Dynamic Evolution of Cardiac Function and Glucose and Lipid Metabolism in Ovariectomized Rats and the Intervention Effect of Erxian Decoction

Aims Abnormal changes in cardiac function have been reported in menopausal women, but there are few clinical studies on this topic. Erxian decoction (EXD) is a classic prescription that is widely used in the treatment of female menopausal diseases. The purpose of this study was to investigate the dynamic evolution of cardiac function and glucose and lipid metabolism in ovariectomized (OVX) rats and the intervention effect of EXD. Materials and Methods The OVX climacteric rat model was established by bilateral ovariectomy. After successful modeling, the rats were randomly divided into four groups: the sham operation (SHAM) group (equal volumes of purified water), OVX group (equal volumes of purified water), estradiol (E2) group (1.8 × 10−4 g/kg), and EXD group (9 g/kg). Each group of rats was treated for 16 weeks. At the 4th, 8th, 12th, and 16th weeks after treatment, the cardiac function of the rats in each group was evaluated by ultrasound. The coaxial method was used to measure blood pressure (BP). Serum endothelin-1 (ET-1) and angiotensin-2 (Ang II) levels were determined by the enzyme-linked immunosorbent assay (ELISA). The strip method was used to measure fasting blood glucose (FBG). The serum total cholesterol (TC) and triglyceride (TG) levels of rats were measured with the oxidase method. Direct methods were used to measure serum high-density lipoprotein (HDL-C) and low-density lipoprotein (LDL-C) levels. At week 16 of dosing, serum E2 levels were determined by E2 radioimmunoassay. The myocardium and uterus of the rats in each group were stained with HE (hematoxylin-eosin). The ultrastructure of the rat myocardium was observed by electron microscopy. Results After the 16th week of treatment, the serum E2 level decreased (P < 0.05), and the uterus was atrophied in OVX rats. The cardiac ejection fraction (EF%) decreased at 4 weeks after treatment, and systolic and diastolic function decreased after 12 weeks. After the 16th week, the EF%, which reflects the “pump” function of the heart, decreased significantly (P < 0.05 or P < 0.01). At the 4th, 8th, 12th, and 16th weeks of treatment, the systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean pressure (MBP) of the rats in the OVX group increased with time (P < 0.05 or P < 0.01). The serum ET-1 and Ang II levels of rats in the OVX group increased (P < 0.05 or P < 0.01). In the OVX group, FBG was increased (P < 0.05 or P < 0.01), and blood lipids, especially LDL-C, were significantly increased (P < 0.05 or P < 0.01). After the 16th week of treatment, the myocardial tissue of OVX rats showed obvious pathological changes. EXD significantly increased serum E2 levels (P < 0.01), decreased ET-1 and Ang II levels (P < 0.01), reduced the cardiac function risk factors BP, FBG, and blood lipids, and significantly improved cardiac function and structural changes in OVX rats (P < 0.05 or P < 0.01). Conclusions EXD can improve abnormal cardiac structure and function in OVX rats.


Introduction
Globally, cardiovascular disease (CVD) is the most common cause of death, accounting for 31% of all deaths worldwide [1]. In addition to causing deaths, CVD places a large economic burden on society, accounting for a considerable proportion of healthcare expenditures and lost productivity worldwide [2]. Although CVD mortality has declined signifcantly over the past 40 years, CVD remains the leading cause of death among women [3]. Researchers have noted a link between E 2 and CVD [3]. After menopause, risk factors for CVD are greatly increased in women due to the decrease in E 2 levels [4]. Menopause-induced E 2 reduction combined with aging can lead to unique female cardiac dysfunction [5,6]. It has been reported that more menopausal women than men have heart failure (HF) with preserved EF%, that is, a left ventricle ejection fraction (LVEF) greater than 50% [5,6]. A recent cross-sectional study showed that postmenopausal women undergo a series of pathological changes, such as infammatory responses and vasodilatory dysfunction, due to E 2 defciency that leads to a decrease in myocardial contractile function [7]. However, it has also been reported that the decline in E 2 levels in women in early menopause (within 1 year) actually increases LVEF and decreases diastolic function [8]. Specifcally, in a certain period of menopause, the cardiac function status of women may vary with the time of menopause. However, the current literature that reports on abnormal cardiac function in menopausal women is inconsistent. Terefore, we speculate that women experience fuctuations in E 2 changes during menopause with pathological dynamic changes in their cardiovascular system and cardiac function. In addition, there are few basic studies on cardiac function in menopausal model animals. Terefore, it is of great signifcance to explore the dynamic evolution of cardiac function in animal models of menopause.
Metabolic abnormalities are also typical symptoms of menopause [9,10]. Blood lipid profles, changes in blood glucose [9], and elevated BP levels [10] are common metabolic abnormalities in menopausal women, all of which are related to abnormal cardiac function [11]. All common pathological processes that afect left ventricular structure or function, such as hypertension [12], diabetes, and obesity, will afect left ventricular diastolic function [5]. However, the conclusions of clinical and basic research are not consistent. In a study of 350 perimenopausal women, abnormal HDL-C levels accounted for more than 50% of metabolic abnormalities, and high TG levels accounted for up to 40% of these abnormalities [13]. Compared with those in premenopausal women, both TG and TC levels were elevated in menopausal women [13]. A meta-analysis also showed that there was no signifcant diference in HDL-C levels in premenopausal and postmenopausal women, but TG, TC, and LDL-C levels were signifcantly higher in postmenopausal women than in premenopausal women [14]. A study on lipid levels in perimenopausal and postmenopausal women in China showed that postmenopausal women had lower HDL-C levels and higher TC, TG, and LDL-C levels than perimenopausal women [15]. Studies on the dynamic evolution of blood glucose and blood lipid profles during menopause in OVX rats are rare. Terefore, we need to study the evolution of cardiac function in OVX rats and the dynamic evolution of its risk factors-BP, blood glucose, and blood lipids.
Hormone replacement therapy (HRT) was developed in response to the series of pathological changes caused by hormonal changes in menopausal women [16]. In basic research, E 2 supplementation can also reduce pathological myocardial hypertrophy [16]. However, HRT has had mixed results. Tere is clinical evidence that exogenous E 2 may reverse or delay menopause-related diastolic dysfunction and improve indicators of cardiac diastolic function [17]. However, primary and secondary clinical trials suggest that HRT increases the early risk of coronary heart disease [18]. In addition, some researchers have noticed that HRT in women can reduce the risk of CVD in the frst year after menopause and that HRT after many years of menopause is inefective or even harmful [19].
Phytoestrogens are found in a variety of herbs and are considered an alternative to HRT due to their structural similarity to E 2 [20]. Erxian decoction (EXD) is a classic prescription that is widely used in the clinic for the treatment of menopausal syndrome [21]. EXD consists of six commonly used herbs, namely, Curculigo orchioides Gaertn (Xianmao), Epimedii Folium (Yinyanghuo), Morinda ofcinalis Radix (Bajitian), Angelicae Sinensis Radix (Danggui), Phellodendri Chinensis Cortex (Huangbo), and Anemarrhenae Rhizoma (Zhimu) [22]. Some studies on OVX rats show that EXD has the efect of increasing serum E 2 [22,23]. One research group assessing the efect of EXD on the cardiovascular system of OVX rats in the early stage found that the cardiac electrical activity of female OVX rats was abnormal, the electrophysiological properties of the myocardium were remodeled, the myocardium also had tissue structure remodeling, and myosin and other genes were also changed [24]. EXD can improve abnormal cardiac electrical activity in OVX rats, can regulate the expression of genes related to myocardial contraction, and has a protective efect on myocardial injury in OVX rats [24]. Terefore, this study used OVX rats as the model to discuss the dynamic evolution of cardiac function, BP and its traditional risk factors, blood glucose, and blood lipids in rats after ovariectomy. Te function and efect of EXD interventions on glucose and lipid metabolism were also observed.

Drug Preparation. Curculigo orchioides Gaertn
Phellodendri Chinensis Cortex (Huangbo), and Anemarrhenae Rhizoma (Zhimu) were purchased from Beijing Qiancao Traditional Chinese Medicine Co. Ltd.. We decocted the ingredients of EXD according to a mass ratio of 2 : 2 : 2 : 2 : 1 : 1. We added 10× and 8× (v/w) distilled water for the frst and second times, respectively. It was heated by a slow fre until boiling and maintained for 1 hour after boiling. Te medicinal solution was mixed and concentrated to 0.9 g (raw herbs)/mL after mixing twice and stored in a refrigerator at 4°C until use. For estradiol valerate tablets (Bayer Health Care Co. Ltd., Guangzhou Branch, batch number: 398A), 9.2 mg was dissolved in 500 mL of normal saline to prepare liquid medicine with a concentration of 0.018 g/L and stored in a refrigerator at 4°C until use. . After 1 week of adaptive feeding, rats in the modeling operation group (n � 24) were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (1.3 mL/kg), and the ovaries were removed. In the SHAM group (n � 8), small pieces of adipose tissue on both sides of the abdominal cavity were removed, and the ovaries were preserved. Te rats were intraperitoneally injected with penicillin sodium 240,000 U/kg/d for 3 consecutive days after the operation to prevent infection. One week after the operation, the rats were tested by the vaginal smear for 5 consecutive days. Te estrous cycle remained intact for the rats in the SHAM group but had disappeared in the OVX group. Vaginal smears were used to confrm the success of the model [23]. Te successfully modeled rats were randomly divided into the model group (OVX group, n � 8), E 2 group (n � 8), and EXD group (n � 8). Two weeks after ovariectomy, the rats were intragastrically administered their treatment once a day. It was stopped for 6 days for 1 consecutive day for a total of 16 weeks. Te doses of each group were as follows: EXD group, 9 g/kg; E 2 group, 1.8 × 10 −4 g/kg; and SHAM group and OVX group, equal volumes of purifed water. Te mental state, posture, fur color, mobility, eye cleft mucosa color, auricle color, and feces and other general conditions of rats were carefully observed every day. Te rats in each group were weighed and recorded at a fxed time each week.

Detection of Cardiac Function.
After the 4 th , 8 th , 12 th , and 16 th weeks of treatment, 6 rats in each group were fasted for 12 hours. Echocardiography (UCG) was performed using an ultrahigh resolution small animal ultrasound imaging system (VisualSonics, Canada, Vevo2100) to assess cardiac structure and cardiac function. After the depilatory cream (Nair Australia, LL6084) was applied to the clavicle and the xiphoid process of rats, rats were laid in a supine position and fxed on the operating table. Ten, the coupling agent was applied. Te rats were anesthetized by inhalation of a mixture of isofurane and pure oxygen using a ventilator inhalation anesthesia machine (VisualSonics, Canada, VS4083), and the heart rate of the rats was maintained at approximately 350 beats/min. Ten, we used a Vevo2100 ultra-high-resolution small-animal ultrasound imaging system and the MS-250 21 Hz probe to obtain the short axis view of the left ventricle of the rats and made the M-type UCG sampling line at the level of the papillary muscle. At least 10 cardiac cycles were continuously recorded, and the following parameters were measured and calculated: EF%, left ventricular short axis shortening rate (FS%), left ventricular end systolic diameter (LVIDs), left ventricular posterior wall end systolic thickness (LVPWs), left ventricular end systolic volume (LVVols), left ventricular end diastolic diameter (LVIDd), left ventricular posterior wall end diastolic thickness (LVPWd), and left ventricular end diastolic volume (LVVold). All indicators were the average of 3 cardiac cycle parameters.

Detection of BP and Heart Rate (HR).
After the 4 th , 8 th , 12 th , and 16 th weeks of treatment, a noninvasive BP meter for rats (Japan Softron Corporation, BP-98A) was used to measure the tail artery SBP, DBP, MBP, and HR of the rats in each group using the coaxial method. During the experiment, rats were allowed to adapt to the dark experimental environment in advance, and the insulation cylinder was kept at 38°C. After rats were in a stable state, BP and HR were measured. Each rat was continuously measured 3 times, and each data point was the average of 3 measurements. A BP-98A noninvasive BP acquisition system was used to collect data.

Biochemical Analysis.
After the 4 th , 8 th , 12 th , and 16 th weeks of treatment, each group of rats fasted for 12 hours in advance. FBG was measured using a blood glucose meter and test strips (USA Johnson & Johnson). Ten, blood samples (0.2 mL) were collected by inserting capillary tubes into the periorbital plexus. After the 16 th week of treatment, the rats in each group were anesthetized with an intraperitoneal injection of 3% sodium pentobarbital (1.3 mL/ kg) after 12 h of fasting, and abdominal aortic blood was collected. Serum was isolated by centrifugation (4°C, 5000 rpm for 15 min) using a low-temperature centrifuge (American Termo Company, Multifuge X1R) and stored at −80°C until use. Serum TC and TG levels in rats were measured by the oxidative enzyme method (Intech Innovation (Xiamen) Technology Co. Ltd, batch numbers: 671030E12, 671031E12). Serum HDL-C and LDL-C levels were measured by a direct method ((Intech Innovation (Xiamen) Technology Co., Ltd, batch numbers: 671016E12, 671024E12)). Serum ET-1 and Ang II contents were detected by using ELISA kits (Nanjing Jiancheng Institute of Biological Engineering, batch numbers: 20181121, 20181214), and an automatic microplate reader (American Termo Company, Multiskan EX Primary EIA V. 2.3) was used to measure absorbance A. After the 16 th week of treatment, serum E 2 levels were detected by using an E 2 radioimmunoassay kit (Beijing North Institute of Biotechnology Co., batch number: 20190320) and using a gamma radioimmunoassay counter (Xi'an Nuclear Instrument Factory, XH-6080c).

Histological Staining.
After the 16 th week of treatment, rats were anesthetized by an intraperitoneal injection of 3% sodium pentobarbital (1.3 mL/kg). Te rat heart cross section was obtained. It was immediately rinsed with ice-cold

Efect of EXD on Body Weight and Uterine Coefcient in OVX Rats after the 16 th
Week of Treatment. Compared with that in the SHAM group, the body weight of the rats in the OVX group increased signifcantly, and the uterine coefcient decreased signifcantly (P < 0.01). Compared with that in the OVX group, the body weight of the rats in the E 2 group decreased (P < 0.05), and the uterine coefcient increased signifcantly (P < 0.01). However, there was no signifcant diference in the body weight or uterine coefcient of the rats in the EXD group. Body weight and uterine coefcient graphs are shown in Figures 1(a) and 1(b).

Efect of EXD on Pathological Changes in the Uterus after the 16 th
Week of Treatment. After the 16 th week of treatment, the morphological changes in the uteri of the rats in each group were observed under a light microscope. Te endometrium of the rats in the SHAM group was thicker, with abundant glands. Te monolayer columnar epithelium was neatly arranged (Figure 2(a)). In the OVX group, uterine atrophy occurred, the wall of the tube became signifcantly thinner, and the lumen narrowed (Figure 2(b)). Compared with the OVX group, the endometrium in the E 2 group was covered by a single layer of cuboidal epithelium. In addition, the single layer of columnar epithelium was irregularly arranged, and the number of endometrial glands was higher (Figure 2(c)). Te endometrium of rats in the EXD group was covered with a single layer of squamous epithelium or a single layer of cuboidal epithelium. It had fewer endometrial glands and occasional secretions (Figure 2(d)).

Efect of EXD on Serum E 2 Content after the 16 th
Week of Treatment. Compared with that in the SHAM group, after 16 weeks of treatment, the serum E 2 content of the rats in the OVX group decreased (P < 0.05). In addition, compared with that in the OVX group, the serum E 2 content of the rats in the E 2 group increased (P < 0.05). Furthermore, the serum E 2 content of the rats in the EXD group increased signifcantly (P < 0.01). Te above E 2 results are shown in Figure 3.

Efect of EXD on Pathological Changes in the Left Ventricular Myocardium after the 16 th Week of Treatment.
After the 16 th week of treatment, the results of myocardial HE staining and myocardial transmission electron microscopy showed that in the SHAM group, myocardial stripes were arranged regularly and clearly, myocardial cells were arranged neatly and densely, myocardial cell flaments were neat, and sarcomeres were regular and clear. Te myocardial mitochondrial membrane was intact, and the mitochondrial crista structure was clear in the SHAM group. Compared with the SHAM group, the myocardial tissue of the OVX group showed atrophy and thinning of myocardial fbers, enlarged myocardial cell space, obvious focal eosinophilic changes in myocardial cells, and deep staining of the nuclei. Te ultrastructure in the OVX group showed that the arrangement of myocardial myofbrils was disordered, the arrangement of myoflaments was not neat, the structure of sarcomeres was blurred, the nuclei of myocardial cells were pyknotic, and the cristae of myocardial mitochondria appeared obviously broken and sparse. Compared with those in the OVX group, the rats in the E 2 group had obvious myocardial striations, occasional atrophy of myocardial cells, focal interstitial hyperplasia, a neat arrangement of myoflaments, a clear mitochondrial crista structure, and no obvious swelling. Atrophy and stromal hyperplasia of myocardial cells in the EXD group were lighter, the mitochondrial membrane was intact, and the mitochondrial crista structure was clearer than that in the OVX group. Te above results indicate that both E 2 and EXD can improve the pathological changes in myocardial structure in OVX rats. Te above pathological fndings are shown in Figures 4(a) and 4(b). In the OVX group, LVIDs, LVVols, LVIDd, and LVVold were signifcantly increased after the 12 th week of treatment (P < 0.01). At the 16 th week of treatment, both EF% and FS% were signifcantly decreased in the model group (P < 0.01), and LVIDs and LVVols increased signifcantly (P < 0.01). Compared with those in the OVX group, after the 4 th week of treatment, EF%, FS%, and LVPWs of the E 2 group were signifcantly increased (P < 0.01), and the LVIDs, LVVols, LVIDd, and LVVold were signifcantly decreased (P < 0.01).

Efect of EXD on Cardiac
EF%, FS%, and LVPWs of the EXD group were increased to diferent degrees (P < 0.05 or P < 0.01), and LVIDs, LVVols, and LVIDd were decreased to diferent degrees (P < 0.05 or P < 0.01). After the 12 th week of treatment, the systolic and diastolic function indices of the rats in the E 2 group and the EXD group showed a trend toward improvement, but there was no signifcant diference. After the 16 th week of treatment, EF% and FS% of the E 2 group and EXD group were signifcantly increased (P < 0.01), LVIDs was signifcantly decreased (P < 0.01), and LVVols was decreased (P < 0.05). Te above pathological fndings are shown in Figures 5(a) and 5(i).     week of treatment (P < 0.01) and after the 8 th week of treatment (P < 0.05). In addition, there was a trend toward an increase in FBG in the OVX group after the 12 th and 16 th weeks of treatment, but the change was not signifcant. Compared with that in the OVX group, the FBG of rats in the E 2 and OVX groups decreased signifcantly after the 4 th and 8 th week of treatment (P < 0.01). Te above results show that E 2 and EXD can reduce the FBG levels of OVX rats after the 4 th and 8 th week of treatment. Tere was no signifcant diference in the FBG levels of the rats in each group after the 12 th and 16 th week of treatment. Te above results are shown in Figure 8.

Efect of EXD on Serum TC, TG, HDL-C, and LDL-C Levels at the 4 th , 8 th , 12 th , and 16 th Week of Treatment.
Compared with that in the SHAM group, after the 4 th week of treatment, the level of serum LDL-C in the OVX group increased signifcantly (P < 0.01). After the 8 th week of treatment, there was no signifcant diference in serum TC, TG, HDL-C, and LDL-C levels. After the 12 th week of treatment, the serum TC and HDL-C levels increased signifcantly (P < 0.01), as did serum LDL-C levels (P < 0.05). After the 16 th week of treatment, serum TC levels signifcantly increased (P < 0.05), as did serum LDL-C levels (P < 0.01). Compared with those in the OVX group, after the 4 th and 8 th week of treatment, there were no signifcant diferences in the serum TC, TG, HDL-C, and LDL-C levels in the E 2 group. After the 12 th week of treatment, the serum TC, HDL-C, and LDL-C levels were signifcantly reduced (P < 0.01). Furthermore, after the 16 th week of treatment, the serum TC and LDL-C levels were signifcantly reduced (P < 0.01). After the 4 th and 8 th week of treatment, there were no signifcant diferences in the serum TC, TG, HDL-C, and LDL-C levels of the rats in the EXD group. After the 12 th week of treatment, the serum TC and HDL-C levels were Evidence-Based Complementary and Alternative Medicine signifcantly reduced (P < 0.01), as were serum LDL-C levels (P < 0.05). After the 16 th week of treatment, the serum TC level signifcantly decreased (P < 0.05), as did the serum LDL-C level (P < 0.01). Te above results are shown in Figures 9(a)∼9(d).

Discussion
Clinically, cardiac function is closely related to HF. HF is an important cause of increased morbidity and mortality in patients [25]. Te incidence increases rapidly with age [26]. HF is caused by abnormal diastolic ventricular flling and decreased systolic ejection [25]. Te left ventricular HF can be divided into HF with a preserved ejection fraction (HfpEF) and HF with a reduced ejection fraction (HFrEF). HfpEF is defned as LVEF > 50%, and HFrEF is defned as LVEF < 40% [25]. At present, research on HF and heart function in menopausal women has not yet reached a unifed conclusion [6]. Many clinical studies have indicated that menopausal women are more likely to sufer from HfpEF than men, with women being almost twice as likely as men to sufer from it [6]. However, it has also been reported that after adjusting for age and risk factors, the prevalence in women and men does not difer signifcantly [27]. According to the study by Alexanderson-Rosas et al., cardiac systolic function is decreased in menopausal women [7]. Tere are also studies suggesting that LVEF increases in early menopausal women, while diastolic function decreases [8]. Basic research has also addressed the issue of abnormal cardiac function in OVX animal models, and it is believed that various cardiac function parameters of OVX rats have abnormal changes [27,28].
In view of the adverse efects of estrogen on clinical studies, traditional Chinese medicine with estrogen-like efects has become a research hotspot [29,30]. EXD is a classic prescription for the treatment of menopausal syndrome in females. It was developed by the modern and famous doctor Zhang Bone [21]. It can supplement serum E 2 in OVX rats [22]. Tere are abundant E 2 receptors in rodents, and the biological roles of E 2 and E 2 receptors involve . Data are presented as the mean ± SD. * P < 0.05, * * P < 0.01 vs. SHAM group; # P < 0.05, ## P < 0.01 vs. OVX group. n � 6 per group. 8 Evidence-Based Complementary and Alternative Medicine their efects on genomic or nongenomic pathways or the direct activation of estrogen receptors by E 2 in mitochondria [31][32][33].
In this study, we successfully established a rat model of menopause by bilateral ovariectomy [34]. OVX rats showed pathological changes manifesting as decreased E 2 levels, increased body weight, a decreased uterine coefcient, and uterine atrophy due to ovariectomy [35]. EXD may play a role in reducing uterine atrophy by increasing serum E 2 content in OVX rats, which is consistent with the fndings of Vijayanarayana et al. [35]. Vijayanarayana et al. found that curcuma alcohol extract can promote endometrial hyperplasia [35]. Tey also found that it can signifcantly increase vaginal keratinization and uterine quality in rats, thereby exerting estrogen-like efects [35]. In addition, the EXD group rats showed an increase in the uterine coefcient, but the diference did not reach statistical signifcance. Icariin is one of the components of Epimedii Folium (Yinyanghuo) [36]. Icariin increases E 2 and promotes the expression of estrogen receptors [36]. However, in this experiment, EXD had no obvious efect on body weight. Some researchers believe that body composition may play a more important role in cardiovascular disease and that increased lean mass in obese individuals may improve protection in patients with coronary heart disease without infammation [37].
Ultrastructure showed that the arrangement of myofbrils in OVX rats was disordered, the sarcomere structure was blurred, the nuclei of the myocardium were pyknotic, and the mitochondrial cristae of the myocardium were obviously broken and sparse. Te myocardial mitochondrial membrane of rats in the EXD group was intact, and the mitochondrial cristae were clear without swelling. Tese fndings suggest that EXD can improve myocardial energy metabolism, mitochondrial structure, and mitochondrial function in OVX rats. E 2 plays an active role in improving the mitochondrial function process [5]. Icariside II can reduce myocardial fbrosis in spontaneously hypertensive rats, inhibit cardiomyocyte apoptosis, reduce myocardial ROS production, and inhibit mitochondrial apoptosis through the ASK1-JNK/p38 signaling pathway [38]. Te protective efect of EXD in OVX rats occurs through the above pharmacological efects.
Te results of the cardiac function of rats at diferent times after treatment showed that after 4 weeks of treatment, cardiac systolic and diastolic function decreased in OVX rats. After 8 weeks of treatment, the parameters of cardiac function in OVX rats showed no obvious abnormalities.
After 12 weeks of treatment, the cardiac systolic and diastolic functions of OVX rats decreased, and the cardiac systolic function of OVX rats decreased at 16 weeks. EXD can obviously improve the abovementioned changes in cardiac function. After comparing the cardiac function parameters of OVX rats, we believe that the decrease in cardiac function in rats after treatment for 4 weeks is due to surgical reasons and that the cardiac function of rats after treatment for 8 weeks is still in the compensation period. At the 12 th week, the systolic and diastolic functions of OVX rats were decreased. At that time, the OVX rats were at a low E 2 level for a long period. To maintain normal blood perfusion, the myocardium showed compensatory changes in the prolonged ejection time and shortened ventricular diastolic time. Tis process further reduced ventricular diastolic blood perfusion, resulting in left ventricular compensation [29]. Tis compensation increases the inner diameter, and myocardial diastolic dysfunction occurs [29]. At 16 weeks of treatment, the increase in LVVold in OVX rats did not reach statistical signifcance, but the parameters EF% and FS%, refecting cardiac pump function, decreased signifcantly. EXD can increase serum E 2 levels in OVX rats, and E 2 plays a positive role in improving myocardial relaxation, myocardial cell Ca 2+ homeostasis, and antimyocardial fbrosis [5]. Pharmacological studies have shown that favonoid icariside II, the main active component of Epimedii Folium (Yinyanghuo), can improve abnormal ventricular remodeling in spontaneously hypertensive rats [38]. Terefore, EXD may reverse the abnormal cardiac systolic and diastolic function of OVX rats in the abovementioned ways. It also protects cardiac function and improves myocardial pathological changes in OVX rats to a certain extent.
Te incidence of hypertension is increased in postmenopausal women, and hypertension is also an important cause of cardiac left ventricular remodeling [39]. Clinically, the treatment of patients with HFpEF focuses on lowering BP [25]. ET-1 is an important player in the occurrence and development of hypertension [40]. Te renin-angiotensin system (RAS) is an important regulator of renal function and arterial pressure [41]. Ang II can be activated and has a strong vasoconstrictive efect [42]. Ang II can also induce the production of ET, which further produces the efect of vasoconstriction [42]. Elevated BP has been demonstrated in OVX model animals [43], and E 2 defciency is closely related to the occurrence of hypertension [41,44,45]. Te calcium channel antagonist action of E 2 can inhibit ET-1, thereby dilating blood vessels [46]. E 2 also reduces BP by inhibiting the RAS system [41]. In this experiment, after 16 weeks of treatment, the SBP of OVX rats was signifcantly increased, which may be related to the increased serum ET-1 and Ang II levels. EXD may play an important role in lowering BP by reducing serum ET-1 and Ang II levels in OVX rats. Another study showed that intragastric treatment with icariside II reduced blood pressure in spontaneously hypertensive rats [38]. Clinical studies have shown that EXD can further reduce SBP and DBP in postmenopausal female hypertensive patients on the basis of benazepril treatment and can also regulate E 2 levels [47]. Terefore, EXD may play an important role in lowering BP through the above pharmacological efects.
Changes in blood lipid profles and blood glucose levels are common metabolic diseases in menopausal women, and blood glucose and blood lipid abnormalities are related to abnormal cardiac function [11]. Glucose intolerance and insulin resistance have been reported to be associated with diastolic dysfunction even before the development of diabetes [48]. Tere is also downregulation of aortic estrogen receptor expression in patients with diabetes [5]. Lipotoxicity and glucotoxicity can also lead to the impairment of vascular function, cardiac contractility, and oxidative stress, among others [49]. Even in the absence of overt coronary artery disease, metabolic abnormalities can lead to cardiac remodeling and cardiac dysfunction [11]. Minta et al. reported that 6 weeks after oophorectomy, high-fat-diet-fed female rats can develop dysglycemia and dyslipidemia [33].  According to Delgobo et al., the E 2 level will rapidly decrease in the short term in the OVX model without afecting oxidative damage, infammation, or lipid metabolism. In OVX rats with long-term decreases in E 2 levels, TG, glucose, and LDL-C levels are unchanged [50]. From the results of this study, it can be seen that an increase in serum TG levels was never evident in OVX rats. However, after 4 weeks of treatment, the LDL-C level increased signifcantly. After 12 weeks, the TC, HDL-C, and LDL-C levels of OVX rats increased signifcantly. After 16 weeks of treatment, the TC and LDL levels of OVX rats increased signifcantly. Te blood glucose of OVX rats increased signifcantly at 4 weeks and 8 weeks after treatment, and there was no abnormal change at 12 or 16 weeks. Rats in the EXD group had pathological changes in blood lipids and decreased blood glucose levels at diferent stages. We speculated that the abnormal changes in the blood lipid profle of OVX rats after 4 weeks of treatment were due to their postoperative stimulation and that the blood lipids of OVX rats were in a compensatory phase after 8 weeks. One study showed that icariin reduced serum total TC, TG, and LDL levels and increased HDL levels in rats with nonalcoholic fatty liver disease [51]. EXD combined with Xiaochaihu decoction and metformin hydrochloride tablets can efectively improve FBG in patients with type 2 diabetes mellitus and menopausal syndrome [52]. EXD improves the changes in glucose and lipid metabolism in OVX rats at diferent stages, which may be related to the increase in serum E 2 levels and the lipid-lowering efect of icariin on OVX rats.

Conclusions
In this study, OVX rats showed decreased cardiac systolic and diastolic functions at the 4 th week after treatment, and EF% and FS%, refecting cardiac pump function, were signifcantly decreased at the 16 th week. Te BP of OVX rats increased at the 4 th week after treatment, and SBP, DBP, and pulse pressure diference increased gradually with time. In OVX rats, FBG increased in the early stage after ovariectomy, and there was no signifcant diference in FBG levels between the groups in the later period. Serum levels of LDL-C in OVX rats were signifcantly increased at the 4 th week of treatment, and there was no signifcant diference in blood lipid levels between the groups at the 8 th week. Overall, serum levels of LDL-C were elevated in OVX rats throughout the experimental observation period. EXD can improve myocardial histological reconstruction, lower BP, regulate blood hormones and bioactive substances, lower LDL-C in blood, improve cardiac structure and function, and signifcantly improve glucose and lipid metabolism disorders in castrated rats. Tis study provided an experimental basis for an indepth understanding of the pathophysiological mechanism of cardiovascular disease in menopausal women and the clinical application of EXD in the prevention and treatment of cardiovascular disease in menopausal women. In addition, this study also provides preliminary work for further exploring the molecular mechanism of abnormal evolution of cardiac function and glucose and lipid metabolism in OVX rats treated with EXD.

Data Availability
Te data that support the fndings of this study are available from the corresponding author.

Additional Points
Concoction Method. Te method for preparing Morinda ofcinalis Radix (Bajitian) and Epimedii Folium (Yinyanghuo) was as follows: Morinda ofcinalis Radix (Bajitian) was cooked at 90∼100°C for 80∼100 minutes. Ten, it was cooked in the same pot as licorice soup until licorice soup ran out. Next, it was cut into sections and dried. Te method for making licorice soup was as follows: water was added to licorice tablets at 10× by volume. Tis was fried 2 times, with the frst occurring for 1 hour and the second occurring for 30 minutes. Ten, the two fried licorice decoctions were combined. 0.06 kg of licorice was used for each 1 kg of Morinda ofcinalis Radix (Bajitian). For Epimedii Folium (Yinyanghuo), suet oil was heated until it melted. Ten, Epimedii Folium (Yinyanghuo) was added and stirred. Te temperature was set to 200°C, and the simmering powder temperature (80∼120°C) was used. Te samples were fried for 10 minutes until the surface was even and shiny. When it turned yellow-green, it was removed and allowed to cool. For every 1 kg of net Epimedii Folium (Yinyanghuo), 0.2 kg of suet oil was used.

Ethical Approval
Tis animal study was reviewed and approved by the Institutional Ethics Committee of the China Academy of Chinese Medical Sciences (approval number: 2018-058).

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Supplementary Materials
Concoction Method. Te method for preparing Morinda ofcinalis Radix (Bajitian) and Epimedii Folium (Yinyanghuo) is as follows. Morinda ofcinalis Radix (Bajitian) was cooked at 90∼100°C for 80∼100 minutes. Ten, it was cooked in the same pot as the licorice soup until the licorice soup ran out. Next, it was cut into sections and dried. Te method for making licorice soup was as follows: water was added to licorice tablets at 10× by volume. Tis was fried 2 times, with the frst occurring for 1 hour and the second occurring for 30 minutes. Ten, the two fried licorice decoctions were combined. 0.06 kg of licorice was used for each 1 kg of Morinda ofcinalis Radix (Bajitian). For Epimedii Folium (Yinyanghuo), suet oil was heated until it melted. Ten, the Epimedii Folium (Yinyanghuo) was added and stirred. Te temperature was set to 200°C, and the simmering powder temperature (80∼120°C) was used. Te samples were fried for 10 minutes until the surface was even and shiny. When it turned yellow-green, it was removed and allowed to cool. For every 1 kg of net Epimedii Folium (Yinyanghuo), 0.2 kg of suet oil was used. (Supplementary Materials)