ROS-Mediated Necroptosis Promotes Coxsackievirus B3 Replication and Myocardial Injury

Figures

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  Figure 1. CVB3 induces necroptosis in HeLa cells. (A) PI staining (red) of HeLa cells at 24 h post-infection with CVB3 (MOI = 1) or mock treatment. Scale bar = 10 µm. (B) The relative count of PI-positive cells in the CVB3 group was calculated after normalization to total cell count and is presented relative to the mock-treated group. (C,D) Western blot analysis of RIP1, RIP3, and P-MLKL expression in CVB3-infected cells at indicated time points with viral titers measured in parallel. GAPDH was used as a loading control to normalize protein loading. (E,F) Bands in panels C and D were quantified by ImageJ, and the intensity ratios of RIP1, RIP3, and P-MLKL to corresponding GAPDH were shown. Data are represented as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 2. Nec-1 inhibits CVB3-induced necroptosis in HeLa cells. (A) Experimental timeline for Nec-1 treatment. HeLa cells were infected with CVB3 (MOI = 1) and treated with Nec-1 at indicated time points. The arrows indicated the start of each process. (B) HeLa cells infected with CVB3 (MOI = 1) were treated with Nec-1. At 24 hpi, cell morphology was analyzed by PI staining, with images captured using light and fluorescence microscopy. Scale bar = 10 µm. (C) Relative PI-positive cells in the CVB3+Nec-1 group, normalized to total cells and presented relative to the mock group. (D) HeLa cells were pretreated with 20 μM Nec-1 for 2 h before CVB3 infection (MOI = 1). Cells were harvested at 24 h post-infection, and lysates were analyzed by Western blot. (E) Quantitative analysis of RIP1 and RIP3 protein levels by ImageJ, normalized to GAPDH as a loading control. (F) RIP3 expression as assessed by fluorescence microscopy with Immunofluorescence staining of RIP1 and RIP3 in CVB3-infected cells (MOI = 1) at 24 hpi. Cells were stained with anti-RIP1 and anti-RIP3 antibodies, followed by FITC-conjugated (green) or CY3-conjugated (red) secondary antibodies, respectively. DNA was counterstained with…

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  Figure 3. Nec-1 inhibits viral CVB3 production in HeLa Cells. (A) HeLa cells were infected with CVB3 at MOI of 1 and were cultured with Nec-1 for 24 h. Total RNA was extracted and viral CVB3 RNA was quantified by RT-qPCR. Total RNA was extracted and viral CVB3 RNA was quantified determined by RT-qPCR. (B) Total progeny viruses were collected in Nec-1-treated HeLa cells 24 h after infection with CVB3 (MOI = 1) using HeLa cells titration. (C,D) HeLa cells were preincubated with 20 μM Nec-1 for 2 h prior to CVB3 (MOI = 1) infection. Cellular lysates were analyzed by Western blot subjected to immunoblot analysis 24 h post-infection to determine 3D expression, with GAPDH utilized as the loading control. All data were normalized to GAPDH mRNA and are expressed as fold change relative to the CVB3 group, which was assigned a value of 1.0. (E) Fluorescence microscopy images of HeLa cells infected with EGFP-CVB3 (MOI = 5). Images were acquired 24 h post-infection. Scale bars = 10 µm. Data are represented as mean ± SD of three independent experiments. ** p < 0.01.

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  Figure 4. Nec-1 demonstrates cardio-protective effects in CVB3-infected mice. (A) Experimental design. (B) Daily body weight changes in mice. The arrows indicated the time points of virus infection, addition of Nec-1, and sample collection. (C) Histopathological assessment of cardiac lesions by H&E staining at 7 dpi in CVB3-infected mice with Nec-1 treatment. (D) Comparative analysis of serum LDH and CK-MB levels in Nec-1-treated versus untreated groups. (E,F) Myocardial inflammatory cytokines (IFN-α, IL-1β, IL-6, TNF-α) were quantified by RT-qPCR. Total RNA isolation from cardiac tissues was performed. Data are presented relative to the CVB3 group after normalization to GAPDH mRNA levels, with the control group value set to 1.0. (G,H) Cardiac RIP1, RIP3, and CVB3 3D protein expression at 7 dpi with/without Nec-1 treatment. (G) Representative immunoblots. (H) Quantified protein levels normalized to GAPDH. Data are represented as mean ± SD of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.

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  Figure 5. Excessive ROS induces necroptosis. (A,B) Myocardial tissues were, respectively, collected from control and CVB3-infected mice at 5 and 7 dpi. Nrf2 and HO-1 were analyzed by Western blot, using GAPDH as a loading control. (C) HeLa cells were mock-infected (M) or infected with CVB3 at a MOI of 1 (I) and then collected at the indicated times. Nrf2 and HO-1 were detected by Western blot analysis. GAPDH is shown as a loading control. (D) Densitometric quantification of Nrf2 and HO-1 to GAPDH bands was conducted using ImageJ with background subtraction. (E) Intracellular ROS production was detected by fluorescence microscopy with DCFH-DA staining, followed by quantitative analysis of signal intensity using ImageJ. Scale bar = 10 μm. (F) Cell membrane integrity assessment via PI staining in CVB3-infected HeLa cells (MOI = 1) cultured with NAC for 24 h. Phase-contrast and fluorescence imaging were simultaneously acquired. Cell viability was subsequently determined through CCK-8 absorbance measurements at 450 nm. Scale bar = 10 μm. (G) HeLa cells infected with CVB3 (MOI = 1) were treated with NAC (0, 5, 10 mM) for 24 h, and RIP1, RIP3 and CVB3 3D were detected by Western blotting. (H)…

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  Figure 6. The mechanism of necroptosis by CVB3 infection.