Effects and Mechanisms of Silibinin on Influenza A/H1N1 Pathogenesis in a Mouse Model

Abstract

Silymarin is a polyphenolic flavonoid extracted from milk thistle. It has potent immunomodulatory effects and can inhibit the replication of influenza A virus (IAV). The present study aimed to determine the inflammatory and anti-inflammatory cytokine secretion patterns in mice before and after silibinin treatment. For this, bronchoalveolar lavage (BAL) fluids were collected from the thoracic cavity 5 days after the intervention, and viral quantification was performed using TaqMan Real-time PCR. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate IFN-γ and IL-10 levels in serum and BAL samples. Finally, pathological damage to lung tissue was assessed by pathologists. The results reveal that silibinin pretreatment exhibits a dose-dependent immunomodulatory effect on IFN-γ and IL-10 levels. After the virus challenge, silibinin reduced immune cell infiltration in mouse BAL fluid. These data similarly suggest a remarkable immunomodulatory effect of silibinin. Silibinin also decreased lung damage following the virus challenge in the post-treatment group, but its lung protective properties seem to be due to a different mechanism than when it was administered before infection. Finally, high doses of silibinin (post-treatment) significantly reduced viral load in BAL fluid compared to the virus challenge group. These results support the idea that therapies aimed at moderating immune and inflammatory responses are essential to decrease the mortality rate caused by IAV infection. Silibinin has strong immunomodulatory properties, can inhibit IAV infection, and reduces lung tissue damage in a dose-dependent manner.

Keywords: immunomodulatory effect; influenza A virus (IAV); lung damage; silibinin; silymarin.

Figure 1

Effects of silibinin on mice weight. After 7 days of only silibinin administration, there were no significant differences between the silibinin groups and the control group (a). A high-dose silibinin group significantly reduced mice weight after the H1N1 influenza challenge (first experiment) (b). Additionally, in the second experiment, the high-dose silibinin group significantly reduced mice's weight following the virus challenge (c).

Figure 2

Silibinin induced inflammatory and anti-inflammatory responses. Pretreatment with high doses of silibinin significantly increased IFN production (a). In addition, IL-10 levels were significantly elevated following silibinin pretreatment (a). In contrast, upon challenge with the influenza virus (post-treatment), no significant differences were observed in IFN and IL-10 cytokines (a). Pretreatment with silibinin increased IFN-γ production in BAL samples in a dose-dependent manner, with significant increases at 25 and 50 mg doses (b). IL-10 levels in BAL samples also increased in both dose groups, but this was not statistically significant (b). In contrast, after the post-treatment, no significant differences in IFN-γ or IL-10 levels were observed in BAL samples between groups (b). Also, graphs represent the ratio of IFN-γ/IL-10 in serum (c) and bronchoalveolar lavage fluid (BALF) (d) of pretreated and post-treated animals. ⁣^∗p  <  0.05; ⁣^∗∗∗p  <  0.001.

Figure 3

Silibinin reduced immune cells infiltration in BAL fluid. Low-doses silibinin pretreatment unlike high-doses and virus control groups significantly reduced immune cells infiltration in lung tissue (a). Low and high-doses silibinin post-treatment significantly reduced immune cells infiltration in lung tissue (b). Lymphocyte cells were the most abundant infiltrating cells in the lung tissue (c, d). (e) H&E staining showed distribution of immune cells in pre- and post-silibinin treatment. Bar = 100 μm.

Figure 4

Silibinin attenuated lung damage after virus challenging. Lung damage was significantly induced by high-dose silibinin pretreatment (first experiment) (a). Post-treatment with silibinin (in both doses), however, significantly reduced lung damage (second experiment) (b). ⁣^∗p  <  0.05; ⁣^∗∗p  <  0.01.

Figure 5

Silibinin reduced viral load in BAL fluid. Silibinin pretreatment did not have any effects on viral load (a). In contrast, post-treatment with high doses of silibinin significantly reduced the viral load in the BAL fluid (b)⁣^∗p < 0.05.