Melatonin inhibits JEV propagation and neurotoxicity in human neuroblastoma cell line, SK-N-SH.
In this study, SK-N-SH cells were employed because of its neuron-like behavior when investigating potential neuroprotective activity in in vitro models 39–41. To examine the susceptibility of the SK-N-SH cells to JEV infection and virus replication, SK-N-SH cells were infected with JEV at the multiplicity of infection (MOI) of 5.0. We observed that JEV infection increased non-structural protein 3(NS3) and JEV RNA in 24h, primarily using western blot analysis (Figure 1A, B) and real-time PCR analysis (Figure 1C). We identified JEV RNA propagation in culture media using real-time PCR (Figure 1D). JEV infection significantly increased the neuronal cell death time-dependently compared to the controlled group (Figure 1E) and increased cell death to ~55% in the photomicrography and crystal violet assay (Figure 1F, G).
To confirm whether melatonin inhibits JEV production, western blot analysis was performed and real-time PCR was examined. We observed that melatonin treatment in SK-N-SH cells decreased intracellular virus protein expression of NS3 and the phosphorylation form of NF-κB (Figure 2A, B), as well as JEV RNA in cells and media (Figure 2C, D). Moreover, the photomicrograph indicated that melatonin reduced JEV-induced neuronal cell death (Figure 2E), and the crystal violet assay showed that melatonin decreased neurotoxicity of JEV-infected cells (Figure 2F, G). Our data revealed that melatonin is a possible antiviral agent against JEV.
Melatonin attenuates JEV-mediated neuro-inflammation.
In various neurodegenerative diseases, pro-inflammatory cytokines, such as TNF-α and IL-6, act as a crucial role in mediating neuro-inflammation and neurotoxicity 42–44. We examined the influence of JEV infection on the TNF-α and IL-6 using real-time qPCR and ELISA. Both TNF-α and IL-6 mRNA expressions significantly increased in 6, 12, 24 hours p.i. (Figure 3A, B). These secreted pro-inflammatory cytokines were examined in the culture media of SK-N-SH cells using ELISA. In accordance with mRNA data, the release of TNF-α and IL-6 cytokines increased gradually between 6 to 24 hours (Figure 3C, D). Especially, a robust up-regulation was detected in the release of IL-6 cytokines after JEV infection. We examined the effect of melatonin on the expression of TNF-α and IL-6 at both mRNA and released cytokine protein levels. Melatonin treatment significantly reduced mRNA levels of TNF-α and IL-6 (Figure 3E, F) and repressed the production of inflammatory cytokines (Figure 3G, H). Taken together, our data demonstrate that melatonin reduces JEV-mediated production of inflammatory cytokines.
Inhibition of CaN decreases JEV-induced neuro-inflammation and neurotoxicity by melatonin. Recent studies have recommended that CaN is an significant therapeutic target for the potential treatment of neurodegenerative diseases 45,46. To investigate the effect of JEV infection on CaN alteration, we examined the phosphorylation levels of bcl-10 and CaN activity as CaN has the ability to dephosphorylate endogenous phosphor-bcl-10 47. We observed that JEV infection decreased protein expression of phospho-bcl-10 and increased CaN activity time-dependently (Figure 4A, B), while melatonin treatment reversed the JEV-mediated alteration of phosphor-bcl-10 and CaN activity (Figure 4C, D). This demonstrated melatonin’s protective effect against JEV-mediated neurotoxicity and CaN alteration. We also investigated FK506, CaN inhibitor, to determine whether it has a protective effect against JEV-induced neurotoxicity and neuro-inflammation. The results of microscopy (Figure 4E) and crystal violet assay (Figure 4F, G) showed that FK506 treatment attenuated JEV-mediated neuronal cell death. We also provided the evidence that FK506 treatment reversed JEV-mediated upregulation of TNF-α and IL-6 using real-time PCR (Figure 4H, I) and ELISA (Figure J, K).
Melatonin attenuated JEV-induced autophagy impairment through CaN alteration.
Literature have indicated that JEV infection activates autophagy in neurons 4,48,49. To observe JEV’s role in the induction of autophagy, we monitored the levels of LC3-II and SQSTM1/p62 and noted that JEV infection increased LC3-II. This phenomenon becomes indicative of autophagy induction (Figure 5A). JEV infection also remarkably increased the expression levels of SQSTM1/p62, a selective autophagy substrate that forms a scaffold for protein aggregates and causes their autophagic degradation. The increased levels of SQSTM1/p62 are indicative of autophagosomal-lysosomal block in JEV-infected cells.
We investigated the relationships between melatonin, CaN, and autophagy in JEV infection. Melatonin treatment led to the degradation of SQSTM1/p62, indicating that autophagosomes are being turned over by lysosomal proteolysis (Figure 5B, C). We also observed that JEV infection increased GFP-LC3 fluorescence, while melatonin decreased a little GFP-LC3 puncta because of lysosomal degradation (Figure 5D). A number of vesicles, including double-membraned autophagosomes (arrowheads), were induced by JEV infection, which indicated the inhibition of lysosomal degradation (Figure 5E). Melatonin treatment induced autophagic degradation through single-membraned autolysosomes (arrows).
To investigate whether melatonin induces autophagic degradation through CaN, we used CaN inhibitor, FK506. We proved FK506 reversed the increase of SQSTM1/p62 in JEV infected cells (Figure 6A, B). It decreased a little GFP-LC3 puncta because of lysosomal degradation (Figure 6C) and improved autophagic degradation through creating autolysosomes (Figure 6D). These results indicate that melatonin induced autophagic degradation through CaN alteration in JEV-infected cells.