Modulation of TLR4/NFκB Pathways in Autoimmune Myocarditis

Abstract

Myocarditis is an inflammatory and oxidative disorder characterized by immune cell recruitment in the damaged tissue and organ dysfunction. In this paper, we evaluated the molecular pathways involved in myocarditis using a natural compound, Coriolus versicolor, in an experimental model of autoimmune myocarditis (EAM). Animals were immunized with an emulsion of pig cardiac myosin and complete Freund's adjuvant supplemented with mycobacterium tuberculosis; thereafter, Coriolus versicolor (200 mg/Kg) was orally administered for 21 days. At the end of the experiment, blood pressure and heart rate measurements were recorded and the body and heart weights as well. From the molecular point of view, the Coriolus versicolor administration reduced the activation of the TLR4/NF-κB pathway and the levels of pro-inflammatory cytokines (INF-γ, TNF-α, IL-6, IL-17, and IL-2) and restored the levels of anti-inflammatory cytokines (IL-10). These anti-inflammatory effects were accompanied with a reduced lipid peroxidation and nitrite levels and restored the antioxidant enzyme activities (SOD and CAT) and GSH levels. Additionally, it reduced the histological injury and the immune cell recruitment (CD4⁺ and CD68⁺ cells). Moreover, we observed an antiapoptotic activity in both intrinsic (Fas/FasL/caspase-3) and extrinsic (Bax/Bcl-2) pathways. Overall, our data showed that Coriolus versicolor administration modulates the TLR4/NF-κB signaling in EAM.

Keywords: experimental autoimmune myocarditis; immunomodulation; oxidative stress.

Figure 1

Coriolus versicolor modulates TLR4/NFκB signaling. A Western blot analysis of (A) TLR4, (B) MyD88, (C) p-IkB, and (D) RT-PCR with mRNA levels of NFκB. The results were analyzed using a one-way ANOVA followed by a Bonferroni post hoc test for multiple comparisons. A p-value of less than 0.05 was considered significant. Note: # p < 0.05 vs. EAM, ** p < 0.01 vs. control, ## p < 0.01 vs. EAM, *** p < 0.001 vs. control.

Figure 2

Coriolus versicolor modulates inflammatory cell recruitment. Histological analysis (magnification 20×): (A) controls; (B) EAM; (C) EAM + Coriolus versicolor; (D) pathological scores; (E) inflammatory cell infiltration scores. Immunohistochemical analysis of CD4 expression (magnification 40×): (F) controls; (G) EAM; (H) EAM + Coriolus versicolor; Immunohistochemical analysis of CD68 expression (magnification 40×): (I) controls; (J) EAM; (K) EAM + Coriolus versicolor; (L) graphical quantification of CD4 expression; (M) graphical quantification of CD68 expression. The results were analyzed using a one-way ANOVA followed by a Bonferroni post hoc test for multiple comparisons. A p-value of less than 0.05 was considered significant. Note: # p < 0.05 vs. EAM, *** p < 0.001 vs. control, ### p < 0.001 vs. EAM.

Figure 3

Coriolus versicolor modulates pro-inflammatory cytokines levels and oxidative stress. Serum levels: (A) IL-10; (B) IL-6; (C) IFN-γ; (D) IL-2; (E) IL-17; (F) TNF-α. (G) Malondialdehyde (MDA) levels. (H) Nitrite levels. (I) SOD activity. (J) CAT activity. (K) GSH levels. The results were analyzed using a one-way ANOVA followed by a Bonferroni post hoc test for multiple comparisons. A p-value of less than 0.05 was considered significant. Note: # p < 0.05 vs. EAM, ** p < 0.01 vs. control, ## p < 0.01 vs. EAM, *** p < 0.001 vs. control, ### p < 0.001 vs. EAM.

Figure 4

Coriolus versicolor modulates apoptosis. TUNEL assay: (A) controls; (B) EAM; (C) EAM + Coriolus versicolor; (D) number of TUNEL⁺ cells. RT-PCR for mRNA levels: (E) Fas; (F) FasL; (G) caspase-3; (H) Bax/Bcl-2 ratio. The results were analyzed using a one-way ANOVA followed by a Bonferroni post hoc test for multiple comparisons. A p-value of less than 0.05 was considered significant. Note: # p < 0.05 vs. EAM, *** p < 0.001 vs. control, ### p < 0.001 vs. EAM.