Decoding TMAO in the Gut-Organ Axis: From Biomarkers and Cell Death Mechanisms to Therapeutic Horizons

Introduction

The gut is a vital organ of the host. The gut microbiota plays a pivotal role in regulating host metabolism and immune responses and is closely associated with various human diseases. Metabolites produced by the gut microbiota serve as key mediators between the microbiota and the host, interacting with multiple systems in the body to regulate physiological and pathological functions.¹ Trimethylamine N-oxide (TMAO) is a significant metabolite generated from dietary components by gut microbes through microbial enzymes (such as trimethylamine lyases). Dietary choline, L-carnitine, and betaine are metabolized by gut microbiota, generating trimethylamine (TMA); in the liver, TMA is metabolized by the enzyme flavin-containing monooxygenase 3 (FMO3) to produce TMAO, which then enters the bloodstream.^2–6 In recent years, it has gradually been recognized that TMAO may act as an “axis” connecting the gut and host organs. Its research scope has expanded from cardiovascular diseases to other systemic diseases, including pulmonary, kidney, neurological, and pancreatic diseases. Elevated plasma TMAO levels are closely associated with the development and progression of various diseases. TMAO is expected to become a potential biomarker for the diagnosis and prognosis evaluation of specific diseases.

There is widespread recognition that TMAO causes endothelial cell (EC) dysfunction.⁷ TMAO can stimulate ECs to release nitric oxide (NO), adhesion molecules (ICAM-1, VCAM-1, E-selectin, etc), and interleukin-6 (IL-6) to control the adherence of immune cells.⁸ Additionally, while TMAO affects endothelial barrier function, it can also act on various types of cells, including vascular smooth muscle cells, epithelial cells, pancreatic cells, neurons, astrocytes, microglia, and oligodendrocytes, thereby exerting broad impacts on numerous human diseases. Notably, TMAO can induce various types of cell death, including necrosis and programmed cell death (PCD). Necrosis is a kind of cell death that occurs passively. PCD is a manner of cell death actively triggered by internal and external stimuli. Apoptosis is the most common form of PCD, and other kinds of PCD include autophagy, pyroptosis, and ferroptosis, among others.⁹

This paper presents a detailed overview of the biomarker function of TMAO in the “gut-organ” axis, as well as the regulation mechanism of TMAO on the “gut-organ” axis, particularly in relation to the crosstalk mechanisms involved in cell death. Concisely, we first present the generation, synthesis, and metabolic pathways of TMAO. After applying strict criteria for literature screening, we selected clinical trials focused on TMAO to determine its potential as a biomarker for specific disorders through the “gut-organ” axis. We also provide a concise overview of the mechanism by which TMAO triggers PCD. Ultimately, we offer a summary of some medications that specifically aim to inhibit or regulate the generation, synthesis, or metabolism of TMAO. Our review offers new insights into the potential mechanisms of TMAO in disease onset and progression while emphasizing its crucial role in health regulation.

Methods

This narrative review included a literature search using the PubMed and Web of Science databases with the keywords: “(TMAO OR Trimethylamine N-oxide)” AND (“clinical study” OR “animal model” OR “in vitro study”). We included high-quality, peer-reviewed literature over the last 20 years (2004–2024), including cohort studies, randomized controlled trials, systematic reviews, meta-analyses, and original research publications. Studies were rejected if they were not in English, were duplicates, lacked full-text access, or consisted just of abstracts or comments without empirical data. It is essential to emphasize that, in contrast to systematic reviews or meta-analyses, we concentrated on presenting pertinent clinical and preclinical data on TMAO to provide complete insights. In Regulation Mechanism of TMAO in the “Gut-Organ” Axis: Crosstalk in Cell Death, we retrieved pertinent information about clinical features and the assessment of TMAO as a biomarker from chosen clinical trials. Subsequently, we qualitatively discussed the results within the literature in a narrative format, emphasizing significant findings to illustrate the potential use of TMAO in predicting, diagnosing, or prognosticating human illnesses. In Diverse Therapeutic Strategies Targeting TMAO, we curated the most pertinent preclinical and in vitro studies on TMAO, emphasizing findings associated with TMAO-induced cell death to deliver a thorough summary of its role in the development and progression of diseases along the “gut-organ (lung, pancreas, heart, kidney, and brain)” axis. In Conclusion and Future Directions, we reviewed clinical and preclinical data on therapeutic techniques aimed at TMAO, with the objective of elucidating the possible prospects of TMAO-targeted interventions in disease therapy.

Source, Synthesis and Metabolism of TMAO

Choline, L-carnitine, betaine, gamma-butyryl betaine in animal-derived foods (eg, red meat, eggs), and ergothione derived from mushrooms are critical precursors for the production of TMA.^10–13 These compounds are primarily derived from the diet and all contain a trimethylamine group. They can be metabolized into TMA in the gut through the action of various enzymes in interaction with certain gut microbiota. TMAO, abundant in crustaceans, fish, and other seafood products, can also be directly assimilated into the human body through the gastrointestinal tract. Choline is a water-soluble nutrient that is essential for human health. Exogenous supplementation is required to attain the necessary choline levels for human health. Choline is primarily present in animal-derived foods as free, water-soluble compounds (choline phosphate, choline glycerophosphate) and fat-soluble compounds (phosphatidylcholine, sphingomyelin). The phospholipase D enzyme can convert phosphatidylcholine, also referred to as lecithin, into choline. Choline kinase can catalyze the conversion of choline to lecithin, and the process is bidirectional between PC and choline.⁵ Furthermore, the seeds of cruciferous plants contain choline in the form of phenolic cholinesterase. Choline erucate, abundant in cauliflower, is also a precursor of TMAO.^3,4 Choline TMA-lyase (CutC) catalyzes the production of TMA from choline. It is important to note that the choline-TMA conversion process can be substantially influenced by the composition and diversity of the gut microbiome, which in turn impacts the production and accumulation of TMAO. L-carnitine is another precursor of TMA that is typically obtained from meat (red meat is exceptionally high in L-carnitine) and eggs. The primary function of L-carnitine is to transport long-chain fatty acids from the cytoplasm to the mitochondria for β-oxidation, which is essential for the body’s energy metabolism.^2,6 Two critical enzymes involved in the conversion of L-carnitine to TMA are carnitine oxygenase (CntA) and carnitine reductase (CntB).¹⁴ Betaine is a choline derivative that is present in both animal and plant diets. Choline dehydrogenase and betaine aldehyde dehydrogenase can continuously convert choline to betaine. Carnitine dehydrogenase can also catalyze the conversion of L-carnitine to betaine. Betaine is converted to TMA through the action of betaine reductase. Ergothione is exclusively obtained through the diet (beans, livestock products, or mushrooms) and is converted to TMA by degrading the enzyme ergothionase.⁵ Briefly, the conversion of dietary precursors into TMA involves four enzymatic pathways, including the choline-utilizing TMA lyase system (CutC/D), the carnitine Rieske-type oxygenase/reductase system (CntA/B), betaine reductase, and TMAO reductase. Additionally, YeaW/X, a system homologous to CntA/B, can also metabolize carnitine, choline, γ-butyrobetaine, and betaine to generate TMA.¹⁵

In mammals, this process is catalyzed by enzymes encoded by genes such as CntA/B, CutC, and betaine reductase. Although less than 1% of gut microbiota possess the genes required for TMA synthesis, these relevant microbial communities are still sufficient to drive significant TMA production.¹⁶ As a core step in TMAO biosynthesis, the gut microbiota plays a pivotal role in regulating the generation and metabolism of TMA. Key microbial groups involved in this process include Firmicutes, such as Lachnoclostridium, Clostridium hathewayi, Clostridium asparagiformis, and Clostridium sporogenes, as well as Proteobacteria, such as Escherichia coli MS 200-1, Escherichia fergusonii, Proteus penneri, Edwardsiella tarda, and Desulfovibrio desulfuricans.^17,18 Additionally, other gut microorganisms, including Deferribacteraceae, Anaeroplasmataceae, Prevotellaceae, and Enterobacteriaceae, also participate in this process.^19–21 Considering the conserved nature of TMAO metabolism, we posited that fluctuations in TMAO levels throughout many illnesses, including cardiovascular, renal, and neurological disorders, are affected by alterations in gut microbiota. The gut microbiota significantly contributes to the progression of numerous illnesses and exhibits distinct alterations in response to different conditions. However, they may all result in the overexpression of TMAO, which may impact several organs.

TMA is absorbed into the liver through passive diffusion. In the liver, it is processed by FMOs, particularly FMO3, to create TMAO.²² After its formation, TMAO is excreted from the liver into the bloodstream via specialized transporters. ABCB1 (MDR1) and ABCG2 (BCRP), both members of the ATP-binding cassette (ABC) transporter family, play a pivotal role in facilitating the diffusion of TMAO.²³ Notably, FMO3 is not only highly expressed in the liver but also significantly in the lungs, adrenal glands, and aortic tissues.^24,25 This indicates the presence of several locations for the production of TMAO (Figure 1).

Figure 1 Schematic representation of TMAO source, synthesis, and excretion. Initially, foods rich in L-carnitine, choline, betaine, ergothioneine, and γ-butyrobetaine, primarily fish, meat, eggs, and dairy products, are metabolized into TMA by the gut microbiota and their associated enzymes (Source). Most of the TMA ingested or generated in the gut is swiftly absorbed into the portal circulation via passive diffusion across intestinal epithelial cell membranes, subsequently undergoing oxidation to TMAO through the action of FMO3 in the liver. TMAO can be directly sourced from fish, shellfish, and marine invertebrates (Synthesis). Finally, TMAO is filtered by the kidneys and excreted via three main pathways: urinary excretion, fecal elimination, and respiratory clearance (Excretion). Created in BioRender. Ge, P. (2025) https://BioRender.com/k35g185. Abbreviations: TMA, Trimethylamine; TMAO, Trimethylamine-N-oxide; FMO3, Flavin-containing monooxygenase 3.

The Biomarker Role of TMAO in “Gut-Organ” Axis: Clinical Reality

TMAO in Gut-Lung Axis

The gut-lung axis is a bidirectional pathway facilitating interaction between the gut and lungs via symbiotic microbes at a distance. Research on the gut-lung axis has revealed intricate interactions between the respiratory and gut microbiota, as well as between respiratory and gastrointestinal illnesses. The digestive system and the respiratory tract have a common origin in embryonic development. Moreover, in gut microbiota imbalance, the lung becomes the most susceptible organ. The process may include bacteria and their metabolites entering the bloodstream, disseminating throughout the body, initiating an inflammatory cascade, and culminating in sepsis.²⁶ Wang et al found TMAO to be a possible biomarker for endoplasmic reticulum stress (ERS) in lung tissue after mice were given tunicamycin, which caused lung damage.²⁷ Cao et al found that changing the gut microbiota, specifically by lowering the production of TMA and TMAO, may help reduce acute lung injury caused by sepsis by blocking the nuclear factor-κB signaling pathway.²⁸ Chou et al originated the term “paradox of TMAO.” Researchers found a negative correlation between plasma TMAO levels and cardiovascular mortality in individuals suffering from severe sepsis. This indicates that TMAO levels represent the body’s inflammatory condition and nutritional status.²⁹ The respiratory tract has its own microbiota, and pulmonary inflammation may lead to gut dysbiosis.³⁰ Coronavirus disease 2019 (COVID-19) is an infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The virus primarily invades the respiratory system but also affects the gastrointestinal tract. SARS-CoV-2 has been detected in esophageal, gastric, duodenal, rectal, and fecal samples of COVID-19 patients. The gastrointestinal symptoms in COVID-19 patients are mainly manifested as diarrhea, which is associated with a decrease in gut microbiota richness and diversity, immune dysregulation, and delayed clearance of SARS-CoV-2.³¹ TMAO may serve as a potential biomarker for COVID-19, aiding in diagnosis, assessment of disease severity, and prognosis. Terruzzi et al noted that the gut microbiota-derived metabolites TMAO and lipopolysaccharides, which generate a pro-inflammatory microenvironment, may have a positive correlation with the severe risk of COVID-19.³² Marhuenda-Egea et al utilized ¹H NMR to analyze urine samples from COVID-19 patients, revealing metabolic differences between COVID-19 patients and healthy controls. These differences primarily involve energy metabolism (such as glucose, ketone bodies, glycine, creatinine, and citrate), as well as processes related to the bacterial microbiota (such as TMAO and formic acid) and detoxification (such as hippuric acid).³³ Isabel Tristán analyzed 80 serum and urine samples from COVID-19 patients (34 in intensive care and 46 hospitalized) and 32 healthy controls, identifying various metabolites, including TMAO, that could serve as biomarkers for the diagnosis, prognosis and severity assessment of COVID-19.³⁴ Notably, the variation trend of TMAO among COVID-19 patients is not entirely consistent across all studies. In a nutshell, TMAO has shown promise within the gut-lung axis, warranting further exploration of its function (Figure 2).

Figure 2 Application of TMAO in different body fluids as a potential biomarker for human diseases. TMAO levels have been related to various human diseases, such as pulmonary diseases (such as COVID-19), pancreatic diseases (such as diabetes mellitus), cardiovascular diseases, kidney diseases, and nervous system diseases, TMAO may serve as a potential biomarker for disease risk assessment and progression, underscoring its relevance in clinical settings. Created in BioRender. Ge, P. (2025) https://BioRender.com/f31k885. Abbreviations: TMAO, Trimethylamine N-Oxide; TMA, Trimethylamine; FMO, Flavin-containing monooxygenase.

TMAO in Gut-Pancreas Axis

The pancreas has two main functions: endocrine and exocrine. The endocrine function is primarily responsible for producing and secreting hormones that regulate glucose homeostasis, while the exocrine function focuses on producing and secreting enzymes that facilitate digestion. Pancreatic tissue comprises various cellular and non-cellular components, forming a highly organized and complex microenvironment. When cells are stimulated, the dynamic balance of this microenvironment may be disrupted, leading to the development and progression of pancreatic diseases, such as diabetes and pancreatitis (Figure 2).

Diabetes mellitus (DM) is a class of metabolic diseases marked by elevated blood glucose levels. It is the third largest chronic disease in the world, second only to tumors and cardiovascular and cerebrovascular diseases. DM is categorized into T1DM, T2DM, gestational DM, and other special types of DM. Among them, T2DM is the most common type, accounting for more than 90% of DM patients.³⁵ T2DM is mainly characterized by pancreatic β cell dysfunction, insulin resistance leading to glucose metabolism deficiency, and chronic low-grade inflammation.³⁶ Lifestyle, environment, and genetics are closely related to the occurrence and development of T2DM.³⁷ Additionally, diet and gut microbiota are also significantly associated with T2DM susceptibility.³⁸ Accumulated clinical evidence supports a significant association between TMAO and DM (Table 1). A cross-sectional study reported that plasma TMAO levels in T2DM patients were higher than in controls.^39–41 This concurs with the findings of a previous report.⁴² A long-term prospective cohort study involving 2088 participants found that higher serum TMAO levels were associated with an increased risk of T2DM and elevated fasting blood glucose.⁴³ Another meta-analysis also reported that TMAO may be a novel biomarker for DM.⁴⁴ However, a cohort study of 892 participants found that plasma TMAO levels were negatively associated with T2DM risk.⁴⁵ Preclinical studies have shown a significant association between TMAO and the onset and progression of T2DM.^46–48 Mechanistically, TMAO may induce T2DM by inducing pancreatic β-cell dysfunction, chronic inflammation, oxidative stress, and disrupting glucose and lipid homeostasis.^36,49 The latest research indicates that TMAO can impair β-cell function and glucose tolerance, and the mechanism may be related to the promotion of β-cell ERS, dedifferentiation, and apoptosis.³⁶

Table 1 The Effects of TMAO on Clinical Behavior and Prognosis in Diabetes

In acute pancreatitis, the autodigestion of pancreatic acinar cells and the abnormal activation of pancreatic enzymes are the primary pathological features. Yang et al described the damaging effects of TMAO on pancreatic acinar cells.⁵⁰ More and more evidence shows that the abnormalities in intercellular communication within the pancreatic microenvironment play a critical role in the pathophysiological response of the pancreas to various stimuli. TMAO may be a key mediator in the cross-talk between “endocrine cells and exocrine cells” in the pancreas.

TMAO in Gut-Heart Axis

Cardiovascular disease (CVD), including coronary atherosclerotic heart disease (CHD), heart failure (HF), arrhythmia, and cardiomyopathy, is a significant public health problem threatening human life and health. Despite the widespread use of secondary prevention drugs for CVD, the residual cardiovascular risk of patients remains high. Recent epidemiological evidence shows that the residual cardiovascular risk of CVD patients is closely linked to gut microbiota disturbances. Imbalances in the gut microbiota may regulate lipid metabolism by changing the level of bacterial metabolites and then induce the occurrence and development of CVD. In 2011, researchers using non-targeted metabolomics methods found that TMAO was associated with an increased risk of cardiovascular events in subjects.¹² Different groups have demonstrated a strong association between TMAO and CVD and CVD-related risk factors in recent years. Hypertension is a major risk factor for CVD. Recently, the potential link between TMAO and Hypertension has garnered increasing attention. A Mendelian randomization study found a causal relationship between TMAO and its precursors and susceptibility to hypertension.⁵¹ A meta-analysis study reported that elevated TMAO levels were significantly associated with the risk of hypertension in a dose-dependent manner.⁵² Atrial fibrillation (AF) is not only a complication of various cardiovascular diseases but also significantly increases the risk of cardiovascular events. A growing number of preclinical and clinical studies have shown that TMAO is associated with AF^53–56 (Table 2). Compared with AF patients without thrombosis, AF patients with thrombosis had significantly increased circulating TMAO levels. Mechanistically, TMAO may accelerate thrombosis by promoting platelet activation, inducing platelet hyperreactivity, and platelet aggregation.^57,58 Several research groups have previously summarized the role of TMAO in the early diagnosis and prognosis assessment of atherosclerosis (AS),^12,59–65 CHD,^66–76 and HF.^77–88 Therefore, we have generated Tables 2–4 to complement the latest research findings.

Table 2 The Effects of TMAO on Clinical Behavior and Prognosis in Atrial Fibrillation and Atherosclerosis

Table 3 The Effects of TMAO on Clinical Behavior and Prognosis in Coronary Atherosclerotic Heart Disease

Table 4 The Effects of TMAO on Clinical Behavior and Prognosis in Heart Failure

The value of TMAO in CVD is multifaceted. On the one hand, TMAO is a potential diagnostic marker for CVD, including atherosclerosis, coronary artery disease, and acute coronary syndrome. However, circulating TMAO levels are dynamically variable and are tightly regulated by diet, gut microbiome composition, liver enzyme activity, and renal excretion. Therefore, more sophisticated clinical studies are still needed to confirm the value of TMAO in diagnosing CVD. In addition, Additionally, given TMAO’s critical role in the development and progression of CVD, it is significantly associated with adverse events. Follow-up detection of circulating TMAO levels is helpful in screening out high-risk populations. However, existing clinical studies are mostly single-center and subject to racial limitations. Future studies should include larger and more ethnic groups of patients. Based on previous data, lowering plasma TMAO levels may reduce adverse outcomes of CVD. Targeting TMAO is a potential strategy for CVD therapy.

TMAO in Gut-Kidney Axis

Chronic kidney disease (CKD) is a progressive form of kidney structural damage and loss of function. The early clinical symptoms of CKD are not obvious, and the renal function gradually deteriorates in the later stage and eventually develops into uremia, that is, end-stage renal disease (ESRD). Advanced age, hypertension, dyslipidemia, and diabetes are recognized risk factors for CKD (Table 5). The “gut-kidney” axis plays a crucial role in the pathogenesis of kidney diseases.⁸⁹ TMAO, as a metabolite derived from gut microbiota, may be a critical mediator in the “gut-kidney” axis.⁹⁰ In recent years, increased TMAO levels have been found to be a novel risk factor and early diagnosis biomarker for CKD.⁹¹ Compared to healthy individuals, CKD patients’ TMAO levels significantly increased in blood and urine.^92–94 TMAO levels are also significantly associated with several measures of renal function.^95–98 Mechanistically, in the absence of impaired kidney function, more than 95% of TMAO in the human body is excreted through renal tubules or glomerular filtration,^99,100 suggesting a potential mechanism for elevated circulating TMAO levels when renal function is impaired. GFR is an important indicator of renal function. Studies have found a strong negative correlation between plasma TMAO levels and measured GFR (mGFR) in patients with CKD.^93,101 Stubbs et al also described a negative correlation between serum TMAO concentration and estimated GFR (eGFR).¹⁰² In patients with CKD, elevated TMAO levels accelerate the decline of renal function and affect long-term survival.⁹⁴ Consistent with the above findings, Wang et al’s study also suggested that increased TMAO levels are a crucial risk factor for decreased renal function in CKD patients.¹⁰³

Table 5 The Effects of TMAO on Clinical Behavior and Prognosis in Kidney Disease

It is well known that the incidence of adverse cardiovascular events increases with the progression of CKD.¹⁰⁶ The deterioration of renal function is the main reason for accelerating the occurrence and development of adverse cardiovascular events in patients with CKD.¹⁰⁷ TMAO is a potential risk factor for cardiovascular complications in patients with CKD.^25,102 A meta-analysis reported that increased circulating TMAO levels were closely associated with an increased risk of all-cause mortality in patients with CKD.¹⁰⁴ In CKD patients undergoing coronary angiography, TMAO is an independent predictor of coronary atherosclerotic burden and can also predict long-term mortality. A study of 1232 hemodialysis patients revealed no significant variation in TMAO levels between white and black individuals. Whites had 2-fold higher TMAO levels, which were strongly related to cardiac death, first cardiovascular event, and all-cause mortality. However, no significant association was seen among blacks.¹⁰⁵ Mechanistically, on the one hand, TMAO may promote cardiac remodeling in hemodialysis patients by accelerating myocardial fibrosis and myocardial hypertrophy. On the other hand, TMAO may increase the incidence of adverse cardiovascular events in hemodialysis patients by increasing visceral fat.¹⁰⁸ Notably, in patients undergoing peritoneal dialysis, researchers also identified a significant correlation between elevated serum TMAO levels and an increased risk of cardiovascular disease-related mortality. However, no significant association was found between TMAO level and mortality in peritoneal dialysis patients.¹⁰⁹

Overall, there is a close association between TMAO and renal function, CKD, and cardiovascular outcomes in patients with CKD. Specifically, the levels of TMAO exhibit dynamic changes at different stages of CKD patients.⁹⁷ In the early stages of CKD, circulating TMAO levels are relatively low because kidney function has not yet been significantly impaired. As CKD progresses, the TMAO levels in patients gradually increase. This may be related to decreased renal function, leading to reduced excretion of TMAO, along with alterations in the composition and metabolic function of the gut microbiota. In addition, elevated TMAO levels are closely associated with an increased risk of cardiovascular disease in CKD patients, possibly due to TMAO enhancing platelet reactivity and amplifying endothelial inflammatory responses, thereby increasing the risk of thrombosis. In conclusion, TMAO levels hold promise as a potential biomarker for predicting CKD progression and the risk of associated complications.

TMAO in Gut-Brain Axis

The “gut-brain” axis has progressively become a research hotspot. This bidirectional, stable axis connects the gut with the central nervous system. TMAO exists in human blood circulation and cerebrospinal fluid (CSF)^110–112 and serves as a critical mediator in the “gut-brain” axis. Studies have found that high levels of TMAO have damaging effects on the neurovascular unit.^113–115 “Neurovascular unit (NVU)” consists of three fundamental units: vascular cells, glial cells, and neurons, as well as the extracellular matrix in the cerebrovascular system. NVU emphasizes the interdependent symbiotic relationship between brain cells and cerebrovascular in terms of development, structure, and function, as well as the importance of cell-to-cell interactions in maintaining normal brain function and facilitating brain repair after injury. Dysfunction within NVU may lead to the onset and progression of various pathological reactions in the brain.^116–118 Deng et al found that TMAO may induce neuroinflammation and neuronal apoptosis, leading to NVU dysfunction, which in turn exacerbates cognitive impairment and neuropathological changes in vascular dementia rats.¹¹⁹ Blood-brain barrier (BBB) disruption and white matter lesions are precursor events of neurological disorders. White matter primarily consists of myelinated nerve fibers and glial cells responsible for producing myelin sheaths. Oligodendrocytes are the cells in the central nervous system that form myelin sheaths. Myelin sheath surrounds myelin nerve fibers, acts as electrical insulation, and provides structural protection. It is crucial for maintaining physiological functions such as neuronal nutritional metabolism and information processing.¹²⁰ Cell pyroptosis is also involved in oligodendrocyte damage and inflammatory demyelination.^121,122 Ji et al found that oral administration of TMAO significantly exacerbated demyelination in spontaneously hypertensive rats, with the underlying mechanism possibly related to TMAO-induced pyroptosis of oligodendrocytes and inflammation.¹²³ TMAO may influence the crosstalk of different cells in the NVU and participate in the occurrence and development of neurological disorders. This offers a potential direction for the study of the gut-brain axis in neurological diseases.

The potential of TMAO in the early diagnosis, disease monitoring, and prognosis assessment of neurological disorders is equally noteworthy. Plasma TMAO levels accumulate with age. As is well known, aging is a significant risk factor for cognitive deterioration. Therefore, Alzheimer’s disease (AD), characterized by cognitive impairment and memory loss, is inevitably influenced by the accumulation of TMAO. Moreover, TMAO can cross the BBB. Compared with healthy volunteers, CSF TMAO levels in AD patients were also significantly increased. Multiple clinical studies have repeatedly demonstrated a significant causal association between high plasma and CSF levels of TMAO and cognitive decline^{110,111,115,124–126} (Table 6).

Table 6 The Effects of TMAO on Clinical Behavior and Prognosis in Nervous System Disease

As a common neurodegenerative disease, the onset and progression of Parkinson’s disease (PD) are closely related to dysbiosis of the gut microbiota. Gut microbiota-related metabolites (especially TMAO) may be the key mediators that mediate the changes in brain structure and function of PD mediated by gut microbiota.^130,131 On the one hand, TMAO is intimately linked to PD risk factors such as metabolic syndrome and cardiovascular disease.^132,133 On the other hand, TMAO can directly promote alpha-synuclein folding and aggregation, neuroinflammation, mitochondrial dysfunction, and neuronal injury.^134–136 Clinical studies have reported that compared with healthy controls, plasma, CSF, and saliva TMAO levels in PD patients are significantly elevated.^127–129

Stroke seriously affects human health and quality of life.¹³⁷ Among them, ischemic stroke (IS) is the most common type, mainly manifested as brain parenchyma injury, neuroinflammation, and immune response caused by middle cerebral artery occlusion in the affected area.¹³⁸ IS is primarily associated with neuronal excitotoxicity, oxidative stress, neuroinflammation, apoptosis, mitochondrial damage, amyloid production, and tau protein dysfunction.^139–143 Accumulated evidence emphasizes the critical role of gut microbiota and metabolic disorders in the occurrence, development, prognosis, and rehabilitation of stroke.^144,145 Mechanistically, TMAO may mediate the occurrence and development of stroke by promoting endothelial dysfunction,⁷ inflammatory response,¹⁴⁶ oxidative stress, and lipid metabolism disorders.¹⁴⁷ Additionally, multiple clinical studies have reported the associations between plasma TMAO levels and their dynamic changes and stroke risk, severity, and long-term adverse outcomes.^148–152 Additional studies have revealed a nonlinear dose-dependent relationship between circulating TMAO concentrations and stroke risk.^153–155

Regulation Mechanism of TMAO in the “Gut-Organ” Axis: Crosstalk in Cell Death

Cell death is usually divided into two types: accidental cell death and regulated cell death (RCD). RCD includes apoptosis, regulatory necrosis, autophagy-dependent cell death, pyroptosis, ferroptosis, etc.

TMAO and Apoptosis

Apoptosis is a natural, non-inflammatory, programmed form of cell death. TMAO-induced apoptosis is closely related to ERS and mitochondrial dysfunction.¹⁵⁶ The unfolded protein response (UPR) helps to re-establish endoplasmic reticulum (ER) homeostasis and promote cell survival. Moderate ERS can play a protective role through UPR. If ERS persists and does not alleviate, UPR will induce apoptosis.¹⁵⁷ In the mitochondrial pathway, disruption of the outer mitochondrial membrane integrity can lead to mitochondrial outer membrane permeabilization (MOMP). MOMP plays a critical role in the mechanism of disease development by contributing to mitochondrial dysfunction. On the one hand, the occurrence of MOMP will lead to the release of apoptosis-related proteins into the cytoplasm, thus causing the opening of the mitochondrial permeability transition pore, which in turn results in the disruption of mitochondrial respiration, matrix swelling, and rupture of the mitochondrial outer membrane, thereby inducing apoptosis. On the other hand, cytochrome C released during MOMP can activate the caspase cascade and rapidly lead to apoptosis. Preclinical studies have demonstrated that TMAO can induce apoptosis in various target cells, such as human aortic vascular smooth muscle cells,¹⁵⁸ pancreatic acinous cells, renal tubular epithelial cells,¹⁵⁹ human aortic endothelial cells,^160,161 and neurons,¹¹⁹ contributing to the development and progression of cardiovascular, renal, neurological, and pancreatic diseases.

Protein kinase RNA-like endoplasmic reticulum kinase (PERK) is a type I transmembrane protein located in the ER, consisting of an ER lumen domain and a cytoplasmic protein kinase domain. PERK-eukaryotic initiation factor 2 alpha (eIF2α), inositol-requiring enzyme 1 alpha (IRE1α)-X-box binding protein 1 (XBP1), and activating transcription factor 6 alpha (ATF6α) serve as key proteinS involved in ERS-related signaling pathways. Several studies have demonstrated that TMAO can stabilize protein metabolism, thereby reducing PERK activity and alleviating ER stress.¹⁶² TMAO may accelerate the progression or rupture of abdominal aortic aneurysm by inducing VSMC apoptosis through the activation of PERK-mediated UPR. Yang et al found that TMAO may promote pancreatic acinous cell apoptosis by inducing sustained oxidative stress and regulating the IRE1α/XBP-1 pathway. Additionally, TMAO may exacerbate the severity of CKD in rats by promoting renal tubular epithelial cell apoptosis through the activation of the ASK1-JNK pathway. Mitochondria are important components of cell metabolism, and the changes in mitochondrial outer membrane permeability are closely related to apoptosis. The accumulation of TMAO is a potential irritant to mitochondrial dysfunction. Studies have found that TMAO can significantly reduce the number of mitochondria, membrane potential, and ATP content of oocytes in polycystic ovary syndrome rat models, thus accelerating the apoptosis of oocytes.¹⁶³ TMAO in circulation seems to never stop at all in its assault on vascular endothelial cells.^164–167 In addition to direct damaging effects, TMAO-stimulated exosomes released by hepatocytes also play a crucial role in vascular inflammation and endothelial dysfunction.¹⁶⁸ Liu et al demonstrated that exosomes released by TMAO-stimulated hepatocytes can promote HAEC apoptosis, inhibit cell migration, and endothelium-dependent vasodilation.¹⁶⁰ It is noteworthy that TMAO’s role is not a one-way street. It also has an anti-apoptotic effect. In 2009, it was reported that TMAO degraded misfolded G98R mutant CRYAA by activating the ubiquitin-proteasome pathway, alleviating ERS and apoptosis of human lens epithelial B3 cells induced by aggregated G98R CrYAA.¹⁶⁹ High concentrations of TMAO can promote the proliferation of human colon cancer cells, but have no significant effect on apoptosis.¹⁷⁰ We speculate that the above differences are related to TMAO dosage and cell type heterogeneity, but there is currently no direct evidence to prove this. Figure 3 illustrates the regulatory pathways of TMAO-induced cell apoptosis in different cell types.

Figure 3 Mechanisms of TMAO inducing cell Apoptosis. The mechanisms through which TMAO induces cell apoptosis via the endoplasmic reticulum (ER) and mitochondrial pathways. TMAO induces ER stress, activating the PERK/ATF-4 and IRE-1α/XBP-1s signaling pathways, leading to apoptosis, inflammation, and vascular injury. Additionally, TMAO-induced apoptosis may result from reduced SERCA activity and disrupted calcium homeostasis. It also promotes mitochondrial reactive oxygen species (mtROS) production and activates NLRP3 inflammasomes, which upregulate IL-1β and further drive apoptosis. TMAO triggers apoptosis by activating the ASK-JNK and SIRT1 pathways. Conversely, TMAO may inhibit apoptosis by mitigating aggregation and ER stress caused by G98R αA-crystallin. Created in BioRender. Ge, P. (2025) https://BioRender.com/t03q624. Abbreviations: TMAO, Trimethylamine-N-oxide; ERS, Endoplasmic reticulum stress; PERK, RNA-dependent protein kinase-like endoplasmic reticulum kinase; eIF2α, Eukaryotic translation initiation factor 2 subunit-α; ATF4, Activating transcription factor 4; CHOP, C/EBP homologous protein; IRE1α, Inositol-requiring enzyme 1α; XBP1, X-box binding protein 1; GRP78, Glucose-regulated protein 78; UPR, Unfolded protein response; Serca2, Sarco/endoplasmic reticulum ATPase type 2; ROS, Reactive oxygen species; NLRP3, Nod-like receptor family pyrin domain containing 3; ASC, Apoptosis-associated speck-like protein; IL-1β, Interleukin 1β; SIRT1, Sirtuin 1; JNK, C-Jun N-terminal kinase; ASK, Apoptosis signal-regulating kinase; MMP, Mitochondrial membrane permeabilization; LncRNA, Long non-coding RNA; NEAT1, Nuclear enriched abundant transcript 1; STAT3, Signal transducer and activator of transcription 3.

TMAO and Autophagy

Autophagy is a cellular self-degradation process, which is an important process for cells to cope with nutritional stress. Under the regulation of autophagy-associated genes (ATG), misfolded or aggregated proteins and damaged organelles are removed and recycled by lysosomes or mitochondria to preserve cellular homeostasis.^171,172 Moderate autophagy is a degradation system that maintains cellular homeostasis,¹⁷³ and cells can achieve renewal and avoid death through basal levels of autophagy. However, during cell growth and development, autophagy acts as a double-edged sword. Insufficient autophagy may lead to the accumulation of toxic proteins and damaged organelles, affecting normal cell function.¹⁷⁴ Conversely, excessive autophagy can result in the erroneous engulfment of essential proteins or normal organelles such as mitochondria.¹⁷⁵ Insufficient and excessive autophagy are detrimental to cell viability and may cause cell apoptosis.¹⁷⁶ In addition to non-selective autophagy induced under starvation conditions, researchers have found that cells can also target the degradation of damaged or redundant organelles, protein aggregates, and invading pathogens for selective autophagy. Selective autophagy includes mitophagy, endoplasmic reticulum autophagy, proteasome autophagy, ribosome autophagy, etc.¹⁷⁷

Recent studies have found that the interactions between gut microbiota and their metabolites, especially TMAO, and autophagy not only regulate intestinal epithelial function and gut homeostasis but also play a role in regulating physiological functions in distant organs. Epithelial cells, ECs, and vascular smooth muscle cells (VSMCs) in multiple organ systems may be the primary targets of TMAO-induced autophagy-dependent cell death. In this process, mTOR and ATG16L1 may serve as vital regulatory factors. Kidney stone is a common urological disorder, which can cause renal insufficiency, atrophy kidney, and empyema. Calcium oxalate is the major component in the formation of kidney stones, causing tubular epithelial injury. A recent research discovered that serum TMAO levels were significantly higher in hyperoxaluria mice. Exogenous TMAO administration exacerbated kidney stones and inflammation in mice. Mechanistically, TMAO may promote autophagy-dependent cell death in human renal proximal tubular epithelial cells by activating the PERK/ROS pathway, thereby aggravating calcium oxalate crystal deposition and kidney injury.¹⁷⁸ Additionally, Yue et al found that TMAO can inhibit autophagy in colonic epithelial cells, thereby exacerbating inflammatory bowel disease. The mechanism may be related to inhibiting ATG16L1-mediated autophagy of normal colon epithelial cells, promoting ROS accumulation and NLRP3 inflammasome assembly.¹⁷⁹

TMAO mediates the onset and progression of CVD through its interaction with autophagy. Intervention in vascular remodeling is an important strategy to prevent and alleviate atherosclerosis.¹⁸⁰ Vascular remodeling refers to a series of structural and functional abnormalities in the vascular wall, including the proliferation, migration, and apoptosis of cells within the vessel wall, and changes in the matrix components. The phenotypic transformation of VSMCs and vascular ECs plays a significant role in vascular remodeling. Early vascular remodeling is an adaptive protective mechanism, and vascular remodeling during decompensation can promote the progression of atherosclerosis and the occurrence of complications. Moderate activation of VSMC autophagy can significantly alleviate the atherosclerosis process induced by statins or cholesterol.^181,182 PIK/AKT/mTOR signaling pathway is involved in the formation of autophagosomes.¹⁸³ Studies have shown that TMAO can inhibit oxidative low-density lipoprotein (ox-LDL)-induced VSMC autophagy by activating the PIK/AKT/mTOR signaling pathway, thereby aggravating atherosclerosis.¹⁸⁴ Besides VSMCs, the autophagy of ECs is also closely associated with the occurrence of atherosclerosis.¹⁸⁵ Once ECs are damaged, various pathophysiological changes, such as thrombosis, inflammation, and excessive proliferation of smooth muscle cells, will be induced.¹⁸⁶ Studies have shown that ox-LDL may exacerbate atherosclerosis by inhibiting HUVEC autophagy through the activation of the PI3K/AKT/mTOR signaling pathway.¹⁸³ Hong et al found that TMAO may promote neointimal hyperplasia after balloon injury by activating Beclin1-mediated autophagy flow abnormality (the conversion of autophagosomes to autolysosomes).¹⁸⁷ It should be emphasized that moderate autophagy is a positive process in combating atherosclerosis. However, excessive stress or overactive autophagy can still lead to autophagy-related cell death in VSMCs, negatively affecting the cells.¹⁸⁸ In conclusion, the complex mechanisms involving TMAO, autophagy, and the crosstalk between ECs and VSMCs in the progression of atherosclerosis still require further research. The regulatory mechanisms of TMAO-induced autophagy in different cells are shown in Figure 4.

Figure 4 Mechanisms of TMAO inducing autophagic cell death. TMAO upregulates Beclin1, a key regulator of autophagy, but this overexpression impairs autophagic flux, disrupting the normal degradation and recycling processes within the cell. Additionally, TMAO promotes autophagy inhibition by activating the PI3K/AKT/mTOR signaling pathway, a well-known negative regulator of autophagy. TMAO also triggers the PERK/ROS pathway, which paradoxically enhances autophagy, suggesting a complex role for TMAO in modulating autophagic processes. Furthermore, TMAO significantly suppresses the expression of ATG16L1, a critical component of autophagosome formation. This suppression prevents the proper multimerization of the ATG16L1-ATG5-ATG12 complex, further inhibiting autophagy and contributing to cellular dysfunction. These mechanisms highlight TMAO’s dual role in enhancing and impairing autophagy, ultimately leading to autophagic cell death. Created in BioRender. Ge, P. (2025) https://BioRender.com/i74e270. Abbreviations: TMAO, Trimethylamine-N-oxide; PI3K, Phosphoinositide 3-kinases; AKT, Protein kinase B; mTOR, Mammalian target of rapamycin; ERS, Endoplasmic reticulum stress; PERK, RNA-dependent protein kinase-like endoplasmic reticulum kinase; eIF2α, Eukaryotic translation initiation factor 2 subunit-α; ATF4, Activating transcription factor 4; LC3, Light chain 3; ROS, Reactive oxygen species; ATG, Autophagy-related gene.

TMAO and Pyroptosis

ECs form the lumen of capillaries, arteries, and veins via tight junctions between cellular and matrix components. Originally considered an inactive resting layer, the endothelium disseminates throughout the circulatory system. ECs are crucial in the physiological regulation of vascular homeostasis. Pathophysiological processes such as vascular permeability, vasomotor tone, vascular growth, inflammation and immunology, coagulation homeostasis, and low-density lipoprotein oxidation are all under the control of the endothelium. ECs keep blood vessels in balance by releasing substances that narrow and widen blood vessels through autocrine, endocrine, and paracrine pathways. This includes the secretion of nitric oxide for vasodilation and endothelin-1 for vasoconstriction, which modulates vascular tension and diameter, directly influencing the balance of oxygen supply to meet tissue metabolic demands.¹⁸⁹ The barrier function constitutes the fundamental role of ECs. The endothelium acts as a selective permeability barrier, controlling the steady movement of blood cell parts and different macromolecules across the vascular wall. This is made easier by the surface glycocalyx, intercellular junctions, and the basement membrane working together.¹⁹⁰ EC damage or death is the main cause of illness. It can cause vasomotor imbalance, inflammatory responses, oxidative stress, and fat buildup, and it is strongly linked to the prevalence of diseases in many body systems.¹⁹¹ Researchers have recognized blood flow shear stress, cytokines, reactive oxygen species, pathogen-associated molecular patterns, and injury-associated molecular patterns as elements that trigger EC inflammation and dysfunction.¹⁹² Ongoing research on gut microbiota indicates that TMAO may play a crucial role in the stimulation of EC inflammation (Especially pyroptosis).

Pyroptosis is a pro-inflammatory programmed cell death that can be induced by exogenous microbial infection and endogenous injury-related signals. The key event in pyroptosis is the cleavage of gasdermin proteins by caspases. The gasdermin proteins share similar N-terminal and C-terminal domain structures, where the C-terminal domain acts as an inhibitory domain, restraining the activity of the N-terminal domain. However, not all gasdermin proteins can be cleaved by caspases. GSDMD and GSDME are the most extensively studied proteins in the gasdermin family. The linker region of GSDMD is cleaved by inflammatory caspases, releasing its active N-terminal domain. This N-terminal domain of GSDMD can be transported to the cell membrane, assembling into pore structures that release IL-1β and IL-18, leading to excessive inflammation and cell death.¹⁹³ Additionally, caspase-3 cleaves GSDME at its linker region, releasing an N-terminal fragment that activates pyroptosis through a mechanism similar to that of the GSDMD N-terminal domain. Pyroptosis was initially discovered in immune cells, but subsequent studies have shown that other cell types, including epithelial and endothelial cells, can also undergo pyroptosis.¹⁹⁴ Pyroptosis of renal parenchymal cells is considered a critical event exacerbating kidney injury. Yi et al found that TMAO aggravated high glucose-induced pyroptosis in human renal proximal tubular epithelial cells.¹⁹⁵ Endothelial cell pyroptosis is a critical triggering event for vascular dysfunction.^196,197 TMAO contributes to the onset and progression of atherosclerosis by inducing ECs pyroptosis. Mitochondria serve as the powerhouse and energy metabolism center of the cell, playing a pivotal role in sustaining cellular homeostasis and proper function. When mitochondrial homeostasis and integrity are compromised, it can lead to excessive production of ROS.¹⁹⁸ ROS is a critical molecule that triggers pyroptosis.¹⁹⁹ Succinate dehydrogenase complex subunit B (SDHB) is a subunit of the succinate dehydrogenase (SDH) family. A recent study showed that TMAO may induce atherosclerosis by upregulating the expression of SDHB in ECs, promoting the destruction of mitochondrial structure and function, and excessive production of ROS, accelerating the process of cell pyroptosis.¹⁶⁴ Acetaldehyde dehydrogenase 2 (ALDH2) is a nuclear-encoded aldehyde oxidase present in the mitochondrial matrix.²⁰⁰ It has been found to play a protective role in various organ injuries.^201–204 ALDH2 can inhibit the activation of NLRP3 inflammasome by reducing the excessive accumulation of mitochondrial ROS (mtROS) and total intracellular ROS, thereby mitigating high glucose-induced pyroptosis in cardiomyocytes. A study has found that TMAO may induce pyroptosis in HUVECs by inhibiting ALDH2 activity, which subsequently activates the ROS/NLRP3/GSDMD pathway.²⁰⁵ Recently, researchers have found a non-classical pathway of pyroptosis mediated by caspase-3 and gasdermin E (GSDME) in human primary cells and tumor cells.^206–208 Although Caspase-3 is a pro-apoptotic caspase, existing evidence shows that after treatment with chemotherapy drugs, GSDME can be cleaved and activated by caspase-3, leading to pyroptosis.^209,210 Another study reported that TMAO may induce endothelial progenitor cell pyroptosis through the Bax/caspase-3/GSDME pathway in vitro, thus participating in endothelial dysfunction. Figure 5 summarizes the regulatory mechanisms of TMAO-induced pyroptosis in various cells.

Figure 5 Mechanisms of TMAO inducing cell pyroptosis. ROS plays a crucial role in activating the NLRP3 inflammasome, which subsequently triggers Caspase-1-dependent pyroptosis. TMAO can directly induces excessive ROS production and indirectly enhances ROS generation by activating the SDHB pathway and inhibiting the ADLH2 pathway, ultimately triggering cell pyroptosis. Furthermore, TMAO may also induce cell pyroptosis through the Bax/caspase-3/GSDME pathway, highlighting the multifaceted roles of TMAO in mediating inflammatory cell death. Created in BioRender. Ge, P. (2025) https://BioRender.com/i00w506. Abbreviations: TMAO, Trimethylamine-N-oxide; ROS, Reactive oxygen species; NLRP3, Nod-like receptor family pyrin domain containing 3; ASC, Apoptosis-associated speck-like protein; GSDMD, Gasdermin D; GSDME, Gasdermin E; NEK7, (NIMA)-related kinase 7.

TMAO and Ferroptosis

Ferroptosis is a form of PCD characterized by iron dependency and lipid peroxidation, which primarily involves three pathways: iron metabolism, lipid metabolism, and antioxidant system. Iron and oxygen are fundamental drivers of metabolism. Iron metabolism disorders trigger the Fenton reaction and induce ROS production, an inevitable byproduct. The sustained accumulation of ROS and the ROS-induced buildup of lipid peroxides further compromises the integrity of the plasma membrane, and ultimately, the cell will die due to ferroptosis. Therefore, Ferroptosis can be considered as a byproduct of cellular metabolism. Currently, evidence directly linking TMAO to ferroptosis is limited. Wang et al found that in diabetic myocardial ischemia-reperfusion injury (DIR) rats, TMAO promoted the ferroptosis of DIR cardiocytes by up-regulating the expression of ALB, PPARG and HMOX1 (ferroptosis related genes).²¹¹ TMAO is closely related to excessive oxidative stress within cells. Luo et al found that TMAO may inhibit the expression of nuclear factor E2-related factor 2 (Nrf2), HO-1, and glutathione peroxidase 4 (GPX4), resulting in the overproduction of ROS and the reduction of SOD activity, thus promoting oxidative stress.²¹² Similarly, Cristina Gonzalez-Correa et al found that in a mouse model of systemic lupus erythematosus, TMAO may induce elevated NADPH oxidase activity and vascular endothelial oxidative stress by inhibiting Nrf2-mediated antioxidant defense mechanism.²¹³ GPX4 is a negative ferroptosis regulator, playing a critical role in converting lipid hydroperoxides to non-toxic lipids.²¹⁴ In CKD, TMAO may induce ferroptosis-related changes in renal tubular epithelial cells, including increased MDA and iron levels, excessive ROS accumulation, reduced GSH, and decreased GPX4 protein levels.²¹⁵ Nrf2 is a key transcription factor that regulates cellular redox homeostasis and inflammatory response. In many chronic diseases, activation of Nrf2 signaling can also protect cells from ferroptosis.²¹⁶ Additionally, levels of the anabolic reductant NADPH are biomarkers of ferroptosis sensitivity.²¹⁷ It can be seen that TMAO may participate in iron death by regulating the expression of ferroptosis-related genes and inducing oxidative stress (Figure 6).

Figure 6 Mechanisms of TMAO inducing cell ferroptosis. TMAO promotes ferroptosis through two major pathways: Modulation of the GSH/GPX4 axis–TMAO inhibits system Xc−, leading to reduced GSH synthesis and impaired GPX4 activity. Dysregulation of iron metabolism–TMAO alters the expression of key iron-related proteins (eg, TRF-1, FPN-1, and FTH-1) and suppresses the NRF2 signaling pathway, exacerbating iron accumulation and lipid peroxidation. Created in BioRender. Ge, P. (2025) https://BioRender.com/upipqo4. Abbreviations: TMAO, Trimethylamine-N-oxide; SLC7A11, solute carrier family 7 member 11; GSH, glutathione; GPX4, Glutathione peroxidase 4; GSSG, Glutathione disulfide; TFR1, Transferrin receptor protein 1; TF, Transferrin; HO-1, Haem oxygenase-1; NRF2, Nuclear factor-erythroid 2-related factor 2; FPN, Ferroportin; FTH1, Ferritin heavy chain 1; ROS, Reactive oxygen species; STEAP3, Six-transmembrane epithelial antigen of the prostate 3; DMT1, Divalent metal transporter 1; NCOA4, nuclear receptor coactivator 4.

In conclusion, within the “gut-organ” axis, TMAO is closely associated with the pathological changes of various chronic diseases by regulating processes such as apoptosis, pyroptosis, abnormal autophagy, and ferroptosis. Although current research has unveiled multiple mechanisms by which TMAO affects cellular function, its specific roles in different tissues and pathological conditions require further investigation.

Diverse Therapeutic Strategies Targeting TMAO

Probiotics, Prebiotics, and Antibiotics

The role of probiotics in reducing TMAO levels has been in the spotlight, shining brightly over the past decade.²¹⁸ Currently, the most common probiotics include Lactobacillus and Bifidobacterium. Lactobacillus and Bifidobacterium exhibit anti-inflammatory, antioxidant, anti-thrombotic, and endothelial protective effects.²¹⁹ They can prevent and cure illnesses, such as atherosclerosis,²²⁰ cardiovascular diseases,²²¹ kidney diseases,²²² and metabolic syndrome.²²³ Existing studies have indicated that probiotics, including Bifidobacterium longum, Lactobacillus plantarum, Lactobacillus amylovorus, Enterobacter aerogenes, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium lactis, Bifidobacterium breve, Bifidobacterium animalis, and Lactobacillus casei, have a certain downregulatory effect on circulating TMAO levels. Table 7 shows the direct or indirect regulatory effects of probiotics on circulating TMAO level.^{222,224–233} Notably, probiotics can be utilized to modify the gut microbiota targeting TMAO, but the impact of different strains on TMAO levels varies. The effect of probiotics on TMAO is strain-dependent, and the specific outcome is determined by the influence of the particular strain on TMAO metabolism, and not all probiotics exhibit this effect. Therefore, further large-scale clinical studies are needed to provide strong evidence that specific probiotic strains can target TMAO metabolism and improve disease outcomes.

Table 7 The Regulatory Effects of Probiotics on Circulating TMAO Level

Prebiotics are a class of non-digestible food ingredients that exerting positive effects on host health.²³⁸ Prebiotic compounds include oligosaccharides, fructooligosaccharides, galactooligosaccharides, beta-glucans, and inulin. Recent research has also revealed that prebiotics may have the potential to regulate TMAO levels, thus improving related diseases^234–237 (Table 7).

Probiotics and prebiotics have shown great potential in targeting TMAO production. Nevertheless, this field continues to encounter numerous challenges, such as the need to identify safe and effective probiotic strains to ensure that they can significantly reduce TMAO levels. Concurrently, it is also necessary to determine the optimal dosage, duration, and frequency of probiotic and prebiotic administration.

Antibiotics are the most potent agents for inhibiting the production of TMAO. In pediatric patients with trimethylaminuria (FMO3 deficiency), the use of metronidazole, amoxicillin, and neomycin can partially inhibit the conversion of choline to TMA, with neomycin being the most effective, followed by amoxicillin, and metronidazole being the weakest effective among them.²³⁹ It is worth noting that using antibiotics to target TMAO for disease treatment belongs to a strategy of “Pyrrhic victory.” Briefly, the use of broad-spectrum antibiotics such as ciprofloxacin and metronidazole almost completely suppress TMAO levels. However, once antibiotics are discontinued, TMAO levels rise again within a month. The combination of vancomycin, neomycin sulfate, metronidazole, ampicillin, and other broad-spectrum antibiotics can alleviate choline-induced atherosclerosis in mice, and the mechanism may be related to the inhibition of choline conversion to TMA, the reduction of TMAO levels, and the inhibition of macrophage foaming. However, long-term use of antibiotics may lead to the emergence of drug-resistant strains and the depletion of beneficial bacteria.¹² Hence, the rational use of antibiotics, combined with probiotics and prebiotics, or the exploitation of new intervention strategies, should be important directions for future research.

Clinical Compounds

Antidiabetic drugs (such as metformin and Empagliflozin), antiplatelet drugs (such as aspirin and clopidogrel), as well as statins (such as atorvastatin and rosuvastatin) have been shown to target TMAO to exert therapeutic effects. Metformin has been shown to reduce TMA and TMAO levels by remodeling gut microbiota.²⁴⁰ Notably, empagliflozin, unexpectedly gives rise to a rapid and significant elevate in TMAO levels in patients with AMI.²⁴¹ Low-dose aspirin mitigates platelet hyperreactivity linked to high TMAO and may decrease TMAO’s dependence on choline.⁵⁷ Clopidogrel resistance is associated with elevated TMAO levels, as shown by Ge et al, who found that dietary choline and TMAO inhibit the metabolic activation of clopidogrel via the NOX/ROS/Nrf2/CES1 pathway.²⁴² Statins not only significantly improve blood lipid levels but also influence gut microbiota and the production of its metabolite, TMAO. A meta-analysis involving 244 subjects showed a significant reduction in plasma TMAO levels after statin treatment.²⁴² Two independent cohort studies found that patients with dyslipidemia experienced a decrease in plasma TMAO levels after initiating statin treatment, but TMAO levels returned to baseline after a four-week discontinuation of the medication.²⁴³ In patients with atherosclerotic cardiovascular disease, rosuvastatin not only significantly reduced blood lipids but also decreased TMAO levels. Additionally, research also observed a significant elevation in the levels of TMAO precursors during rosuvastatin treatment.²⁴⁴ Therefore, a complex regulatory mechanism may underlie the modulation of TMAO metabolism by statins, necessitating further research to clarify the precise pathways involved and their clinical implications (Table 8).

Table 8 Other Drugs Regulating TMAO Levels and Their Mechanisms

Natural Products

Natural products, including alkaloids, polyphenols, and flavonoids, exhibit various biological activities, such as anti-inflammatory, antitumor, and anti-oxidative stress effects, providing highly valuable lead compounds for new drug research and development. Studies indicate that the bioactivity of natural products may be closely related to their biotransformation by the gut microbiota. Additionally, compared to antibiotics and other drugs, natural products not only inhibit harmful bacteria but also enhance the abundance of certain beneficial bacteria, thereby improving disease conditions. In this section, we summarize the research progress on various natural products that regulate gut microbiota composition and structure, reduce TMAO levels, and ultimately improve disease outcomes (Table 8).

Alkaloids are nitrogenous secondary metabolites found in plants, exhibiting diverse biological activity. Berberine may reduce the risk of thrombosis and improve atherosclerosis by modulating the gut microbiota, inhibiting CutC enzyme activity and decreasing TMAO production.^245–247 Additionally, it protects hypertensive mice by remodeling gut microbiota and preventing TMAO-induced endothelial dysfunction.¹⁶¹ Trigonelline may exert anti-atherosclerotic effects by inhibiting Citrobacter freundii and the activity of FMO3 and blocking the choline-TMA-TMAO metabolic pathway.²⁴⁸ Curcumin (Cur) is a natural polyphenolic compound derived from the rhizome of plants such as Curcuma longa L. (turmeric), which has anti-inflammatory, antioxidant, anti-apoptosis, and anti-cancer effects.²⁵⁷ The concentration of Cur in plasma is relatively low, it is significantly higher in the gut.²⁵⁸ Studies have shown that curcumin can reduce TMAO production by regulate the abundance, diversity, and composition of gut microbiota, thereby regulating macrophage polarization and mitigating cadmium-induced atherosclerosis in mice.²⁵⁰ Docosahexaenoic acid is a n-3 long-chain polyunsaturated fatty acid widely found in Marine animals. Shi and his team recombined DHA and Cur, and demonstrated that DHA-acylated Cur diesters significantly improved cisplatin-induced acute kidney injury in mice compared to a single DHA or Cur treatment group. The mechanism is linked to the regulation of gut microbiota and inhibition of LPS and TMAO-induced PI3K/AKT/NF-κB signaling pathways.²⁵¹ Additionally, Resveratrol (RSV), Polyphenol extracts of hickory nut and Mangiferin can regulate the diversity of gut microbiota, promote the metabolism of TMA, decrease TMAO levels, thus alleviates TMAO-induced atherosclerosis.^249,259 Taurine is a natural sulfur-containing amino acid. Numerous preclinical studies have reported the pharmacological activities of taurine, such as anti-inflammatory, anti-oxidative stress, anti-depression, hepatoprotective, and anti-tumor. Supplementing with taurine can help prevent aging, mitochondrial diseases, metabolic syndrome, cancer, cardiovascular diseases, and neurological disorders. Yang et al showed that taurine may alleviate atherosclerosis by inhibiting TMAO. Specifically, taurine significantly reduced TMAO-induced atherosclerosis lesions and chronic inflammation in mice, and the mechanism may be related to increasing the ratio of conjugated bile acids to unconjugated bile acids in the serum.²⁵³

Satheesh et al reported that the effects of a moderate blueberry (rather than strawberry) diet on TMAO were not mediated by metabolic changes (TMAO levels in the circulation can be reduced without changing choline or TMA). The sequencing results showed that blueberry significantly increased the abundance of bacterial taxa negatively correlated with circulating TMA/TMAO, suggesting that it may affect TMAO synthesis by affecting the composition of gut microbiota.²⁵² Further analysis of freeze-dried blueberries and strawberries revealed a significant increase in phenolic compounds, particularly chlorogenic acid, which may be a critical factor in blueberries’ inhibition of TMAO production. Asparagus contains various phytochemicals, including polyphenols, saponins, asparagusic acid, and alkaloids, which have anti-inflammatory, antibacterial, antioxidant, neuroprotective, anti-anxiety and anti-asthmatic properties. In vitro experiments have demonstrated that asparagus extract can alleviate TMAO-induced endothelial dysfunction, and the mechanism may be related to improving the TMAO-induced decrease in EC proliferation and regulating the molecular circadian clock controlled by NEAT1 and MAPK signaling pathways.²⁵⁴ Other natural plants or plant-derived chemical compounds have likewise been demonstrated to reduce plasma TMAO levels and alleviate TMAO-induced tissue and organ damage. Commelina communis L., a plant widely distributed in tropical regions, is used in its dried aerial part known as Yazhicao (Commelina communis L., YZC). YZC has been shown to improve metabolic function, reshape and regulate gut microbiota, reduce TMAO synthesis, and inhibit NLRP3 inflammasome activation, thereby ameliorating LPS-induced lung inflammation and injury in septic ALI mice.²⁸ In addition, Decaisnea insignis seed oil mitigated liver dysfunction in L-carnitine-fed mice, potentially by modulating the composition of the gut microbiota, reducing the abundance of Firmicutes, Proteobacteria, and Erysipelotrichaceae, as well as the proportions of Lactobacillus and Akkermansia, thereby preventing TMAO generation.²⁵⁵ Similarly, Sandalwood seed oil has been shown to improve insulin resistance in rats induced by a high fat/high sugar diet, with mechanisms related to changes in gut microbiota composition and the production of microbial metabolites (including TMAO).²⁵⁶

Conclusion and Future Directions

TMAO may drive the cross-talk between inflammatory response and oxidative stress by mediating cell “death” (including apoptosis, pyroptosis, autophagy, and ferroptosis, etc) and then induce pathological processes such as foam cell activation, massive secretion of cytokines and adhesion molecules, overaccumulation of ROS, enhanced platelet reactivity and abnormal vascular tone regulation. It is deeply involved in the pathogenesis and progression of diseases across the “gut-organ” axis pulmonary diseases, including cardiovascular diseases (such as atherosclerosis, heart failure, and hypertension), renal diseases, neurodegenerative disorders, and metabolic diseases, including diabetes. As a risk factor for various chronic diseases, the detection of TMAO and related metabolites can provide a potential basis for the prevention, diagnosis, and treatment of cardiovascular diseases, kidney diseases, neurological diseases, and metabolic diseases. We recommend incorporating TMAO and its related metabolites into routine health examinations. Additionally, this paper also summarized a variety of measures to intervene in related diseases by targeting the reduction of TMAO levels, including probiotics, prebiotics, antibiotics, anti-inflammatory drugs, antiplatelet agents, hypoglycemic agents, lipid-lowering drugs, Chinese herbal monomers, herbal extracts, and formulas. The above strategies may lower the circulating TMAO levels by targeting gut microbiota, inhibiting the activity of TMA/TMAO production and conversion-related enzymes, and intervening in downstream effects, thereby providing a theoretical basis for the clinical treatment of TMAO-related diseases.

Notably, there are still many limitations in current research on TMAO. On the one hand, TMAO levels are dynamically changing in human circulation (this physiological concentration range is also controversial) and are strictly regulated by factors such as age, diet, gut microbiota composition, hepatic enzyme activity, as well as liver and kidney function. In the early diagnosis and prognosis evaluation of various human diseases, the specificity and sensitivity of TMAO have constantly been challenged. Moreover, different tissues and organs have varying tolerance thresholds for TMAO, and further clarification is still required on whether the dynamic fluctuations of TMAO exert beneficial roles or detrimental effects in human organs. Second, although numerous observational studies have shown causal associations between TMAO and cardiovascular, kidney, neurological, and metabolic diseases, due to the inherent limitations of statistical methods (bias due to confounding factors and reverse causality), and the causal relationship between TMAO and human diseases still requires validation through multicenter, multi-ethnic randomized controlled trials. Finally, the pathological process of endothelial cell dysfunction induced by TMAO may be regulated by various forms of cell death, yet the specific mechanism of this process is still unclear. To provide a more thorough theoretical foundation for treating disorders associated to TMAO, larger-scale clinical trials are needed to validate TMAO’s potential as a biomarker for various diseases along the “gut-organ” axis. In-depth mechanistic studies are also required to fully understand the interactions between TMAO, cell death, and the “gut-organ” axis.