Compound probiotics regulate the NRF2 antioxidant pathway to inhibit aflatoxin B 1 -induced autophagy in mouse Sertoli TM4 cells

This study investigated the effects of compound probiotics (CP) on AFB -induced cytotoxicity in Sertoli TM4 cells. The L9 (3 × 3) orthogonal test was conducted to determine the optimal CP required for high AFB degradation in the artificial gastrointestinal fluid in vitro. The maximal AFB degradation rate was 40.55 % (P < 0.05) when the final viable count was 1.0 × 10 CFU/mL for Bacillus subtilis, Lactobacillus casein, and Saccharomyces cerevisiae. The effects of CP and the CP supernatant (CPS) on TM4 cell viability were evaluated to achieve the optimal protective conditions. When CPS4 (corresponding to CP viable counts of 1.0 × 10 CFU/mL) was added to the TM4 cells for 24 h, the cell viability reached 108.86 % (P < 0.05). AFB reduced TM4 cell viability in a concentration- and time-dependent manner at an AFB concentration ranging from 0 to 1.5 μM after 48-h AFB exposure. The optimal AFB concentration/times for low- and high damage models were 0.5 and 1.25 μM both for 24 h, which decreased viability to 76.04 % and 65.35 %, respectively. however, CPS4 added to low- and high-damage models increased the cell viability to 97.43 % and 75.12 %, respectively (P < 0.05). Transcriptome sequencing was performed based on the following designed groups: the control, 0.5 μM AFB , 1.25 μM AFB , CPS4, and CPS4+0.5 μM AFB . The Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis was further performed to identify significantly enriched signaling pathways, which were subsequently verified. It was shown that AFB induced apoptosis by blocking the PI3K-AKT-mTOR pathway and upregulating autophagy proteins such as LC3B, Beclin1, and ATG5 while inhibiting autophagic flux. CPS4 promoted AFB degradation, activated the p62-NRF2 antioxidant, and inhibited ROS/TRPML1 pathways, thereby reducing ROS production and inflammation and ultimately alleviating AFB -induced autophagy and apoptosis. These findings supports the potential of probiotics to protect the male reproductive system from toxin damage.