Antiviral and immunoregulatory effects of curcumin on coxsackievirus B3-infected hepatitis

Fig. 1. Curcumin treatment reduced CBV3 infection and cell death and increased HO-1 expression. Huh-7 or HepG2 cells were infected without (-) or with (+) CVB3 at the MOIs of 0.002 (A, D, E) or 2 (B, C), treated with the indicated concentrations of curcumin at 2 h post-infection (hpi.), and collected at indicated hpi. or 24 hpi. (A, D-E) for assays. (A, D, E) The representative western blots (above) and quantitated results (bottom) are shown. In each sample, the level of indicated protein was normalized to that of internal control, β-actin. The values of infected cells without curcumin treatment are set as 100%. The western blotting data represent means + SEM (error bars) of ≥3 samples per group. *, p < 0.05, **, p < 0.01, and ***, p < 0.001 via the Student's t-test. The viral titer data represent means ± SEM (error bars) of ≥3 samples per group. *, p < 0.05 via the Mann-Whitney U test.

Fig. 2. The effects of curcumin treatment on the cytokine and chemokine mRNA levels of CVB3-infected Huh-7 cells. Huh-7 cells were infected without (-) or with (+) CVB3 (MOI = 0.002), treated without or with curcumin (30 μM) at 2 h post-infection (hpi.), and collected at the hpi. to assay for the mRNA levels of IL1B (A), TNF (B), IL6 (C), IFNB (D), and CXCL10 (E) by quantitative real-time RT-PCR. In each sample, the level of indicated mRNA was normalized to that of β-actin. The values of cells without infection are set as 100%. The data represent means + SEM (error bars) of ≥3 samples per group. *, p < 0.05 via the Student's t-test.

Fig. 3. The effects of curcumin treatment on levels of survival rate, clinical score, body weight, and liver damage of CVB3-infected mice. Mice were infected with CVB3, treated with one shot of indicated doses of curcumin (n = 17, 11, and 13 for 0, 10, and 50 mg/kg, respectively) per day for 10 days starting from 2 h post-infection, and monitored for survival rates (A), clinical scores (B), and body weight (C). (D) The liver sections of mock-infected (Mock) or CVB3-infected mice treated with one shot of vehicle or curcumin (50 mg/kg) were harvested on 5 or 7 days post-infection (dpi.), stained with H&E, examined by a microscopy (D), and measured for damage areas (E). Scale bar = 20 μm. The damage area in the liver of mock-infected mice is set as 1. The data represent means ± or + SEM (error bars). (E) n ≥ 3 samples per group. *, p < 0.05 via the log-rank test (A), Mann-Whitney U test (B), or Student's t-test (E) between the 0 and 50 mg/kg groups (B) or indicated groups.

Fig. 4. The effect of curcumin treatment on tissue viral loads of CVB3-infected mice. Mice were infected with CVB3 and treated with one shot of vehicle or curcumin (50 mg/kg) per day starting from 2 h post-infection. The mouse organs or tissue were harvested on indicated times to determine viral titers by plaque assay. The data represent means + SEM (error bars) of ≥3 per group.

Fig. 5. Curcumin treatment reduced the cleaved caspase-3 level and increased the HO-1 level in the liver of CVB3-infected mice. Mice were mock-infected (Mock or -) or infected with CVB3 (+) and treated with one shot of vehicle or curcumin (50 mg/kg) per day starting from 2 h post-infection. Mouse livers were harvested 3 days post-infection to assay for cleaved caspase 3 (A) and HO-1 (B) by western blotting. The representative blots (above) and quantitated results (bottom) are shown. In each sample, the level of indicated protein was normalized to that of β-actin. The values of mock-infected samples are set as 100%. The data represent means + SEM (error bars) of 4 samples per group. *, p < 0.05 via the Student's t-test.

Fig. 6. The effect of curcumin treatment on cytokine and chemokine levels in the liver or serum of CVB3-infected mice. Mice were mock-infected or infected with CVB3. Infected mice were treated with one shot of vehicle or curcumin (50 mg/kg) per day starting from 2 h post-infection. At the indicated day post-infection (dpi.), mouse livers were harvested to quantify (A) IFN-γ, (B) MIG, (C) RANTES, and (D) TNF, and mouse sera were collected to assess (E) IL-8, (F) IL-10, (G) IFN-γ, and (H) TNF by CBA or ELISA. The data represent means + SEM (error bars) of ≥4 per group. *, p < 0.05 and **, p < 0.01 via the Mann-Whitney U test.

Fig. 7. The effect of curcumin treatment on leukocyte levels in the liver of CVB3-infected mice. Mice were infected with CVB3 and treated with one shot of vehicle or curcumin (50 mg/kg) per day starting from 2 h post-infection. Mouse livers were harvested 5 days post-infection and processed to isolate leukocytes, which were stained with CD45, CD11b, CD11c, F4/80, Ly-6 G, and CD3 to quantify (A) neutrophils, (B) dendritic cells, and (C) T cells by flow cytometry. The data represent means + SEM (error bars) of ≥3 per group. **, p < 0.01 via the Student's t-test.