Neuroprotective Role of Probiotics (Bacillus clausii and Lactobacillus fermentum NMCC-14) in Restrain Stressed Mice Model

Abstract

Stress is a non-specific physiological response of our body to adversity. The gut brain/brain-gut axis and probiotics are gaining more interest in neurobiologists to better treat stress and other neurological disorders. In this study, the effects of probiotics (Lactobacillus fermentum NMCC-14 and Bacillus clausii, dose: 1010 colony forming unit/day/animal, per oral) were investigated in acute (up to day 7), subacute (days 8- 14), and chronic (15-21) restrain-stressed and normal mice. In the behavioral paradigms, time spent in the open arms of the elevated plus maze (EPM), time spent in the light compartment of the light dark box (LDB), and movable time and time spent in the center of the open field test (OFT) were significantly (p < 0.05, n = 5) increased in probiotics-treated acute, subacute, and chronic restrain-stressed mice, as compared to the negative group. Compared to the negative group, Lactobacillus fermentum NMCC 14 and Bacillus clausii significantly (p < 0.05, n = 5) decreased the blood levels of cortisol and adrenocorticotropic hormone (ACTH) on day 7, 14, and 21 in restrain stressed mice, in the enzyme-linked immunoassay (ELISA). Treatment of Bacillus clausii also showed a significant (p < 0.05, n = 5) decrease in the serum ACTH and cortisol levels in group PBT-LF and PBT-BC mice on days 14 and 21, as compared to the normal group. Probiotics treatment significantly (p < 0.05, n = 5) increased the level of glutathione (GSH), glutathione S-transferase (GST), and catalase, and a significant decrease (p < 0.05, n = 5) was observed in the production of malondialdehyde (MDA) and nitric oxide (NO) in the liver, colon, hippocampus (HC), and prefrontal cortex (PFC) of the restrain-stressed mice’s brains. Results from ELISA also showed a significant (p < 0.05, n = 5) decrease in the DNA damage indicating enzyme, 8- Hydroxyguanosine (8-OHdG) in the HC and PFC of acute, subacute, and chronic restrain-stressed mice brains. Immunohistochemistry slides showed a significant decrease in the expression of nuclear factor-kB (NF-kB), tumor necrosis factor-α (TNF α), and interleukin-1β (IL-1β) in the brain and intestine of acute, subacute, and chronic restrain-stressed (PBT-LFS and PBT-BCS) mice. Expression of c-Jun N-terminal kinase (JNK) also reduced significantly (p < 0.05, n = 5) in the brain of probiotics treated stressed mice on days 7, 14, and 21. High-performance liquid chromatography (HPLC) and Quantitative polymerase chain reaction (qPCR) were performed to determine monoamines level and mRNA expression of dopamine receptor (D1 and D2), synaptophysin, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), DRSML QAU xii postsynaptic density protein 95 (PSD-95), and vascular endothelial growth factor (VEGF) in mice's HC and PFC. Levels of dopamine, serotonin, and norepinephrine were also significantly (p < 0.05, n = 5) increased in HC and PFC in probiotics-treated acute, subacute, and chronic stressed animals’ brains. Fold expression of mRNA of D1, D2 (except D2 HC, PBT-LFS on day 14 and 21) receptors, synaptophysin, BDNF, NGF, PSD-95, and VEGF were also significantly (p < 0.05, n = 5) increased in the same brain parts in probiotics treated stressed mice on day 7, 14, and 21. Hematoxylin and eosin (H&E) stained hippocampal slides showed the reverting effect of neurodegeneration and increased neuronal arborization in the brain of acute, subacute, and chronic stressed probiotics-treated mice. The current study revealed that spore-forming Bacillus clausii (Enterogermina®) and non-spores-forming Lactobacillus fermentum NMCC-14 strains significantly improved activity and performance and corrected biological functions in restrain-stressed mice. Comparatively, treatment of Bacillus clausii showed more improvement than Lactobacillus fermentum NMCC-14 in non-stressed and stressed mice. Compared to the normal group, no stress-inducing effects were observed in the probiotics (Lactobacillus fermentum and Bacillus clausii) treated groups (PBT-LF and PBT-BC), but they even enhanced the activity of mice. Lactobacillus fermentum NMCC-14 and Bacillus clausii (Enterogermina®) can be safe and better therapeutic entities in stress and good possible alternative for mental disorders