the modle group,the content of GSH-PX in Chinese medicine treatment group was significantly increased than the modle group(P <0.05); 3.4 Comparison of mini-pigs CAT content in each group:compared with the normal control group,the content of CAT in model group was significantly lower than the normal group(P <0.01),compared with the modle group,the content of CAT in Chinese medicine treatment group was significantly increased than the modle group(P <0.01); 3.5 Comparison of mini-pigs Bax protein expression in each group:compared with the normal control group,the express of Bax protein in model group was significantly increased than the normal group(P <0.01),compared with the modle group,the express of Bax protein in Chinese medicine treatment group was significantly lower than the modle group(P <0.05); 3.6 Comparison of mini-pigs Bcl-2 protein expression in each group:compared with the normal control group,the express of Bcl-2 protein in model group was significantly lower than the normal group(P <0.01),compared with the modle group,the express of Bcl-2 protein in Chinese medicine treatment group was significantly increased than the modle group(P <0.01); 3.7 Comparison of mini-pigs Cyto C protein expression in each group:compared with the normal control group,the express of Cyto C protein in model group was significantly increased than the normal group(P <0.01),compared with the modle group,the express of Cyto C protein in Chinese medicine treatment group was significantly lower than the modle group(P <0.01); 3.8 Comparison of mini-pigs Caspase 3 protein expression in each group:compared with the normal control group,the express of Caspase 3 protein in model group was significantly increased than the normal group(P <0.01),compared with the modle group,the express of Caspase 3 protein in Chinese medicine treatment group was significantly lower than the modle group(P <0.01);Conclusion: 1. The same research group has completed the preparation and evaluation the model of pixu tanzhuo bama mini-pig. The recipe of jianpi qutan huyu can reduce the damage of the heart mitochondrial in bama mini-pigs.2. The recipe of jianpi qutan huyu can enhance the activity of the complexes, in mitochondrial electron transport chain, reduce the damage of mitochondrial in myocardial, the DNA of mitochondrial in order to protect the function and structure of the mitochondrial. 3 The recipe of jianpi qutan huyu can reduce the oxygen free radicals of the myocardial, MDA content and increase CAT, GSH-PX expression, reduce the oxidative stress of the mitochondrial, reduce the Bax, Cyto C, Caspase3 expression, increase the expression of Bcl-2 reduce the damage of the myocardial cells in pixu tanzhuo Bama mini pigs. |
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