Chlorogenic Acid Ameliorates CVB3-Induced Viral Myocarditis by Suppressing Viral Replication and ZBP1-Mediated PANoptosis

Figures

• 

  Figure 3. The antiviral effect of CGA is confined to the replication phase of CVB3. HeLa cells infected with CVB3 (MOI = 1)were treated with CGA during different phases of the viral life cycle. The viral genomic RNA was quantified by RT-qPCR. Meanwhile, the Western blotting analysis was performed to detect 3D protein at specified time points, using GAPDH as the loading control. (A) Virus attachment assay. (B) Virus entry assay. (C) Virus replication assay. (D,E) Virus release assay. The results represent the mean ± SD of three independent biological replicates. * p < 0.05; ** p < 0.01; ns, not significant.

• 

  Figure 1. CGA Alleviates CVB3 Myocarditis via Dual Suppression of Viral Replication and Inflammation. (A) Experimental design. (B) The body weight of the mice was monitored daily for changes (n = 10 mice per group). (C) Histopathological assessment of cardiac lesions was performed on CGA-treated mice at 7 days post-infection (dpi) via HE staining (n = 5 hearts per group). Scale bar = 10 µm. (D) Expression of inflammatory cytokines was assessed by RT-qPCR amplification of myocardial tissue-derived total RNA. (E) LDH and CK-MB comparison between CGA-treated and control cohorts in serum. (F) CVB3 genomic RNA load measured by RT-qPCR using virus-specific primers in cardiac tissue-derived total RNA. (G) Comparative analysis of CVB3 3D protein levels in myocardial tissue by Western blotting under CGA-treated and untreated conditions. Data are presented as mean ± SD of three independent biological replicates. ** p < 0.01; *** p < 0.001.

• 

  Figure 2. CGA inhibits CVB3 replication in HeLa cells in a dose- and time-dependent manner. (A) HeLa cells were incubated with various concentrations of CGA for 48 h, and cell viability was assessed using the CCK-8 assay. (B) HeLa cells were infected with CVB3 (MOI = 1) for 2 h and then treated with the indicated concentrations of CGA for 48 h. Cell viability was determined by the CCK-8 assay. (C) Fluorescence microscopy images of HeLa cells infected with EGFP-CVB3 at 24 hpi under three conditions: mock-infected, CVB3-infected, and CVB3-infected with CGA treatment. Scale bar = 10 µm. (D) Viral titers in CVB3 (MOI = 1)-infected HeLa cells were quantified by TCID50 assay at 24, 36, and 48 hpi. (E) HeLa cells infected with CVB3 (MOI = 1) and cultured in CGA-containing medium were harvested at 24, 36, and 48 hpi for total RNA extraction and RT-qPCR analysis. (F) Total protein from CVB3 (MOI = 1)-infected HeLa cells collected at the indicated time points was analyzed by Western blotting for CVB3 3D expression, with GAPDH as a loading control. Data are presented as mean ± SD of three independent biological replicates. * p < 0.05; ** p < 0.01; *** p < 0.001.

• 

  Figure 4. ZBP1 is a critical mediator of CVB3-induced cell death and a target of CGA. (A) Cells were infected with CVB3 (MOI = 1) and harvested at the indicated time points (0, 6, 12, and 24 hpi). Protein levels of ZBP1, C-Caspase3, RIP3, P-MLKL, C-Caspase1, and GSDMD-N were analyzed by Western blotting. GAPDH served as a loading control. (B) HeLa cells were transfected with siZBP1 at a ratio of 1:4. Knockdown efficiency was assessed by Western blotting, and cell viability was evaluated using the CCK-8 assay. (C) HeLa cells transfected with siNC or siZBP1 were infected with CVB3 (MOI = 1) for 24 h. Protein levels of CVB3 3D, ZBP1, C-Caspase3, RIP3, MLKL, C-Caspase1, and GSDMD-N were detected by Western blotting. (D) Total RNA was extracted from CVB3-infected HeLa cells following siNC or siZBP1 transfection, and CVB3 genomic RNA levels were quantified by RT-qPCR. (E) HeLa cells were infected with CVB3 (MOI = 1) and treated with CGA (50 or 100 μM) for 24 h. Protein levels of ZBP1, C-Caspase3, RIP3, MLKL, P-MLKL, C-Caspase1, and GSDMD-N were analyzed by Western blotting. Densitometric analysis of protein bands was performed using Image J software, and relative protein expression levels were…

• 

  Figure 5. CGA downregulates ZBP1 by promoting its ubiquitin–proteasome-dependent degradation. (A) HeLa cells were infected with CVB3 (MOI = 1) and cultured for 24 h in media with or without CGA. Total RNA was extracted, and ZBP1 mRNA abundance was quantified by RT-qPCR. (B) Following 12 h pretreatment with CGA, HeLa cells were cultured in CHX (30 ng/mL)-containing medium for 12 h. Proteins were harvested at indicated post-infection time points for Western blotting analysis of ZBP1 expression. (C) HeLa cells were either treated with MG132 (30 μM) for 24 h or infected with CVB3 (MOI = 1) followed by CGA treatment for 24 h. Cellular lysates were subjected to Western blotting analysis. (D) Quantitative alterations in ZBP1 expression were calculated relative to GAPDH loading control. (E) Following identical treatments as in (C), cell lysates were analyzed by Western blotting. The results represent the mean ± SD of three independent biological replicates. ** p < 0.01; *** p < 0.001; ns, not significant.

• 

  Figure 6. Scheme summarizing the inhibitory effect of CGA on CVB3-induced PANoptosis.