Health Benefits and Side Effects of Short-Chain Fatty Acids

The gut microbiota and their metabolites could play an important role in health and diseases of human beings. Short-chain fatty acids (SCFAs) are mainly produced by gut microbiome fermentation of dietary fiber and could also be produced by bacteria of the skin and vagina. Acetate, propionate, and butyrate are three major SCFAs, and their bioactivities have been widely studied. The SCFAs have many health benefits, such as anti-inflammatory, immunoregulatory, anti-obesity, anti-diabetes, anticancer, cardiovascular protective, hepatoprotective, and neuroprotective activities. This paper summarizes health benefits and side effects of SCFAs with a special attention paid to the mechanisms of action. This paper provides better support for people eating dietary fiber as well as ways for dietary fiber to be developed into functional food to prevent diseases.


Introduction
Short-chain fatty acids (SCFAs) are mainly metabolites of dietary fiber and protein in gut [1,2], and they could also be produced by bacteria of the skin and vagina [3,4]. Dietary fibers are main sources of SCFAs, and they can be classified as soluble (such as pectins and inulin) and insoluble dietary fibers (such as various forms of resistant starches) [5]. The SCFAs have less than six carbon atom numbers and mainly include acetate, propionate, butyrate, pentanoate, malonate, and so on [6]. Among them, acetate, propionate, and butyrate are three major SCFAs, which account for 90% of SCFAs produced by gut microbiota [6]. The chemical structures of three SCFAs are shown in Figure 1. The SCFAs are produced primarily in the cecum and proximal colon, and their concentrations decline from proximal to the distal colon as the substrates used for fermentation are exhausted gradually [7]. Several factors affected the production of SCFAs, such as substrate source, composition of gut microbes, colonic pH value, gut transit time, and site of substrate fermentation [8,9]. For example, a study showed that resistant starch 5 (amylose-lipid complexes) produced more butyric acid than resistant starch 2 (nongelatinized native starch granule) and resistant starch 3 (retrograded starch) [10]. Another study indicated that low colonic pH value promoted butyrate production and increased populations of butyrate-producing bacteria [11]. In addition, host physiology, such as intestinal environment, microbe-host interaction, and even social stress, could affect SCFA production [12]. For instance, a study found that stress exposure reduced the levels of colonic SCFAs in mice through modulating the gut microbiota, such as decreasing the SCFAs producing genera Anaerostipes, Butyricicoccus, Coprococcus, and Parabacteroides, as well as increasing the abundance of Odoribacter [13]. Furthermore, more than 90% of SCFAs are absorbed from the intestinal cavity and metabolized In recent years, many studies have proven that intestinal microbiota and their metabolites play a vital role in human health [16,17]. The gut microbiota-derived SCFAs have shown a variety of biological effects on the host, such as anti-inflammatory, immunoregulatory, anti-obesity, anti-diabetes, anticancer, cardiovascular protective, hepatoprotective, and neuroprotective effects [6,18,19]. The role of SCFAs in human health and diseases has become a research hotspot. This narrative review collects the literature from the Web of Science Core Collection and PubMed databases in the past five years based on keywords in the title and abstract, including short-chain fatty acids, SCFAs, acetic acid, acetate, propionic acid, propionate, butyric acid, butyrate, isobutyric acid, isobutyrate, valeric acid, valerate, hexanoic acid, and hexanoate, and summarizes the bioactivities of SCFAs with special attention paid to their mechanisms of action.

The Bioactivities of SCFAs
The health benefits of SCFAs have been widely studied, and the mechanisms of action have been explored (Tables 1-3 and Figure 2), which are summarized and discussed in detail below. In each section, the results of epidemiological studies are first described, then the results of preclinical studies are discussed, and the results of clinical studies (if any) are finally elaborated. In recent years, many studies have proven that intestinal microbiota and their metabolites play a vital role in human health [16,17]. The gut microbiota-derived SCFAs have shown a variety of biological effects on the host, such as anti-inflammatory, immunoregulatory, anti-obesity, anti-diabetes, anticancer, cardiovascular protective, hepatoprotective, and neuroprotective effects [6,18,19]. The role of SCFAs in human health and diseases has become a research hotspot. This narrative review collects the literature from the Web of Science Core Collection and PubMed databases in the past five years based on keywords in the title and abstract, including short-chain fatty acids, SCFAs, acetic acid, acetate, propionic acid, propionate, butyric acid, butyrate, isobutyric acid, isobutyrate, valeric acid, valerate, hexanoic acid, and hexanoate, and summarizes the bioactivities of SCFAs with special attention paid to their mechanisms of action.

The Bioactivities of SCFAs
The health benefits of SCFAs have been widely studied, and the mechanisms of action have been explored (Tables 1-3 and Figure 2), which are summarized and discussed in detail below. In each section, the results of epidemiological studies are first described, then the results of preclinical studies are discussed, and the results of clinical studies (if any) are finally elaborated.

Anti-Inflammatory Activity
Inflammation is related to the occurrence and development of many diseases. In recent years, many studies demonstrated that SCFAs could reduce the production of inflammatory factors through several signaling pathways. The SCFAs attenuated the inflammatory response by decreasing the production of pro-inflammatory mediators and enhancing the production of anti-inflammatory mediators. A study indicated that propionate and butyrate alleviated the inflammation in cells by inhibiting the expressions of interleukin (IL)-6, reactive oxygen species (ROS), as well as enhancing the expressions of IL-10 [20]. Besides, it was reported that butyrate attenuated the inflammation induced by lipopolysaccharide (LPS) via up-regulating IL-10 in septic shock [21]. Additionally, the evidence showed that acetate effectively resolved neutrophilic inflammation via inducing caspase-dependent apoptosis of neutrophils, decreasing the activity of nuclear factor-kappa B (NF-κB) and enhancing the production of anti-inflammatory mediators, such as IL-10, transforming growth factor-β (TGF-β), and annexin A1 [22]. In the LPS-treated neonatal mice model, pulmonary inflammation and oxidative stress were reduced by sodium propionate; in the LPS-treated human pulmonary microvascular endothelial cells (HPMECs) model, sodium propionate not only accelerated Nrf2 nuclear translocation, protected cells, and promoted angiogenesis, but also reduced inflammatory response via the NF-κB pathway [23]. Moreover, one in vivo study showed that propionate interfered with the production and migration of inflammatory mediators [24]. Furthermore, some SCFAs had complex bidirectional regulatory properties. It was found that the level of acetate negatively correlated with the pro-inflammatory biomarker interferon-γ (IFN-γ), while the levels of butyrate and valerate positively correlated with IFN-γ and tumor necrosis factor-α (TNF-α) [25]. Health benefits and the related mechanisms of SCFAs. ↓ represents decrease, and ↑ represents increase. Abbreviation: COX-2, cyclooxygenase-2; GPR41, G-protein-coupled receptor 41; IL, interleukin; NF-κB, nuclear factor-kappa B; SITR, small intestinal transit rate; WCF, water content of feces.

Anti-Inflammatory Activity
Inflammation is related to the occurrence and development of many diseases. In recent years, many studies demonstrated that SCFAs could reduce the production of inflammatory factors through several signaling pathways. The SCFAs attenuated the inflammatory response by decreasing the production of pro-inflammatory mediators and enhancing the production of anti-inflammatory mediators. A study indicated that propionate and butyrate alleviated the inflammation in cells by inhibiting the expressions of interleukin (IL)-6, reactive oxygen species (ROS), as well as enhancing the expressions of IL-10 [20]. Besides, it was reported that butyrate attenuated the inflammation induced by lipopolysaccharide (LPS) via up-regulating IL-10 in septic shock [21]. Additionally, the evidence showed that acetate effectively resolved neutrophilic inflammation via inducing caspasedependent apoptosis of neutrophils, decreasing the activity of nuclear factor-kappa B (NF-κB) and enhancing the production of anti-inflammatory mediators, such as IL-10, transforming growth factor-β (TGF-β), and annexin A1 [22]. In the LPS-treated neonatal mice model, pulmonary inflammation and oxidative stress were reduced by sodium propionate; in the LPS-treated human pulmonary microvascular endothelial cells (HPMECs) model, sodium propionate not only accelerated Nrf2 nuclear translocation, protected cells, and promoted angiogenesis, but also reduced inflammatory response via the NF-κB path- Health benefits and the related mechanisms of SCFAs. ↓ represents decrease, and ↑ represents increase. Abbreviation: COX-2, cyclooxygenase-2; GPR41, G-protein-coupled receptor 41; IL, interleukin; NF-κB, nuclear factor-kappa B; SITR, small intestinal transit rate; WCF, water content of feces.
In summary, SCFAs exhibit good anti-inflammatory activity, and the main mechanisms of action include inhibiting the production of pro-inflammatory mediators, such as IL-6 and TNF-α, as well as enhancing the production of anti-inflammatory mediators, such as IL-10, TGF-β, and annexin A1. In the future, more in vivo studies are needed to prove the bidirectional regulation of SCFAs on inflammatory factors and discover its mechanisms.

Immunoregulatory Activity
More and more studies have shown that gut microbiota play a vital role in the host's immune system, and the effects are mainly carried out by producing metabolites, such as SCFAs [26]. The SCFAs could regulate the function of innate immune cells to participate in the immune system, such as dendritic cells. In an ovalbumin-induced allergic mice model, dietary supplement with SCFAs could prevent the exacerbation of lung inflammation induced by vancomycin, via attenuating dendritic cells migration and activation [27]. The SCFAs could also regulate the differentiation and function of T and B cells, and then mediate antigen-specific adaptive immunity. For example, SCFAs induced the production of IL-22 by CD4 + T cells through binding G-protein receptor 41 (GPR41) and inhibiting histone deacetylase (HDAC) [28]. Besides, butyrate promoted the production of IL-22 via increasing hypoxia-inducible factor (HIF) 1α binding to the Il22 promoter through histone modification [28]. For B cells, SCFAs promoted B cell differentiation by increasing acetyl-coenzyme A (acetyl-CoA), oxidative phosphorylation, glycolysis, and fatty acid synthesis [26]. Moreover, as efficient HDAC inhibitors, SCFAs could stimulate B cell differentiation via boosting the expression of B cell differentiation-related genes, such as Xbp-1, Aicda, and Prdm1 [26]. The SCFAs could also decrease circulating immunoglobulin (Ig) E level [27]. Furthermore, SCFAs could promote murine and human B10 cell generation via inhibiting HDAC [29].
In short, SCFAs participate in the function of immune system via reducing the migration and activation of dendritic cells to relieve allergy, as well as promoting T and B cells' differentiation to regulate antigen-specific adaptive immunity. The immunoregulatory action of SCFAs are mainly achieved by directly binding SCFA-specific G-protein-coupled receptor (such as GPR41) on the cell surface and entering cells to regulate cell metabolism and inhibit HDAC.

Anti-Obesity Activity
Obesity is a metabolic disorder and mainly caused by an imbalance between energy intake and expenditure [19,30]. Recently, many studies indicated that SCFAs played a vital role in the management of obesity. For example, one epidemiological study suggested that human milk SCFAs exerted a protective effect against excess weight gain in infants [31].
SCFAs play an important part in obesity and energy metabolism by regulating the appetite. For example, a study showed that the mechanism of SCFAs suppressing food intake was related to vagal afferent, and the efficacy was butyrate > propionate > acetate. Moreover, butyrate exerted an anorexigenic effect through activating vagal afferent neurons and their projection sites, such as nucleus tractus solitaries (NTS) neurons, and directly increasing Ca 2+ concentration in nodose ganglion neurons (NGNs) [32]. Besides, serum glucagon-like peptide 1 (GLP-1), peptide YY (PYY), and leptin participated in the shortterm signal of satiety transferring to the appetite center of the brain. A 28-day experiment of pigs showed that acetate and propionate increased the concentrations of serum GLP-1, PYY, and leptin, and then reduced the appetite [33]. In addition, SCFAs reduced appetite and fat accumulation via modulating relevant genes and hormones, such as mitochondrial transcription factor A, tumor necrosis factor receptor superfamily member 9, cytochrome-C oxidase IV, and free fatty acid receptor 2 [34].
The SCFAs could also exert an anti-obesity effect via other different pathways. It was reported that SCFAs could restrain lipogenesis [35]. The SCFAs mediated the suppression of de novo lipogenesis in male rats by suppressing hepatic acetyl-CoA carboxylase-1(Acc1) expression [35]. Furthermore, propionate repressed the triglyceride (TG) accumulation via modulating the expression levels of PPARα-responsive genes, such as carnitine palmitoyl transferase II (CPTII) and trifunctional protein alpha (TFPα) [36]. Another study suggested that SCFAs could protect against high-fat diet-induced obesity and suppress lipid synthesis [37]. Besides, propionate reduced obesity-associated metabolic disturbances via decreasing the hepatic TG and increasing odd-chain fatty acids (OCFAs) formation [38]. Additionally, acetate could decrease lipid accumulation, inhibit white adipose tissue lipolysis and induce browning of white adipose tissue, which could reduce the body adiposity by increasing thermogenesis [39]. Furthermore, a randomized, placebo-controlled, single-blind crossover study showed that inulin-propionate ester (10 g/day) significantly decreased the striatal blood oxygen level-dependent signal, high energy food picture appeal, and energy intake at an ad libitum meal [40].
In other words, SCFAs have an obvious anti-obesity effect, and the mechanisms involve suppressing appetite, restraining the lipogenesis, and inducing browning of white pose tissue lipolysis and induce browning of white adipose tissue, which could reduce the body adiposity by increasing thermogenesis [39]. Furthermore, a randomized, placebo-controlled, single-blind crossover study showed that inulin-propionate ester (10 g/day) significantly decreased the striatal blood oxygen level-dependent signal, high energy food picture appeal, and energy intake at an ad libitum meal [40].

Cardio-Protective Activity
Cardiovascular disease (CVD) is a chronic non-communicable disease with high morbidity and mortality on a global scale [41]. SCFAs have good protective effects on cardiovascular system [42], and the related mechanisms of action are discussed below.
SCFAs could protect cardiovascular system by decreasing atherosclerosis. For example, a study showed that butyrate inhibited the progression of diet-induced atherosclerosis by decreasing intestinal cholesterol absorption via regulating related transporters expression, such as Niemann-Pick C1-like 1 (Npc1l1, a major intestinal cholesterol transporter) and ATP-binding cassette (ABC) transporters G5 and G8 [43]. Moreover, propionate reduced intestinal cholesterol absorption and aortic atherosclerotic lesion area via increasing levels of regulatory T (Treg) cell and IL-10 and reduced the expression of Npc1l1 [44]. Moreover, the elevation of plasma total cholesterol (TC) is an important risk factor for atherosclerosis. The SCFAs significantly reduced plasma TC via enhancing fecal excretion of bile acids and up-regulating the gene expressions of sterol-regulatory element-binding protein 2 (SREBP2), low-density lipoprotein (LDL) receptor, and cholesterol 7 alpha-hydroxylase (CYP7A1) in the liver [45]. Furthermore, a randomized, double-blind clinical trial suggested that propionate intake (500 mg, twice daily) significantly reduced levels of LDL and nonhigh-density lipoprotein cholesterol, which were effective targets for atherosclerotic CVD prevention [44].
SCFAs could exert a cardiovascular protective effect by inhibiting hypertension. For instance, one study suggested that acetate, butyrate, and propionate could reduce blood pressure, and acetate showed the most powerful antihypertensive effect among these three SCFAs [46]. Moreover, acetate kept the balance of vasoconstriction and vasodilation shifts through up-regulation of SCFAs receptors, Olfr78, GPR41, and GPR43, in which way to prevent high-fructose diet-induced hypertension [47]. Besides, an in vivo study showed that propionate alleviated cardiac hypertrophy, fibrosis, vascular dysfunction, and hypertension in both wild-type NMRI and ApoE −/− mice models, and the mechanisms mainly depended on Treg cells [48].
The evidence indicated that SCFAs had protective effects against ischemia/reperfusionrelated injuries (IRI). Butyrate significantly improved myocardial IRI via the gut-brain neural circuit and might be mediated by the paraventricular nucleus (PVN)-superior cervical ganglion (SCG) sympathetic pathway [49]. Besides, acetate, butyrate, and propionate repaired the myocardial infarction (MI) impairment by reducing the infiltration of CX3CR1+ monocytes to the peri-infarct zone after MI and played an important part in maintaining host immune composition [50].
In summary, SCFAs could be a potential therapeutic strategy to prevent and manage CVD through reducing blood lipid and blood pressure, alleviating IRI, and repairing MI injury.

Hepatoprotective Activity
Liver diseases are generally divided into non-viral liver diseases and viral liver diseases [51]. Recent studies have found that gut microbiota and its metabolites, such as SCFAs, could prevent and manage several liver diseases, particularly non-viral liver diseases [52,53]. An epidemiology study found that fecal SCFAs were negatively correlated with cirrhosis disease severity [54].
Non-viral liver diseases mainly involve alcoholic liver disease (ALD), nonalcoholic fatty liver disease (NAFLD), and drug-or pollutant-induced liver injury. ALD could be caused by a long-term heavy alcohol intake, which involved alcoholic hepatitis, fibrosis, and cirrhosis [55]. A study found that propionate alleviated the ethanol-induced hepatic steatosis and enhanced hepatic function through maintaining the intestinal epithelial barrier function and inhibiting hepatic toll-like receptor 4 (TLR4)-NF-κB pathway [56]. The occurrence of NAFLD was closely related to intestinal flora disturbance, and gut microbiotaderived SCFAs could be a valuable strategy for preventing and treating NAFLD [57]. The SCFAs could regulate mucus secretion, microbial homeostasis, and intestinal epithelial tight junction to reduce the spread of intestinal endotoxin to the liver, thereby reducing the oxidative pressure and the level of inflammation in the liver and then delaying the development of NAFLD [58]. In addition, SCFAs changed the intestinal micro-ecology to protect the gut barrier, which could slow down the development of NAFLD-related diseases [58]. Another study suggested that SCFAs could inhibit hepatic steatosis by activating AMPK and PPAR signaling pathways and down-regulating the expression of genes related to lipid synthesis, such as sterol-regulatory element-binding protein 1 (SREBP-1), FAS, stearoyl-CoA desaturase 1 (SCD1), ACC1, and liver X receptor (LXR) [59]. Furthermore, pectin, Jaboticaba berry peel, and fu instant tea could alleviate fatty liver disease by regulating intestinal SCFAs [60][61][62]. As for drug-or pollutant-induced liver injury, SCFAs also exerted hepatoprotective effects. Cytochrome p450 (CYP) maturation in the liver is important for metabolic activity and xenobiotic detoxification. An in vitro study showed that the mixture of acetate, propionate, and butyrate increased the expression of CYP3A4 and ALB in human-induced pluripotent stem cell-derived liver organoids, which improved the hepatic maturation and enhanced metabolic activity and xenobiotic detoxification [63]. Furthermore, it was reported that acetate reduced serum levels of aspartate aminotransferase and alkaline phosphatase, which indicated that it improved hepatic function. Meanwhile, acetate increased mitochondrial efficiency and adenosine triphosphate production [39].
In brief, SCFAs play a positive role in non-viral liver diseases. The mechanisms of action involved maintaining the intestinal epithelial barrier, regulating the lipid metabolism and inflammatory response in liver, increasing mitochondrial efficiency, and promoting CYP maturation.

Anti-Diabetic Activity
Diabetes mellitus is characterized by hyperglycemia caused by decreasing insulin secretion or insulin resistance, and 592 million people will have diabetes mellitus by the year 2035 worldwide according to the International Diabetes Mellitus Federation prediction [64,65]. The effects of SCFAs on diabetes mellitus have been widely studied. A microbiome-wide association study on large population cohorts showed that butyrate and acetate had a causal relationship with type 2 diabetes using bidirectional Mendelian randomization analyses [66].
Propionate attenuated high-fat diet-induced insulin resistance and improved insulin sensitivity, and the mechanism of action might relate to stimulating OCFA production [38]. Besides, acetate and propionate improved insulin sensitivity and glucose tolerance [67]. In addition, the combination of acetate and propionate effectively improved insulin sensitivity in high-fat diet-fed mice via reducing inflammation through decrease of T helper 1 (Th1) and Th2 cells and increase of Treg cells in the spleen and mesenteric lymph node [67]. Moreover, butyrate could promote the growth of intestinal epithelial cells, strengthen the intestinal tight connection, and regulate the activities of gut microbiota and immune cells, in which way to maintain the gut barrier and ultimately prevent and treat type 1 diabetes mellitus [58]. Several studies also showed that oral and dietary supplementation of butyrate, as well as human acetate colonic infusions and vinegar administrations, could prevent highfat diet-induced insulin resistance and improve glucose homeostasis [68,69]. Additionally, propionate could activate AMP-activated protein kinase (AMPK) by binding GPR43 and increasing intracellular Ca 2+ . Besides, propionate suppressed hepatic gluconeogenesis via down-regulating gluconeogenic enzymes, such as glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK), through activation of AMPK [70]. Furthermore, acylated starch had a greater effect on the improvement of type 2 diabetes indexes compared to native resistant starch, such as fasting blood glucose, serum insulin level, and amino acid metabolism [71]. In addition, a randomized clinical trial based on 29 overweight/obese individuals suggested that after the Mediterranean diet intervention for 8 weeks, postprandial plasma butyric acid incremental area under the curve (IAUC) was significantly increased, which was negatively correlated with plasma insulin IAUC and oral glucose insulin sensitivity [72].
Overall, SCFAs could prevent and manage diabetes mellitus via increasing insulin sensitivity, improving glucose homeostasis, and suppressing hepatic gluconeogenesis. Furthermore, more high-quality clinical studies are also needed to determine the effect of SCFAs on glycemic control and diabetic mellitus.

Effects on Inflammatory Bowel Diseases
The inflammatory bowel diseases (IBDs) are complex immune-mediated diseases characterized by chronic inflammation in the gastrointestinal tract, and caused by the interaction among genetic, immunologic, microbial, and environmental factors [13]. SCFAs are believed to play a beneficial role in human gut health and prevent IBDs via different pathways [73], and the mechanisms of action are shown in Figure 4. The gut epithelium consists of a layer of constantly renewing epithelial cells and becomes the first line of defense against enteric infection [74]. A study showed that propionate promoted intestinal epithelial cell migration and increased cell speed and persistence in a HDAC inhibition, GPR43, and the signal transducer and activator of transcription 3 (STAT3) in a dependent manner [74]. Another study showed that butyrate promoted intestinal integrity and homeostasis via affecting metabolism, intestinal barrier function, and transporter expression [75]. Heat shock proteins (HSP) play a crucial role in maintaining and protecting the integrity of intestinal epithelial cells. Propionate contributed to intestinal homeostasis via increasing the level of Hspa1a (a major transcript of HSP70), up-regulating HSP70, and phosphorylating heat shock factor 1 [76]. Moreover, the transcriptional activation of HSP70 was proven to be related to the activation of mitogen-activated protein kinase (MEK)/ extracellular signal-regulated kinase (ERK) and mTOR pathways, as well as the inhibition of HDAC [76]. Besides, acetate increased the expression of MUC2 and CDX2, as well as the production of mucin proteins, in mucus-secreting colon epithelial cells (HT29-MTX), which indicated that it could improve the intestinal epithelium protective function [77]. SCFAs in an optimal dose could modulate the structure of gut microbiota, regulate the activities of immune cells and intestinal epithelial cells via regulating the gene expression of intestinal cells in an HDAC-dependent way, and subsequently improve the gut barrier function [58]. It was reported that acetate, propionate, and butyrate not only increased transepithelial electrical resistance (TER) and improved the formation of tight junction, but also inhibited the activation of NLRP3 inflammasome and autophagy induced by LPS to protect intestinal barrier function [78]. The SCFAs contributed to intestinal homeostasis via regulating the immune system and reducing inflammation. For example, butyric acid elicited αdefensin secretion by Paneth cells and improved enteric innate immunity through potent microbicidal activities, which contributed to intestinal homeostasis [79]. Besides, propionate repressed IL-17-and IL-22-producing γδ T cells to relieve intestinal inflammation [80]. Moreover, SCFAs transported by monocarboxylate transporter (MCT)-1 could suppress inflammatory responses in Caco-2 cells induced by TNF-α via decreasing IL-8 and IL-6 expression levels and inhibiting the activation of NF-κB, ERK, p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and spleen tyrosine kinase (Syk) [81]. Additionally, SCFAs prevented the development of intestinal inflammation via inhibiting dual-specificity phosphatase 6 (DUSP6) through the up-regulation of microRNA-145 (miR-145) by decreasing the CCAAT enhancer-binding protein beta (CEBPB) expression, and they also improved the disease activity index of LPS-treated mice [82].

Effects on Constipation
Constipation is a multifactorial intestinal disorder with high incidence and difficult treatment, leading to a serious decline in quality of life [83]. Studies have identified SCFAs as important regulatory factors in constipation. For example, an epidemiological study found that acetate, propionate, and butyrate were negatively correlated with the severity of constipation [84]. Another study with analysis of human feces found that the concen- In short, SCFAs, especially butyrate, generally showed protective effects on IBDs. The mechanisms of action involved enhancing intestinal barrier function, regulating immune system, and reducing inflammation.

Effects on Constipation
Constipation is a multifactorial intestinal disorder with high incidence and difficult treatment, leading to a serious decline in quality of life [83]. Studies have identified SCFAs as important regulatory factors in constipation. For example, an epidemiological study found that acetate, propionate, and butyrate were negatively correlated with the severity of constipation [84]. Another study with analysis of human feces found that the concentrations of SCFAs, particularly butyrate, in the feces of constipated patients, were lower than those of healthy people [85]. Furthermore, studies showed that acetic acid and butyric acid, but not propionic acid, could relieve constipation [86,87]. A study indicated that acetic acid increased water content of feces and small intestinal transit rate, and butyric acid decreased the transit time through the gut [86]. Besides, butyrate attenuated constipation symptoms by promoting the secretion of colonic hormones and maintaining intestinal barrier integrity [85]. Moreover, butyrate promoted defecation, improved intestinal mobility, and promoted Cajal cells proliferation via activating the AKT-NF-κB signaling pathway [88].
In brief, SCFAs, especially butyrate, play a key role in regulating colonic motility and might be a novel therapy of constipation.

Neuroprotective Activity
The gut microbiota and brain communicate with each other in a variety of ways and involve microbial metabolites, such as SCFAs [89]. Recent studies have shown that SCFAs are important mediators of the microbiota-gut-brain axis and play a crucial role in regulating the physiology and behavior of the central nervous system [90,91]. A study based on 116 Polish women suggested that depressive women had significantly lower levels of acetate and propionate compared with healthy individuals [92]. A study showed that acetate improved cognitive impairment, decreased the cluster of differentiation 11b (CD11b, a tight junction protein) level, and suppressed neuroinflammation in the Alzheimer's disease model mice. For BV2 cells, acetate exerted anti-neuroinflammatory effects via inhibiting the phosphorylation of NF-κB, p65, ERK, and JNK; decreasing cyclooxygenase-2 (COX-2) and IL-1β levels; and increasing the GPR41 level [93].
In summary, some SCFAs, such as acetate, may help to prevent and manage neurodegenerative and neuropsychiatric diseases.

Anticancer Activity
Cancer is a serious public health problem worldwide, and cancer cells exhibit specific characteristics compared with normal cells, such as infinite proliferation, weak adhesion, and strong agglutination [94,95]. SCFAs could exert anticancer activities through different pathways. For example, SCFAs inhibited proliferation of human cervical cancer HeLa cells via down-regulating free fatty acid receptor 2 (FFAR2) expression [96]. Another study showed that acetate and propionate inhibited the carcinogenesis process of colorectal cancer via inhibiting cancer cell proliferation and inducing cell cycle arrest [97]. The SCFAs decreased the incidence and size of colitis-associated colorectal tumor in azoxymethane/dextran sodium sulfate-treated mice by improving colon inflammation and decreasing cell proliferation [98]. Besides, propionate and butyrate reduced the lung metastasis of melanoma cells by increasing the expression of chemokine (C-C motif) ligand 20 (CCL20) in lung endothelial cells and reducing the recruitment of Th17 cells [99]. It was reported that valeric acid suppressed colony formation, migration, and invasion of liver cancer cells in vitro, and suppressed hepatocellular carcinoma development in vivo, as well as improving the survival rate of mice with liver cancer [100]. Additionally, pentanoate and butyrate enhanced the effect of cancer immunotherapy by enhancing cytotoxic T lymphocytes and chimeric antigen receptor T cells via epigenetic and metabolic reprogramming [101]. Furthermore, the anticancer mechanisms of SCFAs also involved inducing apoptosis, activating autophagy, and modulating hematopoiesis [18,102,103].
In short, SCFAs could be a potential agent against several cancers, such as cervical, colorectal, melanoma, and liver cancers. The mechanisms of action mainly included inhibiting cancer cell proliferation, arresting cell cycle, decreasing inflammation, reducing metastasis, and enhancing effect of immunotherapy.

Anti-Bacterial Activity
SCFAs have been shown to inhibit bacterial growth and viability. A study showed that butyrate could enhance the antimicrobial activity of macrophages without tissue damaging inflammation [104]. Moreover, bacterial infection is one of the main causes of diarrhea. Enterotoxin-producing Bacillus cereus, Clostridium difficile, Clostridium perfringens, Escherichia coli, Staphylococcus aureus, and Vibrio cholerae are the major pathogenic bacteria that secrete highly toxic proteins, which could induce diarrhea [105]. The SCFAs could inhibit these bacteria, decrease enterotoxin cytotoxicity via mimicking the structure of toxin receptors and inhibiting toxin adherence to host cells, and promote the production of beneficial microbiome [105]. In addition, SCFAs could inhibit other foodborne pathogenic bacteria, such as Campylobacter, Salmonella, and Shigella in a pH-, dose-, and complexity-dependent manner [106]. Furthermore, SCFAs could be used to prevent food spoilage due to its antimicrobial activity. The evidence showed that repeated treatments with acetic acid vapors could preserve table grapes' fruit quality, which was safer compared with SO 2 [107].
Overall, SCFAs could prevent and treat bacterial diarrhea via inhibiting the pathogenicity of enteropathogens and decreasing enterotoxin toxicity. Moreover, SCFAs could also play a vital role in prevention of food spoilage.

Effects of SCFAs on Other Diseases
SCFAs also have other health benefits besides the bioactivities mentioned above. For example, a study found that propionate could alleviate mitochondrial dysfunction, oxidative stress, and apoptosis induced by free fatty acids via up-regulating peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α) [108]. Besides, SC-FAs could contribute to infant sleep. A study indicated that the higher the proportion of propionate in total fecal SCFAs, the longer the infant at 7 and 8 months of age slept uninterrupted [109]. Moreover, SCFAs could mediate the effects of intestinal microbiota on the metabolism and function of skeletal muscle. It was reported that SCFAs could regulate lipid, carbohydrate, and protein metabolism in skeletal muscle tissues, and they could also increase skeletal muscle mass retention. The mechanisms of action might be related to the activation of AMPK, PPAR-δ, and PGC-1α, as well as the inhibition of HDACs [110]. Moreover, acetate and propionate might contribute to maintaining a positive balance of bone turnover by up-regulating differentiation in primary osteoblasts [111]. Another study suggested that propionate and butyrate increased systemic bone mass under steady-state conditions via inducing the reprogramming of osteoclasts metabolism, enhancing glycolysis, expensing oxidative phosphorylation, and down-regulating several osteoclast genes, such as TRAF6 and NFATc1 [112]. Besides, SCFAs could also prevent bone loss after menopause [112]. Additionally, acetate, propionate, and butyrate synergistically alleviated rheumatoid arthritis by regulating B cells differentiation via FFAR2 receptors [113]. Furthermore, high concentrations of acetate and butyrate could suppress periodontal disease by decreasing the accumulation of neutrophil granulocytes in the gingival pockets via binding FFAR2 and triggering neutrophil Ca 2+ signaling and chemotaxis [114].
In brief, SCFAs also exerted beneficial effects on sleep, skeletal muscle, bone loss, arthritis, and periodontal disease. Neuroprotective activity Cross-sectional study 116 women Depressive women had lower levels of acetate and propionate [92] Abbreviation: SCFAs, short-chain fatty acids.

The Side Effects of SCFAs
Although most studies showed that SCFAs exerted health benefits, several studies found that some SCFAs could be useless or exhibit side effects in some conditions. Although SCFAs from inulin fermentation had potential to prevent or treat type 1 diabetes, direct SCFAs oral administration alone did not provide a significant impact on the diabetes, which might be attributed to oral administration did not allow for enough SCFAs to reach the site of action (cecum and colon) [116]. Moreover, a meta-analysis including 44 randomized controlled trial studies found that acetate, propionate, butyrate, and the mixed SCFAs had no effect on insulin and blood glucose in humans [120]. Another study showed that SCFAs accelerated the 3T3-L1 adipocyte differentiation and promoted lipid accumulation via modulating the expression of fatty acid metabolism-related enzymes in vitro, such as lipoprotein lipase, adipocyte fatty acid-binding protein 4, fatty acid transporter protein 4, and fatty acid synthase [115]. Additionally, a study suggested that, for those who had NAFLD and poor metabolic health, the gut-derived acetate could provide an extra precursor for de novo lipogenesis in the liver, which could increase hepatic lipid accumulation [15]. In a multi-organ model of ulcerative colitis (UC) ex vivo, SCFAs improved UC severity via increasing the production of ketone bodies, glycolysis, and lipogenesis, and significantly decreased innate immune activation of UC gut, but SCFAs also induced gut barrier disruption through metabolic reprograming during acute T cell-mediated inflammation [121]. Furthermore, some SCFAs could induce brain neurochemistry impairment. For example, an epidemiological study found that depressive women had higher concentrations of isocaproic acid than healthy subjects [92]. Another study showed that propionate caused brain neurochemistry impairment by increasing the levels of IFN-γ and caspase-3, as well as decreasing nor-adrenaline, dopamine, and 5-HT [118]. Moreover, propionate increased the level of glutamate and the glutamate/glutamine ratio, and decreased gamma amino butyric acid (GABA), glutamine and the GABA/glutamate ratio, which showed characteristics of autism [119].
In short, different SCFA types might exert different effects, and the bioactivities of SCFAs might depend on the health condition of hosts. When this section was compared with previous sections, the results from epidemiological studies could be inconsistent. In the future, more high-quality, large sample epidemiological studies are needed to verify the effects of SCFAs on humans.

Conclusions
SCFAs show various bioactivities, such as anti-inflammatory and immunoregulatory effects, as well as preventive and therapeutic effects on several diseases, including obesity, cardiovascular disease, liver disease, diabetes mellitus, IBDs, diarrhea, constipation, neurodegenerative diseases, neuropsychiatric diseases, cancers, arthritis and periodontal disease, and so on. Moreover, the underlying mechanisms are diverse, among which NF-κB, Nrf2, GPR41, and HDAC signaling pathways are highlighted. Furthermore, it is worth noting that, due to differences in SCFAs types and concentrations as well as health condition of hosts, some SCFAs could exhibit a double-sided effect. Therefore, in the application of SCFAs, attention should be paid to the selection of SCFA type, as well as the influence of their concentration and body condition. In the future, more bioactivities of SCFAs should be evaluated, and their mechanisms of action should be further studied. Furthermore, because most of the results comes from preclinical models, more clinical trials should be carried out to verify these potential effects of SCFAs on human beings. This paper is helpful for supporting people eating more dietary fiber, and for some SCFAs and dietary fiber to be developed into functional food to prevent and manage several diseases.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.