Fig 1.
Anti-IAV activity of rhein in vitro.
(A) The cytotoxicity of rhein was determined by a MTT method, *P < 0.05 vs. the blank control (0 μg/mL). (B) Inhibition of rhein on IAV (ST169, H1N1) replication was determined by a plaque inhibition assay. In the negative control (NC), MDCK cells were infected with IAV (MOI = 0.001) but not treated with any drugs; in the positive control (PC) and rhein-treated groups, MDCK cells were infected (MOI = 0.001) and treated with ribavirin (25 μg/mL) and rhein (1.25, 2.5, 5, and 10 μg/mL), respectively. After 48 h p.i., the supernatants were harvested and the titers were determined by a plaque formation assay. (C) The results of the time-of-addition assay, which contained four tests: (a) direct inactivation assay, (b) influence-on-cell assay, (c) influence-on-viral adsorption assay, and (d) different-time-points p.i. assay. MOI = 2.0. 0.5% DMSO was used as the negative control (NC). After 12 h p.i., the supernatants were harvested and the viral titer was determined by a plaque formation assay. The experiment was repeated five times and each experimental condition was performed in triplicate (n = 5). All data shown were mean ± SD. *P < 0.05 vs. the NC group.
Table 1.
Effect of rhein on IAV-induced oxidant stress.
Fig 2.
Effects of rhein on TLRs, Akt, MAPK and NF-κB signaling pathways after IAV infection.
The treatment of the blank control (BC), negative control (NC), positive control (PC), and rhein-treated group (rhein) was same with that of the “antioxidant assay”. After 48 h p.i., the cells were harvested. The expressions of TLR2, TLR3, and TLR4, the phosphorylations of Akt, ERK, p38, JNK MAPK, and the nuclear translocation of NF-κB p65 were determined by western blotting assay. The experiment was repeated five times (n = 5). All data shown were mean ± SD. #P < 0.05 vs. the BC group, *P < 0.05 vs. the NC group.
Fig 3.
Counteracting effects of different agonists on the antiviral activity of rhein.
A549 cells were infected with IAV (MOI = 0.001) and treated with or without ribavirin (Rib, 25 μg/mL), rhein (10 μg/mL), TLR2 agonist LAM-MS (10 μg/ml), TLR3 agonist Poly(I:C) (10 μg/ml), TLR4 agonist LPS-B5 (1 μg/ml), oxidant H2O2 (100 μM), Akt agonist IGF-1 (IGF-1 100 ng/ml), ERK agonist EGF (100 ng/ml), p38/JNK agonist anisomycin (10 μM), and NF-κB agonist PMA (1 μg/ml). Ater 48 h p.i., the antiviral activity was determined by a SRB method (A), IAV replication was determined by a qRT-PCR assay (B). The experiment was repeated five times and each experimental condition was performed in triplicate (n = 5). All data shown were mean ± SD. # P < 0.05 vs. the only virus-infected control, * P < 0.05 vs. the virus + rhein control.
Fig 4.
Effect of rhein on the production of cytokines after IAV infection determined by qRT-PCR (A) and ELISA (B) assays. The treatment of the blank control (BC), negative control (NC), positive control (PC), and rhein-treated group (rhein) was same with that of the “antioxidant assay”. The experiment was repeated five times and each experimental condition was performed in triplicate (n = 5). All data shown were mean ± SD. #P < 0.05 vs. the BC group, *P < 0.05 vs. the NC group.
Fig 5.
Effect of rhein on the production of MMPs after IAV infection determined by qRT-PCR (A) and ELISA (B) assays. The treatment of the blank control (BC), negative control (NC), positive control (PC), and rhein-treated groups (Rhein) was same with that of the “antioxidant assay”. The experiment was repeated five times and each experimental condition was performed in triplicate (n = 5). All data shown were mean ± SD. #P < 0.05 vs. the BC group, *P < 0.05 vs. the NC group.
Fig 6.
Anti-IAV activity of rhein in mice.
In the blank control (BC), mice were not infected with IAV (PR8) but shammed with VGM medium and treated with DMSO (0.5%). In the negative control (NC), positive control (PC), and rhein-treated groups (Rhe75 and Rhe150), mice were infected with 10× MLD50 of IAV (PR8) and treated with DMSO (0.5%), oseltamivir (10 mg/kg/day) and rhein (75 mg/kg/day and 150 mg/kg/day), respectively. (A) The survival rate was observed for 14 days and analyzed by using Kaplan-Meier analysis with Log-rank and Breslow tests. (B) The lung index was assessed by determining the percent of lung wet weight (g) to body weight (g) (lung index = lung wet weight (g) ÷ body weight (g) × 100%). (C and D) The pulmonary viral load and cytokines were determined by TCID50 and ELISA assays, respectively. Data were mean ± SD. Ten mice were used in the survival rate assay (n = 10) and six mice were used in the lung index, pulmonary viral load, and pulmonary cytokines assays (n = 6). #P < 0.05 vs. the BC group, *P < 0.05 vs. the NC group.
Fig 7.
Influence of rhein on the histopathological changes.
Mice were treated as mentioned in Fig 6. At day 6 p.i., six mice from each group were sacrificed. The right lungs were used for H&E staining assay. (A) Blank control (BC), (B) Negative control (NC), (C) Positive control (PC), (D and E) Rhein-treated groups (Rhe75 and Rhe150, respectively). (→) alveolar wall, (▼) inflammatory exudation, (▽) hemorrhage (erythrocytes). The original magnification was 200×.
Fig 8.
Schematic diagram of rhein on inhibition of IAV infection and IAV-mediated ALI.
Rhein can inhibit IAV adsorption and replication. Rhein suppresses IAV replication by inhibiting IAV-mediated oxidative stress and activations of TLR4, Akt, p38, JNK MAPK, and NF-κB signal pathways. Meanwhile, inhibition of these signal pathways further reduces the production of inflammatory cytokines and MMPs and finally decreases IAV-induced ALI.