The Inhibition of H1N1 influenza virus-induced apoptosis by functionalized Selenium nanoparticles with β-Thujaplicin through ROS-mediated P53 and AKT signaling pathways

β-Thujaplicin possess a variety of biological activities. The use of modified biological nanoparticles (NPs) to develop novel anti-influenza drugs has increased in recent years. Selenium nanoparticles (SeNPs) with antiviral has attracted increasing attention for biomedical intervention. Functionalized SeNPs by β-Thujaplicin (Se@TP) surface modified with superior antiviral were synthesized in this study. β-Thujaplicin decoration of SeNPs obviously inhibited H1N1 infection and were less toxicity. Se@TP could inhibit H1N1 from infecting Madin Darby Canine Kidney (MDCK) cells and block chromatin condensation and DNA fragmentation. Se@TP obviously prevented MDCK cells from generating reactive oxygen species (ROS). Furthermore, Se@TP prevent lung injury in H1N1 infected mice through eosin staining and hematoxylin in vivo . Additionally, when treated with Se@TP, the DNA damage of lung tissues reduced substantially by TUNEL-DAPI test. Mechanistic investigation revealed that Se@TP inhibited H1N1 influenza virus from infecting MDCK cells through induction of apoptosis via suppression AKT and p53 signaling pathways through Immunohistochemical assay. Our results suggest that β-Thujaplicin modified SeNPs as carriers is an efficient way to achieve antiviral pharmaceutical candidate for H1N1 influenza. antagonism. The results suggest that Se@TP effectively inhibited the proliferation of H1N1 influenza virus.


Introduction
As a segmented RNA virus, Influenza virus affects millions of people and is still a serious contagious pathogen in seasonal epidemics. 1,2 H1N1 influenza virus is a highly infections respiratory disease which belongs to influenza A type viruses. 3 H1N1 Influenza virus was discovered and identified in US and Mexico in 2009 which infected more than 8768 deaths in 207 countries. 4 Due to the arising mutation of antigenic shifts and the genome in different species, influenza virus may emerge a novel influenza among humans in the future. 5 The influenza infection cycle included three steps: First, the influenza viruses attach to the host cell surface receptor and fuse with the endosomal membrane. Second, uncoating of nucleocapsid and multiplication of the genetic material occurs. Finally, the influenza protein and new viron is expressed and released. 6,7 On the surface of influenza virus, there are two important glycoprotein: Hemagglutinin (HA) and Neuraminidase (NA). 8 In the early stage of viral infection, HA combined sialic acid-containing receptors on host cells and mediated the entry and fusion of virus. 9,10 when mature viruses separate from host cell surface, NA plays an important role in assisting virus cleave the linkage between sialic acid and Hemagglutinin. 11 Although, the conventional way to restrain the spread of influenza infections is vaccination. But, the restraining the spread of influenza infections is the long period between the rapid virus evolution and vaccine development. 12 β-Thujaplicin (2-hydroxy-4-isopropyl-2,4,6-cyclohepentatrieneone) is an antimicrobial tropolone derived from geranyl pyrophosphate (GPP) and monoterpene intermedicate. 13,14 β-Thujaplicin have been reported to possess antibacterial and antifungal activities.
However, anti-influenza virus effects of β-Thujaplicin remain unclear. 15,16 The nanotechnology provided a new prospect to solve these problems, and the new antiviral nanodrug should effectively inhibit viral infection with fewer cytotoxicity. 17 23 Zhang et al reported silver nanoparticle treatment ameliorates biliary atresia syndrome in rhesus rotavirus inoculated mice. 24 among of them, selenium nanoparticles (SeNPs) attract much attention due to the unique antimicrobial activities. 25 Selenium is integral component of several selenoproteins which control several crucial biological processes. 26,27 The deficiency of selenium could enhance the susceptibility to infections including respiratory virus infections. 28 Therefore, the aim of the present study was to exposure novel functionalized selenium nanoparticles which can inhibit the infection of H1N1 virus. We hypothesized that β-Thujaplicin modified SeNPs (Se@TP) have excellent antiviral activity against H1N1 virus. Though several research groups have described the antimicrobial effects of SeNPs, the antiviral mechanisms is still unclear. This study was to verify how β-Thujaplicin modified SeNPs to inhibit H1N1 influenza virus in vitro and in vivo.

Determination of cell viability
The cytotoxic of Se@TP nanoparticles was performed as previously reported. 29,30 First, H1N1 influenza virus was added to MDCK cells for 2 h, and then the indicated concentrations of β-Thujaplicin with or without SeNPs were added to MDCK cells for 24 h.
Then, MTT (20 µl/well) was added for 5 h. The formazan crystals were recorded at the absorbance of 570 nm.

Intracellular localization of Se@TP
MDCK cells were treated with lyso tracker for 60 min as previous reported. 31 DAPI and coumarin-6 labelled Se@TP were added for various periods of time incubation.
Fluorescence microscope was used to obtain and analysis the image.

Caspase-3 activity and TUNEL-DAPI co-staining assay
The caspase-3 activity with the wavelengths at 380 nm (excitation) and 460 nm (emission) was detected as previously described. 32 DNA fragmentation was examined with fluorescence staining by the TUNEL apoptosis detection kit. 33 MDCK cells were confirmed with TUNEL for 1 h and incubated of DAPI for 15 min at 37 ℃ for nuclear staining.

Thin sections of MDCK cells and ROS generation
TEM analysis of MDCK cells were detected as previous described. 34 After incubation with H1N1 influenza virus for 2 h, MDCK cells were washed with PBS and incubated with Se@TP.
Se@TP-treated MDCK cells induced ROS accumulation was detected as previously described. 35 The ROS generation was monitored through the fluorescence intensity (excitation 500 nm and emission 529 nm).

Animals infection and treatment
Fifteen female BALB/c mice (aged 4-6 weeks) were randomly divided into five groups as follow: control group, H1N1 virus, virus + TP, virus + SeNPs, virus + Se@TP. All mice were anesthetized with 10% chloral hydrate at a dose of 3 µl/g. Then the control group was treated with 20 µl physiological saline by nasal dripping while the other four groups with 20 µl H1N1 virus by nasal dripping as well. 24 h later, TP, SeNPs and Se@TP were administered to anesthetized mice respectively via intranasal absorption every 24 h thereafter for a total of three times. The lungs were extracted, affused with physiological saline and fixed in paraformaldehyde before hematoxylin and eosin (HE) staining, tunel test and immunohistochemistry staining. All mice experiments were approved and guided by the Ethics Committee of Guangzhou Medical University.

Statistical analysis
All the data are presented as mean ± SD. One-way analysis of variance (ANOVA) was used in multiple group comparisons. Difference with P < 0.05 (*) or P < 0.01 (**) was considered statistically significant.

Results And Discussion
Preparation and Characterization of Se@TP The light images and Tyndall effect of Se@TP were as shown in Fig. 1A, 1B and 1C, the results indicated that Se@TP nanoparticles were synthesized. The morphology of Se@TP presented spherical particles in Fig. 2A shape. TP modified with SeNPs formed more compact and stable globular nanocomposites. As shown in Fig. 2B, EDX indicated the signal of C (10%) and O (8%) that from TP, the percentage of Se atoms was 82%. As shown in (Fig. 2C and 2D), Se@TP was decreased from 200 nm to 80 nm which indicated much smaller size. The zeta potential of SeNPs (-25 mv) was lower than Se@TP (8 mv), demonstrating the higher stability of Se@TP than SeNPs.

MDCK cells infected by H1N1 influenza was showed reduction in cells numbers and loss of
cell-to-cell contract as shown in Fig. 3A. When co-treatment with Se@TP, the cells morphological were slightly changed. In Fig. 3B, the cell viability were 26% (virus), 43%

Inhibition of H1N1 influenza virus infection
Typical apoptotic features with H1N1 influenza virus with nuclear condensation (blue fluorescence) and DNA fragmentation (green fluorescence) was exhibited by MDCK cells in

In vivo antiviral
Mice were infected with H1N1 virus and treated with TP, SeNPs and Se@TP, followed by HE staining, tunel analyses and immunohistochemical test of lungs tissues after being executed (Fig. 9A). As the HE staining present (Fig. 9B)        Apoptotic signaling pathways by TP in H1N1 infect ion of MDCK cells. The main signal ing pathway of ROS mediated AKT and P53 signaling pathways.