The protective effect of Er-Xian decoction against myocardial injury in menopausal rat model

Herba Epimedii, Radix morindae Officinalis, Radix Angelicae Sinensis, Rhizoma Anemarrhenae, Cortex Phellodendri and Rhizoma Curculiginis were purchased from Beijing Tongrentang (Bozhou) Prepared Slices of Chinese Crude Drugs Co., Ltd. (Beijing, China), and by a pharmacist of traditional Chinese medicine (Feng Sui) in Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. All voucher specimens are deposited in the herbarium center of China Academy of Chinese Medical Sciences.

We extracted a mixture of six herbal medicines (250 g of each herbal medicine, one thousand five hundred grams in total) by decoction with 10× (v/w) distilled water at the temperature of 100 °C for 2 h. After filtration, we boiled the residue for another 1 h, and mixed the filtrates together, then preserved at 4 °C after lyophilized using a freeze drier (Labconco, Freezone). The yield of the dried extract from the initial crude materials was 10%. We dissolved the extracts to 30 and 60 mg/ml by adding water, and then administered to the rats orally with a dose of 0.1 ml/kg body weight.

In this study, seventy-two virgin Sprague-Dawley rats aged six months and weighing approximately 310 ± 20.0 g were acquired from the Experimental Animal Center in National Institutes for Food and Drug Control of China (SCXK (JING) 2009–0017, Beijing, China). The protocol involving animals in this study was authorized by The Institutional Ethics Committee of China Academy of Chinese Medical Sciences (Approval number: 2015–010). The sham operation (n = 18, SHAM group) or bilateral OVX (n = 54) using a dorsal incision [25] was performed on the rats after acclimatization. The rats undergoing OVX were divided into three groups based on the treatment agents, including vehicle (OVX group), EXD (EXD group) and 17β-estradiol (E2 group). Each group included eighteen rats. Subsequently, we dissolved 17β-estradiol (Sigma-Aldrich, USA) in absolute ethanol and diluted with olive oil. The preparation was used for daily subcutaneous injection of the E2 group rats at a dose of 30 μg/kg body weight. EXD was dissolved in aqua destillata and orally force-fed to the EXD group rats at a dosage of 7.5 g raw herbs/kg body weight/day, which was calculated using the human recommended dosage proposed by the Chinese Pharmacopeia and the surface area ratio of rats and humans. The OVX and SHAM rats were orally force-fed equal quantities of aqua destillata and standardized rat food was given to all rats throughout the current study. The rats were treated with agents or aqua destillata seven days after operation and the treatment lasted until the 13th week. The body weight of each subject was measured once a week.

On the day after final treatment, xylazine (12 mg/kg) and ketamine (80 mg/kg) were injected intraperitoneally to anesthetize all animals, which were subsequently exsanguinated for sacrifice.

The estradiol concentration in the serum was detected using an electrochemiluminescence immunoassay (ECLIA) Assay Kit purchased from Roche Diagnostics (Mannheim, Germany) in accordance with the instructions. All measurements were performed according to the manufacturers’ protocols.

We removed the rat hearts rapidly and placed in Krebs-Henseleit buffer (oxygenated, 4 °C) for removal of the extracardiac tissue. The sample (4 mm × 4 mm) was cut from the left ventricle and then pinned on a thin silicon disc at the bottom of a perfusion chamber. Thereafter, the sample was perfused (4 mL/min) with Krebs-Henseleit solution which contained (in mM) 121 NaCl, 5 KCl, 1.2 MgSO₄, 2 CaCl₂, 25 NaHCO₃, 1.2 NaHPO₄, and 11 glucose. Finally, the sample was equilibrated with 5% CO₂ in O₂ at 36 ± 0.5 °C and the pH was 7.36–7.42.

The whole process was stimulated at a frequency 1 Hz by 1 ms square pulse 1.5 times threshold voltage. We recorded and analyzed transmembrane action potentials (APs) using intracellular microelectrode technique (Chengyi, China). The variables measured were resting membrane potential (RMP), AP amplitude (APA), the maximal rate of depolarization (Vmax), and AP duration at 50% and 90% (APD₅₀ and APD₉₀).

The fixture of the heart was carried out by dipping into 10% neutral buffered formalin. Parallel to the atrioventricular groove were serial sections for 5 μm made. Standard HE staining was used for the histomorphological evaluation.

At the end of reperfusion, we cut fresh myocardium tissue (1 mm³) off from about the middle of the apex and the ligation point, then prefixed with 2.5% glutaraldehyde and stored at 4 °C until processed. The post-fixture was accomplished using 1% osmium tetroxide. Then, after dehydration with gradient ethanol, we dipped the specimens in propylene oxide, and embedded with epoxy resin. Afterwards, we made them into ultrathin sections (0.1 μm). Finally, we stained the sections with lead-uranium and observed the changes in the ultrastructural organization by a TEM (HITACHI-S3400 N, Japan).

We used the Kyoto Encyclopedia of Genes and Genomes (KEGG) [26] for the pathway analysis to explore the potential role specific genes played in certain biological processes. We imported the differential expression genes between the EXD group and OVX group into the KEGG and the top pathways (P value≤0.01) associated with myocardial morphology or functions were observed.

We purified total RNA with the help of an RNeasy Mini Kit (Qiagen), then reverse-transcribed 4 μg of RNA to cDNA with the help of the Superscript First Strand synthesis system (Invitrogen). Afterwards, with the help of an ABI-7500 Sequence Detection System (Applied Biosystems), we performed qRT-PCR amplification with SYBR-green according to the instructions, which could detect PCR products in real time. Initiation of the PCR program was at 95 °C for 10s before 40 thermal cycles, then at 95 °C for 5 s and at 60 °C for 34 s. The data were analyzed in accordance with the 2^−△△Ct method and in each sample, the results were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (Gapdh). To guarantee the purity of the amplification products, we created melting curves for each of the PCR reactions. Each of the experiments was matched with a no-template negative control.

Figure 1 shows that the SHAM rats weighted remarkably less compared with the OVX rats. Both E2 and EXD could remarkably inhibit weight gain induced by OVX during the twelve-week treatment.

Compared with the SHAM group, OVX caused remarkable atrophy of the uterus, indicating a successful operation. The administration of E2 or EXD remarkably relieved the atrophy of uterine tissue compared with the OVX rats (Fig. 2).

Figure 3 shows the serum concentrations of estradiol in different rats after treatment for twelve weeks. After the 12-week treatment, the levels of estradiol in the SHAM rats were remarkably higher in comparison with the OVX rats (p < 0.01). In addition, the levels of estradiol in the EXD and E2 rats were remarkably lower in comparison with the OVX group rats.

Table 1 shows the effects of EXD on the ventricular action potential variables of model rats after treatment for twelve weeks. Compared with the SHAM group, OVX caused remarkable reductions in APD₅₀ and APD₉₀, and E2 or EXD could inhibit the reduction in APD₅₀ significantly.

A histopathological evaluation of ventricular samples from all groups was performed to explore the structural basis leading to the observed changes in APs. In the myocardium of SHAM rats, the cell alignment was normal and no obvious damage was detected (Fig. 4a). However, marked irregular cell arrangement, degeneration of ventricular myocytes, ruptured myocardial fibers, cytoplasmic vacuolization, and mild myocardial fibrosis were observed in OVX group rats (Fig. 4b). Upon E2 or EXD treatment, some mitigation of these events in the myocardial structure was observed in the rats (Fig. 4c-d).

The ultrastructures of cardiomyocytes from the SHAM rats demonstrated as normal (Fig. 5a), while the ultrastructures of cardiomyocytes from the OVX group rats demonstrated marked damages, which appeared as swollen and damaged mitochondria, intracellular edema, and myofibrillar derangements and ruptures (Fig. 5b). In contrast, significant decreases were observed in multiple characteristics of cardiomyocyte ultrastructural damages in the E2-or EXD-treated rats (Fig. 5c-d). All these features were ameliorated following EXD administration.

The array data turned out that the expressions of 185 genes were changed (≥2-fold, P value≤0.05) between the ventricular muscles of the EXD and OVX rats. More precisely, 28 genes were up-regulated and 157 genes down-regulated (see Additional file 1).

Based on the differential expression genes, we retrieved the top pathways (P value≤0.01) associated with myocardial morphology or function (Table 2) using the KEGG database. No pathway associated with down-regulated differentially expressed genes was found.

To validate the differential expression genes, we chose 7 genes, including myosin light chain 3 (Myl3), myosin heavy chain 7 (Myh7), integrin subunit beta 5 (Itgb5), titin (Ttn), actinin alpha 2 (Actn2), catenin alpha 3 (Ctnna3), and lymphoid enhancer binding factor 1 (Lef1), which were involved in significant pathways in Table 2 and were associated with myocardial morphology or function. The primers of genes were listed in Table 3. The changes of these genes determined by qRT-PCR are shown in Fig. 6. In general, the results from qRT-PCR of all cases were consistent with the results identified in microarray analysis.