After HSV-1 infection, GM130 and TLR3 were down-regulated and the Golgi apparatus was fragmented.
The cell morphology of BV2 cells infected with HSV-1 at different time points was observed under light microscope. The results showed that as the duration of infection increased, the number of cell protrusions increased, and the proportion of cells with spindle morphology and amoebia-like morphology increased; multiple vacuoles could be seen in the cytoplasm of the cells, and the cell spacing was increased; part of the cell is detached and suspended (Fig. 1a).The propagation and propagation characteristics of HSV-1 in BV2 cells were determined by one-step growth curve. The results showed that the virus titer reached a plateau at 36-48h after infection, with a maximum titer of 5.7× 105 PFU/ml (Fig. 1b). At 12 hours after HSV-1 infection, the level of GM130 protein began to decrease (p < 0.001), and it continued to decrease withthe duration of infection. The level of TLR3 protein increased after 6 hours of infection (p < 0.001), but decreased significantly after 12 hours and 24 hours of infection (p < 0.001) (Fig. 1c-e).
After 6h and 12h of HSV-1 infection, IFN-β, TNF-α and IL-6 secreted by microglia were significantly increased (p < 0.001).The secretion of IFN-β, TNF-α and IL-6 were significantly decreased at 24h after infection compared with 12h after infection (p < 0.05; p < 0.01; p < 0.05) (Fig. 1f).In uninfected cells, immunofluorescence showed tightly packed GA structures in the perinuclear area (labelled by GM130 and P115), which did not change significantly after 6h of infection. At 12h of infection, GA was fragmented and dispersed in the cytoplasm. At 24h of infection, GA structural damage was more serious, and the fluorescence intensity of GM130 was significantly decreased (Fig. 2a,b).
Knockdown of GM130 can inhibit the level of TLR3 and lead to structural damage of Golgi apparatus
BV2 cells were transfected with SiRNA for 36h, then infected with HSV-1(MOI = 1) for 12h.The results showed that GM130 was successfully knocked down in the uninfected group and the HSV-1 infected group (p < 0.001; p < 0.001); Meanwhile, the level of TLR3 was significantly decreased (p < 0.001; p < 0.001) (Fig. 3a,b,d,e).In addition, viral titers were significantly higher in the HSV-1 + SiGM130 group than in the HSV-1 + SiCtrl group (p < 0.01) (Fig. 3c).The results showed that GM130 could regulate the level of TLR3 and affect the replication of HSV-1 in the infection of BV2 cells.
The structural changes of GA were observed by immunofluorescence labeled GM130 and P115.The results showed that knockdown of GM130 and infection with HSV-1 for 12h both caused structural destruction and fragmentation of GA in BV2 cells, and the fragmented GA was dispersed in the cytoplasm. At 12h after infection with HSV-1, knockdown of GM130 further caused structural damage to GA (Figure. 4a,b).The structural damage of GA was further observed by transmission electron microscopy. Compared with the uninfected group, the compact and ordered Golgi stacks in the HSV-1 infected group became swollen and disordered. Compared with the compact and orderly Golgi structure in the uninfected group, the Golgi stack became swollen and disordered in the infected group with HSV-1; this is similar to what was observed when GM130 was knocked down without infection (Fig. 4c).
Over-expression of GM130 can up-regulate the level of TLR3 and reverse the damage of Golgi apparatus
BV2 cells were transfected with pcDNA3.1 GM130 plasmid for 36h, and then infected with HSV-1 (MOI = 1) for 12h.The results showed that GM130 was significantly over-expressed in both the uninfected and 12h post-infection groups (p < 0.001; p < 0.001). Under uninfected conditions, the level of TLR3 in the over-expressed GM130 group did not change significantly (p > 0.05), but in the 12h after HSV-1 infection, the over-expressed GM130 significantly increased the level of TLR3 (p < 0.001) (Fig. 5a, b, d, e). After over-expression of GM130, the virus titer of BV2 cells was significantly decreased (P < 0.05) (Fig. 5c).These results suggest that GM130 can up-regulate the level of TLR3 and inhibit the replication of HSV-1 after infection of BV2 cells with HSV-1.
In addition, the structural changes of GA after over-expression of GM130were detected by IF. The results showed that over-expression of GM130 alleviated fragmentation of GA and partially restored compact structure of GA around the nucleus (Fig. 6a, b).The structural changes of GA were further observed by transmission electron microscopy. Over-expression of GM130 can alleviate the swelling and fragmentation of GA caused by HSV-1 infection and can remodel the structure of the GA (Fig. 6c).
TLR3 agonists reverse knockdown of GM130-induced downregulation of TLR3 and secretion of inflammatory factors
In order to verify whether GM130 affects the secretion of inflammatory cytokines in the HSV-1 infected BV2 cells by regulating the level of TLR3.We knocked down GM130 in BV2 cells and then treated them with a TLR3 agonist (Poly (I:C) (PIC)).
Compared with the HSV-1 group, level of TLR3 was significantly increased in the HSV-1 + Poly (I:C) treatment group (p < 0.001) (Fig. 7a,c); meanwhile, the secretion of inflammatory cytokines IFN-β, TNF-α and IL-6 increased significantly (p < 0.001; p < 0.001; p < 0.001) (Fig. 7d-f); however, the level of GM130 was not significantly different between the two groups (p > 0.05) (Fig. 7a,b).Compared with the HSV-1 + SiGM130 group, the level of TLR3 in the HSV-1 + SiGM130 + PIC group was significantly increased (p < 0.001) (Fig. 7a, c), and the secretion of inflammatory cytokines IFN-β, TNF-α, and IL-6 was up-regulated (p < 0.001; p < 0.001; p < 0.001) (Fig. 7d-f).These results suggest that in HSV-1-infected microglia, down-regulation of GM130 reduces inflammatory factors associated with microglia through inhibition of TLR3.
TLR3 inhibitors can reverse the up-regulation of TLR3 and the secretion of inflammatory cytokines caused by over-expression of GM130
We treated BV2 cells with a TLR3 inhibitor (TLR3i) after over-expression of GM130. Level of TLR3 was significantly lower in the HSV-1 + TLR3i than in the HSV-1 group (p < 0.001) (Fig. 8a,c); meanwhile, the secretion of inflammatory cytokines IFN-β, TNF-α and IL-6 decreased (p < 0.001; p < 0.001; p < 0.001) (Fig. 8d-f); there was no significant difference in GM130 levels between the two groups (p > 0.05) (Fig. 8a,b).Compared with the HSV-1 + OEGM130 group, the level of TLR3 in the HSV-1 + OEGM130 + TLR3i group was down-regulated (p < 0.05) (Fig. 8a,c), and the secretion of inflammatory cytokines IFN-β, TNF-α, and IL-6 was significantly reduced (p < 0.001; p < 0.001; p < 0.001) (Fig. 8d-f). These results suggest that over-expression of GM130 can promote the secretion of microglia-associated inflammatory cytokines by up-regulating the level of TLR3 in HSV-1 infected microglia.
Berberine can reverse Golgi damage after HSV-1 infection and reverse the down-regulation of TLR3 and inflammatory cytokines caused by Golgi damage
BV2 cells were treated with BBR at concentrations of 1uM, 3uM and 10uM respectively, and cell viability was observed to be significantly reduced at concentrations up to 10uM (Supplementary Fig. 1a). Therefore, we chose 3uM as the concentration for the treatment. BV2 cells in the HSV-1 infected group were treated with BBR (3uM) and ACV (3uM), respectively. Compared with the HSV-1 group, the levels of GM130 and TLR3 in the HSV-1 + BBR group were increased (P<0.01; P<0.01)( Supplementary Fig. 1b-e), while the secretion of inflammatory cytokines IFN-β, TNF-α and IL-6 were increased (P<0.01; P<0.01; P<0.01) (Supplementary Fig. 1f-h); viral titers were lower (P<0.05) (Supplementary Fig. 1i).Compared with the HSV-1 group, the levels of GM130 and TLR3 in the HSV-1 + ACV group were increased (P<0.01; P<0.01) (Supplementary Fig. 1b-e), as well as the secretion of inflammatory cytokines IFN-β, TNF-α and IL-6 (P<0.001; P<0.001; P<0.001) (Supplementary Fig. 1f-h); viral titers were significantly lower (P<0.001) (Supplementary Fig. 1i).
Compared with the HSV-1 + BBR group, the levels of GM130, TLR3, IFN-β, TNF-α and IL-6 in the HSV-1 + BBR + ACV group were increased(P<0.01; P<0.001; P<0.01; P<0.01; P<0.01) (Supplementary Fig. 1b-h), and viral titers were lower (P<0.01) (Supplementary Fig. 1i).Compared with the HSV-1 + ACV group, GM130, TLR3, IFN-β, TNF-α and IL-6 levels were increased in the HSV-1 + BBR + ACV group(P<0.05; P<0.01; P<0.05; c5; P<0.05) (Supplementary Fig. 1b-h), while viral titers showed a decreasing trend, but did not reach statistical significance (Supplementary Fig. 1i). Immunofluorescence showed that, compared with the HSV-1 infection group, the destruction of the GA structure has been reversed in the HSV-1 + BBR group (GA structure was more compact and clustered), and fluorescence intensity of GM130 was enhanced (Supplementary Fig. 2).Compared with the HSV-1 infected group, the structure of GA in the HSV-1 + ACV group was also protected, and the structure of GA was more compact and clustered, and the fluorescence intensity of GM130 was stronger (Supplementary Fig. 2).The structure of GA in the HSV-1 + BBR + ACV group was improved compared with that of the HSV-1 + BBR group. Compared with the HSV-1 + ACV group, structure of GA in the HSV-1 + BBR + ACV group was improved (Supplementary Fig. 2).