Ischemic stroke is a common central nervous system (CNS) disease and one of the most serious health problems worldwide. The gut microbiota is an important intestinal microecosystem that plays an important role in the homeostasis of the internal environment. Under normal circumstances, intestinal microorganisms and the host maintain a dynamic ecological balance, and gut microbiota homeostasis is closely associated with the health. In recent years, studies have found that the gut microbiota plays a key role in the occurrence and development of ischemic stroke (1–3). An imbalance within the gut microbiota is not only closely related to gastrointestinal diseases, such as irritable bowel syndrome, ulcerative colitis, colon cancer (4–6), but is also associated with the occurrence and development of hypertension, diabetes, obesity, atherosclerosis and metabolic diseases (7–9), all of which are risk factors for ischemic stroke. In this review, we summarized the influence and mechanisms of gut microbiota disorders on the pathogenesis of chronic metabolic diseases and stroke, and provided new proposals for the prevention or treatment of stroke.

The human gut microbiota is a very complex ecosystem with a large number and wide variety of species involved, which consists of approximately 10 times the number of cells in the human body and > 100 times as many genes as the entire human genome (10). The human gut microbiota is mainly composed of bacteria from the phyla Firmicutes and Bacteroidetes with the rest phyla comprising Actinobacteria, Proteobacteria and Verrucomicrobia (11). The gut microbiota is vital to health and plays a key role in many important physiological functions: (1) physiological nutrition: some probiotics can decompose dietary fiber and starch that cannot be digested in the daily diet and can degrade polysaccharides (12); (2) antagonism: some probiotics reproduce in the intestinal tract and generate various metabolites, such as hydrogen peroxide, carbon dioxide, and acetaldehyde, all of which inhibit or kill intestinal Escherichia coli and Streptococcus spp. (13); (3) immune regulation: probiotics, such as Bifidobacterium spp., can improve the body’s immune response and expression level of multiple cytokines, thus improving the disease-resistance of the host (14); (4) anti-cancer: Lactobacillus and Bifidobacterium spp. and other probiotics can enhance intestinal peristalsis, which may reduce the intestinal retention time of harmful or carcinogenic substances (15); (5) other functions: normal gut microbiota can regulate the development of the brain (15, 16).

Normally, the gut microbiota exists in a dynamically balanced ecological environment. The gut microbiota imbalance may occur for several reasons, such as prolonged drug treatments, stress, or infections by pathogenic bacteria and viruses. Under these conditions, the number, species, proportion, location, and biological characteristics of the gut microbiota will be altered (17).

The colonization of human gut microbiota in the infant is related to many factors including: intrauterine contact, delivery method (vaginal or caesarean delivery), feeding method (breast feeding or formula milk), the mother’s diet, external environment, and the use of antibiotics. These factors have a significant impact on the composition of the neonatal gut microbiota (18–20). The controversy over whether colonization of the human gut microbiota begins at birth or in the womb resulted from the discovery of the presence of bacteria in the placenta and amniotic fluid by Jimenez et al. (19). Prior to this, the fetus was considered to be a completely sterile environment. In a study of pregnant women who had caesarean deliveries, Collado et al. found that the characteristics of the microbiota in the placenta and amniotic fluid were consistent with the meconium of the newborn (21). The delivery mode can also affect the colonization of the neonatal gut. During vaginal delivery, the neonate is exposed to microorganisms that mainly come from the mother’s vagina and feces, while the composition of the baby’s intestinal flora is similar to that of the mother’s skin after caesarean delivery. Lee et al. found that on day 1 and 3 after birth, the richness and diversity of the gut microbiota of newborns delivered vaginally was significantly higher than that of newborns delivered by caesarean section, but the differences gradually decreased over time (22).

The intestinal microbiota undergoes a series of maturation processes in infancy and early childhood; however, by 3 years of age, it has established a structure similar to that of the adult intestinal microbiota (23). During the first 3 years of age, there is substantial synaptogenesis and neural development occurring in the brain (24). In addition, fewer types of gut microbes are present in early childhood and adolescence. The gut microbiota eventually reaches a relative maturity in adulthood and is manifested by an increase in the numbers and types of gut microbiota that display stronger resistance to antibiotics and other disturbances (25). Interactions between the host and gut microbiota have been shown to occur during an important window of brain development, referred to as “early life” (26). Disruption of the gut microbiota during this period may interfere with communication between the intestinal microbiota and brain, which may lead to CNS diseases (27, 28).

The traditional methods used to study the gut microbiota mainly include bacterial cultures and molecular biological techniques that are independent of culturing (29). The former method is mainly used for fecal culture; however, under laboratory conditions, 99% of the microorganisms in feces cannot be isolated and cultured, and these procedures are complicated and have low purification efficiency. These methodologies are laborious, and the bacterial strains obtained are limited. In recent years, real time quantitative PCR, fluorescence in situ hybridization, denatured gradient gel electrophoresis, terminal restriction fragment length polymorphism, second generation of sequencing, metagenomics, metabolomics, 16S rRNA sequencing, and other sequencing technologies have been developed (10, 30, 31). Development of germ-free (GF) animal models is an important method to study gut microbiota. GF mice can be colonized with a specific bacterial strain or transplanted with feces (32, 33). Additionally, antibiotics or probiotics can be used. The former interferes with the composition of the normal gut microbiota in mice, while the latter can be used to treat the mice with brain dysfunction. Using these methods, the differences in various physiological indicators and behaviors of mice after different interventions can be analyzed (34).

The immune system, including both adaptive and innate immunity, plays a crucial role in the gut-brain axis. The gut microbiota can affect the maturation, development, and function of immune cells in the CNS, as well as the activation of peripheral immune cells, involved in both cellular and humoral immunity (63). The gut microbiota affects neurophysiological functions, which includes neural development, CNS immune activation, and BBB integrity, by regulating microglia and astrocyte development and function (64). The gut microbiome also regulates peripheral immune responses and plays a key role in brain inflammation, injury, and behavior (65).

Microglia are the most numerous and abundant immune cells in the brain and account for approximately 5%–20% of all glial cells (66). The immune functions of microglia in the CNS include phagocytosis, antigen presentation, cytokine production, and activation of inflammatory responses (67, 68). The gut microbiota can affect the maturation and function of microglia. Compared with those in normal mice, the numbers of immature microglia in the cortex, hippocampus, olfactory bulb, corpus callosum, and cerebellar gray matter and cerebral white matter in the GF mice were significantly increased (69). Moreover, when compared with normal mice and GF mice, brain microglia showed expanded maturity and increased expression of colony stimulating factor 1 receptor, F4/80, and CD31 in normal mice. The expression of these molecules is present in mature microglia and gradually decrease, and the same phenomenon occurred in normal mice after antibiotic treatment (69). The immune response induced by lipopolysaccharide or choriomeningitis virus included decreased activation of brain microglia, delayed development of immune cells, and reduced production of the inflammatory cytokines IL-6, IL-1β and TNF-α in GF mice compared with normal mice (68).

Astrocytes are the most numerous and largest glial cells in the brain and perform various functions, including regulating BBB integrity, neurotransmitter conversion, ion gradient balance, cerebral blood flow adjustment, and nutrient metabolism (70). Astrocytes integrate information from adjacent glial cells, neurons, blood vessels, and immune cells to regulate neural cells excitability and synaptic formation (71). The intestinal microbiota metabolizes tryptophan, including indole-3-aldehydes and indole-3-propionic acid (72), which in turn, activates the astrocyte aromatic hydrocarbon receptors and regulates astrocyte activity (56).

The intestinal microbiota and mucosal cells regulate the activation of immune molecules that affect the CNS, including the pro-inflammatory factors IL-8 and IL-1 and anti-inflammatory factors IL-10 and TGF-β (73–75). Intestinal mucosal pattern recognition receptors include Toll-like receptors that bind to lipopolysaccharide and other microbial-related molecules and activate immune cells, such as dendritic cells, neutrophils, and macrophages (76). Receptor binding results in the production of pro-inflammatory cytokines that include IL-1α, IL-1β, TNF-α, and IL-6, which in turn can cross the BBB and affect brain function directly (77). One study found that filamentous bacteria promoted the development of T-helper cells that produced IL-17 (Th17) in the small intestine of mice, and further study found that mice lacking normal intestinal flora had very few Th17 cells (78, 79). Lee et al. (80) demonstrated that the lack of normal number of Th17 cells in GF mice resulted in an immune deficiency against pathogenic infections, but the mice had increased resistance to the development of autoimmune diseases, such as autoimmune encephalomyelitis. A study on multiple sclerosis showed that IgA⁺ B cells from the intestine crossed the BBB, migrated to brain lesions, and then released IgA antibodies (81). These antibodies mainly reacted with diversified bacteria mainly, including MS related bacteria, without attacking self-brain tissue, which may play a protective role in ameliorating neuroinflammation (81). In conclusion, the immune system plays a very important role in the gut-brain axis.

Among intestinal lymphocytes, CD4 ⁺ T cells are the main cell population mediating various host protection and homeostasis responses (82). The gut microbiota and its metabolites can directly or indirectly induce the differentiation of CD4⁺ T cells, including T-bet⁺ Th1 cells, RORγt⁺ Th17 cells, Foxp3⁺ Treg cells and GATA3⁺ Th2 cells (76). The initial polarization of CD4⁺ T cells is uncertain and dynamic, mainly characterized by the transformation of key transcription factors and characteristic cytokines. The bacteria-related inflammatory response can reprogram RORγt⁺ cells to co-express T-bet or Foxp3, promoting the interconversion between Treg cells and Th17 cells (83). The gut microbiota can influence the formation of T cell subsets through specific cytokine combinations, microbial environment, and biogeography. For example, Th13 cells that secrete IFN-γ are formed by expressing T-bet under the influence of IL-12, IL-18, and IL-23, while Th2 cells are formed under the influence of IL-4 or IL-5, express GATA3, and secrete IL-5 and IL-13 (84). Most microorganisms with epithelial cell invasion ability can be phagocytosed by dendritic cells, and can also stimulate dendritic cells to release inflammatory cytokines such as IL-6 and TNF-α, and bind with OX40 and IL-12. These signals can preferentially polarize Th1 cells (85). Th17 cells and Treg cells are two important lymphocyte subsets with opposite functions. Despite having different functional properties, differentiation from naive T cells into Th17 cells and iTreg cells is dependent on TGF-β expression levels. Low expression levels of TGF-β, IL-23, or IL-6 induced the development of naive T cells into Th17 cells, while high expression levels of TGF-β induced iTreg cells (86). The gut microbiota can induce Th17 cell response, thus inhibiting intestinal flora, while regulating Treg cell response to provide tolerance to gut microbiota (87). One study found that 11 strains of bacteria isolated from the feces of healthy people, when colonized in combination in the intestine, effectively induced IFN-γ -producing CD8⁺ T cells in the intestine and other organs of mice without causing inflammation. The induction is dependent on CD103⁺ dendritic cells and MHC Ia antigen presenting molecules (88).

Ischemic stroke refers to the obstruction of blood supply to brain tissue caused by various conditions, which induces irreversible damage to brain tissue at the site of insult, and leads to brain tissue necrosis (89). The risk factors for ischemic stroke are complex and diverse. The gut microbiota disorders will change the intestinal environment, affect intestinal metabolism and absorption functions, and cause gut microbiota to affect the risk factors for ischemic stroke by different means.

Hypertension, obesity, diabetes, and atherosclerosis are independent risk factors for stroke. Gut microbiota disorders and their endotoxins and metabolites play an important role in the pathophysiological processes of obesity, diabetes mellitus, and atherosclerosis (113). In addition, increasing numbers of studies have shown that gut microbiota disorders are directly related to stroke directly.

The gut microbiota can effectively regulate the number of lymphocytes, such as regulatory T (Treg) and γδT cells, and a gut microbiota imbalance will affect Treg cells and IL-17 ⁺ γδT cells, both of which are involved in an ischemic brain injury (114). The γδT cells are a group of cells with primary innate immune function, and are situated mainly on the surface of the intestinal epithelium (115). The γδT cells can secrete large amounts of IL-17 after an imbalance in the gut microbiota, which leads to chemokine production from surrounding medullary cells (monocytes and neutrophils) and aggravation of ischemic brain injury (116). Effector T cells may induce focal ischemic brain injury, but Treg cells can play a neuroprotective role by inhibiting post-ischemic inflammation (117). Treg cells inhibit IL-17 ⁺ γδT cells by secreting IL-10, thus exerting a neuroprotective effect (118). However, Treg cells have been not shown to enter the brain parenchyma during the acute stage of stroke, indicating that the beneficial role of Treg cells is achieved by regulating the peripheral immune system rather than directly acting on damaged brain tissue (119).

Wang et al. (120) found that oral butyrate reduced brain damage caused by type 2 diabetes. Xu et al. (121) found that cerebral ischemia rapidly caused intestinal ischemia and increased production of nitrate through free radical reactions, which resulted in an intestinal flora disorder accompanied by expansion of bacteria from the family Enterobacteriaceae. Enrichment of Enterobacteriaceae microbes contributes to cerebral infarction injury by enhancing systemic inflammation and represents an independent risk factor for a major adverse outcome in stroke patients. Treatment with superoxide dismutase or aminoguanidine can reduce nitrate production, while treatment with tungstate inhibits nitrate respiration, inhibits overgrowth of Enterobacteriaceae, reduces systemic inflammation, and alleviates cerebral infarction injury.

The beneficial or harmful effect of gut microbiota imbalance on stroke prognosis remains unclear. Pretreatment with antibiotics inhibited the gut microbiota and significantly decreased the α-diversity of the gut microbiota by day 3 after treatment in mice with middle cerebral artery occlusion, resulting in decreased cerebral infarction volume and better prognosis (122). However, another experiment had the opposite result. In the middle cerebral artery occlusion model using C57BL/6 mice, after gut microbiota was inhibited by broad-spectrum antibiotics, the mortality rate of mice with gut microbiota disorders was significantly increased (123). Singh et al. studied the relationship between brain injury, intestinal microbial disorders, and the immune system after ischemic stroke and found that ischemic stroke caused intestinal microbial disorders and impaired gut microbiota function (1). Conversely, changes in the intestinal microbiota affected the prognosis of brain injury caused by inflammation. After ischemic stroke, T cells migrated from the intestinal tract to the damaged brain, suggesting that gut microbiota disorders may be a therapeutic target to alleviate the immune responses after ischemic stroke. Subsequently, Singh et al. used the method of intestinal bacterial transplantation to restore the balance of the gut microbiota, improve the area of cerebral infarction in mice, and reduce the number of Treg cells (1). In a study comparing the effects of oral and intravenous antibiotics on the gut microbiota, both oral and intravenous antibiotics affected the gut microbiota, taking less time to recover in the intravenous antibiotics group than in the oral antibiotics group (124).

The relationship between ischemic stroke and the gut microbiota has been extensively studied in animal models, and progress has also been made in clinical studies. Karlsson et al. studied the genomic composition of the gut microbiota in patients with cerebral ischemic stroke and found that the microbial composition was different from that of normal people. The number of Ruminococcus spp. was significantly increased in patients with cerebral ischemia, while the number of Eubacillus spp. and Bacteroides spp. was significantly decreased (110). The relationship between gut microbiota disorders and ischemic stroke has not been fully clarified. According to current research, gut microbiota disorders may promote the pathological disease processes and the formation of atherosclerotic plaques. A clinical study compared the gut microbiota and plasma TMAO levels in patients with asymptomatic atherosclerosis, transient ischemic stroke, and stroke. The researchers found that plasma TMAO levels and microflora composition were independent of carotid atherosclerotic plaque formation in the asymptomatic atherosclerosis group. However, the differences in microflora composition and plasma TMAO content between the transient ischemic attack and stroke groups were statistically significant (125). A study shows that patients with acute ischemic stroke can experience significant gut microbiota disturbance that lasts for more than 3 weeks, after 4 weeks, the disturbed gut microbiota can gradually recover with a significant decrease in microbiota diversity (3). The alterations in the gut microbiota may be an indication of ischemic stroke incidence, progression, and prognosis ( Table 1 ).

Gut microbiota disorders and intestinal dysfunction can affect the prognosis after a stroke through various mechanisms that include microflora migration, intestinal bacterial metabolites, and immune regulation. After an ischemic stroke, intestinal motility decreases and intestinal permeability increases, and gut microbe translocation into extraintestinal organs leads to local and systemic infection (2). Endotoxins derived from the gut microbiota, such as lipopolysaccharide and peptidoglycan, enter the blood through the highly permeable intestinal wall, activate the innate immune response of the body, and aggravate the inflammatory reaction. Infection and inflammation are detrimental to stroke outcomes. After cerebral apoplexy, the host immune system is severely suppressed (131), and the immune barrier function of the intestinal tract is reduced (132). Intestinal bacteria may be transferred into the blood and external organs, thereby, inducing a systemic inflammatory response and aggravating the adverse outcome of cerebral infarction injury. Crapser et al. found that, in aged mice, ischemic stroke induced gut permeability and enhanced bacterial translocation that resulted in sepsis, while young mice were able to resolve the infection (133).

The gut microbiota can interact with the brain through various mechanisms, and gut microbiota imbalance can promote the occurrence of strokes, which in turn may aggravate the gut microbiota dysbiosis. The prognosis after a stroke can be improved through multiple interventions ( Figure 1 ). The large number of gut microbes and variety of metabolites may change the impact of the gut microbiota on stroke outcomes. At present, the treatment of stroke by altering gut microbiota has substantial limitations, and more in-depth research will still be required.

There are many symbiotic microorganisms in the gut whose composition and abundance may affect both the autonomic nervous system and CNS. In recent years, many studies have confirmed that intestinal microbiome disorders are associated with certain central nervous system diseases, such as stroke, Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis (146, 147). A stroke is the consequence of many complex factors, including obesity, hypertension, hyperlipidemia, insulin resistance, and atherosclerosis. Gut microbiota disorders are not only involved in the pathological processes of these risk factors, but are also directly related to strokes. A diet high in fat and sugar leads to changes in the proportion of symbiotic bacteria and imbalance of the gut microbiota (148). Therefore, maintaining a reasonable diet may reduce the incidence of ischemic stroke. After the occurrence of an ischemic stroke, the application of antibiotics for gut microbiota treatment and the restoration of intestinal homeostasis may control the development of the disease and improve its prognosis.

Although many studies have been conducted to evaluate the relationship between the gut microbiota and the occurrence and development of strokes, the following limitations remain: (1) Many studies have shown that different bacterial populations are associated with particular clinical symptoms; however, in most cases, it is not clear whether these differences cause disease. (2) Few breakthroughs have been made. Most studies have focused only on the correlations between the gut microbiota and diseases. Further studies are still needed to determine the mechanisms by which intestinal microbiota dysfunction affects the brain. (3) There is no research on chemical drugs that can effectively regulate the gut microbiota. Furthermore, the causal relationships between specific intestinal flora and specific diseases as well as detailed mechanisms should be further clarified to provide theoretical support for clinical disease prevention or treatments related to gut microbiota regulation.

The drug research on microbial flora has been performed for a considerable length of time and the present study mainly includes the following four types: (1) the FMT, which involves extracting gut microbes from healthy donor stools through different preparation processes, such as adding a protective agent to intestinal soluble capsules; (2) bacteria formula, which involves isolating strains one by one through artificial intelligence calculations and functional screenings and creating combinations of strains to treat diseases; (3) bacterial metabolites, small molecule regulators, and drugs similar to small molecules and peptides; and (4) artificial modification of bacteria through synthetic biology and genetic engineering methods to enhance the pharmaceutical function. These disease-specific microbiological drug studies require a deep understanding of the gut microbiota and the pathogenesis of various diseases, including stroke.

Microbe-targeted interventions, such as antibiotics, probiotics, and FMT, have been shown to have a beneficial effect on host health. External interventions with intestinal flora may change the outcome of some refractory diseases, including stroke, indicating that the gut microbiota has substantial therapeutic potential in clinical practice. Restoring gut microbiota homeostasis to treat ischemic stroke may represent a major breakthrough.

Future research will include defining mechanisms of microbe-microbe and microbe-host interactions in complex microbial-human gut ecosystems, using high-throughput sequencing of gut microbial genomes, and developing drugs based on these data. All of these studies will have a significant impact on treatment potential for various diseases, including stroke. Finally, it will be necessary to perform tightly designed, prospective, and longitudinal studies to transform these studies to clinical applications.

JW, HZ, and XX edited the manuscript. JH directed the project, and revised the manuscript. All authors read and approved the final manuscript.

This work was supported by the National Natural Science Foundation of China (no. 82171336, 81870939 to XX) and Health Commission of Jiangxi Provincial (no.202130032 to JH).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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