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Compound probiotics regulate the NRF2 antioxidant pathway to inhibit aflatoxin B1-induced autophagy in mouse Sertoli
Hongwei Guo1, Taiyang Liu2, Jie Li2
1Second Department of Urology, Affiliated Central Hospital of Huanghuai University, Zhumadian 463000, China; College of Biology and Food Engineering, Huanghuai University, Zhumadian 463000, China.
Abstract:
This study investigated the effects of compound probiotics (CP) on AFB1-induced cytotoxicity in Sertoli TM4 cells. The L9 (3 × 3) orthogonal test was conducted to determine the optimal CP required for high AFB1 degradation in the artificial gastrointestinal fluid in vitro. The maximal AFB1 degradation rate was 40.55 % (P < 0.05) when the final viable count was 1.0 × 105 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 × 104 CFU/mL) was added to the TM4 cells for 24 h, the cell viability reached 108.86 % (P < 0.05). AFB1 reduced TM4 cell viability in a concentration- and time-dependent manner at an AFB1 concentration ranging from 0 to 1.5 μM after 48-h AFB1 exposure. The optimal AFB1 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 AFB1, 1.25 μM AFB1, CPS4, and CPS4+0.5 μM AFB1. 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 AFB1 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 AFB1 degradation, activated the p62-NRF2 antioxidant, and inhibited ROS/TRPML1 pathways, thereby reducing ROS production and inflammation and ultimately alleviating AFB1-induced autophagy and apoptosis. These findings supports the potential of probiotics to protect the male reproductive system from toxin damage.
Insights
Compound probiotics (CP) effectively degrade aflatoxin B1 (AFB1) and protect Sertoli TM4 cells from AFB1-induced damage. CP activates antioxidant pathways and reduces inflammation, offering potential protection for the male reproductive system against toxin-induced apoptosis.
Area of Science:
- Reproductive Toxicology
- Microbiology
- Cell Biology
Background:
- Aflatoxin B1 (AFB1) poses a significant threat to male reproductive health.
- Sertoli cells are crucial for spermatogenesis and are vulnerable to toxic insults.
- The protective effects of probiotics against AFB1-induced cytotoxicity require further investigation.
Purpose of the Study:
- To investigate the efficacy of compound probiotics (CP) in degrading AFB1.
- To evaluate the protective effects of CP and its supernatant (CPS) against AFB1-induced cytotoxicity in Sertoli TM4 cells.
- To elucidate the molecular mechanisms underlying the protective effects of CP.
Main Methods:
- L9 orthogonal test to optimize CP for AFB1 degradation.
- In vitro assessment of CP and CPS effects on TM4 cell viability.
- Transcriptome sequencing and KEGG pathway analysis to identify molecular targets.
- AFB1-induced damage models in TM4 cells.
Main Results:
- Optimized CP achieved a maximal AFB1 degradation rate of 40.55%.
- CP supernatant (CPS4) significantly enhanced TM4 cell viability under AFB1 exposure.
- AFB1 induced apoptosis by inhibiting the PI3K-AKT-mTOR pathway and dysregulating autophagy.
- CPS4 activated the p62-NRF2 antioxidant pathway and inhibited ROS/TRPML1, reducing oxidative stress and inflammation.
Conclusions:
- Compound probiotics demonstrate significant AFB1 degradation capabilities.
- CP confers substantial protection to Sertoli TM4 cells against AFB1-induced damage.
- CP exerts its protective effects by modulating key cellular pathways involved in oxidative stress, inflammation, autophagy, and apoptosis, highlighting its therapeutic potential for male reproductive protection.
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