| ObjectiveOvariectomized rats were used as animal models to explore the changes in cardiac function,blood pressure,fasting blood glucose(FBG)and blood lipid at different time points after ovarian removal in rats,and the myocardial energy metabolism related protein expression.We instincted the effect of Er-xian decoction on the above changes,in order to provide experimental evidence for the prevention and treatment of cardiovascular diseases during menopause.Method80 healthy female SD rats of SPF grade were selected,and after one week of adaptive breeding,they were randomly divided into groups according to body weight,20 in the sham operation group,and 60 in the model group.Rats with successful modeling were randomly divided into model group,estrogen group and Er-xian decoction group,and 20 patients in each group were started dosing.Estrogen group:Rats received treatments of orally force-fed estradiol valerate at a dose of 1.8×10-4g·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 aqua distilled water.Rats in each group were administered once a day,and the administration was stopped for 6 days and 1 day for 16 weeks.At the end of the 4th week,8th week,12th week and 16th week after administration,cardiac function of each group were measured by echocardiography,the blood pressure(BP)and heart rate(HR)were detected by the tail method,the FBG levels in rats were detected by test strip method,and levels of total cholesterol(TC),triglyceride(TG)S high-density lipoprotein(HDL-C)and low-density lipoprotein(LDL-C)in rats by were detected by oxidase method and direct method.When taking samples at the end of the 16th week,the LDL subfractions in serum was detected by gel electrophoresis system.The levels of estrogen(E2)and follicle stimulating hormone(FSH)in rats serum were measured by radioimmunoassay.The levels of endothelin-1(ET-1)and angiotensin 2(Ang?)in rats serum were measured by enzyme-linked immune sorbent assay(ELISA).Levels of adenosine triphosphate(ATP)and adenosine diphosphate(ADP)in myocardial tissue of each group by liquid chromatography-mass spectrometry.Western-blotting method was used to detect myocardial protein expression of phosphorylated adenylate-activated protein kinase(p-AMPK),adenylate-activated protein kinase(AMPK-a),peroxisome proliferator activated receptor ?(PPAR-?),glucose transporter 4(Glut-4).And the pathological changes of myocardium and uterus in rats were detected by lignin-eosin(HE)staining.The left ventricular microstructure in rats was observed by transmission electron microscope.Periodic acid Schiff(PAS)staining was used to detect ventricular glycogen deposition of ventricular glycogen deposition in rats.Results(1)Canges in vginal smear and pathological uterusThe vaginal smear regularity of the ovariectomized rat disappears during the estrous cycle,and the vaginal smear of the sham-operated model rat shows the pre-estrous period-estrous period-late estrus period periodic changes in the period.The experimental model was successful.In the sham operation group,the endometrium was thicker,and the glands were abundant.In the model group,the uterine atrophy,the wall of the tube became significantly thinner,and the lumen narrowed.The endometrium of rats in the estrogen group and Er-xian decoction group was covered with a single layer of flat epithelium or a single layer of cubic epithelium.(2)Canges in E2 level in rats serumAt the 16th week after administration,compared with the sham operation group,the E2 level in serum of the model group was reduced(P<0.05).Compared with the model group,the E2 level in serum of the estrogen group increased(P<0.05),and the Er-xian decoction group increased remarkably(P<0.01).(3)Changes in cardiac functionCompared with the sham operation group,the ejection fraction(EF%)in the model group was reduced and the left ventricular end systolic volume(LVVols)was increased in the model group at the 4th week(P<0.05).At the 8th week,no significant difference was found in the cardiac function of the model group rats.At the 12th week,the left ventricular end systolic diameter(LVIDs)of the model group rats increased significantly(P<0.01),LVVols,the left ventricular end diastolic diameter(LVIDd),and the left ventricular end diastolic volume(LVVold)all increased(P<0.05).At the 16th week,the EF%,the left ventricular short axis shortening rate(FS%)of the model group were significantly reduced,and the blood pumping function was significantly reduced(P<0.01).Compared with the model group,the EF%,FS%,left ventricular posterior wall end systolic thickness(LVPWs)increased,and LVVols decreased in the Er-xian decoction group at the end of the 4th week(P<0.05).At the 12th week,there was no significant difference in the cardiac function of the rats in the Er-xian Decoction group.At the end of the 16th week,the EF%and FS%of the rats in the Er-xian decoction group increased significantly(P<0.01).The cardiac function of rats in estrogen group also improved at different time points.(4)Changes in BPCompared with the sham operation group,the systolic blood pressure(SBP)increased in the model group at 4th week,8th week and 12th week(P<0.05).The SBP increased significantly at 16th week(P<0.01).The diastolic blood pressure(DBP)in the model group increased at 4th week,8th week and 12th week.It showed an upward trend but no significant difference.The DBP increased significantly at 16th week(P<0.01).At 8th week,the mean pressure(MBP)in the model group increased(P<0.05).At 16th week,the MBP increased significantly,showing an overall upward trend(P<0.01).At the 4th week,compared with the model group,the SBP of Er-xian decoction group did not decrease significantly.At the 8th week,12th week and 16th week,the SBP in Er-xian decoction group decreased significantly(P<0.01).At the 8th week,the DBP in the Er-xian decoction group decreased(P<0.05).At 8th week,12th week and 16th week,MBP in the Er-xian decoction group decreased significantly(P<0.01);SBP,DBP,and MBP in the estrogen group also decreased at different time points.There was no significant difference in HR in each group at different time points.(5)Changes in levels of ET-1 and Ang ? in serumCompared with the sham operation group,the levels of ET-1 and Ang? in the model group serum were significantly increased(P<0.01).Compared with the model group,the levels of ET-1 and Ang ? in the estrogen and Er-xian decoction groups were significantly reduced(P<0.01).(6)Changes in FBGCompared with the sham operation group,the FBG of the model group was significantly increased at the 4th week(P<0.01),and increased at 8th week(P<0.05).FBG were increased at the 12th and 16th week,but there was no statistical difference.Compared with the model group,the FBG was significantly reduced at 4th week and 8th week in rats of the estrogel group and the Er-xian decoction group(P<0.01).(7)Changes in BPCompared with the sham operation group,the LDL-C in rats serum of model group increased significantly at the 4th week(F<0.01).There was no significant difference in blood lipids at the 8th week.The TC,HDL-C increased significantly(P<0.01)and LDL-C increased(P<0.05)in rats serum at the 12th week.The TC increased(P<0.05)and LDL-C increased significantly(P<0.01)in rats serum at 16th week.Compared with the model group,the rats in the estrogen group and Er-xian decoction group had no significant difference in blood lipids at the 4th week and the 8th week.And the TC,HDL-C,and LDL-C in rats serum of the estrogen group were significantly reduced at the 12th week(P<0.01).The TC and HDL-C were significantly reduced(P<0.01)and the LDL-C was reduced(P<0.05)in rats serum of the Er-xian decoction group at the 12th week.The TC and LDL-C were significantly reauced(P<0.01)in rats serum of the estrogen group at the 16th week.The TC was reduced(P<0.05)and the LDL-C was significantly reduced(P<0.01)in rats serum of the Er-xian decoction group at the 16m week.The reason is worth further investigation.(8)Changes in LDL subfractions in serumAt the 16th week,small particles of LDL-3 subfractions appeared in the serul of the model group rats.Compared with the model group,the proportion of large-particle LDL-1 and LDL-2 subfractions in the rats serum of the Er-xian decoction group increased,and no small particle LDL-3 subfractions appeared.(9)Changes in myocardial energy metabolismAt the 16th week,compared with the sham operation group,the ATP and ADP in the myocardium of the model group were significantly reduced(P<0.01).Compared with the model group,myocardial ADP increased significantly in the estrogen group(P<0.01).And myocardial ATP increased(P<0.05),ADP increased significantly(P<0.01)in the Er-xian decoction group.(10)Changes in myocardial energy metabolism related protein expressionAt the 16th week,compared with the sham operation group,the expression of p-AMPK and AMPK-a protein was significantly increased(P<0.01),and PPAR-a protein was increased(P<0.05)in the myocardium of the model group.And there was no significant difference in Glut-4 protein expression.Compared with the model group,the expression of myocardial p-AMPK protein was significantly reduced(P<0.01),and AMPK-a,PPAR-a protein was reduced(P<0.05).The myocardial p-AMPK protein expression was significantly reduced(P<0.01)and the AMPK-a protein expression was decreased(P<0.05)in Er-xian decoction group.There was no remarkable difference in expression of PPAR-a and Glut-4 protein in Er-xian decoction group.(11)Changes in mocardial pathologyAt the end of the 16th week,from the results of myocardial HE staining and myocardial transmission electron microscopy,it can be seen that the myocardial stripes of the sham operation group are regularly arranged,the myocardial cells are arranged neatly,the myocardial mitochondrial membrane is intact,and the mitochondrial crest structure is clear.There was no obvious glycogen deposition in rats myocardial tissue.In the model group,the myocardial rhabdomyolysis was focally lysed,and the myocardial cells were focally atrophic and interstitial hyperplasia.The nucleus was condensed and the mitochondria were thin and swollen.In the model group,there had more glycogen deposition in the myocardial tissue.The estrogen group and Er-xian decoction group had obvious myocardial transverse striation,and myocardial cells occasionally atrophied.The structure of mitochondria was clearer,and no obvious swelling was seen.The myocardial glycogen deposition in the two groups were reduced.ConclusionIn ovariectomized rats,the cardiac ejection fraction decreased at the end of the 4th week,the cardiac function was compensated at the 8th week,the systolic and diastolic function decreased at the 12th week,and the EF%reflecting the pump function of the heart decreased significantly at the 16th week.The SBP increased significantly at the 4th week,and continued to increase with time.The DBP increased significantly at the 16th week,and its increase was significantly later than the SBP.There are obvious pathological changes in ovariectomized rats myocardial tissue,mainly myocardial focal atrophy and mitochondrial vacuolation.Er-xian decoction can significantly increase the level of E2 in the blood and reduce the levels of ET-1 and Ang ?.It has a significant improvement on the above heart function and structural changes,and its effect on reducing SBP is obvious,which is superior to estrogen.Ovariectomized rats showed a disorder of glucose and lipid metabolism at the 4th week,and the disorder of lipid metabolism was significantly worse by the 16th week,especially the serum LDL-C level was significantly increased.It showed a deposition in myocardial glycogen.The content of myocardial ATP and ADP decreased,and the expressions of myocardial p-AMPK,AMPK-? and PPAR-? protein increased.Although Glut-4 showed an increasing trend,there was no statistical difference.Er-xian decoction can reduce serum LDL-C levels,reduce the proportion of small particles of LDL in serum.It can reduce FBG levels in ovariectomized rats,reduce myocardial glycogen deposition,increase myocardial ATP and ADP content,regulate myocardium p-AMPK,AMPK-? protein expression,improve myocardial energy metabolism.It can obviously improve the disorder of glucose and lipid metabolism in ovariectomized rats.At various stages after ovariectomy,rats show different degrees of cardiac contraction and diastolic dysfunction,and glucose and lipid metabolism disorders.Er-xian decoction can directly affect the target organs such as the heart and blood vessels and regulate the endocrine system and glucose and lipid metabolism.It plays a protective role in the cardiovascular of ovariectomized rats.This study provides an experimental reference for in-depth understanding of the pathophysiological process of cardiovascular disease in menopausal women and the clinical application of Er-xian decoction in the prevention and treatment of cardiovascular disease in menopause. |
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