CARDIOVASCULAR JOURNAL OF AFRICA • Volume 33, No 5, September/October 2022 AFRICA 229 β-catenin pathway.18-20 Whether PRELP increases myocardial fibrosis post AMI by the wnt/β–catenin signalling pathway remains undetermined. In our study, we determined PRELP’s role in myocardial fibrosis post AMI and the underlying mechanisms involved in the process. Methods Male SD mice, aged eight weeks, were subjected to MI by ligation of the left anterior descending coronary artery, as previously described.21 After the mice were anaesthetised, the left anterior descending coronary artery was ligated. Mice in the sham group were also anaesthetised and their hearts were exposed. However, ligation of the coronary artery was not performed in this group. PRELP over-expressing lentiviral vector and PRELP interference-expressed lentiviral vector were generated by Genechem Corporation (Shanghai, China). All mice (n = 60) were randomly divided into four groups: SHAM, MI, MI + PRELP shRNA, and MI + PRELP over group. In the MI + PRELP over group, mice were injected with 1 × 106 PRELP over-expressing lentiviral vector in the myocardium around the ligation point. Additionally, 1 × 106 PRELP interference-expressed lentiviral vector was injected into the myocardium around the point of ligation of the mice in the MI + PRELP shRNA group. All experimental protocols were carried out in accordance with guidelines. All procedures were under approval from the animal care and use committee of Shandong University Qilu Hospital. The cardiac fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% foetal bovine serum, 100 units/ml penicillin and 100 mg/ml streptomycin at 37°C with 95% air and 5% CO2. To identify the fibroblasts, microscopy and immunofluorescence analysis were used. The cells were also randomly divided into four groups: SHAM, MI, MI + PRELP shRNA, and MI + PRELP over group. To mimic the ischaemic conditions, the cells were cultured in DMEM without foetal bovine serum (FBS), and with 0.1% O2 for 24 hours. PRELP over-expressing lentiviral vector was transfected into the cells in the MI + PRELP over group. PRELP interference-expressed lentiviral vector was transfected into the cells in the MI + PRELP shRNA group. Transfection efficiency was assessed by Western blotting, immunohistochemical staining and real-time polymerase chain reaction (PCR). The left ventricular internal diameter in diastole and the left ventricular internal diameter in systole of all the mice were measured with motion-mode (M-mode) echocardiography. Left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) were automatically calculated according to the formula. The heart tissues and the cells were lysed in RIPA buffer. The concentration of total proteins for each sample was quantified using a BCA kit, and 30 μg of each sample was electrophoresed in SDS-PAGE gel and transferred onto polyvinylidene difluoride membranes. After blocking with 5% fat-free milk at room temperature for one hour, the membranes were incubated with primary antibodies overnight at 4°C on a refrigerator shaker. All primary antibodies were dissolved in a 5% solution with a dilution of 1:1 000. Anti-wnt1 antibody (ab15251), anti-PRELP antibody (ab229719), anti-GSK3β antibody (ab93926), antiMM9P antibody (ab228402), anti-β-catenin antibody (ab6302), anti-c-myc antibody (ab32072) and anti-TIMP-1 antibody (ab81282) were purchased from Abcam Biotechnology. AntiGAPDH antibody (200306-7E4) was purchased from Zen Bioscience. Next, the membranes were washed three times at room temperature. Following the addition of the anti-rabbit (or antimouse, anti-goat) secondary antibodies (1:5 000, A0208, A0216, A0181, Beyotime), the membranes were incubated for two hours, followed by three washes at room temperature. Finally, protein expressions were visualised by an enhanced chemiluminescence system. The myocardial tissues were paraffin-embedded, sectioned and dewaxed. The cardiac fibroblasts were fixed in 4% paraformaldehyde for 10 minutes, and then treated with 0.5% Triton X-100 for 10 minutes. Next, they were incubated with primary antibodies overnight at 4°C. After three phosphatebuffered saline (PBS) washes, the slices were incubated with secondary antibodies for 50 minutes at 4°C. This was followed by three PBS washes. Finally, the sections were counterstained with haematoxylin to observe the cellular nuclei and were viewed under a microscope. The heart was sectioned into 1-mm-thick transverse slices and stored at –20°C for 20 minutes. The slices were then incubated with 1% triphenyltetrazolium chloride solution (TTC) at 37°C. The viable tissues stained red, while the infarcted tissues appeared pale. The percentage of infarcted area was quantified using image analysis software (Image-Pro Plus). The heart tissues were fixed, dehydrated and then sectioned into 4-μm sections. After they were dewaxed, hydrated and stained with haematoxylin and eosin (HE), and Sirius red (SR), the slices were dehydrated by gradient ethanol and finally viewed under a microscope. The total RNA content was extracted from cells using Trizol reagent. The RNA was quantified by a Nanodrop One, and the obtained RNA (0.6 μl) was subjected to reversetranscription reaction using the RT reagent kit. Real-time PCR of cDNA was done using a SYBR Premix Ex Taq kit, with GAPDH as the internal control. The primer sequences of PRELP were CTTCTGGTTCCTTCCACTTCTC (forward) and GGCCTTGGCTTGGGTTTA (reverse), and the primer sequences of GAPDH were GGGAAACCCATCACCATCTT (forward) and CCAGTAGACTCCACGACATACT (reverse). Results were calculated based on the 2Ct −ΔΔ method. Statistical analysis The experimental data for the three separate experiments are represented as mean±standard deviation (SD). SPSS 23.0 was used for statistical analyses; p-values < 0.05 were considered statistically significant. The t-test was used to compare the data between the two groups. Results As shown in Fig. 1, the ELISA assay, Western blotting and immunohistochemical staining analysis showed that the PRELP expression in the myocardial tissues was upregulated following AMI compared to that in the control mice. The PRELP expression in the myocardial tissues was upregulated in PRELP
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