Cytotoxicity of MA was evaluated against RAW264.7 cells by the cck-8 assay. The result showed that the MA was largely non-toxic in Raw264.7 cells (Fig. 2A). The CC50 value was 72.44 µM. In the following experiment, 1, 5 and 10 µM concentrations were chosen to investigate the anti-H1N1 effect of MA in Raw 264.7 cells.
When virus proliferates in host cells greatly, it usually leads to a cytopathic effect (CPE) in host cells, which can cause injury and death of infected cells (Laghlali G et al., 2020). Therefore, the CPE can reflect the amount and interaction of virus in host cells. To investigate the protection effect of MA on RAW264.7 cells infected by H1N1, we observed and photographed the CPE of RAW264.7 cells under inverted microscope, and detected the viability of infected cell by cck-8 assay. As shown in Fig. 2B, compared to virus control group (Fig. 2C(b)), MA could markedly increase the survival rate of H1N1-infected cells, and there were no visible CPE in MA treatment groups (Fig. 2C (d, e, f)). The results indicated that MA was able to protect infected cells from viral apoptosis and increase cells viability.
To attest the anti-influenza virus activity of MA, we performed the plaque and hemagglutination assay to evaluate the viral load of RAW264.7 cells after MA treatment, furtherly. The hemagglutination assay was performed with the supernatant of 2-fold serial dilutions of H1N1 and treated with MA in different concentrations (1–10 µM). As shown in Fig. 3A, the HA was significantly reduced when the cells were treated with gradient concentrations (1, 5 and 10 µM) of MA for 24 h. The Fig. 3B showed that RBCs sedimented to the bottom of U-bottomed 96-well plate and exhibited obvious agglutination in the NC group without virus, whereas VC group had an epinephelos appearance and exhibited hemolysis with no sedimentation, since the IAVs can induce hemagglutination of RBCs. From Fig. 3B we can see the obvious agglutination in RBCs, which indicates that the viral particles in the supernatant of MA groups have been significantly reduced, particularly at the concentrations of 5 and 10 µM.
The plaque assay results verified the anti-influenza virus activity of MA, furtherly. The plaque assay was performed with the supernatant of 10-fold serial dilutions of H1N1and treated with MA in different concentrations (1–10 µM). As shown in Fig. 3C, H1N1 of MOI 1 induced an average 4.60 ± 0.43 Log progeny infectious particles per ml in virus control group. In the presence of 1, 5 and 10 µM MA, the viral particle production was reduced by 1.16 ± 0.18, 0.83 ± 0.35 and 0.29 ± 0.22 Log when compared to VC group, respectively. Correspondingly, the lysis plates inhibition of three concentrations was 74.8%, 82% and 93.7%, respectively.
The EC50 of MA against H1N1 in vitro were calculated and presented in Fig. 3E. When the RAW264.7 cells were infected by H1N1 of MOI 1, the EC50 and EC90 values were 1.27 and 5.30 µM MA, respectively, and when the RAW264.7 cells were infected by H1N1 of MOI 10, the EC50 and EC90 values were 2.39 and 8.38 µM, respectively.
The different types of treatment experiments were performed to complement the analysis of the antiviral activity of MA (Mayra D.T et al., 2019). Here, Raw264.7 cells were infected with H1N1 at MOI 1, and MA at a concentration of 5 µM was added at different phase of infection (Fig. 4A). In the Pre- experiments, MA was added 1 or 3 h before infection. In the during- experiments, both the virus and the MA were added simultaneously during the infection for 3 h. The post-infection means the MA was added at 3, 6, 9, 12, 24hpi after H1N1infection, respectively. At 24 hpi cells were harvested and subjected to PFU detection. The results showed that MA exhibited an inhibition to H1N1 when added pre, during and up to 24 hpi (Fig. 4B). These findings also suggested that the anti-H1N1 activity of MA is likely due to its activity against both the stages of intracellular replication of H1N1 and early stages of its replication cycle such as virus attachment or entry. In addition, We noticed a minimum production of virus particle was decreased by of 65% in MA pretreated cells when compared to the control. When the MA was added to cells at 3, 6, 9, 12, 24hpi, maximum reduction of virus particle can reach almost 95%. Obviously, MA post-treatment is more effective than pre-treatment after infection, which suggest that MA might act on the late stages of H1N1 infection.
TFEB is a member of the MiTF/TFE (microphthalmia-transcription factor E) family of transcription factors of the leucine zipper bHLH-LZ, which is involved in the regulation of many important cellular physiological processes, and now it has been demonstrated that it is the master regulators of macroautophagy/autophagy and lysosome function raises the possibility that it may be of central importance in linking autophagy and lysosome(Settembre C et al., 2019). However, the autophagy/lysosome pathway is important for viruses to replicate and escape in infected cells (Wang Y et al., 2018), so the TFEB may have important effects on the replication and spread of viruses. Thus, MA may clear H1N1 virus by inhibiting TFEB. To verify this, TFEB protein expression in H1N1 infected (MOI = 10) RAW 264.7 cells was detected. The result in Fig. 5A displayed that H1N1 increased TFEB levels by 1.5, 2.0 and 3.1-fold at 12, 24 and 36 hpi when compared to uninfected cells, respectively. Yet, After MA intervention, the TFEB levels of infected cells were reduced up to 70% (Fig. 5B). To reveal the affect of TFEB on H1N1 replication, the expression of TFEB was silenced with siTFEB, meanwhile the blank siRNA was transfected as the negative control (siNC). Finally, the PFU was determined. As shown in Fig. 5C, compared to the siNC transfection cells, the TFEB levels was reduced by 85% after 48 h siTFEB transfection. Base on this, H1N1 of MOI 1 was added into transfection or MA treatment cells. At 24 hpi, the PFU was determined. As shown in Fig. 5D, compared to the virus control group and the siNC group, the viral particle production was reduced by 4-fold in siTFEB-transfected group. While after MA treatment, We can also see 3.2-fold decrease of virus particle production. These results indicated the expression of TFEB protein is positively correlated with the increase of virus particle production. Yet, when the TFEB levels was decreased by MA in infected cells, the virus particle production was prominently reduced.
TFEB overexpression promotes the expression of multiple lysosomal genes, thereby regulating the production and efflux function of autolysosomes and increasing autophagic flux. TFEB subcellular localization and its transcriptional activity are strictly regulated by many protein molecules, of which the mTORC1 is the foremost regulator. It mainly affects the nuclear entry and transcriptional activity of TFEB by means of phosphorylation (Noda T. 2017). Therefore, the nuclear transfer of TFEB can reflect the autophagy status. To reveal the efficacy of MA on the nuclear transfer of TFEB and autophagy, we detected the nuclear transfer rate of TFEB and autophagy flux in H1N1 infected cells (MOI = 10) using immunofluorescence. As shown in Fig. 6A, after MA treatment, the nuclear transfer rate of TFEB is statistically significant compared to virus control group, and we observed a concentration-dependent decrease in the levels of TFEB in the nucleus of MA treatment groups (Fig. 6B). To determine whether MA inhibited autophagy induced by H1N1, the autophagic flux of infected cells was determined. The results showed that MA observably suppressed the autophagy in H1N1- infected cells, since the autophagic vesicles stained by MDC, a lysosomotropic compound used to label lysosomes and autophagosomes, were dramatically reduced after MA treatment (Fig. 6C). The autophagic vacuole formation was observed by fluorescence microscope (Fig. 6D).
Influenza virus infections usually cause severe interstitial pneumonia and induce an uncontrolled host-immune response, leading to a life-threatening condition called cytokine release syndrome (CRS) (Liu D et al., 2018). CRS represents an emergency scenario of a frequent challenge, which highlights the complex and interwoven link between infections and autoimmunity, and it has been believed to be a primary cause of death in IAV H1N1-infected patients (Downey J et al., 2018; Peteranderl C et al., 2016). Of note, autophagy has been revealed to be critical for the generation of cytokines from innate immune cells (Deretic V., et al., 2018), which provides possible targets for immunotherapy to combat IAV infection via inhibiting autophagy. Base on the suppressive effect of MA on autophagy of RAW264.7 cells infected by H1N1 by modulating TFEB, we investigated the anti-inflammatory effect of MA. Raw264.7 cells were infected with IAV at MOI 10, and MA was added. Meanwhile, cells transfected with the siTFEB or siNC was seted as the contrast. At 48 h post-transfection or treatment, MA could inordinately decrease the levels of TNF-α, IL-6, IL-1β and IFN-β in the H1N1 infected RAW264.7 cells,as is shown in Fig. 7A, Fig. 7B, Fig. 7C and Fig. 7D, respectively. These four cytokines were also inordinately decreased after the silence of TFEB. This experiment suggested that MA might decrease the levels of inflammatory cytokines induced by H1N1-infection by inhibiting TFEB.