Endocrine organs of cardiovascular diseases: Gut microbiota

Abstract Gut microbiota (GM) is a collection of bacteria, fungi, archaea, viruses and protozoa, etc. They inhabit human intestines and play an essential role in human health and disease. Close information exchange between the intestinal microbes and the host performs a vital role in digestion, immune defence, nervous system regulation, especially metabolism, maintaining a delicate balance between itself and the human host. Studies have shown that the composition of GM and its metabolites are firmly related to the occurrence of various diseases. More and more researchers have demonstrated that the intestinal microbiota is a virtual ‘organ’ with endocrine function and the bioactive metabolites produced by it can affect the physiological role of the host. With deepening researches in recent years, clinical data indicated that the GM has a significant effect on the occurrence and development of cardiovascular diseases (CVD). This article systematically elaborated the relationship between metabolites of GM and its effects, the relationship between intestinal dysbacteriosis and cardiovascular risk factors, coronary heart disease, myocardial infarction, heart failure and hypertension and the possible pathogenic mechanisms. Regulating the GM is supposed to be a potential new therapeutic target for CVD.

TMAO can affect lipid metabolism causing accumulation of cholesterol in cells and it can also have a direct impact on platelet function, promote an inflammatory response and so on. The effect of the TMAO will accelerate the development of atherosclerosis and the level of plasma TMAO and its associated metabolites are directly proportional to the risk of cardiovascular disease (CVD), which will be pointed out in the following sections. [12][13][14][15][16]

| Bile acids
Bile acids are amphipathic molecules synthesised from cholesterol in the liver. This process is an important way to eliminate cholesterol from the human body. After eating, BAs stored in the gallbladder are secreted into the intestine to assist the emulsification of dietary fats and assist the intestinal absorption of lipid nutrients and lipid-soluble vitamins. 17,18 BA itself regulates metabolism, mainly through two receptors, namely the nuclear farnesyl X receptor (FXR) and the G protein-coupled receptor TGR5. 17,19 BAs can control intestinal bacterial overgrowth, in turn, intestinal bacteria can regulate host metabolism by metabolism BAs. 20 Recent studies have shown that BA has a pleiotropic and hormonal activity that regulates lipid and glucose metabolism, controls inflammation and fibrosis, maintains vascular integrity, restores the intestinal barrier and controls ageing and circadian rhythms. 19 Further studies have confirmed that FXR activation can reduce intestinal ischemiareperfusion injury and preserve internal structure and permeability.
Also, FXR agonists decrease the release of pro-inflammatory cytokines, reduce autophagy inhibition and modulate obesity and related metabolic phenotypes. 21,22

| Short-chain fatty acids
Short-chain fatty acids primarily originate from the fermentation of dietary fiber in the gut and they are subsequently absorbed into the bloodstream of the host, in which they interact with host proteins, thereby affecting host physiology. 23 Acetate, propionate and butyrate (roughly 60:25:15 in the colon) account for 80% of the SCFA produced by intestinal microflora. 24 In addition to their important role as fuel for colonic epithelial cells, SCFAs also modulate different cell signal transduction processes via G protein-coupled receptors GPR43 and GPR41. 23,25 Recent evidence has suggested that SCFAs also play an important role in glucose and lipid metabolism and butyrate has been demonstrated to decrease LPS translocation, inhibit macrophage activation, thus reducing the production of inflammatory factors and reactive oxygen species (ROS) production. [26][27][28][29] SCFAs can also inhibit the inflammatory reaction by reducing the migration and proliferation of immune cells, reducing various cytokines and inducing apoptosis. Therefore, SCFA is considered to have an anti-inflammatory effect. 30

| Protocatechuic acid
Protocatechuic acid is chemically known as 3,4-dihydroxybenzoic acid and is one of the main metabolites of complex polyphenols such as anthocyanins and procyanidins that are normally found in high concentrations in vegetables and fruit, such as onions, plums, gooseberries and grapes. 31,32 More and more evidence support that PCA can play diverse biological effects by acting on different molecular targets, such as antioxidant, anti-inflammatory and anti-hyperglycemic and neuroprotective activity. 32 PCA has been implicated in the progression of atherosclerosis. Some scholars concluded that PCA could inhibit OA-induced vascular smooth muscle cells proliferation, improve the endogenous antioxidant capacity of macrophages and promote cell proliferation and cell survival via IGF-I signalling. [33][34][35] Besides, studies have demonstrated that PCA can improve cardiac function and cardiac autonomic balance, prevent cardiac mitochondrial dysfunction and increase anti-apoptotic protein. 36 In a recent experimental study on Type 2 diabetes mouse models it was found that, PCA could significantly stimulate glucose metabolism in skeletal muscle, regulate glycemic and lipid status, reduce the secretion of pro-inflammatory cytokines. 37

| Phenylacetylglutamine, P-cresyl sulfate, IS, enterolactone and H 2 S
Proteins in the intestine are metabolized by harmful microorganisms (such as Clostridium perfringens) to produce harmful amines, phenols and ammonia, causing human diseases and discomfort, which have a great impact on the human cardiovascular system. 38,39 Studies have found that phenylacetylglutamine and p-cresyl sulphateare lower in patients with non-coronary heart disease and p-cresyl sulphate is a significant independent predictor of carotid plaque burden. 40 Escherichia coli-based intestinal flora produce a large amount of quinone by metabolizing the tryptophan in food and further oxidize it to indophenol under the action of microbial oxidase. Similar to TMAO, indophenols in the liver are sulphonatedto form IS. 41 Excessive accumulation of IS may affect oxidative stress in cardiomyocytes, induce myocardial cell damage and cause vascular endothelial cell damage to inhibit self-repair and increase vascular access thrombosis. 42 Enterolactone is produced primarily by intestinal digestion of fibre-rich foods. In a meta-analysis, it was found that the higher risk of acute coronary accidents was lower in patients with higher serum enterolactone and therefore the lower enterolactone may be another risk factor for coronary heart disease (CHD). 43 In addition, some sulphate-reducing bacteria in the human intestinal tract produce a large amount of H 2 S using sulphate as a substrate. Studies indicate that H 2 S acts as a gas transmitter in the human body. H 2 S is an essential mediator of various physiological processes in the human body including cell protection, vasodilation, angiogenesis, blood pressure regulation and heart rate reduction. It plays a major role in CVD ( Figure 1). 44,45

| INTE S TINAL DYS BAC TERI OS IS AND C ARD I OVA SCUL AR RIS K FAC TOR S
Hyperlipidaemia, obesity, diabetes and atherosclerosis are important risk factors for CVD. Plenty of evidence indicates that the GM is closely related to these four risk factors.

| Hyperlipidaemia
A recent survey found that the GM composition can explain 6.0% of the variation in triglycerides and 4.0% of that in HDL-C and 4.5% of that in BMI, independent of age, sex and genetics at the human population level. 46 Studies have also found that individuals with low microbial richness have increased fasting triglycerides and decreased HDL-C. Reverse cholesterol transport (RCT) is a key pathway involving the return of excess cholesterol from peripheral tissues to the liver to excretion of bile and eventually to faeces. 47 In a recent article, a researcher found that TMAO reduced the expression of cholesterol 7-alpha-hydroxylase 1 (CYP7a1), the major BA synthetic enzyme in the catabolism of cholesterol, whose reaction is the ratelimiting step. This finding may in part explain the impact of TMAO on RCT and upregulation of CYP7a1 has been reported to lead to increasing RCT and reduced atherosclerosis in susceptible mice. 48,49 Recent research shows that BA-mediated activation of FXR inhibits the activity of CYP7a1 leading to RCT disorders and elevated cholesterol, but reduced triglycerides. 17,50 However, there are also studies showing that FXR knockout mice exhibit high-density lipoprotein metabolism and RCT leading to elevated cholesterol. 51 Recent studies have found that TMAO can cause cholesterol accumulation in cells by increasing the expression of the scavenger receptors, CD36 and SR-A1 and the formation of foam cells. 16,30 In conclusion, intestinal flora metabolites are closely related to lipid metabolism, but its mechanism of action needs further investigation. Animal study showed 57 that high-fat-fed mice had increased LPS Gram-negative bacteria in the intestine, elevated plasma LPS levels, causing metabolic endotoxemia and obesity and continued subcutaneous injection of LPS in mice also induced the above reaction. CD14

| Obesity and type 2 diabetes mellitus
gene knockdown can reduce the inflammatory state of mice caused by LPS; TLR4 gene knockout can also prevent obesity and insulin resistance caused by high-fat diet, thereby reducing obesity. 58,59 Obesity is often a leading factor associated with type 2 diabetes . 62 Related study 63 shows that FGF-19 can improve glucose tolerance, reduce weight gain and increase the metabolic rate. So BA plays a major role in the occurrence and development of diabetes mellitus.

| Atherosclerosis
Hyperlipidaemia is an independent risk factor for atherosclerosis. In the above discussion, we have discussed the effects of GM on lipid metabolism, which we will not state it here. In addition, studies have shown that TMAO can also directly affect platelet function and increase thrombosis risk. When platelets are directly exposed to high levels of TMAO, the intracellular storage of Ca2+ is released, which enhances platelet activation via a variety of agonists (thrombin, ADP, or collagen). 15 There are also studies showing that TMAO enhanced the platelet reactivity and thrombosis risk and promoted vascular inflammation by activating the NLRP3 inflammasome. 16 Using pyrosequencing to analyse the microbial species of atherosclerotic plaques and intestinal microflora in the same atherosclerotic patients, the results show that several bacteria exist simultaneously, suggesting that bacteria found in the plaque may be derived from intestinal flora. 64 In another study using shotgun sequencing method it was found that atherosclerosis patients were rich in Collinsell, and healthy people were rich in Roseburia and Eubacterium. 65 The above results suggest that the GM is directly related to the occurrence of atherosclerosis.
In the above section, we have already mentioned that BA-mediated FXR and SCFA, PCA have anti-inflammatory effects and can regulate glucose metabolism; so the intestinal flora can also inhibit the development of atherosclerosis. However, the core mechanism that causes atherosclerosis remains to be further clarified.

| Coronary heart disease
Coronary heart disease is one of the most common CVD in clinical practice, which is extremely harmful to human health. Some scholars have found that plasma TMAO levels are elevated in patients with coronary heart disease and the structure of intestinal flora is also changed and manifested by the decrease of Lactobacillus and Bacillus. 66 A research showed that the structural changes in the GM could be used as a diagnostic marker for coronary heart disease. 67 Using a high-choline diet to feed ApoE-/-C57BL mice, results display that higher the serum TMAO level in mice, larger was the area of the sizeable atherosclerotic plaque . 68  disease. TMAO can also be used to assess the degree of plaque burden of coronary heart disease. 70 In short, the current research on TMAO is thorough and the level of TMAO is expected to become the standard for diagnosis and risk assessment of patients with coronary heart disease. patients, the researchers found that after STEMI, the abundance and diversity of blood flora were increased and more than 12% of the blood bacteria were from the intestinal flora. This indicates that a significant increase in intestinal flora in plasma is associated with systemic inflammation and cardiovascular adverse events after STEMI. 71 Animal studies showed that AMI rats were parallel to intestinal barrier injury and intestinal flora richness was significantly higher than the sham group. Therefore, the intervention of intestinal flora structure to improve the clinical prognosis of AMI may be a new method of AMI treatment. 72 Study has shown that probiotics can reduce cardiac hypertrophy in MI rats. 73 Further study also showed that antibiotics could improve the area of MI in AMI mice, which may be related to the regulation of intestinal flora. However,

| Heart failure
As shown in recent studies, changes in intestinal microecology can directly damage cardiac muscle cells and cause cardiac dysfunction. In the mouse model of dietary intervention, it was found that elevated serum TMAO levels in mice resulted in cardiac injury and fibrosis and increased TMAO levels were prone to heart failure (HF). 76 There are also studies which found that the levels of TMAO, choline and betaine were involved in left ventricular diastolic dysfunction. 77 Also, studies found that, higher the TMAO level in patients with HF, higher the mortality rate in 5 years. Compared with the risk factors for HF, heart and kidney index and systemic inflammatory markers, TMAO can better evaluate the prognostic value. 78 After a 1-year follow-up of 972 patients with acute HF, investigators found that serum TMAO levels could predict adverse prognostic events, whereas TMAO+NT-proBNP could predict a higher value. 79 A clinically controlled cohort study showed that serum TMAO levels were elevated in patients with chronic HF, and TMAO levels were associated with cardiac function and survival. 80 Besides, chronic HF patients had significantly more pathogenic bacteria and Candida than healthy controls, which is a result concluded by multiple researchers. The inflammatory, intestinal permeability and right atrial pressure of these chronic HF patients were significantly increased and these were signals of venous congestion. Also, these correlations were stronger in patients with moderate to severe HF (NYHA heart function III-IV) than those with mild HF (NYHA cardiac function I-II). 81 Therefore, improving intestinal flora and lowering TMAO level are both expected to improve the prognosis of patients with HF.

| Hypertension
Recent findings show that SCFA bind the G protein-coupled receptors GPR41 and GPR43 and olfactory receptors olfO78 in the kidney, heart, sympathetic ganglia and blood vessels to modulate blood pressure. 82,83 SCFA can induce the release of renin from the afferent arteriole and increase the blood pressure, which is mediated by Olfo78. This, in turn can be counteracted by the vasodilator action of GPR43. 82,84 GPR41 can increases energy expenditure by stimulating the sympathetic nervous system, but this could also lead to an increase in blood pressure. 85 Other researchers have found that TMAO can increase blood pressure and hydrogen sulphate can directly act on blood vessels to modulate blood pressure. 86 (Table 1). 92

| THE ROLE OF G UT MI CROB I OTA IN C ARDIOVA SCUL AR DIS E A S E
Gut microbiota is an essential micro-ecosystem in the human body and even some scholars believe it is the 'virtual endocrine organ' of the human body. Summarizing the above and combining current (c) intestinal flora disorder promotes oxidative stress in the body and aggravates the development of CVD. As the first two mechanisms have already been discussed in the previous discussion. In next section, the discussion would be concentrating on oxidative stress.
Gut microbiota is also involved in the metabolism of purine and uric acid (UA). For example, xanthine dehydrogenase, the key enzyme responsible for the oxidative metabolism of purines is generated by the secretion of Escherichia coli in intestinal bacteria. 93 Therefore, the decomposing activity of GM on UA is positively related to the content of Escherichia coli. The study found that the number of E. coli in patients with coronary heart disease increased, blood UA levels increased significantly, whereas high concentrations of UA showed prooxidation. Increased blood UA levels can lead to high level nitrite/nitrate in the blood, decreased bioavailability of NO and oxidative stress. 68,94 There are also studies showing high UA decreased cardiomyocyte viability and increased ROS production in cardiomyocytes. 95 In addition, carotenoids, as antioxidants have an anti-angina effect. The carotenoid genes were increased in healthy patients compared with arteriosclerosis patients. 65 The

| THE TARG E T OF FUTURE TRE ATMENT
At present, the improvement or reversal of intestinal microflora has become a hot spot for CVD, which includes the faecal microbiota transplantation, dietary regulation, probiotics, prebiotics, antibiotic intervention and so on.

| Dietary regulation
Dietary regulation has been proven to be an effective strategy to   They has a beneficial effect on the host by selectively stimulating the growth and activity of bacteria. A recent study has

| Probiotics and prebiotics
shown that probiotics can improve metabolic syndrome and infiltration of small intestinal cells. 104 However, probiotics remain at risk of infection and their safety needs further study.

| Antibiotics
Antibiotic therapy has greatly disrupted the short-term and long-

| TMA inhibitors and other intervention methods
Research shows that choline structural analogues (3,3-dimethyl-1-butanol) can block the choline metabolic pathway, reduce TMA production and play a role in the prevention and treatment of CVD. 112 However, studies have shown that drugs that inhibit production of TMA-FMOs can lead to acute hepatitis and fish odour syndrome. 68 Another recent study shows that resveratrol can stimulate the growth of beneficial bacteria in the intestinal tract through the reconstitution of intestinal microflora, thus decreasing the production of TMAO. 113 In addition, for colectomy, a study showed that patients with an average age of 45 years of age survived 1000 days after colectomy and compared with other five groups of non-gastrointestinal surgery patients, colectomy did not reduce the risk of CVD and only reduced the risk of hypertension. 114

| CON CLUS ION
Intestinal microecology is the largest microorganism system repre- the internal mechanism is not fully understood. Therefore, the primary and clinical study of intestinal microecology and CVD needs to be further carried out, especially for guiding treatment of coronary heart disease and HF.

ACK N OWLED G EM ENT
This work was supported by the National Natural Science Foundation of China (no. 81603559).

CO N FLI C T S O F I NTE R E S T
The authors confirm that there are no conflicts of interest.