This work aims to explore the effect of glycolysis on the replication of porcine reproductive and respiratory syndrome virus (PRRSV) in porcine alveolar macrophages (PAMs). The changes of glucose metabolism, PRRSV protein levels, PRRSV titers, and the relative expression levels of genes and proteins in PAMs were analyzed by ELISA, qPCR, virus titration, and Western blotting after PRRSV infection. The effect of hypoxia-inducible factor-1α (HIF-1α) on PRRSV replication was subsequently assessed by specific siRNAs targeting to HIF-1α. The results showed that PRRSV infection enhanced glycolysis, elevated the levels of glucose uptake and lactate in the supernatant (P<0.05 and 0.01, respectively), reduced ATP production (P<0.05), and up-regulated the expression of hexokinase 2 (HK2), 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3), and pyruvate kinase isozyme type M2 (PKM2) in PAMs (P<0.05 and 0.01, respectively). Glycolysis inhibitors down-regulated the expression of PRRSV proteins and reduced virus titers (P<0.01). The knockdown of HIF-1α by siRNAs inhibited glycolysis and lowered PRRSV titers (P<0.05). In addition, the interferon pathways inhibited by PRRSV infection were reversed by the inhibition of glycolysis. These findings may facilitate further investigation of the role of glycolysis in PRRSV replication.
Porcine respiratory and reproductive syndrome virus/physiology* ; Glycolysis ; Swine ; Animals ; Virus Replication ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Macrophages, Alveolar/metabolism* ; Porcine Reproductive and Respiratory Syndrome/virology* ; Cells, Cultured ; RNA, Small Interfering/genetics*

In order to investigate the apoptosis triggered by porcine circovirus type 2 (PCV2) in lymphocytes and the underlying mechanism, the levels of apoptosis and the expression levels of miRNA were examined by flow cytometry, Western blotting and real-time PCR (qPCR). The mimics or inhibitors of miR-125a-5p, an apoptosis-related miRNA, were transfected into PK-15 cells, and the apoptosis rate was examined upon overexpression or inhibition of mir-125a-5p. The target gene of mir-125a-5p was predicted by bioinformatics method, and the regulation of mir-125a-5p on the target gene was analyzed by luciferase reporter assay. The expressions of Bcl-2, Bax, cytochrome C and caspase-3 were detected by Western blotting. The results showed that exosomes secreted by PK-15 cells infected with PCV2 significantly increased the lymphocyte apoptosis rate, which was dose-dependent in certain concentration range. The expression of miR-125a-5p was dramatically increased. The apoptosis rate was increased significantly in the cells transfected with miR-125a-5p. It was predicted that there were binding sites of miR-125a-5p at Bcl-2 3'UTR by TargetScan. The luciferase activity of wild-type pmir-Bcl-2 3'UTR was inhibited significantly by miR-125a-5p mimics, but that of mutant pmir-Bcl-2 3'UTR was not changed. By Western blotting, Bcl-2 was reduced significantly, while Bax, cytochrome C and caspase-3 increased significantly, and the ratio of Bcl-2/Bax was significantly decreased. These results showed that PCV2 up-regulated the expression of miR-125a-5p through exosomes, then inhibited the expression of Bcl-2 at both mRNA and protein level, activated mitochondrial apoptosis pathway and induced apoptosis in lymphocytes.
3' Untranslated Regions ; Animals ; Apoptosis/genetics* ; Caspase 3/genetics* ; Cell Proliferation/genetics* ; Circovirus/genetics* ; Cytochromes c/genetics* ; Luciferases/genetics* ; Lymphocytes/metabolism* ; MicroRNAs/metabolism* ; Swine ; bcl-2-Associated X Protein/genetics*

The purpose of this study was to explore the effects of seneciphylline on the proliferation and autophagy of cervical cancer HeLa and Caski cells and the possible mechanisms of autophagy. MTT assay was used to evaluate the effect of seneciphylline on the proliferation of cervical cancer cells. Immunofluorescence assay was applied to investigate the formation of autophagosomes in GFP-LC3/HeLa and GFP-LC3/Caski cells. The effect of seneciphylline on the expression of autophagy-related proteins was checked by Western blotting. In addition, fluorescence colocation assay was used to detect the fusion of autophagosomes and lysosomes. Human cervical cancer subcutaneous xenografts in nude mice were used to evaluate the effect of seneciphylline on the growth of the tumor in vivo. Results showed that HeLa cells proliferation was inhibited by seneciphylline in a dose- and time- dependent manner. Seneciphylline could induce formation of autophagosomes, increase the expression of LC3-II and decrease the expression of P62, suggesting that seneciphylline induced autophagy in HeLa and Caski cells. Compared with seneciphylline alone, seneciphylline combined with later-stage autophagy inhibitor chloroquine significantly increased the expression of LC3-II and P62. Moreover, and fluorescence colocation assay showed that autophagosomes induced by seneciphylline could colocate with lysosomes, indicating that seneciphylline could induce the complete autophagy flux. Compared with seneciphylline alone, seneciphylline combined with earlier-stage autophagy inhibitor 3MA significantly increased the expression of LC3-II and significantly decreased HeLa and Caski cells viability, suggesting that seneciphylline induced protective autophagy. Compared with seneciphylline alone, seneciphylline combined with MEK inhibitor significantly decreased the expression of P-ERK1/2 and formation of autophagosomes, suggesting that autophagy induced by seneciphylline activated MEK/ERK1/2 signal pathway. In addition, seneciphylline showed a significant inhibitory effect on growth of human cervical cancer cells subcutaneous xenografts.