Next, we detected proteins that are representative of inflammation and apoptosis. In our research, we examined the levels of p65 and TNF-α to understand the inflammatory response in the context of doxorubicin-induced cardiotoxicity. These indicators served as crucial markers in elucidating the inflammatory pathways involved in our experimental model. Western bolt analysis indicated that DOX promoted the phosphorylation and nuclear accumulation of p65, and shikonin administration mostly reversed this alteration (Fig. 3A,B). Cardiac TNF-α levels detected by ELISA showed that shikonin decreased the elevlated cardiac TNF-α induced by DOX (Fig. 3D). The mRNA expression of cardiac inflammation biomarkers were also examined, and the result showed that DOX administration promoted the mRNA expression of pro-inflammatory genes (il-6, il-1β, mcp-1 and tnf-α), which were prevented by shikonin treatment (Fig. 3E). In our study, we investigated the expression of key apoptotic regulators, including Bax and Bcl-2, along with assessing caspase-3 activity, to elucidate the underlying mechanisms of cell apoptosis in response to doxorubicin-induced cardiotoxicity. Shikonin attenuated DOX-induced upregulation of Bax and the down-regulation of Bcl-2 (Fig. 3A,C). Besides, shikonin significantly reduced the level of apoptotic cardiomyocytes in DOX-treated mice, as evidenced by Tunel staining and caspase3 activity (Fig. 3F–H).

Shikonin attenuated DOX-induced cardiomyocyte inflammation and apoptosis. (A–C) Western blot and quantitative analysis showing the protein levels of p-P65, t-P65, Nuc-P65, Bax, Bcl-2 in four groups (n = 6). Original blots/gels are presented in Supplementary Fig. S4. (D) Cardiac TNF-α levels as detected by ELISA (n = 8). (E) The relative mRNA levels of il-6, il-1β, tnf-α, and mcp-1 normalized to gapdh in mice (n = 8). (F) Activity of caspase-3 of mice in four groups (n = 8). (G,H) Myocardial apoptosis measured by TUNEL staining in heart sections (n = 8, bar = 50 μm). **p < 0.01, ****p < 0.0001, significantly different as indicated.

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