Clinical case presentation- acute coronary syndrome (ACS) combined with ET
A 58-year-old woman was admitted to the hospital because of sustaining chest pain for 8 hours, she had a history of stable angina for 5 years and was diagnosed essential thrombocythemia 2 years ago, but she didn’t receive any treatment in this duration. Biochemical examination showed blood platelet level at 1140 × 1012/L (normal range at 125–350 × 1012/L), and myocardial injury markers and other biomarkers were at normal ranges. Electrocardiogram showed inverted T wave in V1-V6 Leads. The patient was diagnosed ACS and underwent a coronary angiography (CAG) as soon as admitted, result showed a severe diffuse stenosis in proximal left anterior descending coronary artery (LAD) and moderate stenosis in distal right coronary artery (RCA)(Fig. 1). After consultations with hematologists and cardiac surgeon, the patient underwent coronary artery bypass grafting (CABG).
After the surgery, she started oral medical treatment of ET and ACS using aspirin 100 mg QD, atorvastatin 20 mg QD and hydroxyurea 100 mg BID and have herself a reexamination once in a while.
The data and information of the case was approved by the institution ethnic committee of the First Hospital of the Jilin University and was published with the patients’ consent.
Chemicals and Reagents
Atorvastatin calcium, aspirin, hydroxyurea (purity > 98%) for animals were purchased from Solarbio Biotechnology, Co., Ltd. (Beijing, China). Clopidogrel bisulfate (purity > 98%) was provided by the TCI chemical industry development co., Ltd (Shanghai, China). Berberine (98%) was obtained from J&K scientific Co., Ltd. The saturated oil red O solution, OCT frozen section embedding agent, 4% tissue cell fixation solution and hematoxylin dye solution were purchased from Solarbio Biotechnology. Co., Ltd (Beijing, China). Deionized distilled water was obtained from Hangzhou Wahaha Group Co. Ltd. (Hangzhou, China). Chromatography grade methanol and isopropanol were obtained from Thermo Fisher Scientific, Co., Ltd. (Fair Lawn, NJ, United States). Other chromatographic reagents were obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Serum glucose (Glu), triglycerides (TG), total cholesterol (CHO) and low-density lipoprotein cholesterol (LDL-C) kits were purchased from Biosino Bio-Technology & Science Inc. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine kits were obtained from Nanjing Jiancheng Bioengineering Institute. Proprotein convertase subtilisin/kexin type 9 (PCSK9) ELISA kits were purchased from Abcam Co., Ltd. (Cambridge, MA).
Eight 8-Week old wild-type (C57BL/6) and forty eight Apolipoprotein E knockout (ApoE−/−) mice (8 weeks) were acquired from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animals were housed in the SPF-grade rooms with a temperature of 22–24 ℃, a humidity of 45%, and a 12-h light/dark cycle (light time from 8:00 to 20:00). All the mice had free access to food and water during the treatment. All experiments were conducted in accordance with institutional and ethics guidelines and were approved by the Laboratories Institutional Animal Care and Use Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College (No. 00001020).
Grouping and medication
The grouping rules were as following: wild-type C57 mice were used in normal control groups and ApoE-/- mice were used in atherosclerosis model and medication groups. After acclimatization with normal diet for a week, the model groups and medication groups were fed with high-fat diet (HFD: 21% fat, 0.15% cholesterol and 78.85% regular diet) for 4 weeks, then, all the groups were treated with saline or drugs for 8 weeks for efficacy study. The normal control groups were fed with normal diet. Fasting serum and aortic vessels were obtained after experiment. Six aortic vessels in each group were preserved for pathological staining and the rests were preserved for aortic full-length staining.
All the mice were separated into 7 groups: (1) The normal control group: normal diet and normal saline (oral, 0.2 mL); (2) Atherosclerosis model group: high-fat diet and normal saline (oral, 0.2 mL); (3) Dual antiplatelet therapy group: high-fat diet, aspirin (oral, 5 mg/kg/day), and clopidogrel bisulfate (oral, 25 mg/kg/day); (4) Low-dose HU therapy group: high-fat diet, aspirin (oral, 5 mg/kg/day), clopidogrel bisulfate (oral, 25 mg/kg/day), and HU (oral, 10 mg/kg/day); (5) High-dose HU therapy group: high-fat diet, aspirin (oral, 5 mg/kg/day), clopidogrel bisulfate (oral, 25 mg/kg/day), HU (oral, 20 mg/kg/day); (6) Positive medicine group: high-fat diet, aspirin (oral, 5 mg/kg/day), clopidogrel bisulfate (oral, 25 mg/kg/day), and atorvastatin calcium (oral, 5 mg/kg/day); (7) Combined medicine treatment group: high-fat diet, aspirin (oral, 5 mg/kg/day), clopidogrel bisulfate (oral, 25 mg/kg/day), atorvastatin calcium (5 mg/kg/day), and HU (oral, 10 mg/kg/day).
Oil red O staining
The proximal aorta that attached the heart was used for cross-sections. The collected aortic tissue was fixed in 4% paraformaldehyde for 24 hours, and dehydrated overnight. Then, the tissue was embedded in the embedding agent OCT to prepare frozen slices (5 µm) under − 20℃. Saturated oil red O solution that dissolved in isopropanol was mixed with distilled water by a ratio of 3: 2 (V: V). After filtration, the frozen slices were stained in the diluted oil red O working solution at room temperature for 6 hours. Then, rinse the slices in 60% isopropanol-water solution for 3 times and once in running water. The slices were restrained in hematoxylin solution for 1–2 min and washed by running water. The dyed slices were sealed by neutral gelatin, photographed and recorded by microscope system (Leica, Germany).
Hematoxylin-eosin (H & E) staining
The aortic tissue was fixed in 4% paraformaldehyde for 24 hours after collected and dehydrated for 16 hours by an automatic dehydrator. After routine embedded with paraffin in automatic embedding machine, cut the tissue into sections (5 µm). Then, place the sections in an oven (60℃) for 1 hours and dewax by xylene, ethanol and water. The sections were further stained by hematoxylin for 10 minutes and placed under running water to remove residual color. 5% acetic acid was used to differentiate, and then stained by eosin. Finally, seal the sections with neutral gelatin after dried and captured by the microscope system. The corrected plaque areas were calculated by: Area of plaques / Area of vascular × 100% in the corresponding cross section. The statistical results of corrected plaque area were the average values of the plaque area obtained by the oil red O staining and H&E staining of each mouse.
Full length staining of aorta by oil red O
The aortic tissue that attached the heart of the animals were removed after sacrifice, and dehydrated in a 20% sucrose solution overnight. After rinsed with PBS solution, remove the excess tissue on the aorta under the microscope. The tissue was stained for 6 hours at room temperature in oil red O working solution. Then, the plaques were stained red. After that, the stained aorta was taken out, and rinsed for 3 times in 60% isopropanol-water. Soak the tissue in PBS, cut the blood vessel longitudinally and pin it flat on a black silicone pad. The image of the aorta tissue was record by camera (Canon EOS 700D, New York, USA).
Serum and Plasma analysis
Serum and plasma samples were collected from fundus venous plexus and the tubes for collecting plasma were pretreated by heparin sodium. Serum glucose (Glu), triglycerides (TG), total cholesterol (CHO) and low-density lipoprotein cholesterol (LDL-C) were measured according to the instructions of kit. Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine in plasma were determined by test kits and the experiment steps referred to the instructions. Analysis of proprotein convertase subtilisin/kexin type 9 in plasma was performed by ELISA kits according to the instructions.
Culture of hepG2 cells
HepG2 cells (European collection of cell cultures, Wiltshire, UK) were cultured in dulbecco's modified eagle medium (DMEM, Gibco), containing penicillin, streptomycin, and 10% fetal calf serum (Gibco) in 37 °C (with 5% CO2). After culturing overnight, vehicle, berberine (20 µg/mL) or increasing concentrations of (10, 20 and 50 mM) of HU were added to the culture media and maintained for 16 h. The concentrations of HU were defined by MTT results. Then, the cells and media were collected into tubes and centrifugation (2, 000 g × 10 min), the supernatant was used for the analysis of PCSK9. For the analysis of PCSK9, the ELISA kits were used.
All the data were analyzed by Graphpad Prism 5.0 version and presented as the means ± standard deviation (SD). The atatistical analyses were conducted using two-way Student’s t-test and P values less than 0.05 were considered statistically significant.