Virucidal Activity of Moringa a From Moringa Oleifera Seeds Against In uenza A Viruses by Regulating TFEB

Yongai Xiong Shenzhen University https://orcid.org/0000-0003-1280-2468 Muhammad Shahid Riaz Rajoka shenzhen university MengXun Zhang Shenzhen University Ning Liang Shenzhen University Zhendan He (  hezhendan@126.com ) Department of Pharmacy, Shenzhen Key Laboratory of Novel Natural Health Care Products, Innovation Platform for Natural Small Molecule Drugs, Engineering Laboratory of Shenzhen Natural Small Molecule Innovative Drugs, School of Medicine, Health Science Center, Shenzhen University, Shenzhen 518060, China


Introduction
In uenza virus is the most threatening respiratory virus to human health, causing millions of people to suffer upper respiratory tract infections every year. In the past ten years, the large-scale outbreaks of SARS, MERs, H1N1, H7N9 and other viruses have brought serious harm to human life and health (Atkin-Smith G.K et al., 2018). The "cytokine storm" triggered by in uenza virus infection can cause lung in ammation and acute respiratory distress syndrome, which is an important reason for mortality (Zhang Y et al., 2020;Xiaoyong C et al., 2018). So far, the prevention and control of in uenza virus remains a worldwide problem. However, due to the high mutation rate of in uenza viruses and the occurrence of recombination between viruses, it is di cult for existing anti-in uenza drugs to cope with the changing u viruses (Mazhar H et al., 2017). The M2 ion channel blockers and neuraminidase inhibitors, which are Moringa oleifera Lam., also known as the Drumstick tree, is a cultivated species from the genus Moringa, (family Moringaceae) and the order Brassicales. It is geographically distributed across South Asia, and in particular, India, Sri Lanka, Pakistan, Bangladesh, and Afghanistan. The Moringa seeds and leaves have broad uses in both the food industry and as therapeutics. Previous pharmacological investigations have shown M. oleifera has anti-in ammatory, anticancer, antioxidant, and anti-obesity properties, etc (Bhattacharya et al. 2018).
M. oleifera seeds are a promising resource for both food and non-food applications, as they have both edible and medicinal value. They have been shown to be rich in nutrients such as oils, proteins, and vitamins (Bhattacharya et al. 2018; Kc et al. 2020). The M. oleifera seeds also contain numerous bioactive compounds, such as avonoids, phenolic acids, alkaloids, isothiocyanates, and thiocarbamate In recent years, research into the M. oleifera seeds has focused on their active antiviral ingredients. Guided by antiviral activity, our team has obtained some new compounds from Moringa seed, of which the Moringa A(MA) has been proved to have good activity of anti-H1N1 virus (Yongai X et al. 2020). The chemical structure of Moringa A is shown in Fig. 1.
Research in this paper is a continuation of our previous work. In this paper, we aimed to study the antiviral properties of MA against IAVs by regulating transcription factor EB(TFEB), an important transcription regulator of autophagy-lysosomal pathway (Napolitano G et al., 2016). In recent years, the complex interplay between IAVs and host autophagy machinery has been extensively revealed, and many studies testify that the infection of IAV strains such as H1N1, H3N2, H9N2 can activate autophagy in host cells, the mechanism is reportedly involved in viral replication and immune escape (Wang Y et al., 2018;Choi Y et al., 2018). Mounting evidences have demonstrated that autophagy is an important aproach for viral passage and virus particle production in host cells (Wang R  TFEB is a key transcription molecule that regulates autophagy at the transcriptional level. It can promote the transcription of multiple genes in the lysosome pathway, and regulate the production and e ux of autophagy lysosomes (Napolitano G et al., 2016). Therefore, inhibiting TFEB to block the production of lysosome may be an important strategy for host against viruses.

MA preperation
The MA with a purity of 99.8% was prepared according to our previous method (Yongai X et al. 2020). It's a highly polar compound and esay to be dissolved in water, therefore the cell culture medium was used to prepare the different concentrations of MA solution.

Cell lines and IAV strains
Raw264.7 and the MDCK cells were obtained from Mingzhou biotechnology co. LTD (Ningbo, China).
Cells were cultured in DMEM containing 10% fetal bovine serum with 100 U/ml of penicillin and 100 µg/ml of streptomycin at 37 °C with 5% CO 2 . In uenza A/Weiss/43(PR8, mouse passaged H1N1) virus was provided by Wuhan institute of virology, Chinese academy of sciences (Wuhan, China). New viral stocks of IAV were passaged in 10 days old embryonic chicken eggs for 48 to 72 h. Allantoic uid was collected and stored at -80 °C until required. Experiments were conducted in a physical containment level 3(PC3) laboratory.

Virus ampli cation
IAV was ampli ed as described before (Gao Li et al., 2017). Brie y, when MDCK cells grew to a density of 90% in cell culture asks, 2 ml DMEM containing TPCK tyrisin (2 µg/ml) were added. When the cells were suspended, 2 ml in uenza virus stoste (MOI = 10) were added into the cell culture ask. 4 hours later, 7 ml DMEM was added to the ak. Cytopathic changes were observed every 12 hours. 72 hours later, the mixture of virus and cells were collected and centrifuged, and the supernatant was collected and stored in the refrigerator at 4 °C for later use.

Cell viability assay
Cytotoxicity of MA was assessed by cck-8 assay. Brie y, RAW264.7 cells were seeded in 96-well plates at a density of 1 × 10 5 per well. At 24 h post-seeding, cells were treated with gradient concentrations of MA ranging from 10 to 320 µM. At 48 h, 10 µl cck-8 solution was added into every well and incubated for 2 h.
The OD values was estimated by measuring absorbance at 450 nm in a microplate reader (MK3, Thermo, USA).

Viral cytopathic effect (CPE) assy
RAW264.7 cells were seeded in a 96-well plate at a density of 1 × 10 5 per well. Experimental groups were set with the following conditions: normal control group (NC, saline + DMEM), virus control group (VC, H1N1 + DMEM), positive control group (H1N1 + DMEM supplemented with10µM Oseltamivir (OST)), and treatment group (H1N1 + DMEM supplemented with 1, 5, and 10 µM MA). Except the NC group, cells in other groups were infected with H1N1 (MOI = 1). After treatment for 24 h, the CPE in RAW264.7 cells was observed and photographed under inverted microscope (CKX53, OLYMPUS, Japan), and the cell viability was assessed by the CCK-8 assay.

Virucidal activity assay in vitro
The virucidal activity of MA in vitro was evaluated by hemagglutination assay and plaque assay, respectively. For hemagglutination assay, RAW264.7 cells were inoculated in 96-well plates at a cell density of 5 × 10 3 cells per well. 24 h later, 2-fold serial dilutions of H1N1 viruses were added to infect cells. After 24 h post-infection and treatment, the supernatant was collected and hemagglutination was detected using cavy red blood cell (RBC). Brie y, 50 µL supernatant was added to each well of a U-

Western blot
At the end of the experiment, cells were lysed in RIPA Lysis Buffer and proteins were extracted. The protein concentration was quantitated by using BCA kit (Sigma). An equal amount of protein was subjected to SDS-PAGE and transferred onto a PVDF membrane. After blocking with 5% skim milk, the membrane was incubated with primary antibody diluent (The antibody against TFEB 1:1000; The antibody for β-actin1:5000) and secondary antibodies (HRP-conjugated antibodies and anti-mouse). The membrane blots were developed with the enhanced chemiluminescence method and visualized by using the ChemiDoc XRS camera system (Bio-rad, USA).
2.9. Immuno uorescence assay 3 × 10 4 RAW264.7 cells per well were plated in 24-well plates on 13-mm coverslips, transfected/infected/treated as designated in the scheme and incubated for 48 h. The cells were xed with 4% paraformaldehyde in PBS and followed by permeabilisation in 0.2% Triton X-100 in PBS for 10 min at room temperature with agitation. Coverslips were then blocked in 5% skim milk-TBS-T blocking solution. Primary antibody anti-TFEB was diluted (1:400) into 4% BSA in PBS and added to coverslips. After incubation for 20 min, the secondary antibody goat anti-rabbit (1:1000) was added (1:500) in a solution of 4% BSA in PBS. DAPI was added to the secondary antibody solution to stain nuclei (1:10000 in PBS). The confocal images were acquired by using a OLYMPUS-HGLGPS uorescence microscope and accompanying Cellsens standard software (OLYMPUS, Japan) (Rusmini P et al., 2017).

Autophagic ux assay
The autophagic ux in RAW264.7 cells was detected by cell autophagy staining kit (MDC methods) (Dai S et al., 2017). Raw264.7 cells were infected with IAV at MOI 1 and treated with MA. After 48 hrs, autophagic vacuoles were labelled with MDC by incubating cells with 0.05 mM MDC in DMEM at 37 °C for 10 min. After incubation, cells were washed three times with wash buffer and immediately photographed and analysed by using a uorescence microscopy (Nikon Eclipse TE 300, Japan) and accompanying excitation/emission (380/420 nm) lters. Images were captured with a CCD camera and the mean uorescence intensity was quanti ed by the Image Pro Plus 6.0.

Cytokines assay
Raw264.7 cells were seeded in 96-well plates at a cell density of 5.0 × 10 3 cells per well. The siTFEB or siNC was transfected at 24 hpi, then cells were infected with IAV of MOI 10 at 48 hpi. The MA was added at 72 hpi. After treating and incubating, culture supernatant was harvested. The inhibition of TNF-α, IL-6, IL-1β and IFN-β production was detected according to the manufacturer's instruction of Elisa kits, respectively.

Statistical analysis
All data were analyzed in OriginPro 2017 software. Data are presented as mean ± SEM from ve samples. The signi cance of the statistical difference between the means was determined using ANOVA (*p < 0.05, **p < 0.01). The level of signi cance was determined using Duncan's multiple-range test (*p < 0.05, **p < 0.01).

Results
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 CC 50 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 re ect 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-in uenza 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 signi cantly 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 signi cantly reduced, particularly at the concentrations of 5 and 10 µM.
The plaque assay results veri ed the anti-in uenza 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.  (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 ndings 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 posttreatment 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 e ux function of autolysosomes and increasing autophagic ux. 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 re ect the autophagy status. To reveal the e cacy of MA on the nuclear transfer of TFEB and autophagy, we detected the nuclear transfer rate of TFEB and autophagy ux in H1N1 infected cells (MOI = 10) using immuno uorescence. As shown in Fig. 6A, after MA treatment, the nuclear transfer rate of TFEB is statistically signi cant 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 ux 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 uorescence microscope (Fig. 6D).
In uenza virus infections usually cause severe interstitial pneumonia and induce an uncontrolled hostimmune 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-in ammatory 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 in ammatory cytokines induced by H1N1-infection by inhibiting TFEB.

Discussion
M. oleifera seeds have become a focus of research in recent years, as they have extensive pharmacological activities and abundant levels of valuable chemical components. However, there have been relatively few antiviral studies on the chemical compositions of M. oleifera seeds. Our team has obtained many compounds from the M. oleifera seeds, the MA is one of them. In this paper, we reported that MA has good antiviral activity against H1N1 in RAW264.7 cells, as it can inhibit RBC hemolysis and plaque in H1N1-infected cells. The EC50 and EC90 values in RAW264.7 cells relate to 1.27 and 5.30 µM MA when infected by H1N1 of MOI 1, respectively. When cells were infected by H1N1 of MOI 10, the EC50 and EC90 values were 2.39 and 8.38 µM, respectively. In addition, the MA protects cells from cytopathic effect induced by H1N1 and increases cell survival. The different types of treatment experiments showed that the main anti-H1N1 activity of MA is likely due to its activity against the late stages of infection.
Autophagy has been con rmed to be a important mechanism of RNA viruses immune evasion, replication and release from infected cells. Double-membrane compartments formed during autophagy can provide a physical platform for the viral replication machinery, locally concentrate essential intermediates and protect viral RNAs from detection by innate immune sensors and degradation (Arrey F et al., 2009; Zhou Z et al., 2009). IAV also triggers the accumulation of autophagosomes for viral replication. Our study showed that the autophagic ux of RAW264.7 cells was markedly enhenced after H1N1 infection, meanwhile the virions increased dramatically too. Hence it was suggested that the induction of autophagosome during IAV infections might promote virus replication within infected cells. The MA could repress H1N1-induced autophagic ux, therefore it blocked the important pathway of virus ampli cation and restricted viruses replication and virulence. The suppressive effect of MA on autophagy of RAW264.7 cells infected by H1N1 was positive related to the reduction of TFEB protein, which is the master regulator for transcription of genes involved in autophagy and lysosome biogenesis.
In this paper, it was proved that cellular factor TFEB may contribute to the replication of H1N1 virus. We revealed the expression of TFEB was upregulated gradually with the extension of H1N1 infection time, so the H1N1 virus could promote the TFEB expression in infected cells, and it was a possible target for H1N1 virus triggered autophagy. In fact, the production of infectious virus particles were reduced by 80% after the silence of TFEB. What's most important, the nuclear transfer rate of TFEB in H1N1-infected cells was markedly increased. The nuclear transfer of TFEB can promote the transcription of multiple genes in the lysosomal pathway, and regulate the production and e ux function of autophagy lysosomes (Napolitano G et al., 2016). Therefore, the nuclear transfer promotion of TFEB was another evidence for H1N1 virus inducing antophagy in infected cells. It may be an important mechanism for H1N1 to complete its life cycle by utilizing autophagy.
MA was found to inhibit TFEB expression in H1N1-infected cells, and it could also decrease the nuclear transfer rate of TFEB. Accordingly, MA could repress autophagic activity caused by H1N1 infection. This inhibition was shown to be positively correlated with the antiviral activity of MA. However, whether MA acted directly on TFEB or regulated other proteins in relevant pathway needs further study.
What's more frightening than the virus is the cytokine storm, which is an uncontrolled excessive immune response and an important pathogenesis of viral pneumonia. The virus usually hijacks the immune system, leading to the loss of negative feedback in immune regulation, abnormally increasing a variety of cytokines, impairing the alveolar diffusion function and causing multiple organ dysfunction (Gerlach T et al., 2019; Gounder A. P et al., 2019). In addition to investigating the antiviral activities of MA, we also focused on its anti-in ammatory activity. The result showed that MA could reduce the levels of in ammatory cytokines TNF-α, IL-6, IL-1β and IFN-β in H1N1-infected cells. These four cytokines have been con rmed to play important roles in in ammation and apoptosis, which are responsible for organ dysfunction. The protection of MA on infected cells might be related to the reduction of in ammatory cytokine levels.
Mounting evidence have con rmed that many in ammatory diseases may fundamentally be caused by autophagy-lysosome pathway dysfunction (Ahmad L et al., 2019; Racanelli A C et al., 2018). As the master regulator of macroautophagy /autophagy and lysosome function, the TFEB may be a bridge in linking autophagy and lysosome dysfunction with in ammatory disorders. Our study also demonstrated the TFEB has a close link with cell in ammatory induced by H1N1. In the H1N1 infected cells with high TFEB expression, we found that higher levels of in ammatory cytokines compared to normal or siTFEB transfected cells. Yet, following the TFEB expression was down-regulated by MA, the in ammatory cytokines of infected cells was dramatically decreased.

Conclusion
This study indicated MA is a new compound from M. oleifera seeds which exhibit antiviral activity towards H1N1. The possible mechanism of MA anti-H1N1 virus was described in Fig. 8. The observed EC50 and cytotoxicity together with the different types of treatment data are hopeful and indicate that MA is a potential compound for the prophylaxis and treatment of in uenza virus infection. Availability of data and material Data and materials are available upon request by the corresponding author.