Abstract:

The study aims to explore the impact and mechanism of Radix pseudostellariae polysaccharide (RPP) in regulating miRNA Let-7d-3p on the inflammation caused by pseudorabies virus (PRV) infection. PK-15 cells were cultured in vitro and treated with various concentrations of RPP (0.5-32.0 mg·mL^-1). The cell viability was then assessed using the CCK-8 assay. And PK-15 cells were divided into the control group, PRV group, 0.5 mg·mL^-1 RPP group, 1.0 mg·mL^-1 RPP group, and 2.0 mg·mL^-1 RPP group. The expression levels of inflammatory cytokines IL-6, TNF-α, and IL-17 in cells from each group were detected using real-time fluorescent qPCR. Let-7d-3p inhibitors were transfected into PK-15 cells and then subjected to intervention with 0 or 0.5 mg·mL^-1 RPP. The expression levels of Let-7d-3p and inflammatory factors were examined using qPCR. In the vivo experiment, 15 healthy mice were randomly assigned to the control group, PRV group, and RPP (10.0 mg·kg^-1) group, with 5 mice in each group. The expression levels of Let-7d-3p and inflammatory factors in mouse kidney tissues were detected using qPCR. The results showed that within the concentration range of 0 to 4.0 mg·mL^-1, RPP has no cytotoxicity. Compared to the control group, the transcription levels of the inflammatory factors IL-6, TNF-α, and IL-17 mRNA in PK-15 cells infected by PRV are significantly up regulated (P < 0.01), while the transcription level of Let-7d-3p is significantly down regulated (P < 0.01). In comparison with the PRV group, the expression of Let-7d-3p was significantly up regulated (P < 0.01) after the cells were treated with three concentrations of RPP, while the transcription levels of the three inflammatory factors were concurrently and significantly down regulated (P < 0.01). After the inhibition of Let-7d-3p, compared with the PRV group, the transcription levels of IL-6, TNF-α, and IL-17 in the Let-7d-3p inhibition group were significantly up regulated (P < 0.01). Compared with the PRV+RPP group, after the suppression of Let-7d-3p and subsequent intervention with RPP, the transcription levels of cellular inflammatory factors were significantly up regulated (P < 0.01). In vivo results indicate that, in comparison with the control group, the transcriptional level of Let-7d-3p in the kidneys of mice in the PRV group was significantly decreased (P < 0.01), and the transcriptional levels of inflammatory factors IL-6, TNF-α, and IL-17 were significantly up regulated (P < 0.01). Compared with the PRV group, the expression of Let-7d-3p in the kidneys of mice treated with RPP was significantly increased (P < 0.01), and the transcription levels of the inflammatory factors IL-6, TNF-α, and IL-17 were significantly decreased (P < 0.01). Results from both in vivo and in vitro experiments indicate that RPP can downregulate the transcription levels of inflammatory genes induced by PRV infection through Let-7d-3p, providing a rationale for subsequent studies on the alleviation of PRV-induced inflammation by RPP.

Key words: Radix pseudostellariae polysaccharide, pseudorabies virus, Let-7d-3p, inflammatory