Lactobacillus Plantarum GKM3 Promotes Longevity, Memory Retention, And Reduces Brain Oxidation Stress in SAMP8 Mice Part 2

3.4. Effect of L. plantarum GKM3 on Brain Oxidative Stress Reduction

Glycoside of cistanche can also increase the activity of SOD in heart and liver tissues, and significantly reduce the content of lipofuscin and MDA in each tissue, effectively scavenging various reactive oxygen radicals (OH-, H₂O₂, etc.) and protecting against DNA damage caused by OH-radicals. Cistanche phenylethanoid glycosides have a strong scavenging ability of free radicals, a higher reducing ability than vitamin C, improve the activity of SOD in sperm suspension, reduce the content of MDA, and have a certain protective effect on sperm membrane function. Cistanche polysaccharides can enhance the activity of SOD and GSH-Px in erythrocytes and lung tissues of experimentally senescent mice caused by D-galactose, as well as reduce the content of MDA and collagen in lung and plasma, and increase the content of elastin, have a good scavenging effect on DPPH, prolong the time of hypoxia in senescent mice, improve the activity of SOD in serum, and delay the physiological degeneration of lung in experimentally senescent mice With cellular morphological degeneration, experiments have shown that Cistanche has the good antioxidant ability and has the potential to be a drug to prevent and treat skin aging diseases. At the same time, echinacoside in Cistanche has a significant ability to scavenge DPPH free radicals and has the ability to scavenge reactive oxygen species and prevent free radical-induced collagen degradation, and also has a good repair effect on thymine free radical anion damage.

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TBARS is a by-product of lipid peroxidation which could describe the level of oxidative stress. The concentration of TBARS was detected in SAMP8 mice brains after the sacrifice. Both male and female SAMP8 mice fed with probiotic GKM3 showed a significant reduction in the concentration of brain TBARS when compared to the control mice (Figure 4A). Another marker of oxidative stress, 8-OHdG, in the brain was also significantly lower in the GKM3 treatment group in comparison to the non-treatment (Figure 4B). This indicates that less DNA damage occurred in the probiotic group. There were no dose effects of GKM3 observed in brain TBARS and 8-OHdG levels in either both genders, suggesting the low dosage of probiotic GKM3, which was 1.0 × 109 CFU/kg BW/day, was enough for the antioxidant defense in aged mice.

3.5. Effect of L. plantarum GKM3 on Amyloid-β Participation in SAMP8 Mice Brains

Amyloid-β (Aβ) precipitation is one of the characteristics in SAMP8 mice which shared the same features with dementia at the clinical observation. Figure 5 shows the immunohistochemical result of the SAMP8 brain tissue. The Aβ precipitation was observed in control male and female SAMP8 mice brains (Figure 5A, D), but little protein appeared on the samples of those treated with a low dose of GKM3 (Figure 5B, E), and even less protein accumulated on those with the treatments with a high dose of GKM3 (Figure 5C, F). The percentage of the Aβ precipitation area in SAMP8 mice fed with probiotic GKM3 presented a significant reduction compared to the controls in both genders (Figure 5G). This evidence revealed that L. plantarum GKM3 could prevent age-related Aβ precipitation, which may contribute to a neurological disorder.

3.6. Effect of L. plantarum GKM3 on Hippocampus Histology in SAMP8 Mice

The hippocampus of SAMP8 mice brains was analyzed by hematoxylin and eosin staining (Figure 6). There was hyperchromic staining with the shrinking of nerve cells present in the control and GKM3 low-dose groups. However, the neuron in the hippocampus of SAMP8 fed with a high dose of GKM3 showed a tight arrangement. In addition, there was no abnormal observation of the cell structure and morphology in the GKM3 high-dose group. This indicates that probiotic GKM3 could delay the neuron damage caused by aging in the hippocampus of mice brains.   

4. Discussion

There were no significant differences in body weight or food intake between the control group and the probiotic groups (Table 1). This indicates that the probiotic GKM3 is safe and non-toxic to the mammal. Under the same metabolic parameter and energy consumption, the effects of anti-aging in GKM3-fed SAMP8 can be discussed in the following section.

Through observation of the lifespan of SAMP8, we note the death started from 6–7 months; however, both males and females in the probiotic GKM3-H group tended to present low death rates (Figure 1). Especially up to 11 months, the survival rates were very different. The GKM3-H group still kept 90–95% of the survival rate but the control group only presented 60–40% of the survival rate. Compared with other reported nutrients such as plant extract or marine sources on anti-aging effects, probiotic GKM3 provided a better result, especially in terms of a longer survival rate [32,33]. It is possible that those anti-aging materials contained lipids, organic acids, polyphenols, or vitamins, which also can be easily found from the probiotic’s fermentation, and resulted in a longer lifespan [34–36].

Behavior avoidance tests are usually used for evaluating learning and memory in subjects. Memory is defined as a behavioral change caused by an experience, while learning is defined as a process for acquiring memory [37]. Both memory and learning were formed and achieved by being involved with nerve transmission and the nerve cell. Hence, the process of aging could increase the accumulation of ROS in neurons and damage the cell,  resulting in poor memory and learning ability [38]. The passive avoidance test is a fear motivated in which subjects are required to inhibit a previously exhibited response [39]. Mouse with a tterbetterory and learning ability can avoid entering dangerous areas. Conversely, active avoidance requires subjects to emit a response such as running to a safe ace to avoid various stimuli. Good memory and learning skills help the mice respond to alert events and avoid incoming dangers. Evidence from our study showed that probiotic GKM3 could contribute to learning and memory by inhibiting the occurrence of undesirable responses, while there was no contribution in the control group even after e training procedure (Figure 2). Interestingly, the control group showed an increased tendency in the successful avoidance followed by the training days in the active avoidance test; however, the group fed with probiotic GKM3 showed a stronger significant successful dance (Figure 3). This could be explained by the trial-designed base [40]. Mice were forced to learn what to do in the active avoidance test but were relatively unstimulated in ing required to learn what not to do in the passive avoidance test. InDespitehe the different actions presented by the control mice, the effect of probiotic GKM3 in improving learning d memory could not be denied with the stimulations of both different mechanisms [41].

Similar behavior results were reported by Yong et al. with chicken extract as a diet edition and Su et al. with the supplementation of yam for SAMP8 mice [40,41]. As the active compounds should exhibit a tremendous difference between the meat extract d plant origin, it could be speculated that the effect of cognitive maintenance was highly related to the alteration of gut microbiota [42,43]. Probiotics can produce metabolic compounds that enhance or suppress the growth of certain gut microorganisms [44]. These metabolic compounds, such as peptides, cortisol, or SCFAs, can also modulate the nervous stem and maintain brain functions through the microbiome-brain interaction. It s found that the gut microbiota in centenarians was very different from the aging population [45]. In particular, the relative abundance of Firmicutes was found. Even ough we did not analyze the microbiota in this study, several papers pointed out that the ministration of probiotics altered the gut microbiota [46–50]. In addition, our unrevealed data concerning a probiotic mixture mainly contained L. plantarum GKM3 in a clinical trial that showed an increase of several Bifidobacterium species and several Lactobacillus species in the stool analysis after four weeks of consumption. It is suggested that L. plantarum GKM3  may maintain brain functions by changing the composition of gut microbial flora [51].

SAMP8 is a neuropathological model of accelerated brain aging derived from an AKR/J  breeding colony by Professor Toshio Takeda at Thoto University [52]. Regarding engage-associated morphological alteration, early amyloid accumulation in the hippocampus was und in SAMP8 mice, which resulted in learning disturbances and impaired memory [53]. Aβ was considered to induce ROS formation, lipid peroxidation, and neurotoxicity in hippocampal neurons [54]. Our results reveal that probiotic GKM3 not only inhibited oxidative stress in the brain (Figure 4) but also was involved in the upper inhibition of amyloid formation (Figure 5). TBARS is formed as a by-product of lipid peroxidation and MDA is formed as its end-product. 8-OHdG is a common end-product of deoxyribonucleic id (DNA) oxidation. That is, high levels of TBARS and 8-OHdG both represent strong ideation and result in cognitive impairment [55].

Pyramidal cells, a type of populous neuron involved with the sensory and motor cues in the hippocampus, could contribute to information processing, learning, and memory [56]. The disordered arrangements of pyramidal cells in the hippocampal CA1 region were found in Alzheimer’s disease-affected mice [57]. It is indicated that L. plantarum GKM3 could alleviate the functional decline of neuron-transmitting by maintaining cell morphology (Figure 6). That is, long-term administration of probiotic GKM3 could enhance a better consciousness and encourage appropriate actions in life.

5. Conclusions

In this study, we examined the dose-dependent effect of long-term administration of L. plantarum GKM3 on longevity in both male and female SAMP8 mice. In addition,  supplementation of probiotic GKM3 showed the improvement of memory and learning ility by being involved in anti-oxidative stress, by lowering Aβ accumulation, and by maintaining the arrangement of nerve cells in the hippocampus. These results suggest at probiotic L. plantarum GKM3 could act as an antioxidant for delaying the aging process and preventing-related cognitive impairment. With its desirable functions and fe consumption history, L. plantarum GKM3 is a promising probiotic supplementation for e elderly.

Author Contributions: T.J.F. and Y.-L.C. provided the ideas and conceived the experiments. M.-F.W. operated the animal model and conducted the biochemical analysis. W.-H.L. contributed to the statistics. C.-C.C. advised discussion. S.-W.L. collected the data and wrote the paper. Y.-S.T. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding: This research received no external funding.

Institutional Review Board Statement: The study was conducted according to the guidelines of the Institutional Animal Care and Use Committee at Providence University (Taichung City, Taiwan) with the number 20170629-A02.

Informed Consent Statement: Not applicable.

Data Availability Statement: All data can be assessed from W.S. Lin via the email address. 

Acknowledgments: We thank Jiunn-Wang Liao from the Graduate Institute of Veterinary Pathobiology, National Chung Hsing University (Taichung, Taiwan), for helping us with the tissue slicing and theological examination. We also give a special thanks to Tseng Andrew, Chief Executive Officer, at Grape King Bio Ltd. (Taoyuan, Taiwan) for his permission on providing probiotics dry powder with ss production as the sample in this experiment

Conflicts of Interest: The authors declare no conflict of interest.

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