PART ONE Phenylethanol Glycosides Protect Myocardial Hypertrophy Induced By Abdominal Aortic Constriction Via ECE-1 Demethylation Inhibition And PI3K/PKB/eNOS Pathway Enhancement
Mar 06, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
Qiong-Ling Fan,1 Jia-Wei Wang,1 Shi-Lei Zhang, & et al.
Phenylethanoild glycosides (CPhGs) are the core material basis of pharmacological activity in Cistanche tubulosa and have a variety of pharmacological effects. However, it is unclear whether CPhGs(Phenylethanoild glycosides from cistanche) have an ameliorative effect on pressure overload-induced myocardial hypertrophy. In this study, male SD rats weighing (200 ± 20) g were established cardiac hypertrophy models by abdominal aortic coarctation (AAC). After the operation, the rats were gavaged with corresponding medicine for 6 weeks (CPhGs(Phenylethanoild glycosides from cistanche) 125, 250, and 500 mg/kg/d and valsartan 8.3 mg/kg/d). Echocardiography, heart weight index (HWI), cross-sectional area of cardiomyocytes (CSCA), fibrosis area, plasma endothelin 1(ET-1), and proinflammatory factors levels were detected. Our results showed that different CPhGs(Phenylethanoild glycosides) dosage decreased left ventricular posterior wall thickness (LVPWT), left ventricular end-diastolic diameter (LVED), HWI, CSCA, fibrosis area, ET-1, proinflammatory factors, arterial natriuretic peptide (ANP), brain natriuretic peptide (BNP), endothelin converting enzyme 1(ECE-1) mRNA levels, cyclooxygenase 2 (COX-2), high mobility group box 1 (HMGB-1) protein levels, and ECE-1 demethylation level while increasing left ventricular ejection fractions (LVEF), left ventricular fractional shortening (LVFS), phosphorylated phosphatidylinositol 3-kinase (p-PI3K), phosphorylated protein kinase B (p-PKB), and phosphorylated endothelial nitric oxide synthetase (p-eNOS). -e indexes of CPhGs(Phenylethanoild glycosides from cistanche) 250 and 500 mg/kg group were significantly different from AAC group; compared with the valsartan group (AV), the indexes of CPhGs 500 mg/kg group were not significantly different. In conclusion, CPhGs(Phenylethanoild glycosides from cistanche) ameliorated myocardial hypertrophy rats by AAC, which may be related to ECE- 1 demethylation inhibition and PI3K/PKB/eNOS enhancement.
2. Materials and Methods
2.1.Animals.
70 male SPF SD rats weighed 200 ± 20 g (10 per group) were provided by the Experimental Animal Centre of Xinjiang Medical University (Urumqi, China). The animal production license number was SYXK(XIN)2016-0003 and the certificate number was SCXK(XIN)2016-002. -e study was approved by the Animal Ethics Committee of the First Affiffiffiliated Hospital of Xinjiang Medical University (approval number 20180223-183). -ey were maintained with free access to water and food in plastic cages and kept in a 12 h light/dark cycle at a temperature of 22 ± 2°C and humidity of 45–55% in our Experimental Animal Centre.
2.2. Materials.
CPhGs(Phenylethanoild glycosides from cistanche) , whose content of echinacea was more than 40% and acteoside was more than 16%, was purchased from Hetian Di Chen Medical Biotechnology Co., Ltd. Valsartan was purchased from Beijing Novartis Pharma Co., Ltd. Carboxymethylcellulose sodium (CMC-Na) was provided by Xian Zhengtian Pharmaceutical Accessories Co., Ltd. And CPhGs were dissolved in 0.5% CMC-Na.
Phenylethanoild glycosides from cistanche
2.3. Abdominal Aortic Coarctation (AAC).
After being anesthetized by 2.5% pentobarbital sodium (2.5 ml/kg) (Merck, Darmstadt City, Hessen, Germany), the rats were shaved and kept warm on heating pads. -en the abdominal cavity of the rat was opened for 2 cm in the middle of the abdomen in a sterile environment, the abdominal viscera were pushed to the right side, and the abdominal aorta was fully exposed at 0.5 cm above the right renal artery. Number 8 needle was ligated with number 2 wire in parallel to the abdominal cavity being sutured [18]. The abdominal aortas of rats in the sham group were separated without ligation and there was no control group for the surgery. -e day after the operation, AC 125 mg/kg, AC 250 mg/kg, and AC 500 mg/kg were fed with CPhGs(Phenylethanoild glycosides from cistanche) by gavage; AV group were fed with valsartan by gavage; and control group, AAC group, and the sham group were not fed with CPhGs or valsartan, once a day for 42 consecutive days. Drug dose was set on the basis of previous references and preliminary experiments [8].
2.4. Echocardiography.
Six weeks after AAC, the rats were anesthetized with 2.5% pentobarbital sodium and performed the echocardiography (Philips HD11 XE, Amsterdam, Netherlands). LVPWT, LVED, LVEF, and LVFS were measured four times to calculate the averages as the final indicators.
2.5. HeartWeight Index (HWI).
After echocardiography, the rats were executed by collecting blood from the abdominal aorta. -e hearts of rats were removed from the chest and weighed after being washed by normal saline (4°C) 3 times and drained with clean filter paper. -e HWI was calculated through the following formula: HWI � heart weight body weight. (1)
2.6. Hematoxylin-Eosin and Masson Staining.
-e myocardium from left ventricular was fixed by 4% paraformaldehyde embedded in parafin, crosscut with a section thickness of 4 μm, dried in an oven at 60°C for 2 hours, and then stained with hematoxylin-eosin or Masson (Solaibio, Beijing, China). -e structure of the myocardium was observed with an inverted phase-contrast microscope (Leica, Frankfurt, Germany). Image J software was used to calculate the CSAC and fibrosis area.
2.7. Enzyme-Linked Immune Sorbent Assay (ELISA).
Abdominal aortic blood was extracted from rats and placed in an anticoagulation tube for 2 h at room temperature and then centrifuged at 3000 r/min speed. -e supernatant was absorbed with pipettes (Eppendorf, Hamburg, Germany) and stored at 80°C for testing. Plasma ET-1, interleukin 1β(IL-1β), interleukin 6 (IL-6), tumor necrosis factor α Evidence-Based Complementary and Alternative Medicine
(TNF-α) (Bioss, Beijing, China), and cyclooxygenase 1 (COX-1) (Cusabio, Wuhan, China) levels of rats were detected according to ELISA kit instructions.
2.8. Methylation-Specific PCR.
DNA was extracted from the left ventricular myocardial tissue of rats by DNA extraction kit (Tiangen Biotech, Beijing, China). -e absorption value (A value) was equal to OD260/OD280 (1.8∼1.9). 20 μl genomic DNA (500 pg-1 μg) was modified with sulfurous acid by Methylation-Gold Kit (ZYMO, Orange County, CA, USA). -e MSP reaction system was 25 μL, including 3.0 μL of sulfite modified DNA template, 2.5 μL of 10 × buffer, 2.5 μL of 2.5 mm dNTPs, 1.0 μL of 10 μm primer, 0.1 μL of Hotstar Taq DNA polymerase (TAKARA Biotechnology), and 14.9 μL of ddH2O. -ere were 45 cycles in total: denaturation at 95°C, annealing at 55°C, and extension at 72°C for 30 s. After PCR, 5 μL product was separated by 2% agarose gel electrophoresis. -e DNA was treated by M. Sssl methyltransferase (New England Biolabs, Ipswich, MA, USA) was used as the positive control of methylation, and the DNA of the normal control group was used as the positive control of demethylation. Finally, the agarose gel was photographed and analyzed by a Bio-Rad gel imaging system. -e demethylation level was calculated through the following formula:
All primers designed in Methprimer (Applied Biosystems, MA, USA) and synthesized by Sangon Biotech (Shanghai, China) are listed in Table 1.
2.10. Western Blot.
Proteins were extracted from the left ventricular myocardium with Ripa Buffffer (Scientific) and protease inhibitor was added. Protein concentration was determined by the BCA method (Solarbio, Beijing, China). Proteins were taken at 50 μg and denatured at 100° C for 5 mins. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime, Shanghai, China) and then transferred to a polyvinylidene fluoride (PVDF) membrane by electrophoretic equipment (Bio-Rad, CA, USA). After being blocked with 3% BSA for 2 hours at room temperature, the membranes were incubated with anti-PI3K antibody, antiphospho-PI3K antibody, antieNOS antibody, anti-phospho-eNOS (1:1000, Bioss, Beijing, China), anti-COX-2 antibody, anti-HMGB-1 antibody (1: 1000, Proteintech, Chicago, IL, USA), and anti-ECE-1 antibody (1: 2000, Signalway Antibody, College Park, MD, USA), primary antibodies overnight at 4°C. The next day, after being washed with TBST 3 times, the membranes were incubated with HRP-labeled goat anti-rabbit IgG (1: 25000, 1 h, Zhongshanjinqiao, Shanghai, China) at room temperature. After being washed with TBST 3 times, the membranes were visualized by Pierce™ ECL Western Blotting Substrate and imaged using FluorChem E Imaging System (Protein Simple, USA), which were normalized to β-actin as an internal control.
The benefit of Phenylethanoild glycosides from cistanche
cistanche benefit
3. Results
3.1. CPhGs(Phenylethanoild glycosides from cistanche) Improved Cardiac Function in Rats after AAC.
-e rats received gavage from the day after AAC. -e positive control group (AV) was given gavage with valsartan 8.3 mg/kg/d, and the CPhGs group (AC) was given gavage with CPhGs 125, 250, and 500 mg/kg/d, respectively. After six weeks’ continuous dosing, the rats have performed echocardiography under 2.5% sodium pentobarbital anesthesia. Two rats died after AAC: one in the model group died on the third day after AAC from pulmonary edema; another one in AC 500 mg/kg died the day after surgery suspected to be caused by surgical stress. As shown in Figure 2, compared with the sham group, LVED, LVEF, and LVFS in the AAC group were decreased significantly but LVPWT in the AAC group was increased significantly. Compared with the AAC group, LVED, LVEF, and LVFS were increased and LVPWT was decreased to varying degrees in each AC dose group; for LVPWT, a significant change was not found in AC 125 mg/kg group but was found in AC 250 and 500 mg/kg groups. Compared with the AV group, the LVPWT and LVED in AC 125 mg/kg group were observed a significant difference, while LVPWT, LVED, LVFS, and LVFS in AC 250 and 500 mg/kg groups were not.
3.2. CPhGs(Phenylethanoild glycosides from cistanche) Decreased HWI, CSAC, and Fibrosis Area in Rats after AAC.
-en we weighed the heart of each rat to calculate the HWI and took the myocardial tissue for HE and Masson staining to observe the changes of the cross-sectional area (CSAC) and fibrosis area in cardiomyocytes. As shown in Figures 3(a), 3(d), and 3(e), compared with the sham group, the heart weight (HW) and HWI in the AAC group were reduced significantly; compared with the AAC group, HW and HWI in AC250 and 500 mg/kg groups were reduced significantly; compared with AV group, HW and HWI were increased significantly in AC 125 mg/kg group but were not in AC 250 and 500 mg/kg groups. In Figures 3(b) and 3(f ), compared with the sham group, the Masson staining results showed that the fibrosis area in the AAC group was increased significantly. For the fibrosis area, a significant decrease was found in AC 125, 250, and 500 mg/kg groups when compared to the AAC group, while significant increase in AC 125 and 250 mg/kg groups and a significant decrease in AC 500 mg/kg group were found when compared to AV group. In Figures 3(c) and 3(g), the HE staining results showed that CSAC in the AAC group was increased significantly in comparison with that in the sham group; compared with the AAC group, CSAC in AC 250 and 500 mg/kg groups was reduced significantly; compared with that in AV group, however, CSAC was increased significantly in AC 125 and 250 mg/kg groups, and no significant difference was found in the AC 500 mg/kg group
3.3. CPhGs(Phenylethanoild glycosides from cistanche) Reduced mRNA Levels of Myocardial Hypertrophy Genes in Myocardial Tissue of Rats after AAC.
ANP, BNP, and myosin heavy chain β (β-MHC) are important cardiac hypertrophy factors [19, 20]. We detected the relative expression levels of these three cardiac hypertrophy genes in the myocardial tissues. As shown in Figure 4, compared with the sham group, the relative expression levels of ANP, BNP, and β-MHC mRNA in the AAC group was increased significantly; however, compared with the AAC group, the relative expression levels of the three myocardial hypertrophy genes in AC 250 Evidence-Based Complementary and Alternative Medicine 5.
