| Coronary Microvascular Dysfunction(CMD)is the common cause of microvascular angina pectoris,PCI postoperative no-reflow phenomenon and hypertrophic cardiomyopathy and other heart diseases.CMD became one of the hot issues of heart vascular disease prevention research.There were inflammatory cells infiltration and increased proinflammatory factor in the process of CMD,it reducing blood vessel function and coronary blood flow reserve,ultimately affecting myocardial systolic function.And Macrophage phenotype M1/M2 polarization played an important role in this process,meanwhile miR-155 played an important role in the M1 / M2 polarization.But there was no Chinese medicine study of macrophage polarization in CMD.Bushenhuoxue recipe was not only good for the treatment of postmenopausal CMD,but also for the prevention and treatment of coronary microvascular endothelial cells morphology and function damaging of ovariectomized rats,and for the antagonism of endothelial cell oxidative stress injury and inflammation.Meanwhile this kind of anti-inflammatory effects that was the most obvious in traditional Chinese medicine recipe concentration of aqueous solution containing crude drugs for 3 g/m L was associated to block the NF-kappa Bp50(I kappa B alpha)-IL-1 beta signaling pathways.Because of IL-1 beta is a mark inflammatory factor of M1 macrophage,so that number of macrophage and(or)different phenotype M1 / M2 might be associated with CMD.Therefore,this study proposed to observe coronary microvascular density and the expression of macrophage phenotype M1 / M2 marks inflammatory factors by creating ovariectomized mice CMD model,exploring the effects of Bushenhuoxue recipe on myocardial macrophage phenotype M1 / M2 polarization,for providing reliable experimental basis about the treatment of postmenopausal women CMD.First Part To establish ovariectomized Coronary Microvascular Dysfunction mice modelObjective: To establish ovariectomized coronary microvascular dysfunction mice model by observing the myocardial tissue structure,coronary microvascular density and the myocardial systolic function.Motheds: 1)Eight weeks old sexual maturity C57 BL / 6 female mice were divided into 6W control group,6W sham group,6W model group,8W control group,8W sham group,8W model group.After ovariectomized,6W and 8W model group were conventionally breeded for 6 weeks and 8 weeks.6W sham group and 8W sham group were only removed a little fat around the ovarian tissue,and then conventionally breeded for 6 weeks and 8 weeks.6W control group and 8W control group didn't make any processing and were conventionally breeded for 6 weeks and 8 weeks.2)Weighing each group of female mice body quality once four days,comparing the change of body quality.3)Using small animal ultrasound imaging system collecting echocardiography to analysis and compare each group of female mice myocardial LVFS,LVEF,evaluating the myocardial systolic function.4)Using HE staining to observe the structure of myocardial tissue.5)Using fluorescent staining to test each group of female mice cardiomyocyte cross-sectional area and coronary microvascular density.6)Using real-time quantitative PCR technique to detect the expression of vascular endothelial growth factor(VEGF)of each group.Results: 1)Ovariectomized 6 W,compared with normal group,control group showed no statistical difference(P > 0.05);compared with control group,model group mass increased,and the difference was statistically significant(P < 0.05).But ovariectomized 8 W,compared with normal group,control group showed no statistical difference(P > 0.05);compared with control group,model group mass increased,and the difference was statistically significant(P < 0.05).2)Ovariectomized 6 W,the myocardial LVEF,LVFS between normal group,control group and model group were no significant difference(P > 0.05);ovariectomized 8 W,compared with normal group,myocardial LVEF,LVFS of control group was no significant difference(P > 0.05);compared with control group,myocardial LVEF,LVFS of model group were decreased(P < 0.05).3)Ovariectomized 6 W,three groups of female mice myocardial are abundant cytoplasm,complete structure,neat cells;ovariectomized 8 W,the normal group and the control groups of female mice myocardial tissue structure is complete,neat rows,abundant cytoplasm;and model groups of female mice cardiomyocyte was swelling,horizontal stripes blur,myocardial tissue degeneration,inflammatory cells infiltration,myocardial interstitial hyperemia.4)Ovariectomized 6 W,there were no obvious change between normal group,control group and model group(P > 0.05);ovariectomized 8 W,compared with normal group,control group showed no obvious change(P > 0.05);compared with the control group,model group myocardial cross-sectional area significantly increased(P < 0.05)5)Ovariectomized 6 W,there were no obvious change between normal group,control group and model group(P > 0.05);ovariectomized 8 W,compared with normal group,control group was no significant difference(P > 0.05);compared with the control group,coronary microvascular density of model group was decreased significantly(P < 0.05).6)Ovariectomized 6 W,there were no obvious change of the expression of VEGF m RNA between normal group,control group and model group(P > 0.05);ovariectomized 8 W,compared with normal group,the expression of VEGF m RNA of control group was no obvious difference(P > 0.05),compared with control group,model group VEGF m RNA expression levels of myocardium was decreased(P < 0.05).Conclusion: Ovariectomized 8W,the female mice myocardial appeared damage and degeneration,decreased coronary microvascular density,disordered arrangement of myocardial cells,inflammatory cells infiltration,myocardial hypertrophy,and myocardial systolic function reduced.This suggested ovariectomized 8W,CMD model was successfully established.Second Part The role of macrophage phenotype M1 / M2 in ovariectomized CMD mice modelObjective: To observe the relationship between the number of macrophage or its phenotype M1 / M2 polarization and myocardial injury,coronary microvascular density in ovariectomized CMD mice model.Motheds: 1)Eight weeks old sexual maturity C57 BL / 6 female mice were divided into model group and macrophages clear group.With reference to the first part of experiment,ovariectomized CMD mice model was established.Macrophages clear group were intraperitoneally injected Clophosome,10 ul/g,once a week,from ovarian 8W,for another 8 W.Model group were intraperitoneally injected the same volume of physiological saline for 8 weeks.2)Weighing each group of female mice body quality once four days,comparing the change of body quality.3)Using small animal ultrasound imaging system collecting echocardiography to analysis and compare each group of female mice myocardial LVFS,LVEF,evaluating the myocardial systolic function.4)Using HE staining to observe the structure of myocardial tissue.5)Using fluorescent staining to test the expression of CD31,CD68 in myocardial tissue,evaluating coronary microvascular density and myocardial macrophage infiltration degree.6)Using real-time quantitative PCR technique to detect the expression of IL-1?,TNF-?,IL-10,VEGF m RNA of myocardial tissue.Results: 1)Compared with model group,the body quality of clear macrophage group was not statistically significant(P > 0.05).2)Compared with model group,the myocardial LVEF,LVFS of clear macrophage group was not statistically significant(P > 0.05).3)Model groups cardiomyocyte was swelling,horizontal stripes blur,myocardial tissue degeneration,inflammatory cells infiltration,clear macrophage group myocardial was also swelling,organizational structure,myocardial cells,nucleus pycnosis,but infiltration of inflammatory cells was decreased.4)Compared with model group,the expression of CD68 positive number in myocardial was decreased significantly(P < 0.05);coronary microvascular density of clear macrophage group was no statistical difference(P > 0.05).5)Compared with model group,the expression of IL-1 beta,TNF-alpha m RNA of clear macrophage group was decreased obviously(P < 0.05);the expression of IL-10 and VEGF m RNA levels of clear macrophage group was no significant difference(P > 0.05).Conclusion: CMD to clearing macrophage decreased proinflammatory factor of M1 in clear macrophage group,but failed to improve the model coronary microvascular lesion.The relationship between coronary microvascular dysfunction and the proinflammatory role of M1 macrophage was closely in ovariectomized CMD mice model.The increased of marked inflammatory cytokines of M1 macrophage and decreased of marked inflammatory cytokines of M2 macrophage is the main mechanism of inflammatory response.Third Part The effect of Bushenhuoxue recipe on macrophage M1 / M2 polarization of myocardium in ovariectomized CMD mice modelObjective: To explore the effects of Bushenhuoxue recipe on macrophage M1/M2 polarization of inflammatory response in coronary microvascular.Motheds: 1)Eight weeks old sexual maturity C57 BL / 6 female mice were divided into model group and Bushenhuoxue group.With reference to the first part of experiment,ovariectomized CMD mice model was established.Bushenhuoxue group gavaged granules solution 5 m L/kg/d,1/d from ovarian 8W,for another 8 W.Model group were gacaged the same volume of physiological saline.2)Weighing each group of female mice body quality once four days,comparing the change of body quality.3)Using small animal ultrasound imaging system collecting echocardiography to analysis and compare each group of female mice myocardial LVFS,LVEF,evaluating the myocardial systolic function.4)Using HE staining to observe the structure of myocardial tissue.5)Using fluorescent staining to test cardiomyocyte cross-sectional area,super oxygen anion accumulation degree,the expression of CD31,CD68 in myocardial tissue,evaluating coronary microvascular density and myocardial macrophage infiltration degree of each group.6)Using real-time quantitative PCR technique to detect the expression of IL-1?,TNF-?,IL-10,VEGF m RNA of myocardial tissue.Results: 1)Compared with model group,the body quality of Bushenhuoxue group was not statistically significant(P > 0.05).2)Compared with model group,the myocardial LVEF,LVFS of Bushenhuoxue group was increased(P < 0.05).3)Model groups cardiomyocyte was swelling,horizontal stripes blur,myocardial tissue degeneration,inflammatory cells infiltration,Bushenhuoxue group myocardial tissue structure was relatively complete,and infiltration of inflammatory cells was decreased.4)Compared with model group,cardiomyocyte cross-sectional area of Bushenhuoxue group was increased obviously(P < 0.05).5)Compared with model group,myocardial superoxide anion of Bushenhuoxue group was decreased obviously(P < 0.05).6)Compared with model group,the expression of CD68 positive number in myocardium of Bushenhuoxue group was no obvious difference(P > 0.05);coronary microvascular density of Bushenhuoxue group was increased obviously(P < 0.05).7)Compared with model group,the expression of IL-1 beta,TNF-alpha m RNA of Bushenhuoxue group was decreased obviously(P < 0.05);the expression of IL-10 and VEGF m RNA levels of Bushenhuoxue group was increased obviously(P < 0.05).Conclusion: Bushenhuoxue group could inhibite coronary microvascular inflammatory reaction improving the myocardial systolic function and coronary microvascular,which might be relative to inhibiting myocardial macrophage phenotype to M1,promoting to M2.Fourth Part The effects of Bushenhuoxue recipe on miR-155 expression of myocardium in ovariectomized CMD mice modelObjective: To explore the effects of Bushenhuoxue recipe on miR-155 expression,the key control factor of macrophage M1 / M2 polarization,of myocardium in ovariectomized CMD mice modelMotheds: 1)Eight weeks old sexual maturity C57 BL / 6 female mice were divided into sham group,model group,miR-155 inhibitor group and Bushenhuoxue group.With reference to the first part of experiment,ovariectomized CMD mice model was established.Bushenhuoxue group were deal with the third part of experiment.miR-155 inhibitor group were injected by tail vein,2ug/m L,once a week,from ovarian 8W,for another 8 W.2)Using real-time quantitative PCR technique to detect the expression of miR-155,IL-1?,TNF-?,IL-10,VEGF m RNA of myocardial tissue.Results: 1)Compared with sham group,the expression of miR-155,IL-1 beta,TNF alpha m RNA of model group was increased obviously(P < 0.05);the expression of IL-10 and VEGF m RNA levels of model group was decreased obviously(P < 0.05).2)Compared with model group,the expression of miR-155,IL-1 beta,TNF alpha m RNA of Bushenhuoxue group was decreased obviously(P < 0.05);the expression of IL-10 and VEGF m RNA levels of Bushenhuoxue group was increased obviously(P < 0.05).3)Compared with model group,the expression of miR-155,IL-1 beta,TNF alpha m RNA of miR-155 inhibited group was decreased obviously(P < 0.05);the expression of IL-10 and VEGF m RNA levels of miR-155 inhibited group was increased obviously(P < 0.05).4)There were no significant differences between miR-155 inhibited group and Bushenhuoxue group in the expression of miR-155,IL-1 beta,TNF alpha,IL-10 and VEGF m RNA levels of myocardial tissue.Conclusion: The expression of miR – 155 was closely related to the M1 macrophage,and positively correlated.The expression of miR – 155 was increased in ovariectomized CMD mice model.Interposed by miR-155 inhibitor,the model's myocardial miR-155 decreased,inhibiting myocardial macrophage phenotype to M1,promoting to M2.Interposed by Bushenhuoxue recipe,the model's myocardial miR-155 decreased.Therefore Bushenhuoxue recipe inhibiting myocardial macrophage phenotype to M1,promoting to M2 might be relative to it down-regulating the expression of miR – 155. |
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