## 3 Results  
Hesperetin Protects and Maintains the Structural and Ultrastructular Integrity of Cardiac Tissue Affected by Epirubicin-Induced Damage  
Heart tissue sections from the control group and those treated with HSP displayed typical cardiac histology. However, heart cells in the group treated with Epi exhibited changes such as cytoplasmic vacuolization, myofibril loss, and disarray of fibers (Figure 1). Additionally, light microscopy identified inflammatory cell infiltration and a slight increase in interstitial collagen fibers. The heart tissue of the group receiving both treatments largely resembled that of the control group. The distribution of desmin, a key intermediate filament crucial for maintaining cell structure, was examined in the context of cardiotoxicity caused by epirubicin. In the control mice, immunostaining for desmin was present in the cardiomyocytes. Following Epi treatment, there was a noticeable disorganization of the intermediate filaments, as indicated by the weakened and sporadic expression of desmin. This contrasted with the control and was shown to be improved by co-treatment with HSP, as shown in Figure 1.  
Electron microscopy showed that the control and HSP-treated tissues maintained normal ultrastructure, with mitochondria appearing to be mostly round or polygonal and  
Figure 1: Hesperetin protects and maintains the structural architecture of the cardiac tissue affected by epirubicin-induced damage. H&E—hematoxylin and eosin stain; FG—[PERSON] stain (collagen-red); immunohistochemical (IHC) expression and specific cardiac distribution of desmin; magnification \(\times 20\) (H&E); \(\times 40\) (desmin and FG). containing densely arranged cristae. In contrast, Epi treatment resulted in disruption of normal structure, including vacuolization, myofibrillar degradation, mitochondrial cristae loss, and localized accumulation of lipids and collagen (Figure 2). The group that received the combined treatment showed alleviated ultrastructural architecture. Hesperetin Provides Protection for Cardiomyocytes against Apoptosis Caused by Epirubicin-Induced Damage  
Additionally, we investigated the impact of Epi on the regulation of Bcl-2 family proteins, which are associated with the mitochondrial membrane. Treatment with Epi markedly upregulated the expression of the pro-apoptotic protein Bax in heart tissue (as shown in Figure 3; \(p<0.001\)) and resulted in a decreased expression of the anti-apoptotic protein Bcl-2 (Figure 4). Furthermore, staining for caspase-3 with immunoperoxidase highlighted considerably immunostaining of caspase-3 in the EPI-treated group, indicative of pronounced apoptotic activity in the cardiac tissue (Figure 5) when compared with control tissue (\(p<0.001\)). The immunohistochemical pattern was restored after the treatment with flavonoid. Figure 2: Hesperetin protects and maintains the ultrastructural integrity of the cardiac tissue affected by epirubicin-induced damage. (**a**) Control group; (**b**) epirubicin group; (**c**) epirubicin and HSP group; (**d**) HSP group; M—normal aspect of mitochondria; \({}^{*}\)—mitochondria with rarefied cristae; L—lipids; C—collagen. Figure 3: (**A**) Expression of BAX in cardiac tissue by immunofluorescence (magnification \(62\times\)). (**B**) Bar graphs showing semi-quantification of fluorescence intensity for Bax. *** statistical significance at \(p\) < 0.001 as compared to control; *** statistical significance at \(p\) < 0.001 as compared to Epi group. Figure 4: (**A**) Expression of Bcl-2 in cardiac tissue as revealed by immunofluorescence (magnification 62\(\times\)). (**B**) Bar graphs showing semi-quantification of fluorescence intensity for Bcl-2. *** statistical significance at \(p<0.001\) as compared to control; *** statistical significance at \(p<0.01\) as compared to control; *** statistical significance at \(p<0.001\) as compared to Epi group. Epirubicin treatment increased TUNEL-positive nuclei significantly compared to control (\(p<0.001\)).