Cells, viruses, compounds and antibodies
Non-PCV-infected pig kidney epithelial cell lines (PK-15) was cultured, passaged, and frozen in liquid nitrogen.
The PCV2-SH strain was gifted by Professor Jiang Ping of Nanjing Agricultural University. PK-15 cells were used for virus propagation. 106.4 TCID50/mL of virus titer was determined by IFA.
Matrine and Osthole were purchased with a defined plant origin, chemical structure, concentration, and specific biological activity. Matrine (98.7% purity) was purchased from the National Institutes for Food and Drug Control (lot no. 110805-201709). Osthole (98.0% purity) was purchased from Nanjing Zelang Biotechnology Co., Ltd, China (lot no. ZL20171212SCZS). Ribavirin with 99.0% purity was purchased from Beijing Solarbio Technology Co., Ltd, China (CAS: 36791-04-5). The chemical structures of Matrine and Osthole are shown in Fig. 1.
Antibodies against Cap were respectively purchased from Biorbyt LLC. (San Francisco, CA, USA). GRP78, PERK, eIF2ɑ, p-eIF2ɑ, ATF4, CHOP, Bcl-2, Bax and cleaved caspase-3 were purchased from Abcam (Cambridge, MA, USA). Cleaved caspase-9 and p-PERK were purchased from Bioworld Technology Co., Ltd. (Beijing, China) and Immunology Biology Technology Co., Ltd. (Beijing, China), respectively; Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and horseradish peroxidase-conjugated secondary antibodies were purchased from Wuhan Sanying Biology Technology Co., Ltd.(Wuhan,China) and ComWin Biotech Co., Ltd. (Beijing, China) , respectively.
Cytotoxicity assay
PK-15 cells were seeded onto a 96-well plate at 1×106 cells/mL and cultured with Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Waltham, MA, USA) containing 10% fetal calf serum (FCS; 10% DMEM). When cell confluency reached 80%-90%, the proper concentration of compounds was added. Osthole was dissolved in 1% dimethyl sulfoxide (Solarbio, China), and then diluted in DMEM containing 2% FCS (2% DMEM), and eight dilutions of the compounds in twofold serial dilutions were prepared. Ribavirin (positive control) was dissolved in 2% DMEM and then prepared in serial dilutions. In this laboratory, Sun et al. [27] confirmed that 0.5 mg/mL of Matrine has an anti-PCV2 replication effect in PK-15 cells. Keeping this in view, the maximum dose of Matrine was set to 0.5 mg/mL in the study. The dilution of Matrine combined with Osthole is shown in Table 1. The equipment used for cytopathic image acquisition was Olympus IX81 (objective lenses: LUCPLANFLN20X/PH1). These micrographs were photographed at a resolution of 4080 ´ 3072 with the cameras of DP71, and the image acquisition was obtained using CELLsens software, which enhanced the images to 300 dpi. The halogen lamp used for transmitted light Phase- contrast imaging was 12V, 100W.
Cell were cultured for 60 h, 100 μL of the fresh DMEM with 10% Cell Counting Kit-8 (Boster, Wuhan, China) was added and cells were then incubated for another hour at 37 °C. The OD at 450 nm was measured using a microplate reader (Spectra Max M5, Molecular Devices, San Jose, CA, USA). The cytopathic ratio was calculated based on the OD value with the following formula: [(A - B)/A × 100], in which A and B were the OD value of control and treated cells, respectively. Then, the maximum non-cytotoxic concentration (MNTC) and CC50 of PK-15 cells were calculated using GraphPad PrismTM 5.0 (GraphPad, Inc., LaJolla, CA, USA).
qRT-PCR
The PCV2 propagation was determined by qRT-PCR. When cell confluency reached 80%-90% in the 24-well plate, cells were infected with 104.4 TCID50 of PCV2 for 2 h. The virus was removed from the wells and the wells were washed with PBS for two times, following by addition of 2% DMEM. Cells were incubated for 6, 12, 24, 48, 72 and 96 h. Samples were collected and the DNA was extracted from the PK-15 cell. The copy number of the Cap gene was detected by qPCR. Primers 5¢- TAC ATT TCC AGC AGT TTG and 5¢- CTC CCG CCA TAC CAT AA were used to amplify the PCR products with 148 bp.
The inhibition of PCV2 replication by compounds was also detected by qPCR. The proper concentration of Matrine, Osthole, Matrine combined with Osthole, and Ribavirin was added to the cells after PCV2 infection for 2 h. DNA was extracted and the Cap gene was detected after incubation for 48 h. The concentrations used are shown in Table 2.
IFA
Cells were infected with 104.4 TCID50 of PCV2 for 2 h. Then, Matrine (0.5 mg/mL), Osthole (0.01 mg/mL), Matrine combined with Osthole (0.5+0.01, 0.25+0.01 and 0.125+0.01), and Ribavirin (0.5 mg/mL) were added. Cells were cultured for 48 h at 37℃. A mixture of ice-cold acetone and methyl alcohol (1:1) was used to fix the cells for 30 min at −20 °C and cells were washed twice with phosphate-buffered saline (PBS). PCV2 IFA kit (China Institute of Veterinary Drug Control, Beijing, China) was used to stain the cells. The specific information on IFA image acquisition and processing was the same as the cytopathic test. Besides, fluorescein isothiocyanate (FITC)-labeled cells were sequentially excited with a WU excitation block and a WB excitation. Mercury lamp was used for reflection fluorescence imaging.
Annexin V/propidium iodide (PI) staining for apoptosis
Different concentrations of Matrine combined with Osthole (high, medium, and low) were added after infection with 104.4 TCID50 of PCV2 for 2 h. Cells were cultured for 48 h and then collected for staining with Annexin V-FITC/PI detection kit (Keygen Biotech, Nanjing, China). The samples were analyzed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).
Western blotting
The anti-PCV2 activity and antiapoptotic mechanisms of the combined compounds were determined by Western blotting. Cells in six-well plates were infected with 104.4 TCID50 PCV2 for 2 h and then treated with the compounds for 48 h. The total protein was extracted and was determined using the BCA protein concentration detection kit (Beyotime Biotechnology, Jiangsu, China). The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked for 2 h with 5% skim milk and then incubated with the primary antibody at 4°C overnight. After washing three times with TBS-Tween 20 (TBST), the membrane was incubated with the secondary antibody at room temperature for 1.5 h and then the bands were detected using the eECL Western Blotting detection kit (Cwbio, Beijing, China) and chemiluminescence imaging system (BIO-RAD, Hercules CA, USA).
Effects of the drugs combination on MMP detected by JC-1
Cells in six-well plates were incubated with 104.4 TCID50 of PCV2 for 2 h and then treated with the compounds for 48 h. The harvested cells were treated with JC-1 MMP detection kit (Beyotime Biotechnology, Jiangsu, China) and analyzed by flow cytometry.
Cap recombinant plasmid construction, cell transfection, and its mechanism on apoptosis as analyzed by Western blotting
The optimizing Cap gene sequence was inserted into plasmid pCDNA3.1 at the KpnI and XhoI cutting sites. The plasmid was transformed into the Escherichi coli. Bacteria with the right Cap sequence were used for the plasmid extraction with GoldHi EndoFree Plasmid Maxi Kit (CW2104M, Cwbio, China). The plasmid concentration was determined using a biological mass spectrometer (D30, Eppendorf, Hamburg, Germany).
When cells confluency in six-well plates reached 70%-90%, the pCDNA3.1-Cap (p-Cap) and pCDNA3.1-vector (p-vector) alone were transfected using 7.5 μL Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), respectively, and 2.5 μg p-Cap were mixed with 125 μL Opti-MEM. The mixture was incubated for 15 min at room temperature. Then, 50 µL of the mixture were added dropwise to the cells, and cells were incubated for 6 h. Matrine combined with Osthole were added and cultured for 48 h, p-vector and Ribavirin were used as control. The proteins expression levels were analyzed by western blotting for the Cap protein and the key apoptins of the PERK pathway.
Statistical analysis
CC50 was calculated using nonlinear regression. The results of ‘log (inhibitor) vs. response-variable slope’ and the data of generated by qPCR, IFA, Western blotting, Annexin V-FITC/PI, and JC-1 were all analyzed by GraphPad PrismTM 5.0. ImageJ (National Institutes of Health, Bethesda, MD, USA) was used to measure the grey intensity of protein bolts. All data are expressed as the mean ± standard error of the mean (SEM) of at least 3 repeated experiments. The statistical significance threshold was set at p<0.05.