Background: Myocarditis is a rare, potentially fatal condition that particularly affects young people, with diverse causes ranging from viral infections (the most common in developed nations) to autoimmune diseases and toxins. Although acute viral myocarditis (VMC) is frequently subclinical and may resolve spontaneously, persistent inflammation causes continual myocyte damage, leading to intractable heart failure or death. Treatment options for chronic myocarditis remain inadequate. The primary purpose of this study was to investigate whether C-28 methyl ester for 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO-Me), a potent Nuclear factor erythroid 2-related factor 2 (Nrf2) activator, alleviates coxsackievirus B3 (CVB3)-induced myocardial injury in viral myocarditis, potentially through activating the Nrf2/heme oxygenase-1 (HO-1) signaling axis to exert anti-inflammatory and antioxidant effects.

Methods: Male BALB/c mice were subjected to CVB3 treatment to establish a CVB3-induced VMC mouse model, and body weight loss and survival were documented. Then, an enzyme-linked immunosorbent assay (ELISA) for measuring myocardial injury marker and inflammatory indicator contents in plasma was conducted. Echocardiography was done to detect the mouse cardiac function. To observe alterations in myocardial tissue morphology, hematoxylin-eosin (H&E) staining was conducted. Furthermore, nucleotide oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing protein 3 (NLRP3), apoptotic-associated speck-like protein containing a CARD (ASC) and Caspase-1/Cleaved contents in VMC mice and H9C2 cells were measured through Western blotting (WB), immunofluorescence analysis, and quantitative real-time polymerase chain reaction (qRT-PCR). Nrf2, P-Nrf2, HO-1 and NAD(P)H quinone oxidoreductase 1 (NQO-1) levels were also examined through immunofluorescence, WB, and qRT-PCR in mice and H9C2 cells after treatment with CVB3. Moreover, for exploring how Nrf2 affected CVB3-treated VMC in vitro, Nrf2 was downregulated in H9C2 cells by small interfering RNA (siRNA) transfection.

Results: In vivo experiments revealed that bardoxolone methyl (CDDO-Me) intervention significantly improved survival, body weight loss, myocardial injury, inflammatory reactions and ventricular systolic dysfunction in CVB3-induced VMC (p < 0.05, p < 0.01, or p < 0.001 vs. CVB3 group, where applicable). Immunofluorescence, WB, and qRT-PCR analyses further revealed that the expression of inflammasome-associated proteins such as NLRP3, ASC and Caspase-1/Cleaved Caspase-1 was significantly increased in CVB3-induced VMC mice (p < 0.001), and this change was reversed by CDDO-Me intervention (p < 0.01 or p < 0.001). Notably, the downregulation of Nrf2/HO-1 pathway-related proteins in CVB3-induced VMC mice was reversed by CDDO-Me treatment (p < 0.05, p < 0.01, or p < 0.001). In vitro, we observed that CDDO-Me intervention ameliorated the CVB3-induced decrease in viability and inflammatory reactions in H9C2 cardiomyocytes (p < 0.001). Correspondingly, NLRP3, ASC and Caspase-1 were highly expressed in H9C2 cells co-incubated with CVB3 (p < 0.001), and this high expression could be reversed by CDDO-Me intervention (p < 0.05, p < 0.01, or p < 0.001), suggesting that CDDO-Me intervention could reduce inflammation levels in H9C2 cells co-incubated with CVB3. Moreover, similar to the in vivo experimental results, our in vitro study suggested that the downregulating Nrf2/HO-1 pathway-related proteins in H9C2 cells co-incubated with CVB3 was also significantly reversed by CDDO-Me intervention (p < 0.05, p < 0.01, or p < 0.001). We then transfected H9C2 cells with Nrf2-specific siRNA to silence Nrf2, as Nrf2 knockdown significantly suppressed H9C2 cell survival and enhanced the inflammatory response (p < 0.05, p < 0.005, or p < 0.001).

Conclusion: CDDO-Me reduces myocardial injury and inflammatory reactions in CVB3-induced viral myocarditis, resulting from a reduction in the NLRP3 inflammasome induced by the regulation of Nrf2/HO-1.