PEG-SeNPs as therapeutic agents inhibiting apoptosis and inflammation of cells infected with H1N1 influenza A virus

Abstract

The rapid variation of influenza challenges vaccines and treatments, which makes an urgent task to develop the high-efficiency and low-toxicity new anti-influenza virus drugs. Selenium is one of the essential trace elements for the human body that possesses a good antiviral activity. In this study, we assessed anti-influenza A virus (H1N1) activity of polyethylene glycol (PEG)-modified gray selenium nanoparticles (PEG-SeNPs) on Madin-Darby Canine Kidney (MDCK) cells in vitro. CCK-8 assay showed that PEG-SeNPs had a protective effect on H1N1-infected MDCK cells. Moreover, PEG-SeNPs significantly reduced the mRNA level of H1N1. TUNEL-DAPI test showed that DNA damage reached a high level but effectively prevented after PEG-SeNPs treatment. Meanwhile, JC-1, Annexin V-FITC and cell cycle assay demonstrated the apoptosis induced by H1N1 was reduced greatly when treated with PEG-SeNPs. Furthermore, the downregulation of p-ATM, p-ATR and P53 protein, along with the upregualation of AKT protein indicated that PEG-SeNPs could inhibit H1N1-induced cell apoptosis through reactive oxygen species (ROS)-mediated related signaling pathways. Finally, Cytokine detection demonstrated PEG-SeNPs inhibited the production of pro-inflammatory factors after infection, including IL-1β, IL-5, IL-6, and TNF-α. To sum up, PEG-SeNPs might become a new potential anti-H1N1 influenza virus drug due to its antiviral and anti-inflammatory activity.

Keywords: Apoptosis; Cytokine; Influenza virus; PEG-SeNPs; Signaling pathways.

Fig. 1

The effects of different concentrations of PEG-SeNPs on MDCK cells with or without virus infection. (A) The cytotoxicity of PEG-SeNPs. (B) The antiviral activity of PEG-SeNPs was determined by CCK8 assay. (C, D) The antiviral activity of PEG-SeNPs was determined by TCID 50 assay. The concentration of PEG-SeNPs was 16 μM in C. (E) The morphological changes of cells infected with H1N1 virus and treated with different concentrations of PEG-SeNPs observed with phase-contrast microscope. Experiments were repeated three times. P < 0.05 was considered to be statistically significant. # represents comparison with the control group, while * represents comparison with the virus group.

Fig. 2

The effects of SeNPs on virus proliferation. Compare to the H1N1 virus group, the mRNA level of control group, PEG-SeNPs, and PEG-SeNPs + H1N1 group. The concentration of PEG-SeNPs was 16 μM. The level of viral RNA was detected in the cell culture supernatant using the Real-Time PCR method. Experiments were repeated three times. P < 0.05 was considered to be statistically significant. # represents comparison with the control group, while * represents comparison with the virus group.

Fig. 3

PEG-SeNPs inhibited the reduction of mitochondrial membrane potential caused by virus infection. (A) Detection of mitochondrial membrane potential by flow cytometry. (B)The change of mitochondrial membrane potential compared with the H1N1 group. (C) The green fluorescence of the JC-1 monomer and red fluorescence of JC-1 aggregates were observed by fluorescence microscope. The concentration of PEG-SeNPs was 16 μM. Experiments were repeated three times. P < 0.05 was considered to be statistically significant. # represents comparison with the control group, while * represents comparison with the virus group.

Fig. 4

The effect of PEG-SeNPs on the translocation of phosphatidylserine induced by virus infection. (A) Detection of apoptosis by flow cytometry. The upper right quadrant indicates the percentage of early apoptotic cells, and the lower right quadrant the percentage of late apoptotic or necrotic cells. The lower left quadrant represents cell survival. (B) The percentage of total apoptotic cells, including both early and late apoptotic cells. (C) The apoptosis of MDCK cells were photographed by fluorescence microscope. Experiments were repeated three times. P < 0.05 was considered to be statistically significant. # represents comparison with the control group, while * represents comparison with the virus group.

Fig. 5

The effect of PEG-SeNPs on the apoptosis of MDCK cells infected by the H1N1 virus. (A) The sub-G1 apoptotic cell population in the control, H1N1, PEG-SeNPs, H1N1 + PEG-SeNPs group was 1.80%, 24.35%, 3.93%, 8.10%, respectively. (B) Cell cycle assay involved quantitative detection of DNA content by flow cytometry and the analysis of cell cycle distribution, especially the proportion of apoptosis peaks after different treatments. Experiments were repeated three times. P < 0.05 was considered to be statistically significant. # represents comparison with the control group, while * represents comparison with the virus group.

Fig. 6

DNA fragmentation and nuclear coagulation examined using TUNEL-DAPI staining. (A) Compared to H1N1 group, the mean green fluorescence intensity was significantly reduced after PEG-SeNPs treatment. (B) Apoptotic cells were positively stained by TUNEL green fluorescence. Experiments were repeated three times. P < 0.05 was considered to be statistically significant. # represents comparison with the control group, while * represents comparison with the virus group.

Fig. 7

PEG-SeNPs inhibited the production of ROS induced by the virus. (A) The reactive oxygen levels of the H1N1 virus group, PEG-SeNPs, H1N1 + PEG-SeNPs group were 265%, 105%, 125%, respectively, compared with the control group (100%). Experiments were repeated three times. P < 0.05 was considered to be statistically significant. (B) The production of reactive oxygen species between different groups detected by a fluorescence microscope. # represents comparison with the control group, while * represents comparison with the virus group.

Fig. 8

The protein expression of ATM, ATR, P53 and AKT by Western blot. PEG-SeNPs downregulated the protein expression including, p-ATM, p-ATR, P53, and C-PARP proteins. Meanwhile, PEG-SeNPs upregulated the protein expression of Bcl-2, AKT and PARP.

Fig. 9

The level of inflammatory factors after virus infection by flow cytometry. PEG-SeNPs suppressed the expression of inflammatory factors, including IL1β, IL-5, IL-6, and TNF-α. P < 0.05 was considered to be statistically significant. # represents comparison with the control group, while * represents comparison with the virus group.

Fig. 10

The schematic diagram of apoptosis signaling pathways participated in the inhibition of H1N1 by PEG-SeNPs. PEG-SeNPs inhibited H1N1-induced apoptosis and secretion of pro-inflammatory cytokines.