Pterostilbene protects against doxorubicin-induced cardiotoxicity in canines via a gut microbiota–6AN–NOX2 axis

Doxorubicin (DOX) is a powerful chemotherapeutic agent, but its clinical use is restricted by cumulative and irreversible cardiotoxicity. Intestinal dysbiosis has been linked to DOX-induced cardiac injury, yet the underlying mechanisms and therapeutic targets remain elusive. This study aimed to investigate whether pterostilbene (PTE), a natural prebiotic plant extract, alleviates DOX cardiotoxicity by regulating gut microbiota and their metabolites. Eighteen beagles were randomized into control, DOX (30 mg/m² weekly for 7 weeks), and PTE (50 mg/kg daily for 9 weeks) + DOX groups. Fecal microbiota transplantation (FMT) from canine donors to microbiota-depleted rats, 16S rRNA sequencing, metabolome analysis, and in vitro H9C2 cell experiments were conducted. Main outcomes included survival rate, cardiac function parameters, cardiac injury biomarkers, microbial diversity, and oxidative stress-related indicators. In beagles receiving cumulative DOX (30 mg/m 2 weekly for 7 weeks), PTE cotreatment (50 mg/kg daily for 9 weeks) significantly improved survival (83.3% vs. 50.0%, n=6/group) and attenuated myocardial injury, evidenced by reduced plasma CK and LDH activities (both p<0.01 vs. DOX). Echocardiography revealed PTE restored LVEF and LVFS while reducing EPSS and LVIDd (p<0.05). 16S rRNA sequencing demonstrated PTE reversed DOX induced loss of α-diversity (ACE, Shannon, Chao indices, p<0.05) and enriched beneficial Faecalibacterium while suppressing proinflammatory Corynebacterium and Allobaculum (q<0.05). Fecal microbiota transplantation confirmed microbiota dependent cardioprotection. Metabolomics identified 6-aminonicotinamide (6AN) as a key microbial metabolite inversely correlated with cardiac damage. In H9C2 cells, 6AN (1 µM) replicated PTE’s protection by restoring antioxidant enzyme activities, reducing ROS and MDA, and attenuating apoptosis (all p<0.01), effects abolished by NOX 2 overexpression. PTE mitigates DOX cardiotoxicity via restructuring gut microbiota, increasing microbial metabolite 6AN, and suppressing NOX 2 -mediated oxidative stress. Targeting the microbiota-6AN-NOX 2 axis represents a promising strategy to preserve cardiac function during anthracycline chemotherapy. These findings establish a mechanistic basis for PTE as a safe, natural adjunctive therapy in cancer patients receiving DOX. Not applicable. • PTE improves survival and cardiac function in DOX-treated beagles. • PTE reverses DOX-induced gut dysbiosis and restores beneficial taxa. • FMT from PTE-treated donors protects against DOX cardiotoxicity in rats. • 6AN, a microbial metabolite, reduces DOX-induced oxidative stress and apoptosis. • PTE exerts cardioprotection via microbiota–6AN–NOX 2 axis modulation.