Short-Chain Fatty Acid Butyrate Induces Cilia Formation and Potentiates the Effects of HDAC6 Inhibitors in Cholangiocarcinoma Cells

Cholangiocarcinoma (CCA) is a deadly form of liver cancer with limited therapeutic approaches. The pathogenesis of CCA involves the loss of primary cilia in cholangiocytes, an important organelle that regulates several key cellular functions including the regulation of cell polarity, growth, and differentiation, by a mechanism involving increased expression of deacetylases like HDAC6 and SIRT1. Therefore, cilia restoration may represent an alternative and novel therapeutic approach against CCA. Butyrate is produced by bacterial fermentation of fibers in the intestine and has been shown to inhibit SIRT1, showing antitumor effects on various cancers. Herein, we investigated the role of butyrate on CCA cell proliferation, migration, and EMT and evaluated the synergistic effects with specific HDAC6 inhibition. When CCA cells, including HuCCT1 and KMCH, were treated with butyrate, the cilia formation and acetylated-tubulin levels were increased, while no significant effects were observed in normal human cholangiocytes. Butyrate treatment also depicted reduced cell proliferation in HuCCT1 and KMCH cells, but on the other hand, it affected cell growth of the normal cholangiocytes only at high concentrations. In HuCCT1 cells, spheroid formation and cell migration were also halted by butyrate treatment. Furthermore, we found that butyrate augmented the previously described effects of HDAC6 inhibitors on CCA cell proliferation and migration by reducing the expression of CD44, cyclin D1, PCNA, Zeb1, and Vimentin. In summary, butyrate targets cancer cell growth and migration and enhances the anti-cancer effects of HDAC6 inhibitors in CCA cells, suggesting that butyrate may have therapeutic effects in CCA and other ciliopathies.

Roseburia hominis Increases Intestinal Melatonin Level by Activating p-CREB-AANAT Pathway

Intestinal melatonin exerts diverse biological effects on the body. Our previous research showed that the abundance of the butyrate-producing bacteria, Roseburia, is positively related to the expression of colonic mucosal melatonin. However, the detailed relationship is unclear. Therefore, we aimed to explore whether Roseburia regulates intestinal melatonin and its underlying mechanisms. Male Sprague–Dawley germfree rats were orally administered with or without Roseburia hominis. R. hominis treatment significantly increased the intestinal melatonin level. The concentrations of propionate and butyrate in the intestinal contents were significantly elevated after gavage of R. hominis. Propionate or butyrate treatment increased melatonin, 5-hydroxytryptamine (5-HT), arylalkylamine N-acetyltransferase (AANAT), and phosphorylated cAMP-response element-binding protein (p-CREB) levels. When pretreated with telotristat ethyl, the inhibitor of tryptophan hydroxylase (TPH), or siRNA of Aanat, or 666-15, i.e., an inhibitor of CREB, propionate, or butyrate, could not promote melatonin production in the pheochromocytoma cell line BON-1. Metabolomics analysis showed that propionate and butyrate stimulation regulated levels of some metabolites and some metabolic pathways in BON-1 cell supernatants. In conclusion, propionate and butyrate, i.e., metabolites of R. hominis, can promote intestinal melatonin synthesis by increasing 5-HT levels and promoting p-CREB-mediated Aanat transcription, thereby offering a potential target for ameliorating intestinal diseases.

Profiling and Characterization of microRNAs Responding to Sodium Butyrate Treatment in Gastric Cancer Cells

Background: Short-chain fatty acids exert anti-cancer effects on tumor cells. Objective: We aimed to reveal the signaling network altered by butyrate in Gastric Cancer (GC) using small RNA sequencing (sRNA-seq). Methods: The effects of butyrate on the biological behavior of NCI-N87 and KATO III cells in vitro were assessed by functional assays and half-maximal inhibitory concentrations (IC50) of butyrate in KATO III cells were calculated. sRNA-seq was performed on KATO III cells. Differentially expressed miRNAs (DE-miRNAs) were identified between butyrate treatment and control groups using DESeq2, and miRNA targets were predicted. A protein-protein interaction (PPI) network of DE-miRNA targets was created using Metascape. Key MCODE complexes were identified using the MCODE algorithm and cluster Profiler. The relationship between DE-miRNA and GC overall survival (OS) was evaluated using Kaplan-Meier curves. Results: Butyrate dose-dependently inhibited NCI-N87 and KATO III cell viability. KATO III cells were more sensitive to butyrate than NCI-N87 cells. Butyrate promoted apoptosis and inhibited KATO III cell migration. Total 324 DE-miRNAs were identified in KATO III cells, and 459 mRNAs were predicted as targets of 83 DE-miRNAs. Two key protein complexes were identified in a PPI network of the 459 targets. A key signaling network responding to butyrate were generated using targets in these key complexes and their miRNA regulators. The DE-miRNAs in the key signaling network were related to the OS of GC. Conclusion: Butyrate altered the biological behavior of GC cells, which may be achieved by regulating miRNAs and related oncogenic pathways.

Live Monitoring of Inflammation Reveals Tissue and Sex-specific Responses to Western Diet and Butyrate treatment

Obesity is a current epidemic, affecting millions of individuals worldwide. Chronic obesity is characterized by a low grade systemic inflammation besides not being a classic inflammatory disease. Many studies have tried to identify inflammatory insults dysregulated by a Westernized diet consisted of high fat, high sucrose, and high cholesterol mainly focusing on production and secretion of inflammatory cytokines. The gut microbiome and derived metabolites, including the short-chain fatty acid butyrate, have received increased attention as underlying some of the obesogenic features. In the present work, we utilized a novel biosensor mouse model capable of monitoring in vivo inflammation. We observed tissue- and sex- specific caspase 1 activation patterns in obese mice and treated with butyrate. Our work utilizing a caspase-1 biosensor mouse model, flow cytometry and computational analyses and offers new mechanistic insights underlying the effect of butyrate in obesity and its complications.

Oral Supplementation With Butyrate Improves Myocardial Ischemia/Reperfusion Injury via a Gut-Brain Neural Circuit

Objective: Butyrate, a short-chain fatty acid (SCFA) produced by the intestinal microbiota, plays a protective role in cardiovascular diseases (CVDs), but the mechanisms involved in this process remain unelucidated. We aimed to explore the effect of butyrate on myocardial ischemia/reperfusion (I/R) injury through the gut-brain neural circuit.Methods: Rats were randomly divided into four groups: sham group (sham), I/R group (I/R), I/R+ butyrate group (butyrate), and I/R+ butyrate+ vagotomy group (vagotomy). The rats were treated with sodium butyrate for 4 weeks, and the gut-brain neural circuit was investigated by subdiaphragmatic vagotomy.Results: Butyrate treatment significantly reduced the infarct size and decreased the expression of creatine kinase (CK), creatine kinase myocardial isoenzyme (CK-MB), and lactate dehydrogenase (LDH) compared with the values found for the I/R group. In addition, the I/R-induced increases in inflammation, oxidative stress, and apoptosis were attenuated by butyrate. However, the above-mentioned protective effects were diminished by subdiaphragmatic vagotomy. The RNA sequencing results also revealed that the butyrate-induced protective changes at the cardiac transcription level were reversed by vagotomy. An analysis of the heart rate variability (HRV) and the detection of norepinephrine (NE) showed that butyrate significantly inhibited the I/R-induced autonomic imbalance, but this inhibition was not observed in the vagotomy group. Butyrate treatment also suppressed the neural activity of the paraventricular nucleus (PVN) and superior cervical ganglion (SCG), and both of these effects were lost after vagotomy.Conclusions: Butyrate treatment significantly improves myocardial I/R injury via a gut-brain neural circuit, and this cardioprotective effect is likely mediated by suppression of the sympathetic nervous system.

Microbiota induces aging-related leaky gut and inflammation by dampening mucin barriers and butyrate-FFAR2/3 signaling

Increased chronic inflammation is one of the key risk factors of aging-related disorders although its precise etiology remains elusive. Here, we demonstrate that aged, but not young, microbiota triggers inflammation by promoting gut permeability (leaky gut) via disruption of mucus barriers. Levels of the beneficial short-chain fatty acid, butyrate, are suppressed in the aged gut. Consistent with feedback regulation, the expression of butyrate-sensing receptors, free fatty acid receptor 2/3 (FFAR2/3), are also reduced in aged gut. Butyrate treatment of aged mice revereses the reduced mucin production, increased gut permeability and inflammation associated with low butyrate levels. In agreement, intestine-specific FFAR2/3 knockout mice manifest a compromised gut phenotype typically seen in aged mice, such as increased gut permeability and inflammation with reduced mucin production. Taken together, our results demonstrate that an aged gut microbiota causally instigates inflammation by increasing gut permeability due to reduced butyrate levels, FFAR2/3 expression, and mucin barriers. Thus, butyrate-FFAR2/3 agonism could ameliorate the deleterious effects seen in aged gut and their implications on metabolic health.

Impact of Sodium Butyrate Treatment in LPS-Stimulated Peripheral Blood Mononuclear Cells of Poorly Controlled Type 2 DM

Type 2 diabetes mellitus (T2DM) is associated with chronic low-grade inflammation, which is marked by the dysregulation of innate and adaptive immune responses. Therefore, reducing inflammation, possibly through an immunoregulatory agent, may play a role in T2DM treatment. Butyrate is the most potent short-chain fatty acid (SCFA), and it exerts anti-inflammatory properties by inhibiting histone deacetylase activity. As an immunoregulatory agent, sodium butyrate can inhibit nuclear factor kB (NF-kB) activation and reduce the production of pro-inflammatory cytokines in immune cells. The aim of the study was to measure the level of plasma butyrate in poorly controlled T2DM and normoglycemic participants and to compare the response of peripheral blood mononuclear cells (PBMCs) to sodium butyrate treatment between the groups by measuring production of the following cytokines: tumor necrosis factor (TNF)-α, interleukin (IL)-6, interferon (IFN)-γ, IL-13, and IL-10. The in vitro study examined the PBMCs of 15 participants with poorly controlled T2DM and 15 normoglycemic participants. PBMCs were cultured with the following stimulations for two days at a temperature of 37°C and 5% CO2: 100 ng/mL lipopolysaccharide (LPS), 1 mM sodium butyrate, or a combination of 100 ng/mL LPS and 1 mM sodium butyrate. Plasma butyrate was measured using gas chromatography-mass spectrometry, and cytokines from culture supernatant were analyzed using magnetic beads multiplex assay. Plasma butyrate levels in participants with poorly controlled T2DM did not significantly differ from those in normoglycemic participants (p = 0.105). Compared to treatment with an LPS-stimulated PBMC culture, treatment with 1 mM sodium butyrate reduced the levels of TNF-α (p < 0.039) and IFN-γ (p < 0.038) in normoglycemic participants. The same general trend was seen in PBMC from participants with poorly controlled T2DM, but higher variability appeared to preclude statistical significance. These data suggest that butyrate may modulate inflammatory cytokine production in human PBMCs, but more research is needed to determine if butyrate is anti-inflammatory in poorly controlled T2DM.

Butyrate improves cardiac function and sympathetic neural remodeling following myocardial infarction in rats

Increased inflammation is found in cardiac sympathetic neural remodeling with malignant ventricular arrhythmia (VA) following myocardial infarction (MI). Butyrate, as a microbiota-derived short-chain fatty acid, can inhibit inflammation and myocardial hypertrophy. However, the role of butyrate in sympathetic neural remodeling after MI is unknown. This study aimed to investigate whether butyrate could improve cardiac dysfunction and VA following MI by regulating inflammation and sympathetic neural remodeling. MI rats were randomized to administrate the butyrate or vehicle through intraperitoneal injection to undergo the study. Our data demonstrated that butyrate treatment preserved the partial cardiac function at 7 days post-MI. Butyrate downregulated the expression of essential for inflammatory response in the infarct border zone at 3 days post-MI. Particularly, butyrate promoted expression of M2 macrophage markers. Increased expressions of nerve growth factor and norephinephrine at 7 days after MI were inhibited in butyrate-treated rats. Furthermore, butyrate significantly decreased the density of nerve fibers for growth-associated protein-43 and tyrosine hydroxylase and resulted in fewer episodes of inducible VA. In conclusion, butyrate administration ameliorated cardiac function and VA after MI possibly through promoting M2 macrophage polarization to suppress inflammatory responses and inhibit sympathetic neural remodeling and may present an effective pharmacological strategy for the prevention of MI-related remodeling.