Preparation and Characterization of [email protected]
The light images and Tyndall effect of [email protected] were as shown in Fig. 1A, 1B and 1C, the results indicated that [email protected] nanoparticles were synthesized. The morphology of [email protected] 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), [email protected] was decreased from 200 nm to 80 nm which indicated much smaller size. The zeta potential of SeNPs (-25 mv) was lower than [email protected] (8 mv), demonstrating the higher stability of [email protected] 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 [email protected], the cells morphological were slightly changed. In Fig. 3B, the cell viability were 26% (virus), 43% (virus + TP), 67% (virus + SeNPs) and 88% (virus + [email protected]). The results indicated that the antiviral of SeNPs was effectively amplified by TP. Minimum inhibitory concentration (MIC) of drug A combination present in [email protected] of Se (125 µM); MIC of drug B combination present in [email protected] of TP (2.5 µM); MIC of drug A alone corresponded to free SeNPs (1 mM); MIC of drug B alone corresponded to free TP (10 µM). Fractional Inhibitory Concentration (FIC) was calculated as (MIC drug A combination/MIC drug A alone) + (MIC drug B combination/ MIC drug B alone) = 125 µM / 1 mM + 2.5 µM /10 µM = 0.375. FIC was 0.375, below 0.5, indicating synergy. In this study, the FIC index was basically interpreted as follows: FIC < 0.5, synergy; FIC between 0.5 and 2, indifference; FIC > 2, antagonism. The results suggest that [email protected] effectively inhibited the proliferation of H1N1 influenza virus.
The lysosomes was found and increased in a time-dependent manner in MDCK cells as showen in Fig. 4. After 30 min, [email protected] escapes from lysosomes and transported in the cytosol. Then distributed into the cells after 60 min. This result showed the target organelle of [email protected] was lysosome.
Detection of Caspase-3 activity
The caspase-3 activities were 451% (virus), 332% (virus + TP), 290% (virus + SeNPs) and 190% (virus + [email protected]) as shown in Fig. 5. The treatment of H1N1 influenza virus infected MDCK cells remarkably increased the activity. TP and SeNPs slightly inhibited the caspase-3 activity, [email protected] significantly decreased the caspase-3 activity. The result showed that the [email protected] inhibits the H1N1 influenza virus through capase-3 activity.
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 Fig. 6. After treatment with H1N1 virus for 24 h, the number of TUNEL-positive cells was significant increased. Co-treatment with [email protected] obviously inhibited the H1N1 influenza virus-induced changes. The results indicated that [email protected] inhibited the apoptosis of MDCK cells by H1N1 influenza virus.
Microvilli and mitochondria were observed in MCK cells. As shown in Fig. 7A When MDCK cells was incubated with H1N1 influenza virus, TEM image indicates distorted organelles, shrinking cytoplasm and condensed chromatin. The mitochondria of MDCK cells recovered the shape after treatment with [email protected] in Fig. 7B. The ROS generation was conducted by DCF determination to indicate the action mechanisms of [email protected] The intracellular ROS generation were 430% (virus), 332% (virus + TP), 260% (virus + SeNPs) and 130% (virus + [email protected]) as shown in Fig. 8A. TP and SeNPs slightly inhibited the ROS generation. However, [email protected] remarkably decreased the ratio of ROS generation. The fluorescent intensity of DCF treated with H1N1 influenza virus was much stronger than TP, SeNPs and [email protected] in Fig. 8B. The results indicate the ROS participated in the antiviral action.
In vivo antiviral
Mice were infected with H1N1 virus and treated with TP, SeNPs and [email protected], followed by HE staining, tunel analyses and immunohistochemical test of lungs tissues after being executed (Fig. 9A). As the HE staining present (Fig. 9B), H1N1 infected group manifested as alveolar collapse, perivascular and peri-bronchiolar edema. When treated with TP or SeNPs, the symptoms lessened. [email protected] attenuated the histopathological manifestations substantially. The result indicated that [email protected] protected the lungs from being injured. [email protected] nanoparticles prevent the DNA damage during H1N1 infection. Meanwhile, the HE stain and tunel analysis illustrated that [email protected] inhibited MDCK cells infection by H1N1 influenza virus. Caspase-3, P-AKT, T-AKT, P-P53 and P53 proteins were detected and positive after H1N1 virus infection as showed in Fig. 10. The results demonstrated that [email protected] inhibited H1N1 influenza virus-induced MDCK cell apoptosis by ROS mediated AKT and p53 signalling pathway (Fig. 11).