Figure 5.

Overexpression of TRIM21 in vivo significantly reduces viral load and alleviates CVB3-induced viral myocarditis. (A) Male BALB/c mice were retroorbitally injected 50 μg TRIM21 plasmids using in vivo-jet PEI and were sacrificed daily till day 4. Protein level of cardiac TRIM21was measured by IHC assay. (B) Mice were injected retroordibitally with 2 doses of 50 μg PEI-packaged mock or TRIM21 plasmids on day −2 and 1 and subjected to 1000TCID₅₀ CVB3 on day 0(n = 6). The survival rate (C) and body weight change (D) were monitored daily until day 7 p.i. (E) Representative image of HE-staining hearts of CVB3-infected mice (day 7 p.i.) treated with PEI-TRIM21 or PEI-vector, showing intra-cardiac immune infiltrates (marked with arrows). Scale bar: 100 μm. Pathological scores of the heart of mice are shown. Results are presented as mean ± SEM; ^*p < 0.05. (F) Protein levels of inflammatory cytokines in the homogenates of heart were measured by ELISA. Data were presented as mean ± SEM of three representative independent. (G) The cardiac CVB3 titer at day 3 p.i. were determined by TCID₅₀ assay. Data represent mean values of CVB3 PFU per gram of the heart tissues. Results are presented as mean ± SEM; Data pooled from 3 independent experiments. ^*p < 0.05; ^**p < 0.01. (H) Relative mRNA level of IFN-β (day 3 and 7p.i.) was detected by Q-PCR. Data were normalized to GAPDH expression and presented as mean ± SEM of three representative independent. (I) Hearts of mice at day 3 and 7 p.i. were OCT-embedded and cyrosections (5 μM) were subjected to fluorescent staining. Composite confocal represented images show dsRNA (red, anti-dsRNA Ab) and nuclear (blue, DAPI).Low magnification (magnification, × 100) and higher magnification of the boxed areas (magnification, × 200) are shown. The number of red-stained viral-infected cells in the heart sections of mice were numerated. Data are expressed as mean ± SEM from three repeated experiments (n = 3). ***p < 0.001.