Antioxidative Effects of Lactobacillus Acidophilus against Zearalenone- Induced Oxidative Stress in the Reproductive System of Mice

Introduction: Zearalenone (ZEN) is an estrogenic mycotoxin produced by a specific species of Fusarium fungus. This experimental animal study investigated the antioxidant effects of Lactobacillus aci-dophilus on oxidative stress caused by ZEA in the ovarian and uterine tissues of an animal model. Methods: This experimental animal study was conducted on 40 female mice randomly divided into four groups (n=10). The groups received normal saline, ZEN (40 μg/kg/day, IP), L. acidophilus [5×10^9 CFU/ml, intraperitoneally (IP)], or a combination of ZEN and L. acidophilus for one week. At the end of the intervention, blood samples were collected under deep anesthesia, and the uterine and ovarian tissues were isolated. Biochemical markers (MDA, TAC, thiol groups, sex hormones) and histological changes were evaluated. GraphPad Prism 5 was used for statistical analysis. results: 3. RESULTS 3.1. Effects of Probiotic and ZEN Treatment on TAC in Ovary and Uterus Tissues In the ovarian tissue, the control group (C) exhibited the highest TAC level, with a mean value of approximately 8 µmol/mg protein. Treatment with L. acidophilus alone (P group) resulted in a slight reduction in TAC (around 6 µmol/mg protein) compared to the control, though this difference was not statistically significant (p > 0.05). Exposure to ZEN (Z group) significantly decreased the TAC level to approximately 3 µmol/mg protein, indicating a marked reduction in antioxidant capacity compared to the control group (p < 0> 0.05). ZEN exposure (Z group) led to a significant reduction in TAC (p < 0> 0.05). Exposure to ZEN (Z group) significantly elevated MDA levels to approximately 10 µmol/mg protein, reflecting a substantial increase in lipid peroxidation compared to the control group (p < 0> 0.05). ZEN exposure (Z group) markedly increased MDA levels to approximately 7 µmol/mg protein, indicating heightened oxidative stress compared to the control group (p < 0> 0.05). In the uterine tissue (Figure 3B), a similar pattern was observed. The control group displayed a thiol level of approximately 0.006 µmol/mg protein. The probiotic-only group (P) showed a significant increase in thiol levels to around 0.007 µmol/mg protein compared to the control (p < 0> 0.05) and higher than the ZEN-only group, though the difference was not statistically significant (p > 0.05). 3.4. Effects of probiotic & ZEN treatment on the Progesterone and Estrogen level For progesterone levels (Figure 4A), the control group (C) exhibited a baseline concentration of approximately 30 ng/mL. Treatment with L. acidophilus alone (P group) slightly increased progesterone levels to around 35 ng/mL, though this difference was not statistically significant compared to the control (p > 0.05). Exposure to ZEN (Z group) significantly elevated progesterone levels to approximately 38 ng/mL, indicating a disruption in hormonal balance compared to the control group (p < 0> 0.05). ZEN exposure (Z group) significantly decreased estrogen levels to approximately 22ng/mL, reflecting its estrogenic activity as an endocrine disruptor compared to the control group (p < 0.0001). Treatment with L. acidophilus in the ZP group increased estrogen levels to about 31ng/mL, which was significantly above than the ZEN-only group (p < 0.05) but remained significantly different from the control (p < 0.001). 3.5. Histopathological Results Adipose tissue was removed from the ovaries and uterine horn, and saline was used to clean them. Before starting the preparation process for tissue slides, uterine and ovarian samples were fixed in 10% formalin buffer. Samples were blocked in paraffin and 5 μm tissue sections were prepared from the blocks and stained with H&E. Ovarian and uterine tissue slides were examined under a light microscope for histopathological study and measurement of epithelial layer height. Statistical analysis did not show a significant difference in the thickness of the preatrium and myometrium layers of the uterine horn between different groups. Endometrium with normal oval epithelial cells was seen in the normal group and the probiotic group. However, the height of epithelial cells and the endometrium layer in general decreased significantly in the zearalenone group compared to the control group. Relative and mild endometritis was observed as diffuse lymph cell infiltration in this group. Other changes observed in this layer are the integrity and order of epithelial cells, which disappeared after administration of zearalenone, and administration of probiotics with zearalenone greatly reduced the destruction of the uterine endometrium of mice (Figure 5). Examination of the ovaries in the control group and the group receiving probiotic shows the normal growth of ovarian follicles, blood vessels and normal stromal cells. The pre-ovulatory follicles showed normal morphology and the euchromatin nucleus, which was surrounded by normal, dense shingles and several layers of granulosa cells were located in the basement membrane. External and internal motions were clearly distinguishable. The normal primary follicles were surrounded by a layer of cubic granulosa cells and were located on the basement membrane. The most histopathological changes in the ovaries were seen in the group receiving zearalenone as a decrease in the number of ovarian follicles. Other changes in this group include vacuolation of luteal cells as well as granulosa cells of follicles before ovulation. In addition, histological changes indicate thin and irregular shingles in the follicles of this group. Congestion of blood vessels was another change in this group. Micronucleus formation was observed in follicular oocytes before ovulation, indicating DNA damage in the zearalenone-receiving group. Cell nucleus degeneration changes, which are the division of the nucleus into pre-ovulatory follicles, were not present in any of the groups. Probiotic administration with zearalenone had a significant effect on the number and quality of follicles and tissue integrity (Figure 6). Results: ZEN significantly reduced total antioxidant capacity (TAC) and thiol levels while increasing malondialdehyde (MDA) in ovarian and uterine tissues (p<0.0001), indicating heightened oxidative stress and disrupted hormonal balance by elevating progesterone and lowering estrogen levels (p < 0.0001). L. acidophilus treatment mitigated these effects, partially restoring TAC, thiol, and hormone levels (p < 0.01) while reducing MDA (p < 0.01). Histopathologically, ZEN caused a reduction in follicle count in the ova-ries, which was significantly alleviated by L. acidophilus co-treatment. Discussion: The findings highlighted the significant protective role of Lactobacillus acidophilus against ZEN-induced oxidative stress and endocrine disruption in the murine reproductive system. ZEN markedly impaired antioxidant defenses (reduced TAC and thiol levels, elevated MDA) and altered sex hormone profiles (increased progesterone, decreased estrogen), consistent with its known estrogenic and pro-oxidant effects. These adverse outcomes were accompanied by notable histopathological damage in ovarian and uterine tissues. L. acidophilus supplementation effectively attenuated these effects, likely through multiple mechanisms: direct free radical scavenging, activation of the Nrf2-mediated antioxidant pathway, physical binding and detoxification of ZEN, anti-inflammatory actions, and modulation of the gut-reproductive axis via short-chain fatty acids. Conclusion: The present study has demonstrated L. acidophilus to exert significant protective effects against ZEN-induced oxidative stress, hormonal disruptions, and tissue damage in the ovary. Thus, probi-otic supplementation may serve as an effective strategy to mitigate the adverse effects of ZEN exposure on reproductive health; however, further studies are warranted.