The Aging Gut–Brain Axis: Effects of Dietary Polyphenols and Metal Exposure

ABSTRACT

Diet provides essential metals, which are required for the growth, development, and well‐functioning of the body. Nonetheless, some natural and human activities add toxic heavy metals to the diet, consequently introducing them to our bodies, resulting in several disorders and death. The intestine, the metabolism and absorption site of metals, uptakes them through transporters, active processes, and passive processes. It harbors Enteric nervous system (ENS), connecting directly to central nervous system (CNS) and the micro‐organisms acting as an endocrine organ, thus regulating the movement of metals into the host. Gut microbes mediate the uptake of essential metals and prohibit toxic metals. However, the imbalance in their concentration manifests into various neurodegenerative diseases via gut involving Firmicutes/Bacteriodetes ratio, Akkermansia, Bifidobacteria, Escherichia, Enterococcus, Bacteroides, and Clostridium. Most importantly, with population aging, the incidence of neurodegenerative disorders is rapidly flourishing, marking the need for novel theranostic approaches to overcome it. Several research have demonstrated the significance of gut microbial homeostasis and its influence on brain functions, often termed as gut–brain axis, necessary for the sustainment of overall health and well‐being of the human body. This review presents the novel diagnostic potential of microbes for specific disorders and dietary metals in combination with the therapeutic approach. Furthermore, they propose its utilization as a theranostic tool to investigate the links between mentioned dietary metals and neurological illnesses via the gut.

The influence of gut microbiota on brain health

Summary

• An imbalance in metal concentration and gut microbiota composition (e.g., altered Firmicutes/Bacteroidetes ratio, presence of Akkermansia, Bifidobacteria, Escherichia, Enterococcus, Bacteroides, Clostridium) can lead to neurodegenerative diseases.

• With population aging, neurodegenerative disorders are increasing, creating a need for novel theranostic (therapy + diagnostic) approaches.

• Research emphasizes the gut–brain axis, showing how gut microbial balance influences brain health.

• This review proposes using microbial diagnostics and dietary metal modulation as a theranostic tool to explore the link between diet, gut microbiota, and neurological illnesses.

Abbreviations

5HT₃ receptor

serotonin type 3 receptor

AD

Alzheimer disease

ADAM

a disintegrin and metalloproteases

ADHD

attention deficit hyperactivity disorder

ALS

amyotrophic lateral sclerosis

ASD

autism spectrum disorder

ATP

adenosine triphosphate

BDNF

brain‐derived neurotrophic factor

Ca

calcium

CNS

central nervous system

Co

cobalt

Cu

copper

ECC

enterochromaffin cells

ENS

enteric nervous system

ERK1/2 MAP kinase

Extracellular signal‐regulated kinase 1/2 microtubule associated protein kinase

Fe

iron

FXR

farnesoid X receptor

GABA

gamma‐aminobutyric acid

HMGA‐1

high‐mobility group A

HP1–γ

heterochromatin protein 1 γ

HPA axis

hypothalamic–pituitary–adrenal axis

IBD

inflammatory bowel diseases

IL‐6

interlukein‐6

K

potassium

Mg

magnesium

Mn

manganese

Mo

molybdenum

Na

sodium

OTUs

operational taxonomic units

PD

Parkinson's disease

PYY

peptide YY

ROS

reactive oxygen species

SCFA

short chain fatty acids

TGR5

G protein‐coupled bile acid receptor

TNF‐α

tumor necrosis factor‐alpha

USEPA

United Stated Environmental Protection Agency

WHO

World Health Organization

Zn

zinc

1. Introduction

The billions of microorganisms housed in the human body live in mutual relationships. This variety of aerobic and anaerobic bacteria favors humans by protecting them from diseases and providing immunity and energy from fibres [1]. Microbiota in the respiratory system is shown to participate in organogenesis and immune training [2]. Mammary glands also comprise microbes that infer protection from the colonization of pathogenic microbes; thus, protecting the health of both mother and child [3]. Out of all the organs, the gut comprises the majority of the microbe population and is associated with the onset of lethal medical conditions in the case of dysbiosis [1].

Other than the stated effect on metabolic and immune processes, an important bidirectional connection between the gut and the nervous system has been discovered by researchers, named the gut–brain axis. Crowned with the title of “the second brain,” the gastrointestinal (GI) tract serves a pivotal neurological role and is capable of influencing the brain and vice versa through the enteric neurons [4, 5].

The crosstalk impacts significant processes like neurodegeneration, concerned disorders, and tumor. Linking factors include the neural, endocrine, and immune systems of the body. Neurotransmitters, vagus nerve, hypothalamic–pituitary–adrenal (HPA) axis, peptide YY (PYY), microbial metabolites, antigens, neurotoxins, and toxins are specific factors involved in connecting the two organs [6]. Symptoms of impact, in the absence of microbes, are impairment in recognition, memory, cognition, imbalance in neurotransmitters, and development of disorders like Parkinson's disease (PD), autism, multiple sclerosis, Alzheimer's disease (AD), depression, and anxiety [7].

Healthy diets including plant‐based macronutrients are directly or indirectly associated with affecting the nervous system through gut microbes. They metabolize diet intake by fermenting the fibres to release metabolites regulating numerous functions. Alternatively, the microbiota can contribute to increasing the bioavailability of dietary polyphenols and proved to be efficient in protection against cognitive dysfunction and pathologies leading to disorders [8] (Illustrated in Figure 1). The connecting links between the gut and brain are microbial metabolites like short chain fatty acids (SCFA), tryptophan, and so on obtained from the processing of food which stimulates the vagus nerve, the production of neurotransmitters, and neurotrophic factors. Hormonal stimulation by mentioned metabolites and bile acid processing through microbes also influences neurological pathways. Unhealthy diet intake, as proved by researchers, harms the body through reactive oxygen species (ROS) production resulting in neuroinflammation [9] (Figure 1). Several researchers have attributed the curative effect of diet to microbiota, for instance, the successful expression of antiseizure effect from ketogenic diet and analeptic effect from high fructose diet. Psychobiotics are the category of microbes that indicate the effect of probiotics on mental health via gut residents [10].

Figure 1.

An overview describing the effect of diet on the nervous system through gut microbes. Healthy diets that include plant‐based macronutrients are directly or indirectly associated with healthy brain and nervous activity through healthy gut microbes and poor diet decrease macronutrient absorption and increases the pathogenic bacteria in gut that lead to mental disorders.

Several inorganic elements are required by the body, which are denoted as trace elements. These elements are important for numerous physiological activities in the human body such as growth, development, transportation of oxygen, structural role, stability of molecules, and many more. Out of the long list of required elements, ten are considered as essential trace elements due to their requirement for the above roles. These 10 essential elements are Ca, Co, Cu, Fe, K, Mg, Mn, Mo, Na, and Zn [11, 12]. Essential trace metals are solely obtained by the human body from diets. Their usual sources include fruits and vegetables, ingested and metabolized by the digestive system [13, 14]. Gut microbes aid the absorption of metals increasing their bioavailability and absorption [15]. Increasing pollution also contributes to various toxic chemicals and heavy metals in our dietary sources. Chronic intake and accumulation of toxic heavy metals led to numerous disorders affecting various organs which turn out to be lethal as well [16].

Furthermore, there is a strong correlation between the surge in the mean age of the population and an increase in age‐associated brain disorders [17]. Thus, it is critical to comprehend how to maintain brain health and to implement ways to delay the onset of cognitive decline as people age, thereby enhancing their standard of living and tackling a significant public health issue. Consequently, recent research on the gut–brain axis investigated the role of the gut microbiota as the target for dietary and therapeutic approaches for improving the brain‐health during ageing. In line with this, authors in this review explored the impact of dietary metals and (poly)phenols on gut–brain axis and their significance in sustaining overall brain health. Specifically, this review discusses how essential trace metals (iron, magnesium, zinc, and copper) and toxic heavy metals (arsenic and mercury) relate simultaneously with gut microbes and act on the brain. Specifically, effects on neurological development and disorders are considered concerning their concentration, bioavailability, and action via different mechanisms (Figures 2 and 3). Also, the potential theranostic approach of microbes has been linked to specific neurological disorders. The authors anticipate that the current article will let researchers conduct comprehensive analyses of individual metals and gut bacteria to diagnose concerned neurological illnesses. Additionally, therapeutic applications will also be unveiled.

Figure 2.

A healthy diet owes to a healthy brain via the gut–brain axis. Healthy gut increases the absorption of essential macronutrients like iron, magnesium, zinc, and copper that improves cognitive function and prevent mental disorders via an increase in serotonin production, reduced neuroinflammation and maintain healthy β‐microbial diversity. Healthy gut microbes in turn immobilize toxic metals (arsenic, mercury) preventing any brain damage.

Figure 3.

The pictorial representation of microbiota gut–brain axis and its significance in regulating brain health during ageing. The diversity of the gut microbiota can affect the levels of gut‐derived neuroprotective metabolites, neurotransmitters, gut hormones, and signalling pathways that are involved in neuroactivity and cognitive functions. The gut–brain axis, on the other hand, can support a healthy brain during ageing by lowering free radicals and reactive oxygen species (ROS), enhancing antiapoptotic processes, promoting anti‐inflammatory reactions, and combating oxidative stress.

2. Gut Microbiota–Gut–Brain Axis and Aging

Increasing evidence has revealed the potential impact of gut microbiota on several pathologies, particularly on neurological conditions. By manipulating the gut environment by utilizing compounds generated by commensal microbes as growth promoters, pathogenic bacteria may induce an inflammatory response. Hyperpermeability of the intestines allows these harmful bacteria to permeate into the bloodstream and eventually the brain. The following might lead to loss of neurons and synaptic dysfunction, which can subsequently compromise memory and cognitive function [18]. When combined, these mechanisms may lead to premature aging of the brain and potentially cause AD [19].

According to Broderick et al., [20] the consequences of microbial makeup and its associated alterations indicate that bacteria could speed up the aging process (illustrated in Figure 3). Reduced gut microbial diversity in the aged population is attributed to physiological vulnerability and impaired biological functionality resulting in age‐related neurological alterations [21, 22]. Additionally, research has also investigated the connection between the functionality of gut bacteria in rodents and specific age‐related patterns like frailty [23]. Results confirmed that variations in biological and functional patterns, such as the low maintenance of certain host nutrition‐related processes, may be associated with aging [24, 25]. Lately, it has been demonstrated that increasing distinctiveness in the composition of the microbiota can both depict healthy aging and foretell longevity in elderly people [26]. Further research performed using fecal microbial repository data from across five continents showed that changes in the gut microbiome associated with aging are typified by a rise in taxa linked to disease and a loss of stable microbial elements [27]. Dysbiosis is a condition where the gut microbiota's composition and function are manipulated, leading to disease pathogenesis and treatment responses. Dysbiosis disrupts the gut–brain axis, a crucial system for immune, metabolic, and nervous system regulation. This dysfunction is linked to ageing and stroke, causing risk factors like obesity, diabetes, and atherosclerosis. In line with this, age‐related diseases have been associated with dysbiosis in the gut microbiota of Drosophila [28, 29]. The review by Honarpisheh et al. [30] clearly emphasized the connection between the gut–brain axis and aging, stressing that all components of gut–brain axis endure age‐related changes, that may be altered or entirely driven by these microbes. Several clinical studies have demonstrated the impact of gut microbiota on host cognition, and its dysbiosis connected to aging results in neurodegeneration. Since dysbiosis‐induced inflammation in the gut and aging all contribute to the pathogenesis of AD, altering the microbial makeup of the gut through the intake of foods rich in probiotics can serve as a preventive or remedial measure for AD [31].

Further findings confirmed the idea of emphasizing the gut microbiota in upcoming clinical research to combat age‐related diseases and improve life expectancy [32]. Due to the decreased physiological reserve with age, the influence of the microbiota seems to be more noticeable. Some of the primary biological functions implicated in the Hallmarks of Aging are impacted by microbial metabolites along with other bioactive compounds [33]. The phylogenetic makeup of gut microbiota and how it interacts with the immune system are impacted by aging. In an instance of self‐sustaining cycle, age‐related changes to the gut microbiota are linked to immunosenescence and inflammaging, allowing gut microbiota imbalances to be located between the causes and consequences of inflammation‐aging [34]. Dietary approaches that slow down the microbial shifts associated with aging are also believed to improve cognitive function, inflammatory state, and frailty [33].

3. Interplay of Dietary Metals in Gut–Brain Axis

3.1. Iron

Iron availability from animal sources ranges from 1.28 to 11 mg per 100 g and that from plant diet is from 15.70 to 66.36 mg. Biologically available as nonheme and heme iron, the bioavailability of the former is lesser (around 2%–15%) and the latter is comparatively more (around 15%–35%). Average absorption by humans (around 1–2 mg per day) can be escalated through prebiotics and probiotics [35].

Iron exists predominantly in two oxidation states in the body: ferrous (Fe²⁺) and ferric (Fe³⁺) iron. The limited solubility of Fe³⁺ poses challenges to its bioavailability. Fe³⁺ exhibits an affinity for oxygen ligands, whereas Fe²⁺ favors binding with nitrogen and sulphate ligands. The dynamic interplay between iron and cellular processes takes a pivotal turn in aerobic conditions, where the redox properties of iron can lead to the generation of ROS through mechanisms like the Fenton and Haber‐Weiss reactions. The presence of free, redox‐active iron in such settings carries substantial risks. Of particular concern is the highly reactive hydroxyl ion (OH•), a member of the ROS family, capable of inflicting extensive damage upon cells and critical biomolecules. This susceptibility to iron‐mediated oxidative stress holds profound implications for cellular well‐being and functionality, suggesting a critical role for iron in the delicate balance of cellular health [36].

Iron concentration is also seen to have a significant role, through glucose homeostasis, in neural diseases like diabetic neuropathy, impaired cognitive dysfunction, and insulin resistance [37]. Gut microbial metabolites like SCFAs are involved in iron absorption by increasing its solubility, promoting epithelial cell growth, improving absorptive surface, and modifying ionic forms of iron by increasing the expression of reductase enzymes. Gut microbes also influence the storage and transport of iron. The overall reduction of 25% was visible in rats, in the absence of gut microbiota. Propionate synthesis by Propionibacteria and lactic acid synthesis by Lactobacilli enhance iron absorption [38]. The gut witnesses unabsorbed iron from dietary sources, which is capable of disbalancing eubiosis. Strategies to increase iron concentration through fortification result in gut inflammation and dysbiosis evidenced by a decrease in Bifidobacteria and an increase in Enterobacteriaceae, Escherichia coli, and other pathogenic bacteria. This is also accompanied by the initiation of insulin resistance and a rise in intestinal glucose levels [39].

The deficiency of iron has also been linked with changes in microbial composition leading to disorders [38, 40, 41]. Dietary iron impedes the proliferation of the enteric Citrobacter strains and fosters the evolution of asymptomatic Citrobacter strains [39]. While Citrobacter species (Citrobacter freundii) are typically associated with neonatal meningitis, their propensity for the development of brain abscesses further emphasizes their complex association with the host health [42].

Vibrio cholerae's growth hinges upon iron dependence. The Cholera Toxin enhances luminal heme's accessibility, shaping an iron‐scarce gut milieu that propels V. cholerae growth via heme from the host [43]. Furthermore, Cholera Toxin's B (CTB) subunit attaches to neural ganglioside GM1 receptors, potentially activating neural influence over pathways linked to neuronal survival, synaptic adaptability, and neurotransmitter emission. CTB's axonal retrograde transport to the central nervous system (CNS) may yield neural repercussions extending beyond the gut [44, 45, 46].

Chronic inflammation is associated with PD which is characterized by neuronal death primarily responsible for the secretion of dopamine. The Vagus nerve connecting the gastric wall with the Peripheral nervous system is responsible for the transport of Lewy bodies. The latter is associated with PD and dementia. Additionally, increased infectivity in low iron can provide a direct path for pathogens to reach the brain. Dysbiosis conditions accompanied by a reduction in gut microbial metabolite SCFA can result in Parkinson's through inflammation (Figure 4), which further aggravates the effects via the production of the faulty structure of α‐synuclein. The aggregates travel to the brain via the vagus nerve. Studies link dysbiosis with cognitive impairment, lack of social interaction, changes in emotions, and stress responses along with modification in the concentration of neurotransmitters [47, 48]. Eubiosis of Bifidobacteria, which holds anti‐inflammatory and antitumor properties, can be utilized for the treatment of AD [49]. The microbial metabolites, for instance, SCFA, which decreases in the condition of low dietary iron [50], and Bifidobacteria is directly responsible for serotonin production [51]. Serotonin acts upon the Serotonin type 3 receptor (5HT3 receptor) that has significance in the development and proper neurobiological functioning, modulatory action on the release of different neurotransmitters, and therapeutic potential against diminished cognitive functions and neurological disorders [52, 53, 54]. Inflammagens gifted by dysbiosis contribute to inflammation, gut barrier function, and neuroinflammation. Latter results via inflammagen‐associated amyloid fibres, and activation of microglia, macrophages, and astrocytes further cause the onset and progress of PD [55].

Figure 4.

A systematic representation of brain damage due to an unhealthy gut. Poor diet or dysbiosis conditions accompanied by a reduction in gut microbial metabolite short chain fatty acids (SCFA) and poor essential nutrient absorption (zinc, magnesium, iron, etc) that leads to Inflammatory Bowel Syndrome, Chron's disease, neuroinflammation, leaky gut that causes mental disorders (anxiety, depression) and neurodegenerative diseases (Parkinson's disease and Alzheimer's disease) through the gut–brain axis.

3.2. Magnesium

Required for the enzymatic reaction, controlling of muscular organs in association with nerve control, ossification, neurotransmission, and transport of ions, magnesium is crucial for mortality. Other than 10% being received from water, vegetables, fruits, meat products and dairy products are the dietary sources of magnesium. Common intake of the Western diet and methods of food production causes a deficiency of magnesium [56, 57] evidenced by the inability to meet daily requirements by around 60% of adults [58]. Dietary magnesium source is about 24%–76%, which inversely depends on the concentration of magnesium in the diet [59]. There exists no research relating the absorption of magnesium with gut microbes. Although Suliburska et al., 2021 showed an increase in body magnesium levels with the incorporation of probiotics, stating the strong relationship between the two [60, 61]. Multiple species of Bifidobacterium and Lactobacillus were used to improve the absorption.

The probable mechanism was discussed as the decrease in pH by these microbes, further increasing SCFA production thus enhancing magnesium solubility and retention. Also, the concentration of phosphorus influences magnesium absorption as well [61]. These microbes decrease neuro and systemic inflammation by improving gut permeability and mucosal barrier function [62]. High accessibility and bioavailability of magnesium via gut microbes [15] in the condition of penetration in the CNS enhance cognitive function, neural stem cell proliferation, and synaptic density, and decreasing amyloid‐beta plaques thus improvising AD (Figure 2).

The provision of magnesium alleviates substance P adversely impacting neuroinflammation [63]. Dietary intake of magnesium, in increased concentrations, is also linked to reduced risk of PD, impairment in cognitive functioning, lesser hemorrhage outcomes, and total stroke [64]. Varying concentrations of dietary magnesium have been linked with the development of anxiety and neuroinflammation via the gut microbiota. In addition to other direct links, disbalance in gut microbial concentration, affects hippocampal Interleukin‐6 quantity, linking neuroinflammation with mood, behavior, and hence depression [62]. The hypothesis has been further validated by Zhang, et al., where the Firmicutes/Bacteriodetes ratio was improved by the incorporation of an anti‐mouse interlukein‐6 (IL‐6) receptor antibody, thus ameliorating depression condition [65]. Probiotics and magnesium orotate treatments produced encouraging results in people who were resistant to SSRI therapy, which may be explained by their ability to reduce disruptions in the balance of the gut microbiome [66, 67]. Magnesium deficiency is associated with an increase in tumor necrosis factor‐alpha (TNF‐α), monocyte infiltration, substance P, and pro‐inflammatory cytokines, all contributing to systemic inflammation [68] subsequently resulting in a stroke, brain aging, and accelerating neurological disorders and neurodegeneration [69, 70].

3.3. Zinc

Beginning from the importance of zinc, it is required in catalytic reactions, gene expression, zinc fingers, structural functions, apoptosis, immune regulation, proliferation, and as a signalling molecule. These functions are accomplished by around more than 2.6 g of zinc in the body. Dietary zinc intake ranges between 16% and 50% and is found to be conversely dependent on the amount of available zinc in the diet. Mode of availability along with adaptation according to available zinc in the body play a key role in zinc absorption [71].

Zinc is acquired from dietary sources or supplementary intake, typically in the initial segment of the small intestine, encompassing the distal duodenum or proximal jejunum [72, 73]. Inside enterocytes, intricate intracellular transport systems and zinc‐buffering entities like metallothioneins (MTs) govern the movement and release of zinc into the bloodstream. However, diverse agents possess the capability to diminish zinc assimilation. Copper, which maintains an antagonistic rapport with zinc, and calcium, which could potentially impede zinc absorption, are noteworthy examples. There is a decline in zinc absorption when varied calcium forms are consumed subsequent to a zinc dose, indicating an antagonistic association between them. The consumption of increased levels of iron may also influence the absorption of zinc [74, 75, 76, 77, 78].

Common dietary sources of zinc are meat and dairy products, nuts, and seeds [79]. In a study, involving broiler chicken, dietary zinc deficiency was associated with a quantitative increase in Bacteriodetes and Proteobacteria and a decrease in Firmicutes and Actinobacteria. This was accompanied by an increase in Enterobacteriaceae and Enterococcus and a decline in Clostridiales. The addition of zinc to the diet of livestock led to an increase in the levels of acetate and butyrate and mitigation of coliform and E. coli presence, ultimately improving the functionality of the GI system in pigs [80, 81, 82]. Zinc also demonstrated inhibitory effects on pathogenic strains of E. coli by suppressing alpha‐hemolysin expression in murine models. The reduction in alpha‐hemolysin production resulted in the mitigation of barrier dysfunction and increased intestinal permeability [83, 84].

A significant relation between zinc deficiencies was observed with a decrease in macronutrient and micronutrient digestion, absorption, and metabolism besides alteration in secondary metabolite concentration. This study also correlated the requirement of eubiosis for increasing the bioavailability of zinc proved by SCFA concentration. Intestinal pH reduction by SCFA raises solubility subsequently enhancing the absorption, which in the condition of zinc deficiency, will further limit zinc intake [85, 86]. Contrarily concentration of zinc is found to increase microbial β‐diversity. This is accompanied by an SCFA increase, enhancing the zinc status in the body [87].

Zinc supplementation positively impacts immune response, thus guarding against neurological disorders [88] (Figure 2).

Lack of dietary zinc and chronic diseases have been listed as the top causes of zinc deficiency in developing countries. Direct change in the gut composition of Firmicutes, Bacteroides, and Clostridium according to zinc concentration, and indirect effects via chronic GI issues like diarrhea potentially cause neurological disorders. This change has been correlated with symptoms and severity of autism spectrum disorder (ASD) in children. One of the consequences of inflammatory bowel disease (IBD) and Crohn's disease is low zinc absorption and hence concentration in the body [89, 90]. These diseases are comorbid with neurological disorders like depression, panic, and anxiety (Figure 3). Furthermore, inflammatory events are interlinked with ASD and zinc deficiency, as evidenced by the activation of astroglia and microglia. Inflammation allows entry of toxins triggering immune action visible as lasting inflammation. A further chain of events occurs with a drop in nutrient absorption and further affects the brain in the form of a drop in cognitive and behavioral functions [91]. Another study found links between zinc deficiency and a rise in TNF‐α, IL‐1β, IL‐6 levels, and glial fibrillary acidic protein expression, the former responsible for inflammation and the latter for regeneration, structural support of astrocytes, synaptic plasticity, and reactive gliosis [92, 93].

The neurodevelopmental disorder attention deficit hyperactivity disorder (ADHD) witnesses an increase in gut microbial diversity. Supplementation of zinc oxide nanoparticles showed a reduced effect in ADHD children quantitatively and in composition, thus linking this metal with neurological disorders [94]. Zinc is associated with AD by being a component of amyloid plaques, which may result due to an increase in its concentration [95].

Insufficient consumption of zinc during the developmental period in rats resulted in adverse effects on the ubiquitin‐proteasome system, autophagy, and endoplasmic reticulum stress. The decreased presence of brain‐derived neurotrophic factor, impaired autophagic function, and increased gliosis and endoplasmic reticulum stress together initiated apoptosis. These findings highlight the prospect that a decline in intracellular zinc levels could initiate a chain of events leading to the apoptosis of brain cells [96].

3.4. Copper

Transition metal copper has advantages for the body in the form of cofactor required for enzymatic reactions and proper development of various organs. In the case of the unbound state, copper functions as a cellular toxin [97]. Copper absorption is also inversely related to dietary concentration and the quantity varies between 12% and 67%. The general absorptive range can be taken as around 30%–40% [98]. Sources of dietary copper include whole grains, and protein sources like tofu, nuts, mushroom, and shellfish. The role of gut microbes in absorption and its effect on the bioavailability of copper is not well‐researched among researchers. Yet, a study determines the effect of antibiotics on isotopic copper composition in the gut. As per the researchers, antibiotic treatment was accompanied by a decrease in copper importer and transporter, namely, CTR1 and ATP7A respectively, thus stating the probable effect of gut microbiota on copper uptake in the host [97].

High copper levels are linked with IBD, which have been further linked with neurological diseases in the above section. In another link with CNS via oxidative stress, copper is seen to be one of the factors in the production of cellular hydroxyl radical, subsequently affecting neuronal membranes via lipid peroxidation [99]. With constituents like copper, auto‐oxidizing neurotransmitters [100] and consumption ranging to around 20% oxygen, the brain produces ROS and hence oxidative stress contributing to neurological disorders like AD, PD, depression, amyotrophic lateral sclerosis (ALS), Huntington's disease and so on. In AD, copper binds with Aβ to generate ROS [95, 101]. In the realm of in vitro experimentation, copper augments the process of renaturation, leading to the stabilization of misfolded Prion proteins. These abnormally folded proteins are responsible for initiating prion disease. This phenomenon of copper‐mediated intervention sheds light on the intricate involvement of copper in the pathogenesis of Prion disease [102, 103].

Enhanced copper levels in varying organisms are associated with a decrease in Clostridia, SCFA‐producing microbial concentration, intestinal barriers affected by tight junction proteins, and a rise in pathogenic microbes and microbes responsible for intestinal lesions [104]. The metal induces a drop in permeation of tight junction which opens the door for pathogenic microbes, molecules further contributing to diseases. Thus, serving both as a nutrient and toxin gateway depending upon the concentration [105]. A similar connection with the responsible microbe Akkermansia has been proposed by Song et al. [106]. The deficiency also plays an important role, by negatively changing the metabolite concentration, and intestinal barrier and alleviating Verrucomicrobia and Firmicutes which is reflected in the form of diminished Allobaculum, Lactobacillus, Akkermansia, Coprococcus, and Oscillospira. The changes link the gut–brain axis with alteration in microbial communities that are responsible for the proper functioning of the body. The combination of copper with prebiotics or probiotics has shown beneficial results concerning pathogenicity, the probiotic potential of microbes like Lactobacillus, and bodily benefits [104]. Concerning prebiotics, copper along with fructose is seen to alter gut microbiota [106].

3.5. Arsenic

With increased consumption of contaminated food and water, toxic heavy metal intake is quite common. Arsenic is one of the various commonly consumed heavy metals and one of the 10 chemicals listed in major public health concerns, titled as king of poisons. Arsenic is consumed in ionic, metallic, inorganic, organic, and compound forms, with the highest toxicity in the inorganic form [107]. According to World Health Organization (WHO), 10 µg/L remains the recommended limit for Arsenic, which further recommends lowering the concentration as much as possible. On the contrary, reports suggest consumption of Arsenic in concentrations much greater than 100 µg/L.

The toxic form of Arsenic intake is linked with cancer, kidney, lungs, heart, and brain disorders. High Arsenic concentrations are found in rice, vegetables, cereals, fruits, grains, poultry, and seafood through natural and human activities, for instance, mining, fertilizers, coal ash, and pesticides [108]. Organic arsenicals, such as arsenolipids and arsenosugars, are predominantly present in fish, shellfish, and algae. When compared to inorganic arsenic, their toxicity is far lower. Arsenolipids are commonly found in conjunction with arsenic‐containing hydrocarbons and arsenic‐containing fatty acids. Hydrocarbons containing arsenic have neurotoxic properties, displaying comparable cytotoxicity and oxidative stress potential to trivalent inorganic arsenic [109].

The metal absorption in the human body occurs through the passage from tight junctions in the passive process and transport from cell to extracellular fluid in the active process. Both processes lead the metals to blood and amplify the absorption on chronic exposure, providing an opportunity to affect human health. However, gut microbes showed a beneficiary effect concerning heavy metal bioavailability [110]. The flora repairs leaky gut caused by these metals and adsorbs a variety of metals like arsenic, cadmium, lead, and mercury thus immobilizing them, precipitating them or forming complex, which decreases their bioavailability [111]. Thus, gut microbes express retention and adsorption of heavy metals thus limiting their passage into the cells and via tight junctions to blood (Figure 2).

Microbial action on Arsenic has been discussed by other researchers validating its change in chemical form. Gut microbes acted on cacodyl arsenic, and medicinal arsenic, releasing a strong garlic odor and production of trimethyl arsenic gas. Additionally, TMAVO production was seen from DMAsV [112]. Precipitation and complex formation reaction discussed above were further expressed as reduction and methylation of inorganic Arsenic, which leads to both high and low toxic forms of Arsenic [113, 114]. However, Arsenic introduction was certainly associated with alteration in gut microbial composition, further confirmed by Richardson et al. [115]. This alteration was witnessed at the current maximum level set by WHO for Arsenic consumption. Change in microbiota was expressed as the increase in Firmicutes and Gammaproteobacteria and a decrease in Bifidobacterium and Bacteroides. However, researchers remain unsure concerning Enterobacteriaceae concentration. The presence of essential metals zinc and iron discussed above also plays a key role in mediating the disastrous effects of Arsenic [116, 117]. Arsenic‐containing fatty acid treatment increased Escherichia/Shigella and Fusobacterium which are potential pathogens related to dysbiosis [118, 119, 120].

The exposure to inorganic arsenic in vivo led to alterations in the abundance of Faecalibacterium spp [121]. Faecalibacterium prausnitzii is a prominent producer of butyrate in the human colon and can serve as a bioindicator for assessing human health [122]. The presence of Faecalibacterium in the gut has been suggested to have a beneficial effect in protecting against arsenic toxicity but they are negatively associated with the occurrence of IBD or colorectal cancer [122, 123, 124, 125].

Yet, despite various studies stating the protective effects of microbiota on the host, few studies still claim a negative change in neurodevelopment on exposure to Arsenic. Feeding inorganic Arsenic alone and in combination with another neurotoxic compound, Fluorine caused the decline in neurological associate behavior of rats. This was seen in the form of diminished spatial learning and memory in association with degeneration and swelling of neuro fibres, nuclear pyknosis, and cavity formation in the cytoplasm in the hippocampus [126]. Entry of Arsenic into the brain via the blood–brain barrier affects the metabolite concentration. This metabolite concentration was further linked with the gut microbial composition which resulted in disturbing 12 operational taxonomic units (OTUs) including, Erysipelotrichaceae, Lachnospiraceae, Muribaculaceae, and Ruminococcaceae. These in turn are related to neurological disorders like multiple sclerosis, cognitive dysfunction, AD, and PD [127].

3.6. Mercury

Mercury is another non‐essential toxic heavy metal. It is also one of the 10 chemicals, issued by WHO, of major public health concern [128]. The blood usually has 5–10 µg/L of mercury [129]. Mercury is available to humans in three forms, namely, metallic form, inorganic, and organic form. Out of these, organic form, specifically, methyl mercury, is consumed by humans through diet. Methylmercury is highly toxic and can easily pass through cellular membranes. Mercury toxicity has several major effects on the body. The inhibition of enzymes containing sulfhydryl groups vital for cellular metabolism, the increase in the production of ROS and lipid peroxidation as well as the disturbance of intracellular Ca ion homeostasis are the major ones [130, 131, 132, 133].

Major dietary sources include seafood with numerous fish contributing to disastrous mercury levels in humans leading to neurological disorders (United Stated Environmental Protection Agency [USEPA], WHO). Gut microbes play a significant part in mercury absorption and their bioaccessibility by metabolizing them. Data suggests methylation of mercury, on the incorporation of mercuric chloride, by anaerobic bacteria. The responsible microbes were found to be E. coli, Streptococci and Staphylococci [134]. Contrary data suggest catalytic conversion of methyl mercury chloride to both metallic mercury and its sulphur derivative. Comparison between antibiotic‐treated rats and conventional rats stated decreased mercury content in tissues of latter rats in addition to increased content in faeces [135]. According to the previous study by the same researchers, CH3‐HgCl rat tissue content, reduced by gut flora can deteriorate extremity of degradation of the cerebellum's granular layer along with the reduction in the toxicity induced by organic mercury [136]. The mechanism being demethylation of methyl mercury by gut microbes depends upon the diet, as evidenced by different bodily mercury concentrations on intake of pelleted rodent diet, evaporated milk, and synthetic diet [137]. As a result, intestinal microflora has been analyzed as playing a major function in both methylation and demethylation of mercury affecting toxicity in the host.

Mercury is known to have numerous neurotoxic effects: microtubule degeneration, neuroinflammation characterized by increased levels of pro‐inflammatory cytokines, activation of microglial and astrocytic cells, reduced levels of glutathione, inhibition of glutamic acid decarboxylase (GAD) activity, disturbances in GABAergic and glutamatergic homeostasis, impaired methylation, dysfunction of endothelial cells, reduced blood flow to the brain and cerebellum, activation of nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) and ability to induce immunologic changes in the brain (autoimmune dysfunction) [138, 139]. Lactobacillus and Peptococcaceae protect the brain from the hazardous effects of mercury. The former increases SCFA production crucial for the maturation and function of microglia. Latter microbe demethylates methyl mercury decreasing its bioavailability which in turn protects the brain by not being absorbed (Figure 2). Germ‐free microbes expressed a rapid increase in cerebellar mercury increase [140]. Bacterial siderophores form an insoluble complex with mercury and promote its excretion [141]. The process also affects the bioavailability of a few essential metals discussed above like zinc and iron [13]. As predictable, studies also link change in Firmicutes to Bacteroidetes ratio with increased intake of mercuric chloride and indicative of dysbiosis. The imbalance in ratio reflects neurogenesis and abnormal development of the hippocampus, concerned with learning and memory. Proapoptotic events modified intestinal barrier, diminished Bmi‐1, alteration in high‐mobility group A (HMGA‐1), extracellular signal‐regulated kinase 1/2 microtubule associated protein kinase (ERK1/2 MAP kinase), and heterochromatin protein 1–γ (HP1–γ) are some of the events influenced by gut microbes leading to neurogenesis in the hippocampus. Akkermansia increases with a toxic diet and causes abnormality via impairment of the intestinal barrier and mucus layer. This further contributes to inflammation, gut dysbiosis, and the incorporation of pathogenic microbes [141]. The most common neurological diseases AD and PD, with their common proteins and metals they bind are listed in Table 1.

Table 1.

Different essential and toxic metals binding to proteins.

Neurological disorders	Proteins	Effect	Associating metals	Toxic heavy metals	References
AD	GSK‐3β	Tau hyperphosphorylation	Magnesium, beryllium, zinc, copper	Lead, aluminium	[142, 143, 144, 145, 146]
	APP	Aβ production	Iron, copper, zinc	Lead, mercury, aluminium, cadmium, arsenic	[143, 146, 147, 148, 149, 150]
	Tau	Microtubule Assembly and Stability	Iron, copper	Cadmium, aluminium	[143, 144, 146]
	Aβ	Plaque development	Iron, copper, zinc	Cadmium, lead, mercury	[146, 151, 152, 153]
PD	Alpha‐synuclein	Plaque development	Nickel, copper, iron, calcium, cobalt, manganese, zinc	Aluminium, arsenic, cadmium, lead	[154, 155, 156, 157]
	Parkin	Clear unwanted proteins	Zinc		[158, 159]
	PINK1	Protect against oxidative stress‐induced apoptosis	Iron, copper	Molybdenum, cadmium	[160, 161, 162, 163, 164]

Abbreviations: AD, Alzheimer's disease; APP, amyloid precursor protein; GSK‐3β, glycogen synthase kinase‐3β; PD, Parkinson's disease.

3.7. Diagnosis

According to brain–gut axis theory, there is a link between gut microbiota and the brain, and any alterations in gut microbiota leads to alterations in the brain, therefore, leading to neurological disorders. Hence, for understanding the very factors leading to alterations in the gut microbiome we need to understand what factors are responsible for causing alterations in this microbiota. Various components of diet play an important role in causing disturbances to the millions of microbes living in the gut. Above mentioned studies link the essential dietary metals such as magnesium, iron, zinc, and copper with the brain–gut axis [165, 166, 167, 168].

The study conducted by Winther et al. [62] suggests that dietary magnesium can alter the activity of gut microbes through a mice model. They restricted the dietary magnesium and gave a standard diet to mice for 6 weeks and evaluated the behavior and metabolic tests (forced swim test and intraperitoneal glucose tolerance test) of mice. They concluded that magnesium‐deficient mice showed depressive‐like behavior along with dysbiosis conditions. This led to the suggestion that a deficiency of dietary magnesium causes the imbalance of the gut–brain axis thereby leading to depression [169]. Researchers suggest that low magnesium intake causes a decrease in gut microbes Bifidobacterium and Lactobacillus while it has been reported that high concentrations of gut microbes Bifidobacterium and Lactobacillus have beneficial effects on depression and anxiety disorders [170, 171, 172]. Therefore, using 16s rRNA gene sequencing of gut microbes mentioned in the microbial genus can be used as a diagnostic tool for determining the gut–brain axis associated with magnesium intake.

Autism is a neurological disorder characterized by poor brain development and problems in communication and social interaction. Most individuals suffering from autism show GI disorders such as diarrhea, bloating, and constipation, which in turn is directly linked to gut dysbiosis [173]. The study conducted by Srikantha and Mohajeri [174] reveals that individuals with autism show significantly decreased ratios of gut microbes at the phylum level, including Bacteroidetes and Firmicutes, and severe autistic behavior was found to be associated with microbes at the genus level like Clostridium spp. Zinc in diet also plays a role in causing disturbances in resident intestinal flora which further influences the nervous system consequently resulting in neurological disorders [175]. The study conducted by Sauer et al. [176] shows zinc‐restricted mice models showing altered morphological, biological, and pro‐inflammatory pathways resulting in alteration of gut microbiota quality and neuroinflammation leading to ASD. Studies conducted on mice model show that zinc deficiency causes an increase in Coprobacter, Acetivibrio, Paraprevotella, and Clostridium [174, 176, 177]. Also, zinc requiring metalloprotease A Disintegrin and Metalloproteases (ADAM) causes ASD pathogenesis by disrupting axon signalling, neural and gut inflammation, synapse formation, and gut permeability. Infection by Clostridium perfringens releases LPS and delta toxin which increases this protein's activity leading to diseases concerning the CNS [178]. This further indicates the potential of Clostridium levels to be used for the determination of modification in the gut leading to neurological abnormality.

AD is a neurodegenerative age‐related disorder, with the most common consequence seen in the form of dementia. It is characterized by problems in learning, memorizing, and reasoning resulting in cognitive impairment. Patients suffering from Alzheimer's show symptoms of memory loss, motor loss, and strange behavior. Pathogenesis of AD states the causes to be the intracellular accumulation of a protein called tau and neuritic plaques (amyloid‐beta deposition) which are deposited outside cells. However, recently it has been reported that alterations in gut microbes also play a role in causing the mentioned neurological disease. Amyloid is a protein that is responsible for learning processes but in AD this protein is impaired which causes activation of brain innate immunity. The immune cells of the CNS microglia cause the release of cytokines leading to a neuroinflammatory cascade. Chronic continuation of the process involving inflammatory cells causes neurodegeneration [179]. Ping Liu et. al link another causative agent of AD, the tau protein, with gut microbes. His research suggests the reduction in microbiota phylum Firmicutes and Actinobacteria while an increase in Bacteriodetes among individuals with AD [180]. The level of family Enterobacteriaceae of the phylum Proteobacteria is found to be directly associated with an increase in the severity of AD patients compared to normal individuals [181, 182, 183].

Iron is found to be associated with neurodegeneration [184]. Although iron in sufficient amounts is essential for body and brain regulation disturbances in its homeostasis led to complications. The study conducted by Hsieh et al. [185] is suggestive of the role of iron in determining the relationship between cognition and gut microbes. It suggests that iron can play a significant role in the regulation of neurologic development and microbiota alterations. It has been reported that increased levels of iron are associated with high plaque deposition (Amyloid beta) and nerve fibre tangles in AD individuals. Iron is also responsible for causing changes in gut flora. Rusu, et. al. 1993 reported that iron causes alterations in gut microbes, i.e., reduces Bifidobacteria and increases Enterobacteriaceae [41]. Phylum Proteobacteria, the family Enterobacteriaceae, and its constituent genera like Escherichia and Shigella produce toxins in AD [186]. The concentration of family is directly related to severity of the disease. The pathogenesis involves two factors, namely, quantity of endotoxin and serotonin. Released endotoxins, such as LPS, deposit in the hippocampus and neocortex and contribute to increasing pro‐inflammatory cytokines and stimulate fibrillogenesis of Amyloid beta. Serotonin, a neurotransmitter, levels are regulated by E. coli which misbalances both the enteric nervous system (ENS) and CNS in case of dysbiosis [180]. Thus, we can hypothesize that the key microbe, that is, increased Enterobacteriaceae can be used as diagnostic criteria for AD.

PD is another neurodegenerative disease associated with gut flora. It is found commonly in both older and middle‐aged individuals affecting 1% and 5% of the population, respectively [187]. Symptoms of PD include bradykinesia, asymmetric resting tremor, postural instability, and rigidity, which continue to cause dementia in later stages. PD is associated with motor and nonmotor impairment. The latter impairment is mainly related to problems in GI functioning where affected individuals experience nausea, excessive salivation, and dysphagia. Reports suggest that dysbiosis of gut microbiota leads to pro‐inflammatory factors thereby causing PD to develop. Pathological action of gut flora is associated with the alpha‐synuclein which is involved in PD development. Intake of heavy metal toxicity is one of the risk factors in dysbiosis and hence development of PD, thus the two are correlated as suggested by Forero‐Rodriguez et al. [188]. Further [189] link heavy metal toxicity with alterations in gut flora, consequently affecting several organs. Zhang et al., showed that Firmicutes/Bacteroidetes levels were disturbed in Bufo raddie [190]. Lorente‐Picón and Laguna suggest alterations of various species of microbiota associated with PD [156]. The increase in Bacteroidetes is associated with increased pathogens and hence toxins released by them. They activate inflammatory pathways further damaging the nonmotor functioning [191]. It has been hypothesized that Bacteroidetes can be used for the diagnosis of PD correlated with heavy metals toxicity and gut–brain Axis.

3.8. Therapeutic Potential via Diet–Gut–Brain Axis

There lies more connection between diet and the gut–brain axis than assisting in diagnosis and causing disorder. The ENS produces < 30 neurotransmitters, peptides, and hormones which reach their target (brain) through blood circulation. They work in synchronous with the vagus nerve to control hunger and intake of food. The vagus nerve is part of the parasympathetic nervous system, while the latter along with ENS and sympathetic nervous system makes autonomic nervous system. Thus, the autonomic nervous system modulates emotion and cognition, enteric reflux, intestinal permeability, and immune activation through the CNS. Now, the vagus nerve transfers information from a variety of inner organs to the CNS, which in turn regulates mood, memory, cognition, and disorders chronic fatigue syndrome, alcoholism, restless legs syndrome, and fibromyalgia, synthesizing and negatively impacting neurotransmitters, depression, and anxiety [192]. The Vagus nerve in the bidirectional axis is influenced by metabolites (hormones and peptides) produced by gut microbes via diverse mechanical and chemical receptors. The resultant is the alteration in food intake and stomach draining [193, 194, 195, 196].

The intestinal flora communicates with the CNS via hormonal and neurocrine systems. Participating metabolites include generally secreted compounds, namely, SCFA, ketones, tryptophan, and modulated bile acids that impact cognition and memory. Diving into the mechanism, we see the mentioned molecules bind to Enteroendocrine cells through the expressed receptors, namely, Farnesoid X receptor (FXR) and G protein‐coupled bile acid receptor (TGR5). The next step is the passage of these molecules into blood–brain barrier where they eventually reach the hypothalamus in the brain and control the release of neuropeptide Y. The consequence is GLP‐1 secretion, which controls the fate of glucose in cells [197, 198, 199]. Another link of control is through tryptophan levels from gut microbes. They modulate the release of serotonin through enterochromaffin cells (ECC), linking ASD [200]. The visible signs of gut–brain axis are the time taken in the movement of food from the stomach to intestines, alteration in quality and quantity of mucus, and abnormal neuronal activity evident as cecocolonic spike bursts, whose outcome changes in nutrient availability to microbiota [201, 202].

A variety of diets has expressed therapeutic effects concerning neurological diseases. Antibiotic treatment of mice and faecal transplantation showed contrary and positive effects of ketogenic diet on antiseizure effects. The ketogenic diet infers protection via alteration in gut microbes with specific incorporation of ketogenic diet‐associated microbes Akkermansia and Parabacteroides. Changes are further validated by metabolite concentrations such as a rise in gamma‐aminobutyric acid (GABA) and glutamate concentration and alleviation of circulatory gamma‐glutamylated amino acids. The mechanism of effect is suggested to be competition for nutrients by the microbial population. On the other hand, antibiotic‐treated mice did not show any beneficial impact on feeding a ketogenic diet [203, 204]. Various other mechanisms relating to the ketogenic diet and antiseizure effect act via inhibition of oxidative stress, release of neurotransmitters, change in the fate of glucose and its metabolic product tricarboxylic acid, and triggering of adenosine triphosphate (ATP)‐mediated potassium channels [205]. Another method of diet intake, intermittent fasting holds benefits over neurological diseases like PD, multiple sclerosis, and ASD via the gut–brain axis. The effect is the consequence of enriched diversity in the gut microbiome, which due to time restricted fasting or intermittent fasting lowers T lymphocytes and pathogenesis. Also, re‐establishes equilibrium between astrocytes and microglia and elevates brain‐derived neurotrophic factor (BDNF) [206]. Cognitive decline common in Type 2 diabetes seemed to improve through intermittent fasting. Along with the above‐mentioned effects, enhancement in hippocampal gene expression and metabolites concerning cognitive function were the key factors responsible for improvement. Validation by the introduction of related metabolites also improved cognitive function in Diabetes‐affected mice models [207]. Thus, gut microbial composition can be used for theranostic approach.

4. Conclusion

The body requires inorganic elements (essential metals) from exogenous sources, to catalyze several functions concerning its development. The current practice of imbalanced diet intake provides these metals in excess or deficient quantities. The absorption of these metals starts from the organs comprising trillions of microbes. Being living and endocrine in nature, they play a pivotal role in the metabolism and uptake of these metals in the host. A particular study also links the bidirectional effect of the mentioned two variables, with a neurological system in the body. Intestinal flora assists in the absorption of essential metals but restricts toxic heavy metals. There is an interconnection, as dysbiosis affects the concentration of metals thus causing AD, PD, ASD, cognitive dysfunction, anxiety, and depression. Metal concentration directly affects the nervous system, which in turn contributes to dysbiosis characterized by alterations in the Firmicutes/Bacteroidetes ratio, as well as changes in the abundance of Actinobacteria, Gammaproteobacteria, Enterobacteriaceae, Proteobacteria, Akkermansia, Bifidobacteria, Escherichia, Enterococcus, Bacteroides, and Clostridium.

4.1. Future Perspectives

An increasing amount of research from preclinical and to some degree, clinical studies demonstrate the impact of numerous dietary (poly)phenols and metals on aging and brain function by influencing the gut–brain axis. In this respect, the current review presents inflammatory, oxidative, and pathogenic actions of metals such as Iron, Magnesium, Zinc, and Copper and toxic metals such as Arsenic and Mercury. Additionally, specific microbes being altered due to neurological disorders can be used for diagnosis. Diagnosis of neurological disorders, due to altered intake of magnesium through Bifidobacterium, zinc through Clostridium, iron through family Enterobacteriaceae, and heavy metals through Bacteroidetes can be applied. The alteration can be further modified to eubiosis implicating therapeutic effects on neurological disorders. Effects of a ketogenic diet, intermittent fasting, general excess, and deficiency are taken into consideration. The authors hope to provide specific direction to researchers, in linking dietary metals with gut microbes and neurological disorders.

Overall, further study in this domain needs to focus on the mechanisms underlying the intricate relationship between these dietary compounds and the host systems and gut bacteria. More importantly, researchers need to focus on the elderly population since microbial diversity declines with the ageing of the gut, which is a critical component in sustaining overall gut health and functionality. For a better understanding of the protective effect of these natural substances on healthy brain ageing, effort should be made to characterize the brain's health and the systemic biomarkers associated with it, in addition to neuroimaging in tandem with gut microbiota analysis.

Author Contributions

Conceived and designed by L.Q.C., S.K.J., and Z.S.C. Materials collected and artwork done by N.K., X.C., and R.S. Critical evaluation and analysis of data done by S.K.J., L.Q.C., R.S., X.C., L.Y., and R.S. Manuscript written by L.Q.C., S.K.J., N.K., L.Y., R.S., and H.K. All authors read the manuscript and agreed to submit it. Manuscript edited by Z.S.C.

Ethics Statement

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Data sharing not applicable to this article as no data sets were generated or analyzed during the current study. The data supporting the findings of this study are available from the corresponding author upon reasonable request.