ANIMAL AND GROUP
All animal experiments were approved by the animal care and use committee of Henan hospital of Chinese Medicine. (PZ-HNSZYY-2019-021) one hundred male wistar rats(4-week old) were purchased from Beijing Charles River experimental animal technology Co., Ltd༈Beijing,China༉. The rats were randomly divided into the normal group (n = 10) and the DCM group (n = 90). All of them were allowed free access to food and water. DCM group received a gavage of furazolidone (Tianjin Lisheng, China) solution (50 mg/ml, 0.3 mg/g for 8 weeks). Left ventricular end-diastolic diameter (LVEDD) and ejection fraction (LVEF) was measured by echocardiographic examination to confirm the model successful or not[18, 19]. The successful DCM rats (n = 62) were randomly divided into 5 group, including model, KXYX high, KXYX medium, KXYX low and captopril groups. The daily gavage of KXYX was 3.6 g / kg, 1.8 g / kg, 0.9 g / kg, respectively. captopril group was 10.125 mg / kg (Shanghai Sine, China). normal group and model group were given the same amount of normal saline. The indicated treatment was administered orally daily for 4 weeks after furazolidone gavage.
Preparation of KXYX
The KXYX contains the following Chinese medicines: ginseng (Panax Ginseng C. A. Mey) 12 g, astragalus (Astragalus membranaceus Fisch. Bge.)30 g, poria cocos (fungus nucleus of Poria cocos Schw.)15 g, salviae miltiorrhiza(Salvia miltiorrhiza Bge. Labiatae)15 g, atractylodes rhizome (Atractylodes macrocephala Koidz.)15 g, herba lycopi (Aconitum gymnandrum Maxim.) 15 g, leonurus (Leonurus japonicus Houtt)15 g, ophiopogonis ༈Ophiopogon japonicus (Linn. f.) Ker-Gawl.༉12 g, cimicifugae Rhizoma (Cimicifuga foetida L. ) 9 g. Concentrated granule made from nine Chinese medicinal herbs mentioned above. The batch numbers of the above Chinese herbal formula granules are 18080058, 18060154, 18090064, 18050094, 18040048, 18060048,18010423,18720032,18510233. All herbal materials, dispensing granules, and quality control data of HXWTF were supplied by Sichuan Neo-Green Pharmaceutical Technology Development Co., Ltd (Sichuan, China).
Rats were anesthetized with intraperitoneal injection of 1% pentobarbital sodium (30 mg/kg). Cardiac function and dimensions were measured by echocardiography with an Acuson Cypress system and a 7L3 probe (Siemens, Germany) after 4 weeks of KXYX treatment for rats, M-mode echocardiography was used to measure the left ventricular end-diastolic diameters (LVEDD)and left ventricular end‐systolic diameters(LVESD) on the parasternal long axis views. Next, ejection fraction (EF)and fractional shortening (FS) were calculated. EF was calculated by the Teichholtz method. The mean values were obtained from at least three different cardiac cycles.
H&E and masson staining
After echocardiography examination, the hearts were removed by thoracotomy, the remaining blood was washed in pre-cooled PBS, and fixed in 4% paraformaldehyde solution for 48 hours. After dehydration, heart tissue specimens were embedded in paraffin, and cut into5 µm sections. Hematoxylin eosin staining (HE) and Masson staining were performed according to the standard protocol. The myocardial collagen deposition in each group were observed under optical microscope. 5 fields were randomly selected to observe and record the images.
The ventricles of neonatal rats were cut into pieces under aseptic conditions, digested into single cells in 0.1% collagenase, and collected every 5 min. After centrifugation, all the cells were cultured in DMEM (DMEM, Gibco, USA) medium with 10% calf serum (Gibco, Australia), incubated in incubator of 5%CO2 at 37℃ (Thermo Fisher Scientific, USA) for 24 h, and myocardial fibroblasts (CFs) were obtained with differential attachment method. Passage as cells grow to a near-fused state. The adherent CFs were spindle-shaped, transparent cytoplasm, large oval shape nucleus under the microscope. the cultured cells identified by SP staining with vimentin. Fibroblasts of passage two were inoculated into a 6-well plate and cultured for 24 h, and the serum containing KXYX or fasudil were added to the culture for 2 h, washed with PBS, and then treated with Ang II for 24 h, and then samples were collected for further test.
Preparation of medicated serum
SD Rats weighing 220 ± 10 g were chosen and randomly divided into KXYX group (KXYX serum) and normal group (normal serum). Rats in KXYX groups were treated gavage with a KXYX solution at a dosage of 1 ml/100 g/day (n = 10) while the normal group received the same volume of normal saline (n = 10) for 10 days. The blood from abdominal aortic were collected one hour after the final treatment. After that, centrifuged at 3500 rpm for 15 min, and retained the supernatant. Serum was heated at 56 °C for 30 min, filter with a 0.22 µm filter membrane, stored at − 80 °C.
The fibroblasts were fixed in 4% paraformaldehyde for 30 min at room temperature, washed three times with PBS, and permeabilized with 0.3% Triton X-100 for 30 min. After blocking with BSA at room temperature for 30 minutes, slide the slides with anti-αSMA, Vimentin antibodies (Wuhan proteintech Biotechnology Co., Ltd.,) at After overnight incubation at 4℃, washing 3 times with PBS, the slides were incubated with FITC fluorescent secondary antibody (Wuhan proteintech Biotechnology Co., Ltd.,) at 37 ° C, protected from light for 2 h. After counter-staining the nuclei with DAPI, the images were observed and recorded using a fluorescence microscope.
Fibroblasts were grown in 96-well plates, and the cell density in each well was controlled at 1 × 104 cells / well for 24 hours. After that, each well was given different concentrations of KXYX (0–1.0 mg / ml) and cultured for 24 hours. 10 was added to each well. ul of CCK-8, and then detect the absorbance at 450 nm in each well to calculate the growth and viability levels of KXYX cells at different concentrations. The optimal intervention concentrations of angiotensin II and fasudil were measured in the same way.