Rehearsal For Thesis Presentation

Attention deficit/hyperactivity disorder (ADHD) is the most prevalent neurodevelopmental disorder in children and adolescents, characterized by complex, incompletely understood pathogenic mechanisms involving genetic and environmental factors. A mainstream hypothesis points to an imbalance of catecholamine neurotransmitters, potentially linked to brain oxidative stress and chronic inflammation. Despite the efficacy of current ADHD treatments, their side effects and variable responses underscore the need for alternative therapies. Recent research highlights the microbiota-gut-brain (MGB) axis as a potential therapeutic target for ADHD. In this study, nine potential strains K1, K5, K11, K14, K38, K49, K50, K53, and K60 isolated from kefir, a fermented milk product rich in lactic acid bacteria and yeasts, were co-cultured in vitro with human neuroblastoma SH-SY5Y cells. We used their cell-free fermentation supernatant (CFS) and established a neuronal damage model via serum deprivation and 6-hydroxydopamine (6-OHDA) to evaluate effects on cell viability, proliferation, and antioxidant capacity. In vitro, 2.5% and 5% CFS concentrations significantly promoted cell viability in strains K5, K11, K14, K38, K49, K50, K53, and K60 after 24 hours in a dose-dependent manner (p < 0.05). Under serum deprivation for 24 and 48 hours, CFS from strains K1, K5, K11, K14, K38, and K50 significantly enhanced cell viability and proliferation (p < 0.05). After excluding composite genotype strain K1 using random amplified polymorphic DNA (RAPD) technology, 5% CFS from strains K5 and K38 significantly reduced reactive oxygen species (ROS) production under serum deprivation for 24 hours (p < 0.05). Furthermore, in the 10 μM 6-OHDA-induced toxicity model (24 hours), CFS from strains K5, K11, and K38 significantly restored cell viability (p < 0.05), with K11 and K38 CFS also significantly reducing ROS levels (p < 0.05). Following 6 hours of 6-OHDA induction, CFS from strains K5, K11, K14, K38, and K50 significantly reduced malondialdehyde (MDA) levels (p < 0.05). Based on these findings, Liquorilactobacillus nagelii K38 demonstrated robust neuroprotective potential across various in vitro damage models. To further elucidate L. nagelii K38's protective mechanism against 6-OHDA toxicity, we analyzed Nrf2 (Nuclear factor erythroid 2 related factor 2), a key antioxidant defense transcription factor, via Western blotting. Results showed L. nagelii K38 CFS significantly restored 6-OHDA-induced Nrf2 level decreases (p < 0.05). Building on these findings, in vivo experiments utilized spontaneously hypertensive rats (SHRs), a well-established ADHD animal model. Four groups were orally administered 500 μL daily for 24 days: WKY rats (WKY group, MRS), SHR rats (SHR group, MRS), L. nagelii K38 Live bacteria (K38 Live group, 5 × 10⁸ CFU/rat/day) and L. nagelii K38 CFS group. Behavioral assessments revealed neither L. nagelii K38 Live nor CFS significantly improved hyperactivity or impulsivity in SHRs. However, the L. nagelii K38 Live group significantly improved short-term memory in the novel object recognition test (p < 0.05). Nissl staining showed both L. nagelii K38 Live and CFS group significantly increased cortex neuron cell numbers (p < 0.05), although no significant changes were observed in brain-derived neurotrophic factor (BDNF), Nrf2, tyrosine hydroxylase (TH), or blood–brain barrier marker claudin-1 expression. In conclusion, L. nagelii K38, a potential probiotic strain isolated from kefir, exhibited multiple in vitro neuroprotective effects. While it didn't ameliorate core ADHD behavioral symptoms in vivo, it improved short-term memory deficits and increased neuronal cell numbers. These findings suggest L. nagelii K38 holds promise as a supportive therapeutic strategy for ADHD.
 
Keywords: ADHD, kefir, oxidative stress, probiotics, microbiota-gut-brain axis, neuroprotection

• Alvarez-Arellano, L., N. González-García, M. Salazar-García and J. C. Corona. (2020). Antioxidants as a potential target against inflammation and oxidative stress in attention-deficit/hyperactivity disorder. Antioxidants, 9 (2): 176
• Checa-Ros, A., A. Jeréz-Calero, A. Molina-Carballo, C. Campoy and A. Muñoz-Hoyos. (2021). Current evidence on the role of the gut microbiome in ADHD pathophysiology and therapeutic implications. Nutrients, 13 (1): 249.
• Corona, J. C. (2020). Role of oxidative stress and neuroinflammation in attention-deficit/hyperactivity disorder. Antioxidants, 9 (11): 1039.
• Dash, S., Y. A. Syed and M. R. Khan. (2022). Understanding the role of the gut microbiome in brain development and its association with neurodevelopmental psychiatric disorders. Front. Cell Dev. Biol., 10: 880544.
• Khanna, H. N., S. Roy, A. Shaikh and V. Bandi. (2022). Emerging role and place of probiotics in the management of pediatric neurodevelopmental disorders. Euroasian J. Hepatogastroenterol., 12 (2): 102-108.
• Silva, B. S., E. H. Grevet, L. C. F. Silva, J. K. N. Ramos, D. L. Rovaris and C. H. D. Bau. (2023). An overview on neurobiology and therapeutics of attention-deficit/hyperactivity disorder. Discov. Ment. Health, 3 (1): 2.
• Yin, X., W. Liu, H. Feng, J. Huang, Q. Wang, Q. Zhang, J. He and R. Wang. (2024). Bifidobacterium animalis subsp. lactis A6 attenuates hippocampal damage and memory impairments in an ADHD rat model. Food Funct, 15 (5): 2668-2678.