Cells, viruses, antiboties and nature compounds
PCV-free pig kidney endothelial cell line (PK-15) was preserved in our laboratory. Cells were cultured and passed in Dulbecco's modified eagle's medium (DMEM, Hyclone, USA) containing 10% fetal calf serum (FBS, BI, Israel) (10% DMEM) and maintenance in DMEM containing 2% FBS (2% DMEM).
The strain of PCV2-SH (GenBank: AY686763.1) was gifted by Professor Jiang Ping of Nanjing Agricultural University. Virus was replicated and harvest in PK-15 cells infected with PCV2. The titer of 106.4 TCID50/ mL was determined by Indirect Immunofluorescence (IFA).
Antibodies against Cap were purchased from Biorbyt LLC. (California, Britain) cleaved caspase-3、Bcl-2 and Bax were purchased from Abcam (Cambridge, USA); GAPDH and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Wuhan Sanying Biology Technology Co.,Ltd.（Wu Han, China）.
Ribavirin, the positive control drug, was purchased from Beijing Solarbio technology co., LTD; Syringin was purchased from Nanjing Puyi biotechnology co., LTD; Glycyrrhizin aid was purchased from DOSF biotechnology co., LTD; Other tested compounds, such as Curcumol, Paeonol, Oxymatrine, Caffeic acid, Formononetin, Cepharanthine, Apigenin, Psoralen, Lcariine, Curcumin and Astragaloside were purchased from National Institutes for Food and Drug Control. Compounds information and chemical structure were respectively listed Table 1 and Figure 1.
The viability of PK-15 cell was assessed with MTT. 1.0 × 106 cells/mL of cells were seeded into 96-well plates and incubated until 100% confluency was reached. The tested compounds were added with two-fold serial diluted and cultured for another 60 h and cell control group was established. Four repeated wells were set up for each concentration with 100 µL per well. The cytopathic condition of cells was observed every day and photographed. The compounds and dissolution methods are shown in Table 1. Then 25 µL MTT (4 mg/mL in PBS) was added to each well and incubated at 37 ℃ for 4 h. 150 µL of DMSO (Solarbio, Beijing) was added and incubated for 30 min after removing the culture medium. The optical density (OD) were measured at 490 nm using a microplate spectrophotometer (Spectra Max M5, Molecular Devices, USA). Cytopathic ratio (CR) was calculated based on CR = [(OD control - OD test)/ OD] ×100. Then MNTC and CC50 on PK-15 cells were calculated using GraphPad PrismTM5.0 (Inc. California, USA).
Real time quantitative PCR
The anti-PCV2 effect of compounds in PK-15 cell was determined by real time quantitative PCR (qPCR). When cell confluency rate of 24-well plate reached 80%, 104.4 TCID50 of PCV2 was incubated for 2 h and discarded the supernatant. MNTC of every test compound was in two-fold serial dilution with 2% DMEM into 3 gradients and added to 24-well plates (500 μL/well) (Table 2). Meanwhile, the cell control group, PCV2-infected group and Ribavirin positive control group were applied and incubated for 48 h with 5% CO2 at 37 ℃. DNA was extracted according to the instruction of DNA extraction kit (TianGen, Beijing, China) and the DNA concentration was measured using a nucleic acid concentration analyzer (NanoDrop Technologies, Wilmington, DE, USA). The copy number of the Cap gene was detected by qPCR. The Primers 5’ TAC ATT TCC AGC AGT TTG and 5’CTC CCG CCA TAC CAT AA were used for the amplification of 148 bp fragment.
The anti-PCV2 effect and anti-apoptotic mechanism of the compound was detected by Western blot. When cell confluency of 6-well plate reached 80%, 104.4 TCID50 of PCV2 was incubated for 2 h and discarded the supernatant. Paeonol, Cepharaanthine and Curcumin were added in turn, 2mL/well. The cell control group, PCV2-infected group and Ribavirin positive control group were applied and incubated for 48 h. Total cell protein was extracted and protein concentration was determined using the BCA protein assay kit (Beyotime Biotechnology, Jiangsu, China). The protein samples were separated by SDS-PAGE, then transferred to the PVDF membrane. The levels of PCV2 Cap protein and apoptin were detected using an eECL Western Blot detection kit (Cwbio Inc, Beijing, China) and chemiluminescence imaging system (BIO-RAD, California, USA).
Annexin V/PI staining for apoptosis
The anti-apoptotic effect of Cepharaanthine and Curcumin were detected by Annexin V/PI. Cells in 6-well plate were incubated with 104.4 TCID50 of PCV2 for 2 h and discarded the supernatant, then Cepharaanthine and Curcumin were added, the cell control group, PCV2-infected group and Ribavirin positive control group were applied and incubated for 48 h. Samples were collected, centrifugated and treated using Annexin V/PI kit (Keygen Biotech, Nanjing, China), then analyzed by flow cytometry (BD Biosciences, USA).
Detection of MMP by JC-1
Mitochondrial membrane potential (MMP) of cells was detected by JC-1. Cells in the 6-well plate were incubated with 104.4 TCID50 of PCV2 for 2 h and discarded the supernatant, then Cepharaanthine and Curcumin were added. After 48 h incubation, cells were stained with JC-1 using a commercial kit (Beyotime Biotechnology, Jangsu, China) treated and analyzed by flow cytometry (BD Biosciences, USA).
Detection of ROS by flow cytometer assay
The amount of reactive oxygen species (ROS) was measured by flow cytometry. Cells in the 6-well plate were incubated with 104.4 TCID50 of PCV2 for 2 h and discarded the supernatant, Cepharaanthine and Curcumin were added and incubated for 48 h. Cells were treated with ROS assay kit and the changes of ROS were detected by Flow cytometer.
CC50 was calculated using nonlinear regression and the results of “log (inhibitor) vs. response-variable slope” were analyzed using GraphPad Prism. The gray intensity of protein bolts was analyzed by Image J (National Institutes of Health, USA). Data generated were analyzed using“Bonferroni: Compare all pairs of columns”in the GraphPad Prism TM 5.0 software for one-way ANOVA implemented (GraphPad Software, Inc. California, USA), and were expressed as the mean ± standard error of the mean (SEM) of at least 3 repeated experiments. Different letters (a, b, c, d, etc.) indicate statistically significant difference to other groups (p˂0.05).