| Background and ObjectiveThe increased incidence of coronary heart disease in women after menopause has been confirmed.In the past,the focus and emphasis on the research and treatment of coronary heart disease mostly focused on the coronary arteries of the epicardium,while ignoring the pathological changes of myocardial microvascular.Clinically,compared with men,women suffer from myocardial microvascular dysfunction more frequently,which has attracted great attention from clinical and basic research.At present,its pathogenesis is not clear.Our previous research found that the heart of ovariectomized rats has remodeled pathological changes,the myocardial glucose and lipid metabolism is also abnormal,and the heart function is also changed.After ovariectomy,whether pathological changes occurred in the myocardial capillaries of rats that affect the state of cardiac function?So it is necessary to conduct in-depth research on the structure and function of myocardial microvessels in ovariectomized rats.Therefore,this study used ovariectomized rats as a menopausal animal model to explore the changes in the structure and function of myocardial microcirculation after ovariectomy,and the changes in myocardial structure and function.Under the guidance of traditional Chinese medicine theory,combined with the results of previous studies,the therapeutic effect of Er-xian Decoction was explored.It provides experimental basis and new ideas for the prevention and treatment of microvascular angina pectoris in menopausal women with traditional Chinese medicine.MethodsThere are 32 healthy female SD rats of SPF grade,10 weeks old,and after one week of adaptive breeding,24 rats were randomly selected to undergo ovariectomized modeling.Vaginal smears were performed for 5 consecutive days one week after the ovariectomy.After confirming that the model was successfully established,24 rats were randomly divided into four groups:model group,estrogen group,and Er-xian decoction group,8 rats in each group.The remaining 8 rats were removed from the adipose tissue adjacent to the ovary and served as a sham operation group.Administration was started 2 weeks after ovariectomy.Estrogen group:rats received treatments of orally force-fed estradiol valerate at a dose of 1.8×10-4 g·kg-1.Er-xian decoction group:rats received treatments of orally force-fed Er-xian decoction at a dose of 9g·kg-1 Sham operation group and model group:rats received treatments of orally force-fed equal quantities of pure water.Rats in each group were administered once a day,and the administration was stopped for 6 days and 1 day for 16 weeks.After 16 weeks of administration,the cardiac function was measured by noninvasive ultrasound cardiogram(UCG);the blood pressure and heart rate was measured by tail artery detection techniques;the pathological changes of myocardium and uterus were detected by lignin-eosin(HE)staining;the morphological structure of microvessels of myocardial tissue were detected by transmission electron microscope;CD34 in the myocardial tissue was tested by immunofluorescence staining to measure the microvessel density;the levels of estrogen(E2)in rat plasma were detected by radioimmunoassay;the levels of TM(thrombomodulin),EPCR(endothelial protein C receptor),eNOS(endothelial nitric oxide synthase),endothelin-1(ET-1),prostacyclin I2(PGI2),thromboxane A2(TXA2),endothelial growth factor(VEGF),and von Willebrand Factor(vWF)in plasma were detected by enzyme-linked immuno sorbent assay(ELISA);the levels of IL-1β,IL-6 in serum were detected by ELISA;the levels of NRG-1,ErbB2,claudin-5,eNOS,TNF-α,IL-1β,and VCAM-1 in myocardial tissue were detected by western-blotting(WB);four items of coagulation was detected by blood coagulation analyzer;whole blood viscosity and plasma viscosity were detected by hemorheology.Results(1)Successfully prepared ovariectomized rat modelThe successful preparation of the ovariectomized rat model can be seen through vaginal smear,E2 level in plasma and pathological changes in uterus.The vaginal smear regularity of the ovariectomized rat disappears during the estrous cycle,and the vaginal smear of sham-operated rats showed periodic changes,which shows that the experimental model was successful.Compared with the sham operation group,the E2 level of the model group increased(p<0.01).Compared with the model group,the E2 level of the estrogen group increased(p<0.05),and the Er-xian decoction group increased remarkably(p<0.01).In the sham operation group,the endometrium was thicker,and the glands were abundant,the monolayer columnar epithelium is neatly arranged;in the model group,the uterine tissue was significantly atrophied,the endometrium became thinner,and consisted of only 1 to 2 layers of squamous epithelial cells,and the lamina propria glands became smaller and less numerous;in the estrogen group,the uterine endometrium was significantly thickened,with single-layer cubic epithelium and single-layer columnar epithelium,irregularly arranged,and the number of lamina propria glands increased;in the Er-xian decoction group,the uterine endometrium was thickened,with single-layer flat epithelium or single-layer cubic epithelium,and the number of lamina propria glands increased.By observing the postoperative vaginal smear and ovarian absence during the sampling,combined with the plasma E2 level of rats and the pathological changes of the uterus,it is believed that the experimental ovariectomized rat model was successfully prepared.(2)Changes in cardiac function,BP and myocardial pathologyChanges in cardiac functionCompared with the sham operation group,the EF and FS of rats in the model group decreased(p<0.01),and LVVols increased(p<0.01).Compared with the model group,the EF and FS of rats in the estrogen group and Er-xian Decoction group increased(p<0.05),and LVVols decreased(p<0.05).Changes in BPCompared with the sham operation group,the SBP,DBP and MBP of the model group increased(p<0.01).Compared with the model group,the SBP,DBP and MBP of the estrogen group decreased(p<0.01).Er-xian Decoction SBP and DBP of rats in the group decreased(p<0.01).Myocardial pathology changesThe cardiomyocytes of rats in the sham operation group are arranged densely and neatly.Compared with the sham operation group,the cardiomyocytes of rats in the model group were atrophied and the gap increased.Compared with the model group,the cardiomyocytes of rats in the estrogen group had occasional atrophy and focal interstitial hyperplasia,while the atrophy and interstitial hyperplasia of rats in the Erxiantang group were lighter.(3)Changes in myocardial microcirculation1)Changes in the myocardial microvessel density and related factorsChanges in the myocardial microvessel densityCompared with the sham operation group,the myocardial microvessel density in the model group was significantly lower(p<0.01),and the distribution was uneven.Compared with the model group,the myocardial microvessel density of rats in the estrogen group and the Erxiantang group increased significantly(p<0.01).Expression of NRG-1 and ErbB2 proteins in myocardial tissueCompared with the sham operation group,the content of NRG-1 and ErbB2 in the myocardial tissue of the model group increased(p<0.01).Compared with the model group,the content of NRG-1 in myocardial tissue of the estrogen group decreased(p<0.05),and the content of ErbB2 decreased(p<0.01);the content of NRG-1 in myocardial tissue of rats in the Er-xian decoction group decreased(p<0.01),and the content of ErbB2 decreased(p<0.05).Level of VEGF in plasmaCompared with the sham operation group,the plasma VEGF in the model group increased(p<0.01).Compared with the model group,the plasma VEGF of rats in the estrogen group and Er-xian decoction group decreased(p<0.01).2)Changes in the myocardial microvessel morphological structure and related factorsChanges in the myocardial microvessel morphological structureIn the sham operation group,the morphology of myocardial microvascular endothelial cells was relatively normal,the thickness of vascular endothelium was relatively uniform,the cytoplasmic electron density was relatively uniform,and the electron density was medium.There were many transport vesicles and the basement membrane thickness was uniform.Compared with the sham operation group,the myocardial microvascular endothelial cells of the model group were significantly swollen,especially around the nucleus,and the remaining part still showed increased thickness with decreased electron density,cytoplasmic cavitation,and no obvious changes in the basement membrane.Compared with the model group,the myocardial microvascular endothelial edema of the estrogen group and the Erxiantang group was milder and limited,the basement membrane was not changed significantly,and the transport vesicles were clearly visible.Level of TM and EPCR in plasmaCompared with the sham operation group,the plasma TM and EPCR of the model group increased(p<0.05,p<0.01).Compared with the model group,the plasma TM and EPCR content of rats in the estrogen group decreased(p<0.05);the plasma TM content of rats in the Erxiantang group decreased(p<0.05),and the plasma EPCR content of rats in the Er-xian decoction group decreased(p<0.01).Expression of Claudin-5 proteins in myocardial microvascular endothelial cellsCompared with the sham operation group,the content of Claudin-5 in myocardial tissue of the model group decreased(p<0.05).Compared with the model group,the content of Claudin-5 in myocardial tissue of the estrogen group and Er-xian decoction group increased(p<0.05).3)Endothelial factors that regulates myocardial microvasomotor activityeNOS/ET-1 and PGI2/TXA2 in plasmaCompared with the sham operation group,the plasma eNOS/ET-1 of the model group decreased(p<0.05),and the PGI2/TXA2 decreased(p<0.01);Compared with the model group,the plasma eNOS/ET-1 in the estrogen group increased(p<0.01),and the PGI2/TXA2 increased(p<0.05);the plasma eNOS/ET-1 in the Er-xian decoction group increased(p<0.05),and PGI2/TXA2 increased(p<0.01).Expression of eNOS in myocardial tissueCompared with the sham operation group,the eNOS of myocardial tissue in the model group increased(p<0.01).The content of eNOS in the myocardium of rats in the estrogen group increased(p<0.05);the content of eNOS in the myocardium of rats in the Er-xian Decoction group increased(p<0.01).(4)Correlation between cardiac function and coronary microvessel densityThe EF and FS of rats were positively correlated with coronary microvessel density,and the correlation coefficients were 0.706 and 0.699 respectively(p<0.01);LVIDs,LVVols were negatively correlated with coronary microvessel density,and the correlation coefficients were-0.705 and-0.698 respectively(p<0.01).(5)Changes in hemorheology,coagulation system function and related factorsHemorheology resultsCompared with the sham operation group,the whole blood viscosity under the five shear rates,cassone viscosity,and plasma viscosity of the model group increased(p<0.01),whole blood high shear index,whole blood low shear index,and red blood cell(RBC)aggregation index Increase(p<0.05),Compared with the model group,the whole blood viscosity under high shear(200 s-1)in the estrogen group decreased(p<0.05),and the Casson viscosity and plasma viscosity decreased(p<0.01);in the Er-xian Decoction group,the viscosity of whole blood under mid-cut(30 s-1 and 50 s-1)and high-cut(200 s-1)decreased(p<0.05),and the relative index of high-cut whole blood and RBC aggregation index decreased(p<0.05),Casson viscosity and plasma viscosity decreased(p<0.01).four items of coagulationCompared with the sham operation group,APTT in the model group shortened(p<0.01),and FIB increased(p<0.01).Compared with the model group,the APTT in the estrogen group and the Er-xian Decoction group prolonged(p<0.01).Level of vWF in plasmaCompared with the sham operation group,the plasma vWF of the model group increased(p<0.01).Compared with the model group,the plasma vWF of rats in the estrogen group decreased(p<0.05).(6)inflammation changesExpression of IL-1β,TNF-α and VCAM-1 in myocardial tissueCompared with the sham operation group,the levels of VCAM-1,TNF-α,and IL-1β in the myocardial tissue of rats in the model group increased(p<0.01);the levels of TNF-α and IL-1β in the myocardial tissue of rats in the estrogen group decreased(p<0.01);the content of VCAM-1,TNF-α,IL-1β in myocardial tissue of rats in the Er-xian Decoction group decreased(p<0.01).Level of IL-1β and IL-6 in serumCompared with the sham operation group,the serum IL-1β and IL-6 levels in the model group increased(p<0.01);the serum IL-1β and IL-6 levels in the estrogen and Er-xian Decoction groups both decreased(p<0.01).ConclusionOvariectomized rats had myocardial microcirculation dysfunction.Including:myocardial microvessel density decreased,which had a strong correlation with decreased heart function;at the same time,the myocardial micro vascular structure and endothelial function were abnormal;the levels of inflammatory factors in myocardial tissue and serum were elevated,and blood viscosity increased.Myocardial microcirculation dysfunction in ovariectomized rats may lead to the occurrence and development of abnormal cardiac function and myocardial pathological changes.The kidney-tonifying prescription Er-xian Decoction can protect the damaged myocardium after ovariectomy by improving myocardial microcirculation dysfunction in ovariectomized rats.This experiment provides experimental evidence for Er-xian Decoction to treat menopausal myocardial microcirculation dysfunction,and provides new ideas for the prevention and treatment of microvascular angina pectoris in menopausal women with traditional Chinese medicine. |
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