Curcumin prevents dexamethasone-induced activation of the pseudorabies virus in rat pheochromocytoma cells through the miR-155-5p-Aak1-Numb/Notch2 signalling axis

Abstract

Pseudorabies virus (PRV) causes neurological disorders and organ damage in diseased animals. After initial infection, PRV activity is gradually inhibited; however, stress stimulation increases the host's glucocorticoid levels, which overcomes the inhibition of PRV activity. Curcumin (Cur) helps maintain the inhibitory state of the Epstein-Barr virus, although further research is needed to establish whether Cur can prevent PRV activation triggered by stress hormones. In this study, we used PC-12 cells to determine the effects of Cur on PRV activation. The cells were successfully infected with PRV at a multiplicity of infection of 1 for 24 h, resulting in the inhibition of PRV activity. Following incubation with 0.5 µM dexamethasone (DEX) for 4 h, the inhibition of PRV activity was blocked. Further mechanistic analyses using a dual-luciferase assay revealed that miR-155-5p directly targets and regulates Aak1 and its downstream signalling molecules, Numb and Notch2, in maintaining and disrupting PRV inhibition. Moreover, in vitro experiments using miR-155-5p mimics and inhibitors, combined with Aak1 overexpression and interference, confirmed that the miR-155-5p-Aak1-Numb/Notch2 axis prevented DEX-induced disruption of PRV inhibition by Cur. These findings provide a novel regulatory target for preventing stress-activated PRV and provide evidence for the potential use of Cur as a stress modulator in practical applications.

Keywords: DEX; curcumin; miR-155-5p/Aak1; pseudorabies virus.

Figure 1

Effects of DEX treatments on PC-12 cell viability, mitochondrial function, and virus replication-related indicators. A Cell viability (η² = 0.694); B Mitochondrial membrane potential (MMP) (η² = 0.895); C ROS content (η² = 0.983); D Virus copy number (η² = 0.979, η² = 0.974); E PRV protein level determined by IFA (10 × 10); F PRV protein quantification map (greyscale value) (η² = 0.995). Mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant. CON: blank control; DEX: 4 h incubation with 0.5 µM DEX; PRV: PRV infection with MOI = 1 for 24 h; PRV + DEX: PRV with MOI = 1 was infected for 24 h and then incubated with 0.5 µM DEX for 4 h.

Figure 2

Targeted regulation of Aak1 gene by miR-155-5p during the maintenance and destruction of relative inhibited PRV activity. A miRNA-Seq results; B RT-qPCR validation of miRNA expression results (η² = 0.983); C RT-qPCR validation of Aak1 mRNA level (η² = 0.983); D The mRNA level of Aak1 gene after transfection with miR-155-5p mimic/inhibitor (η² = 0.897); E Using GAPDH as the internal reference Aak1 protein level was detected by western blotting after transfection with miR-155-5p mimic/inhibitor; F Relative level of Aak1 protein (η² = 0.998); G Dual-luciferase reporter gene results (η² = 0.019, η² = 0.911, η² = 0.08). Mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant.

Figure 3

Effects of miR-155-5P on the function of PC-12 cells. A Cell viability (η² = 0.956); B Mitochondrial membrane potential (MMP)(η² = 0.894); C ROS content (η² = 0.967); D Virus copy number (η² = 0.951); Mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant.

Figure 4

Effects of Aak1 on the function of PC-12 cells. A Cell viability (η² = 0.973); B Mitochondrial membrane potential (MMP) (η² = 0.912); C ROS content (η² = 0.803); D Virus copy number (η² = 0.981, η² = 0.981); Mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant.

Figure 5

Effects of miR-155-5p on Notch2 and Numb protein levels. A The protein levels of Aak1, Numb and Notch2 in PC-12 cells transfected with miR-155-5p mimic and miR-155-5p inhibitor detected by western blotting, with GAPDH as an internal reference; B Relative protein level of Aak1 (η² = 0.976); C Relative protein level of Notch2 (η² = 0.993); D Relative protein level of Numb (η² = 0.985). Mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant.

Figure 6

Effects of Aak1 on Notch2 and Numb protein levels. A The protein levels of Aak1, Numb, and Notch2 in PC-12 cells transfected with Aak1-OE and shRNA detected by western blotting, with GAPDH as an internal reference; B Relative protein level of Aak1 (η² = 0.979); C Relative protein level of Notch2 (η² = 0.993); D Relative protein level of Numb (η² = 0.954). Mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant.

Figure 7

Cur prevents DEX from activating PRV. A Cell viability (η² = 0.929); B Mitochondrial membrane potential (MMP) (η² = 0.791); C ROS content; D Virus copy Number (η² = 0.997, η² = 0.975); E miR-155-5p level (η² = 0.916); F Aak1 mRNA level (η² = 0.989). Mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant. CON: blank control; DEX: 4 h incubation with 0.5 µM DEX; PRV: PRV infection with MOI = 1 for 24 h; PRV + DEX: PRV with MOI = 1 was infected for 24 h and then incubated with 0.5 µM DEX for 4 h. PRV + Cur5 + DEX and PRV + Cur10 + DEX: the cells were stimulated by DEX after incubation with 5 and 10 µM of Cur following PRV infection.

Figure 8

The protein levels of Notch2, Numb, and Aak1 in the process of Cur preventing DEX-activated PRV. A The protein levels of Aak1 (η² = 990), Numb (η² = 0.981), and Notch2 (η² = 0.964) in PC-12 cells transfected with the miR-155-5p mimic/inhibitor were detected by western blotting, and the intensity was calculated using GAPDH as an internal control to normalise protein loading (B–D); E The protein levels of Aak1 (η² = 969), Numb (η² = 954), and Notch2 (η² = 967) in PC-12 cells transfected with Aak1-OE were detected by western blotting, and intensity was calculated using GAPDH as an internal control to normalise protein loading (F–H). Mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant.