T2 cytotoxicity
We found that the cell activity of PK15 cells treated with various T2 concentrations decreased in a dose-dependent manner at 24 and 48 h, and the cell activity was the lowest when 100 nM T2 was used (Fig 1). Because 10 nM T2 had no significant effect on PK15 cell viability at 24 and 48 h, this concentration was selected for the following experiments examining the interaction between T2 and PRV.
T2 inhibits the cytotoxicity and cell death caused by PRV infection
We observed no significant difference in cellular morphology between the T2 and NC groups (Fig 2). However, the PRV-infected cells were rounded, shedding, and showed obvious shrinkage. Interestingly, a significant decrease in the cytopathy of PK15 cells infected with PRV was observed 24 h after T2 exposure. Furthermore, the viability of PK15 cells cultured with 10 nM T2 after PRV infection for 36 h was significantly higher than that in the group without T2 exposure (Fig 3).
T2 inhibits PRV replication in PK15 cells
The number of cells infected by PRV
The number of PRV-infected cells was determined by the immunofluorescence assay. Red fluorescence indicated cells that were infected with PRV. No fluorescence was observed in the NC group, whereas in the PRV group about 80% of the cells were infected (Fig 4). When T2 was used, 5 and 10 nM T2 significantly decreased the number of PRV-infected cells (Fig 4).
T2 inhibits PRV replication in PK15 cells
Next, we determined the PRV growth curves in T2-treated and untreated PK15 cells (Fig 5A). Compared with cells in the PRV group, the number of PRV copies in the PRV+T2 group was significantly decreased from 10 to 24 h (Fig 5A, B). However, there was no significant difference between these two groups after 36 h.
The mode of action assay
When we investigated the T2 mode of action, we found that there was no significant difference in the PRV copy number between the T2-treated and untreated cells in the PRV inactivation assay, pre-treatment assay, and virus entry assay (Fig 6). However, in the virus replication assay, the PRV copy number in the T2-treated PK15 cells was significantly lower than that in the untreated cells (Fig 6).
Oxidative stress and apoptosis are involved in the inhibition of PRV replication in PK15 cells by low-concentration T2
To evaluate oxidative stress in PRV-infected cells treated with T2, we measured the mRNA expression of Nrf2, Keap1, Gpx-1, and Nqo1 in the four indicated groups (Fig 7). Compared with the NC group, the mRNA levels of Nrf2, Keap1, and Nqo1 in the T2-treated cells were significantly downregulated (Fig 7A, B, D). However, the four genes mentioned above were significantly upregulated in the PRV group and the PRV+T2 group, compared with the NC and T2 groups (Fig 7A–D). Interestingly, compared with the PRV group, in the PRV+T2 group the mRNA levels of these genes were markedly downregulated (Fig 7A–D).
When we evaluated the effect on apoptosis-related genes, we found that compared with the NC group, Caspase-3, caspase-8, and Bax mRNA expression was significantly upregulated in the PRV group and the PRV+T2 group (Fig 8A–C). However, the mRNA expression of Bcl-2 was significantly downregulated in the PRV group and the PRV+T2 group (Fig 8D). Additionally, compared with the PRV group, the mRNA expression levels of Caspase-3, caspase-8, and Bax in the PRV+T2 group were significantly lower (Fig 8A–C), whereas the mRNA expression of Bcl-2 was significantly upregulated (Fig 8D).
To verify that Oxidative stress and apoptosis are involved in the inhibition of PRV replication in PK15 cells by low-concentration T2, we examined the protein expression of Cleaved-caspase-3, Cleaved-caspase-8, Bax, Bcl-2, Nrf2, and GPx-1 by western blotting. As shown in Figure 9, the expression levels of these proteins in PRV-infected PK15 cells were significantly downregulated by T2 treatment.