The Emerging Scenario Of The Gut–Brain Axis: The Therapeutic Actions Of The New Actor Kefir Against Neurodegener Part 3

Currently, we are facing a growing number of studies that are focused on isolated probiotic components. On the other hand, our laboratory faces the future of kefir without ignoring history, since we use traditional whole milk fermented from kefir grains in our experimental and clinical studies. 

In recent years, probiotics have attracted more and more attention. Probiotics are considered to be microorganisms that are beneficial to human health and can improve intestinal function, regulate the immune system, and enhance mental health. With the deepening of research, probiotics have also been found to be closely related to memory.

Probiotics play a great role in human intestinal health. When intestinal health is poor, the digestion and absorption functions of food are affected. These foods contain important nutrients, such as glucose and amino acids, which can reach the brain through blood circulation to provide energy and nutrients. If the transport of nutrients is impeded, the brain will lose the nutrients necessary to support memory function.

At the same time, probiotics can also promote the growth of beneficial bacteria in the human body and maintain the balance of intestinal flora. The flora in the intestines plays an extremely important role in human health. They can produce certain vitamins and enzymes, help digest food, and can also affect the functions of the human nervous system and immune system. When the flora is out of balance, it may lead to the growth of a large number of bad bacteria and the production of toxins, which in turn affects human health and memory.

In addition to their effects on gut health, probiotics can also produce neurotransmitters and metabolites that improve the function of the nervous system and cognitive abilities. For example, probiotics can produce neurotransmitters such as gamma-aminobutyric acid (GABA). GABA is an inhibitory neurotransmitter that can help relieve tension and improve emotional stability. In addition, the metabolites produced by probiotics can also affect the body's internal environment, regulate hormone secretion, improve neurological function, etc.

Taken together, probiotics are very closely related to human health, especially to memory. Therefore, we should focus on maintaining good intestinal health and proper intake of probiotics and dietary fiber. Exercise, a good diet, and sleep are also key to keeping your gut healthy. Let us develop healthy living habits, starting from the intestines, to maintain a healthier body and better memory. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory, because Cistanche deserticola can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors. These substances are very important for memory and learning. In addition, Cistanche deserticola can also improve blood flow and promote oxygen delivery, which can ensure that the brain receives sufficient nutrients and energy, thereby improving brain vitality and endurance.

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An advantage of studying a kefir grain is that it is a high spectrum resembling a whole microbiome. Likewise, in kefir grains, the polysaccharide kefiran (also present in other milk-fermented products) serves as a matrix where bacteria and yeast live and proliferate [199,200]. 

Although the metabolic pathways that lead to the production of kefiran are not well understood, this polysaccharide is composed of repetitive units of hexa- and tetrasaccharides that consist mainly of glucose and galactose, with branches [37,192]. 

To preserve and explore the richness of kefir grains, our group has taken advantage of the synbiotic characteristics of kefir (for a detailed description, see [150]), simultaneously exploring the probiotic (bacteria plus yeasts) and prebiotic (kefiran) components.

9. Targeting the Near Future: Milk Kefir against Oxidative Stress and Inflammation

Strong evidence supports the medicinal applications of kefir. In general, kefir acts on the intestinal microbiota, mediating protective/therapeutic effects through its probiotic microorganisms and/or bioactive compounds [201]. 

In this regard, kefir improves the host's health by providing compounds that will systemically reach target organs and brain integrative areas such as those related to AD and PD [201,202]. 

These potentially beneficial compounds were yielded during the fermentation process, including lactic and acetic acids, vitamins, volatile compounds, nutraceutical components, and, especially, small peptides derived from milk proteins, including "captopril-like effects" [9,164]. Recently, many studies have reported the important role of kefir, most of which associate bioactive compounds with the antioxidant and/or anti-inflammatory properties of beverages [163,182,203–205].

As mentioned above, oxidative stress causes severe damage to biological systems, leading to the development of chronic diseases. In this context, evidence points to the antioxidant properties of kefir. In vitro, kefir showed antioxidant potential measured by DPPH free radicals and ABTS assays [177]. 

In addition to its antioxidant actions, kefiran showed dose-dependent protection of proteins from oxidative injury [173,206], and kefir also demonstrated significant antioxidant effects in vivo. 

Over the past decade, our group has shown that kefir is a likely food for the treatment of oxidative stress-dependent diseases [7,14,40,63,80,101,115]. Kefir intake decreases the production of ROS [7,101] and increases antioxidant enzyme (catalase, superoxide dismutase, and glutathione-peroxidase) activity [207–209]. Therefore, kefir safeguards the cells from the harmful effects of ROS by protecting proteins, lipids, carbohydrates, and nucleic acids, avoiding the apoptosis process [7,40,101,115,210]. 

Previous evidence points to the anti-inflammatory and immunomodulatory potential of kefir. Complications associated with inflammation are a key cause of morbidity and mortality due to chronic diseases. Peptides from kefir inhibited the NF-κB signaling pathway [163], increased anti-inflammatory (IL-10), and decreased the production of proinflammatory cytokines (such as INF, IL-1β, IL-6, and TNF) [40,115,159,204,211]. 

In summary, the beneficial effects of kefir are associated with its anti-inflammatory and antioxidant properties, as previously demonstrated by us and others [40,163,212], preventing apoptosis and, consequently, neuronal degeneration [213,214]. Therefore, this probiotic beverage shows the potential to act as an adjuvant in conventional therapies addressing ND.

10. ND and Kefir: Promise or Reality?

NDs are chronic incurable, debilitating conditions characterized by movement and cognitive disorders due to progressive neuronal dysfunction and disability associated with oxidative stress and inflammation, which are two main systemic conditions that aggravate neurodegeneration [1,2]. 

It is estimated that a new case appears every 4 s, and forecasts point out that in 2050, the number of people suffering from these diseases will reach 115.4 million due to population aging [5]. 

Knowledge about the structure of the nervous system and the molecular processes underlying the functioning of neuronal cells is crucial to understanding the pathophysiology and molecular damage in ND [98]. Despite different etiologies, oxidative stress and inflammation are common features of ND since they are associated with neuronal cell death and shrinkage in specific brain areas [98,215]. 

Neuronal stress is associated with synaptic dysfunction, impaired protein degradation systems, increased ROS production, mitochondrial dysfunction, DNA damage, inflammation, and excitotoxicity by mechanisms including cAMP dependence [98,216–220]. Neurodegeneration has been associated with inflammation and its mediators, which together lead to endothelial dysfunction (leading to BBB disruption) [44], apoptosis [221], necroptosis, [222,223], neuronal autophagy [224,225], and astrogliopathy [226] and accumulation of Aβ and tau protein [227,228]. 

These events associated with ROS trigger neurodegenerative events [227,229–231]. In addition, other pathways were explored. As an example, in 2012, for the first time was described by Iliff et al. [232] an important mechanism of brain metabolic waste clearance called as "glymphatic system" (which stands for glial-dependent lymphatic transport) [233]. 

There is experimental evidence that accumulation of Aβ and tau protein could occur in the brain due to impaired glymphatic clearance, aggravating the ND [228,232,233]. Among the proteins involved in this system, AQP4 seems to play a key role in brain fluid homeostasis and justifies, at least in part, the failure of the glymphatic system [234,235]. 

Although it is an exciting area in research, innovative diagnostic and therapeutic strategies involving this issue are still necessary since the glymphatic system in the human brain needs to be characterized in more detail [228].

Beyond endogenous factors, the environment can influence disease risk and course, contributing to the pathogenesis of ND [236,237]. The type and composition of diet during life have important long-lasting effects on brain function [238]. 

In addition to its known effects on cardiovascular diseases, nutrients induce epigenetic changes in neurons, which are associated with degenerative disorders (see Figure 1) [239,240]. In recent years, an increase in the number of papers linking changes in the gut microbiota to ND was observed (see Figure 1). The microbial composition of the gastrointestinal tract influences neuronal tissue through various pathways, such as immune, neurological, and endocrine signaling [241,242], affecting behavior, BBB integrity, neurogenesis, and neurotransmitter production [243]. 

In response to oxidative stress, gut microbiota diversity is altered, which could even trigger neuroinflammation and, consequently, neurodegeneration [244,245]. Regarding this issue, modern setups (e.g., "intestine-on-chip", organoids, and 3D cultures) would be necessary to monitor the effects of kefir and molecules involved in the gut–brain axis [246,247]. 

In this last section of the review, we will provide for the first time a review highlighting the findings that were published in the last 3 years emphasizing the neuroprotective properties of kefir, which is the object of investigation by our translational research group in Brazil.

10.1. What Is New in Dementia?

Dementia in AD is a progressive, global, and irreversible decline in cognitive functions mainly in elderly patients [227,248]. AD shows characteristics of disseminated neurodegeneration and two classical etiopathogenic biomarkers: neuritic plaques (NPs) and neurofibrillary tangles (NFTs) [98,227,249]. 

NPs are extracellular deposits of β-amyloid peptides, whereas NFTs are formed by the aggregation of hyperphosphorylated tau protein [227,250,251]. The neurodegeneration process in AD is a dynamic and multifaceted biochemical phenomenon. 

The presence of soluble amyloid β oligomers (AβOs) induces synaptic dysfunction due to aberrant activation of N-methyl D-aspartate (NMDA) receptors and abnormal increases in postsynaptic Ca2+ levels, leading to excitotoxicity [227,248]. 

In addition, tau hyperphosphorylation could be the necessary point between dysfunction and neural death [227,252]. According to oxidative stress theory, neuronal death in AD occurs due to ROS that interacts with cellular biomolecules, causing functional changes that precede cardinal neuropathological manifestations of this disease [40,227,249,252]. 

Experimental studies have demonstrated that due to high ROS generation accompanied by a low level of antioxidant compounds, cell regeneration is an early and auto-limited phenomenon before apoptosis and the formation of senile plaques and NFTs [227,252,253]. Neuroinflammation is a process that plays a relevant role in the pathogenesis of AD [254]. 

Previous evidence has identified an increasing number of proinflammatory molecules involved in the cognitive impairment of AD, such as interleukin (IL)-6, tumor necrosis factor-alpha (TNFa), and the inflammasome complex (NLPR3) [255–257]. In addition, other authors have demonstrated positive associations between proinflammatory cytokines (e.g., IL-1, IL-6, TNF-α, IL-8, and IL-12) and AD progression [40,254,258]. 

Furthermore, these neuroinflammatory cytokines can compromise β-amyloid clearance, leading to the accumulation of this protein in the brain [254,259,260]. The classical scenario that prevailed for several decades highlighting oxidative stress and inflammation as pivotal mediators in the pathogenesis of AD was recently joined by an ascending and multifaceted "amazing actor": the gut microbiota (see Figure 1). 

However, the latter actor does not shine alone (as in a monologue) but acts in an intense interaction (dialog) between oxidative stress and inflammation. As an example, bacterial lipopolysaccharides can increase the levels of cytokines and other proinflammatory molecules, which are directly associated with AD [261–263]. On the other hand, different sources of probiotic supplementation can modulate cognitive processes of learning and memory [40,264–267], reducing oxidative stress [40,252,268] and proinflammatory cytokine levels [40,254]. 

For the first time, data from our group published by Ton et al., 2020 [40] demonstrated that supplementation with probiotic kefir for 90 days brings substantial improvements in global cognitive function and immediate and late memory and a significant improvement in functions involving constructive skills. 

In addition, kefir reduced ROS, leading to an attenuation of plasma protein oxidation, proinflammatory cytokines, and apoptosis in AD patients [40]. Although the metabolic and hemodynamic profiles were not evaluated in our study, it is well known that the chronic use of probiotics favorably modifies the cognitive capacity of subjects with dementia, in addition to improving blood pressure, insulin sensitivity, and lipid profile [107,269]. 

It is important to emphasize that other studies have demonstrated that kefir supplementation also contributes to the neuromodulatory process that mediates neuroactive and neuroendocrine syntheses (involving, for example, acetylcholine, dopamine, serotonin, noradrenaline, adrenaline, glutamate, gamma-aminobutyric acid and brain-derived neurotrophic factor (BDNF)) and the expression of its receptors [35,107,270–273].

10.2. Encephalitis and Kefir: A New Insight

Encephalitis is characterized by inflammation of the brain tissue due to direct infection or an autoimmune response and is recognized as a common refractory illness [274]. Among them, Rasmussen encephalitis (RE) is a rare chronic inflammatory ND, defined by progressive and diffuse brain inflammation/deterioration (and consequently unilateral brain atrophy), with significant cognitive decline and hemiparesis and, unfortunately, intractable epilepsy [115,275–277]. 

Although the pathophysiology is still unclear, multifocal inflammation, immune-mediated gliosis restricted to the cerebral hemisphere [277,278], microglial activation [279] and, more recently, dysbiosis was observed [115]. To date, the available treatments with antiepileptic drugs and hemispherectomy present incipient results in the control/reduction of seizures [280]. 

At the same time, previous studies have related dysbiosis with increased release of inflammatory cytokines and increased neuronal excitatory activity, especially in the hippocampal area [115,281], and these factors are considered to trigger the processes of epileptogenesis and neuroinflammation [282,283]. In this context, modulation of the intestinal microbiota could be a therapeutic strategy for epilepsy [284]. 

Among the functional foods that could beneficially alter the intestinal microbiota are probiotics, including kefir, which has shown positive results, such as the ability to restore the composition of the intestinal microbiota in individuals with autoimmune diseases [35,115]. RE has an etiology and pathophysiology that has not yet been explained, but the number of studies relating to neurodegenerative diseases, including epilepsy, associated with dysbiosis has increased in recent years [283,285]. 

In the gut microbiota, there are more than 500 species of microorganisms [286,287]. In this regard, the effects of probiotic foods on the evolution and development of different diseases have been increasingly investigated [7,35,63,101,115]. 

Among the most studied microorganisms are the genera Lactobacillus and Bifidobacterium, which produce lactic, acetic, and propionic acids, reduce intestinal pH, produce bacteriocins, and produce biosurfactants, exerting microbial antagonism [288]. Inflammatory cytokines are biomarkers associated with brain inflammation in patients with epilepsy [289]. 

Recently, Hermann et al., 2001 [290] showed that TNF-α presents neuromodulatory properties that alter neuronal excitability. Confirming these data, RE patients treated with an anti-TNF-α drug (adalimumab) showed a reduction in epileptic seizures [291]. In addition, Kobylarek et al., 2019 [289] demonstrated that IL-1B levels are associated with generalized clonic-tonic epileptic seizures, IL-6 with the severity of the seizures, and IL-8 with partial seizures and severity.

Although the mechanism of action of kefir in the gut-brain axis is not fully understood, clinical evidence suggests that the reduction of dysbiosis represents a possibility of adjuvant treatment for refractory epilepsy, a condition that impacts not only the quality of life but also cognitive and motor functions [115]. Therefore, the screening of nonconventional therapeutic strategies aiming to control seizures appears to be a rising strategy to fight against neurodegenerative diseases. 

For example, the gut maintenance of Lactobacillus and Bifidobacterium was associated with the ability to attenuate serum levels of inflammatory markers such as IL-1B and TNFα [115,292,293]. On this subject, in the current year, our group published findings in Lemos et al. (2021) [115], which demonstrated (for the first time) a significant increase in the number of Bifidobacterium spp. and Lactobacillus spp., in an RE patient, suggesting that kefir can treat dysbiosis by modifying the colonization of the gut microbiota. 

Moreover, the probiotic kefir had a possible neuroprotective effect due to the modulation of the microbiota, which was associated with reduced expression of inflammatory cytokines and ROS production, resulting in less cognitive damage [115]. The increase in these genera of bacteria proved to be an important indicator of gut microbiome reestablishment [250,294,295].

10.3. Fighting PD with Kefir: Current Scenario and Future Horizons

Although the consumption of fermented milk has been linked to health and longevity [296], its association with PD still needs more investigation. Recently, Olsson et al. (2020) [296] published a large cohort study that included approximately 82,000 Swedish adults, and the results confirmed the association between milk intake and an increased risk of PD (as previously observed by others) [297,298]. 

In contrast, fermented milk (soured milk and yogurt) intake was not associated with an increased risk of developing PD [298], opening a timely urgency to test new "stars" among probiotics (but with discovery millenniums ago). To date, as noted in Figure 5, trials involving kefir have not yet been published. Notably, the triad "inflammation-oxidative stress-neurotoxic processes" is involved in PD. 

On the other hand, kefir supplementation can attenuate these related pillars, as previously observed in clinical investigations published for members of our research group (abovementioned). Thus, this probiotic, which exhibits features of synbiotic fermented milk (probiotics and prebiotics that beneficially affect the host, see [299]), must become a potential therapeutic strategy against PD progression in future years.

11. Conclusions and Perspectives for Future Research Advances

Cardiovascular disease and ND can significantly undermine the quality of life of an individual, which indicates the need for scientific research that seeks to discover possible alternatives for treatment or even the improvement of the patient's quality of life. 

In this review, we showed the current landscape and the future horizons of probiotic kefir in chronic diseases, aiming to translate its effects into real-life outcomes, mainly in cardiovascular disease and ND. The opportunities in the field of kefir as a nonpharmacological intervention for chronic diseases stem to a great degree from what we can learn about how it influences the gut microbiota and interacts with the host. 

The gut microbiota plays a key role in the pathogenesis of cardiovascular disease and ND by influencing the prooxidant and proinflammatory status. Although these endpoints have not currently been completely met, we discuss recent insights and promising results from the perspective of possible therapeutic applications of this probiotic. This perspective emerged in recent years when kefir research was driven by the characterization of microorganisms as well as by the postbiotic compounds found in beverages. 

For this circumstance, in vitro, in vivo, and in silico approaches were designed to uncover the effects of kefir on chronic diseases. Understanding the influence of individual differences on clinical outcomes would greatly contribute to the efficacy of kefir supplementation in chronic diseases. However, elucidating the interactions between the intestinal microbiota and kefir continues to present a challenge. In this regard, future research must focus on the stratification of clinical trials based on individual characteristics, including microbiota composition.

Author Contributions: Conceptualization and Review Process, T.M.C.P. and E.C.V.; Figures 1–4, E.C.V. and Figure 5, T.M.C.P.; Tables preparation, B.P.C., T.M.C.P. and E.C.V., Review and editing of final manuscript, L.Z.C., M.C.-T., A.M.M.T., S.S.M. and B.P.C.; Supervision, T.M.C.P., B.P.C. and E.C.V. All authors have read and agreed to the published version of the manuscript. 

Funding: The authors gratefully acknowledge the State Agency for Development of Science and Innovation of Espírito Santo (FAPES) and The National Council for the Development of Science and Technology (CNPq), Agencia Estatal de Investigación. 

Ministerio de Ciencia e Innovación (Spain), contributed to this scientific production with the following Grants: (a) PRONEX (FAPES/CNPq), ECV, Edital 24/2018, Termo Outorga 569/2018; (b) PPSUS (FAPES/CNPq/Decit-MS/SESA), ECV, Edital 03/2018; Termo Outorga 225/2018; (c) Universal (FAPES), BPC, Edital 21/2018, Termo Outorga 120/2019; (d) Agencia Estatal de Investigación, MCT, (PID2020-119178GB-I00); (e) Grant FAPES, BPC: Processo 552/2018; (f) Grant CNPq, ECV: Processo 305740/2019-9; (g) Grant CNPq, TMCP: Processo 309277/2019-1; (h) Grant CNPq, SSM: Processo 312056/2018-5. Conflicts of Interest: The authors declare no conflict of interest.

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