FNB Modulates Oxidative Stress Induced by PRV Infection in RAW 264.7 Cells Through the Mir22hg/miR-365-2-5p/iNOS ceRNA Regulatory Axis

Infection with pseudorabies virus (PRV) can induce intense oxidative stress. The normal butanol fraction of flavonoids extracted from Polygonum hydropiper L. (FNB) shows substantial antioxidant activity. However, the molecular mechanisms through which FNB affects oxidative stress caused by PRV infection remain unclear; moreover, how noncoding RNAs influence this process is unknown. Therefore, based on whole-transcriptome sequencing, we propose the following hypothesis: FNB regulates oxidative stress generated in PRV-infected RAW 264.7 cells through the ceRNA regulatory axis. The results showed that 3549 mRNAs, 62 miRNAs, and 1263 lncRNAs exhibited differential expression in PRV-infected RAW 264.7 cells; in contrast, 1427 mRNAs, 11 miRNAs, and 460 lncRNAs displayed differential expression in FNB-treated PRV-infected RAW 264.7 cells. According to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses, the differentially expressed genes (DEGs) exhibited remarkable enrichment in signaling pathways such as TNF, NF-κB, IL-17, and Toll-like receptors. Based on the DEGs in these pathways, a Mir22hg/miR-365-2-5p/iNOS ceRNA regulatory axis was constructed. The lncRNA Mir22hg is localized in the cytoplasm. Interference or overexpression of Mir22hg can significantly regulate oxidative stress induced by PRV infection by controlling Nrf2/HO-1 signaling pathway activation. Specifically, Mir22hg overexpression markedly upregulated the levels of iNOS, ROS, and NO and remarkably downregulated HO-1, Nrf2, and NQO1 mRNA and protein expression levels; however, Mir22hg interference yielded opposite results. Rescue experiments further indicated that simultaneous miR-365-2-5p interference or overexpression could reverse the effects of Mir22hg interference or overexpression, thereby regulating ROS and NO levels and Nrf2/HO-1 signaling pathway activation. Finally, we identified all flavonoid monomers in FNB by using Flavonoid-Targeted Metabolomics technology, providing a new perspective for utilizing plant flavonoids to prevent PRV infection.