Indoxyl sulfate contributes to colorectal cancer cell proliferation and increased EGFR expression by activating AhR and Akt.

Although patients with chronic kidney disease (CKD) have a higher risk of colorectal cancer (CRC) aggravation, the connection between these two diseases is not well understood. Recent studies have shown that both CKD and CRC aggravation are closely related to an increased abundance of indole-producing Fusobacterium nucleatum in the gut. The indole absorbed from the gut is eventually metabolized to indoxyl sulfate in the liver. Since indoxyl sulfate is involved not only in accelerating CKD progression but also in the initiation and development of its associated complications, the present study aimed to clarify whether indoxyl sulfate induces the proliferation of CRC cells. This study found that indoxyl sulfate induced the proliferation of CRC-derived HCT-116 cells by activating the aryl hydrocarbon receptor (AhR) and the proto-oncogene Akt. The AhR antagonist CH223191 and Akt inhibitor MK2206 suppressed indoxyl sulfate-induced proliferation of HCT-116 cells. We also found that indoxyl sulfate upregulated epidermal growth factor receptor (EGFR) expression, which is associated with poor prognosis of CRC, whereas CH223191 and MK2206 repressed EGFR expression. Furthermore, indoxyl sulfate increased the sensitivity of CRC cells to EGF by upregulating EGFR expression. These findings suggest that indoxyl sulfate may be an important link between CKD and CRC aggravation.

Eicosapentaenoic Acid Induces the Inhibition of Adipogenesis by Reducing the Effect of PPARγ Activator and Mediating PKA Activation and Increased COX-2 Expression in 3T3-L1 Cells at the Differentiation Stage.

Obesity has received increasing attention in recent years because it is a factor in the development of non-communicable diseases. The current study aimed to analyze how representative fatty acids (FAs) such as palmitic acid, stearic acid, oleic acid, α-linolenic acid (ALA), and eicosapentaenoic acid (EPA) affected adipogenesis when/if introduced at the differentiation stage of 3T3-L1 cell culture. These FAs are assumed to be potentially relevant to the progression or prevention of obesity. EPA added during the differentiation stage reduced intracellular triacylglycerol (TAG) accumulation, as well as the expression of the established adipocyte-specific marker genes, during the maturation stage. However, no other FAs inhibited intracellular TAG accumulation. Coexistence of Δ12-prostaglandin J2, a peroxisome proliferator-activated receptor γ activator, with EPA during the differentiation stage partially attenuated the inhibitory effect of EPA on intracellular TAG accumulation. EPA increased cyclooxygenase-2 (COX-2) expression and protein kinase A (PKA) activity at the differentiation stage, which could explain the inhibitory actions of EPA. Taken together, exposure of preadipocytes to EPA only during the differentiation stage may be sufficient to finally reduce the mass of white adipose tissue through increasing COX-2 expression and PKA activity.

Activation of the TLR4-JNK but not the TLR4-ERK pathway induced by indole-3-acetic acid exerts anti-proliferative effects on Caco-2 cells.

We previously found that indole-3-acetic acid (IAA) produced from tryptophan by gut microbiota decreases the expression of tumor necrosis factor α (TNFα), which is implicated in the pathogenesis of colorectal cancer (CRC). The present study aimed to determine IAA involvement in the proliferation of CRC-derived Caco-2 cells. Cell proliferation was suppressed by IAA, whereas IAA-induced aryl hydrocarbon receptor activation had no impact. IAA activated extracellular signal-related (ERK) and c-Jun N-terminal (JNK) kinases, but not p38. Toll-like receptor 4 (TLR4) may be required to activate ERK and JNK, but only the TLR4-JNK pathway might elicit the anti-proliferative effects of IAA. Thus, IAA may be a ligand for TLR4 that contributes to inhibiting CRC cell proliferation by activating TLR4-mediated JNK. Because IAA did not induce cytotoxicity, inhibiting cell cycle progression might affect the anti-proliferative capacity of IAA. Therefore, colonic IAA accumulation might help to prevent CRC development and progression.

Skatole-induced p38 and JNK activation coordinately upregulates, whereas AhR activation partially attenuates TNFα expression in intestinal epithelial cells.

Increased tumor necrosis factor α (TNFα) expression in intestinal epithelial cells (IECs) plays a major role in the development and progression of inflammatory bowel disease (IBD) and colorectal cancer (CRC). The present study aimed to clarify the relationship between TNFα and skatole, a tryptophan-derived gut microbiota metabolite. The aryl hydrocarbon receptor (AhR) antagonist CH223191 promoted, whereas the p38 inhibitor SB203580 suppressed the increase in TNFα mRNA and protein expression induced by skatole in intestinal epithelial Caco-2 cells. The c-Jun N-terminal kinase (JNK) inhibitor SP600125 repressed only the increased TNFα protein expression, whereas the extracellular signal-regulated kinase (ERK) pathway inhibitor U0126 did not affect increased TNFα expression at any level. A neutralizing antibody against TNFα partially inhibited skatole-induced cell death. Overall, these results suggested that TNFα expression is increased by the concerted actions of skatole-activated p38 and JNK, and that TNFα exerts autocrine/paracrine actions on IECs despite partial suppression by activated AhR. Therefore, skatole might play an important role in the development and progression of IBD and CRC via increased TNFα expression.

Arachidonic Acid Added during the Differentiation Phase of 3T3-L1 Cells Exerts Anti-Adipogenic Effect by Reducing the Effects of Pro-Adipogenic Prostaglandins.

A linoleic acid (LA) metabolite arachidonic acid (AA) added to 3T3-L1 cells is reported to suppress adipogenesis. The purpose of the present study aimed to clarify the effects of AA added during the differentiation phase, including adipogenesis, the types of prostaglandins (PG)s produced, and the crosstalk between AA and the PGs produced. Adipogenesis was inhibited by AA added, while LA did not. When AA was added, increased PGE2 and PGF2α production, unchanged Δ12-PGJ2 production, and reduced PGI2 production were observed. Since the decreased PGI2 production was reflected in decreased CCAAT/enhancer-binding protein-ß (C/EBPß) and C/EBPδ expression, we expected that the coexistence of PGI2 with AA would suppress the anti-adipogenic effects of AA. However, the coexistence of PGI2 with AA did not attenuate the anti-adipogenic effects of AA. In addition, the results were similar when Δ12-PGJ2 coexisted with AA. Taken together, these results indicated that the metabolism of ingested LA to AA is necessary to inhibit adipogenesis and that exposure of AA to adipocytes during only the differentiation phase is sufficient. As further mechanisms for suppressing adipogenesis, AA was found not only to increase PGE2 and PGF2α and decrease PGI2 production but also to abrogate the pro-adipogenic effects of PGI2 and Δ12-PGJ2.

Prostaglandin D2 Added during the Differentiation of 3T3-L1 Cells Suppresses Adipogenesis via Dysfunction of D-Prostanoid Receptor P1 and P2.

We previously reported that the addition of prostaglandin, (PG)D2, and its chemically stable analog, 11-deoxy-11-methylene-PGD2 (11d-11m-PGD2), during the maturation phase of 3T3-L1 cells promotes adipogenesis. In the present study, we aimed to elucidate the effects of the addition of PGD2 or 11d-11m-PGD2 to 3T3-L1 cells during the differentiation phase on adipogenesis. We found that both PGD2 and 11d-11m-PGD2 suppressed adipogenesis through the downregulation of peroxisome proliferator-activated receptor gamma (PPARγ) expression. However, the latter suppressed adipogenesis more potently than PGD2, most likely because of its higher resistance to spontaneous transformation into PGJ2 derivatives. In addition, this anti-adipogenic effect was attenuated by the coexistence of an IP receptor agonist, suggesting that the effect depends on the intensity of the signaling from the IP receptor. The D-prostanoid receptors 1 (DP1) and 2 (DP2, also known as a chemoattractant receptor-homologous molecule expressed on Th2 cells) are receptors for PGD2. The inhibitory effects of PGD2 and 11d-11m-PGD2 on adipogenesis were slightly attenuated by a DP2 agonist. Furthermore, the addition of PGD2 and 11d-11m-PGD2 during the differentiation phase reduced the DP1 and DP2 expression during the maturation phase. Overall, these results indicated that the addition of PGD2 or 11d-11m-PGD2 during the differentiation phase suppresses adipogenesis via the dysfunction of DP1 and DP2. Therefore, unidentified receptor(s) for both molecules may be involved in the suppression of adipogenesis.

A diet supplemented with cholic acid elevates blood pressure accompanied by albuminuria in rats.

A diet supplemented with cholic acid (CA), the primary 12α-hydroxylated bile acid, can induce hepatic lipid accumulation in rats without obesity. This study examined the effects of a CA-supplemented diet on blood pressure (BP). After acclimation, WKAH/HkmSlc rats (3 weeks old) were divided into two groups and fed with a control AIN-93-based diet or a CA-supplemented diet (0.5 g CA/kg) for 13 weeks. The CA diet increased systolic and diastolic BP as well as hepatic lipid concentrations in the rats. No changes were found in the blood sodium concentration. Urinary albumin concentration increased in CA-fed rats. An increase was observed in the hepatic expression of ATP-binding cassette subfamily B member 1B that correlated BPs and urinary albumin concentration accompanied by an increase in portal taurocholic acid concentration. These results suggest that 12α-hydroxylated bile acids are involved in increased BP and albuminuria via alteration of hepatic function.

Tropomyosin micelles are the major components contributing to the white colour of boiled shellfish soups.

Basket clam soup, a popular Asian dish, is prepared by boiling clams in hot water. The soup is generally cloudy, and it is considered that increased cloudiness enhances taste. However, the composition of the whitening ingredients and their association with taste enhancement remains unclear. In this study, we aimed to identify the components contributing to the white colour of the boiled soup. The white component upon precipitation with trichloroacetic acid reacted positively with ninhydrin, indicating the presence of proteins. The separation of proteins using sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed an intense band of size 33 kDa. Peptide mass fingerprinting of the identified protein using matrix-assisted laser desorption/ionisation-time-of-flight tandem mass spectrometry revealed the protein as tropomyosin. To validate the involvement of tropomyosin in the turbidity of the soup, tropomyosin was expressed and extracted from Escherichia coli. As expected, the purified protein suspended in water resulted in turbid appearance. To determine whether lipids have any association with the observed cloudiness of the soup, the amounts of fatty acids were measured. The proportion of estimated fatty acids was very low compared to that of proteins. Overall, we identified the major component contributing to soup cloudiness as tropomyosin forming micelles.

Microcystin-LR incorporated into colonic cells through probenecid-sensitive transporters leads to upregulated MCP-1 expression induced by JNK activation.

Harmful algae that inhabit eutrophic lakes produce cyanotoxic microcystins. Therefore, the relationship between chronic exposure to microcystins via drinking water and organ disorders has been investigated. The present study aimed to determine whether representative microcystin-LR is involved in increased monocyte chemoattractant protein-1 (MCP-1) expression in rat colonic mucosa and enterocyte-like differentiated Caco-2 cells. The mRNA expression of MCP-1 was increased in the colons of rats administered with microcystin-LR, compared with controls. Furthermore, mRNA levels of MCP-1 expression significantly and positively correlated with those of Adhesion G Protein-Coupled Receptor E1 (ADGRE1; EMR1; F4/80), an indicator of macrophage infiltration, suggesting that increased MCP-1 expression induced by microcystin-LR promotes macrophage infiltration into the colon. Microcystin-LR increased MCP-1 expression in enterocyte-like differentiated Caco-2 cells, by activating c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase (ERK) or p38. The findings of transporter inhibitors indicated that microcystin-LR is incorporated into cells via ATP Binding Cassette (ABC) or solute carrier (SLC) transporters other than the organic anion transporting polypeptides (OATPs)1B1, 1B3, 2B1, and 1A2, which this leads to increased MCP-1 expression in the colon through activating JNK. Thus, increased MCP-1 expression induced by microcystin-LR might be a trigger for initiating tumorigenesis with inflammation in the colon because increased MCP-1 expression induces inflammation associated with macrophage infiltration into the colon, and chronic inflammation is associated with the initiation of tumorigenesis.

Prostaglandin D2 and its analog, 11d-11m-PGD2, added during the differentiation phase contribute to adipogenic program inhibition in 3T3-L1 cells.

We previously reported that prostaglandin (PG)D2 and its isosteric analog, 11-deoxy-11-methylene-PGD2 (11d-11m-PGD2), promote adipogenesis in 3T3-L1 cells during the maturation phase. Focusing on the differentiation phase, although both PGs inhibited adipogenesis, this effect was canceled out by PGI2 and PGJ2 derivatives. Thus, PGD2 and 11d-11m-PGD2 play different roles during the phases, but do not affect PGI2- and PGJ2-derivative-induced adipogenesis.

TLR4 may be a novel indole-3-acetic acid receptor that is implicated in the regulation of CYP1A1 and TNFα expression depending on the culture stage of Caco-2 cells.

Most studies of indole derivatives such as IAA produced by intestinal microbiota have been based on the premise that binding to AhR leads to biological responses. We previously revealed that IAA binds to more than one receptor, and thus the present study aimed to identify a new receptor for IAA and analyze its mechanism of action. We found that the TLR4 antagonist TAK-242 did not affect the IAA-induced increase in CYP1A1 expression at 3 h and decreased TNFα expression at 8 days. However, TAK-242 alleviated decreased TNFα expression induced by IAA at 2 days and promoted IAA-induced increased CYP1A1 expression by inhibiting JNK activation at 8 days. Taken together, TLR4 may be a novel IAA receptor with signaling pathways that regulate CYP1A1 and TNFα expression depending on the culture stage of Caco-2 cells. Furthermore, our findings offer important clues for elucidating the action mechanisms of indole derivatives that affect hosts.

12α-Hydroxylated bile acid enhances accumulation of adiponectin and immunoglobulin A in the rat ileum.

We previously reported that dietary supplementation with cholic acid (CA), the primary 12α-hydroxylated (12αOH) bile acid (BA), reduces plasma adiponectin concentration in rats. The aim of this study was to examine the distribution of adiponectin in the body of CA-fed rats and its influence on mucosal immunoglobulin A concentration in the intestine. Rats were fed a diet supplemented with or without CA (0.5 g CA/kg diet) for 13 weeks. A reduction in plasma adiponectin level was observed from week 3. At the end of the experiment, the CA diet reduced plasma adiponectin concentration both in the portal and aortic plasma. Accumulation of adiponectin was accompanied by an increase in cadherin-13 mRNA expression in the ileal mucosa of CA-fed rats. No increase was observed in adiponectin mRNA expression in the ileal and adipose tissues of the CA-fed rats. Immunoglobulin A concentration in the ileal mucosa was elevated in the CA-fed rats and was correlated with the ileal adiponectin concentration. 12αOH BAs may modulate mucosal immune response that are involved in the accumulation of adiponectin in the ileum.

Suppression of TNFα expression induced by indole-3-acetic acid is not mediated by AhR activation in Caco-2 cells.

Indole-3-acetic acid (IAA) produced by intestinal bacteria from tryptophan in dietary proteins is considered to suppress the inflammatory response through aryl hydrocarbon receptor (AhR) activation. However, AhR activation was not involved in the downregulation of tumor necrosis factor α (TNFα) expression induced by IAA in Caco-2 cells. The activation of unidentified IAA receptors might attenuate the inflammatory response to TNFα in colorectal cancer cells.

A novel strategy of selective gene delivery by using a uniform magnetic field.

The application of a magnetic field to enhance the transfection efficiency has been reported to be mainly dependent on the magnetic force generated by a magnetic field gradient to attract paramagnetic bead-conjugated carrier and polynucleotide complexes. This strategy has the advantage of targeting a point or an area on the culture vessel. However, it is difficult to target deeply placed tissues in vivo. Uniform magnetic field-correlated effect is applicable to such a purpose. Here, we attempted to establish a novel procedure for uniform magnetic field-dependent enhancement of transfection efficiency. We examined the effect of a 1.5 mT uniform magnetic field on cellular reactive oxygen species (ROS) level and transfection efficiency mediated by a ROS-sensitive transfection carrier. Our experimental results revealed that a 1.5 mT uniform magnetic field transiently decreased cellular ROS levels and strongly enhanced transfection efficiency mediated by polyethylenimine (PEI). The uniform magnetic field-dependent enhancement of PEI-mediated in vivo transfection was confirmed in the livers of mice. Local intensification of a uniform magnetic field in a culture dish resulted in selective gene delivery into cells on the target area. Although further examination and improvement are necessary for this procedure, our findings provide a novel option for spatial control of gene delivery.

12α-Hydroxylated bile acid induces hepatic steatosis with dysbiosis in rats.

There is an increasing need to explore the mechanism of the progression of non-alcoholic fatty liver disease. Steroid metabolism is closely linked to hepatic steatosis and steroids are excreted as bile acids (BAs). Here, we demonstrated that feeding WKAH/HkmSlc inbred rats a diet supplemented with cholic acid (CA) at 0.5 g/kg for 13 weeks induced simple steatosis without obesity. Liver triglyceride and cholesterol levels were increased accompanied by mild elevation of aminotransferase activities. There were no signs of inflammation, insulin resistance, oxidative stress, or fibrosis. CA supplementation increased levels of CA and taurocholic acid (TCA) in enterohepatic circulation and deoxycholic acid (DCA) levels in cecum with an increased ratio of 12α-hydroxylated BAs to non-12α-hydroxylated BAs. Analyses of hepatic gene expression revealed no apparent feedback control of BA and cholesterol biosynthesis. CA feeding induced dysbiosis in cecal microbiota with enrichment of DCA producers, which underlines the increased cecal DCA levels. The mechanism of steatosis was increased expression of Srebp1 (positive regulator of liver lipogenesis) through activation of the liver X receptor by increased oxysterols in the CA-fed rats, especially 4ß-hydroxycholesterol (4ßOH) formed by upregulated expression of hepatic Cyp3a2, responsible for 4ßOH formation. Multiple regression analyses identified portal TCA and cecal DCA as positive predictors for liver 4ßOH levels. The possible mechanisms linking these predictors and upregulated expression of Cyp3a2 are discussed. Overall, our observations highlight the role of 12α-hydroxylated BAs in triggering liver lipogenesis and allow us to explore the mechanisms of hepatic steatosis onset, focusing on cholesterol and BA metabolism.

A low coefficient of variation in hepatic triglyceride concentration in an inbred rat strain.

BACKGROUND: Inbred strains are characterized by less genetic variation, which suggests usefulness of inbred strains for evaluations of various parameters. In this study, experimental reproducibility in several parameters was compared between an outbred Wistar rat and Wistar King A Hokkaido (WKAH/HkmSlc) rat, the inbred strain that is originated from Wistar rats. METHODS: Difference of variations was investigated in parameters of body compositions and liver functions such as body weight, liver weight, liver triglycerides (TG), liver cholesterol and plasma alanine aminotransferase activity (ALT) between WKAH rats and outbred Wistar rats by using the coefficient of variation (CV). RESULTS: There was no difference in the CVs of body weight and relative liver weight between WKAH and Wistar rats. The CVs of body weight and relative liver weight were below 10% in both WKAH and Wistar rats. The CVs of TG, cholesterol, and ALT in Wistar rats were between 30 and 40%, whereas those in WKAH rats were between 10 and 25%. A low CV level of TG was observed in WKAH rats compared to that in Wistar rats regardless of the duration of the experimental period in those rat strains. CONCLUSION: The low CV values in metabolic parameters involved in liver functions in the inbred rats suggested an advantage of using inbred rather than outbred rats for the evaluation of liver lipid metabolism.

Correlation between 12α-hydroxylated bile acids and insulin secretion during glucose tolerance tests in rats fed a high-fat and high-sucrose diet.

BACKGROUND: Previously, we found a significant relationship in a rat study between energy intake and bile acid (BA) metabolism especially 12α-hydroxylated (12αOH) BAs. The present study was designed to reveal relationships among BA metabolism, glucose tolerance, and cecal organic acids in rats fed a high-fat and high-sucrose diet (HFS) by using multivariate and multiple regression analyses in two types of glucose tolerance tests (GTTs). METHODS: Male WKAH/HkmSlc rats were fed with a control or a HFS for 13 weeks. Oral glucose tolerance test (OGTT) and intraperitoneal glucose tolerance test (IPGTT) were performed at week 9 and 11, respectively. BAs were analyzed by using ultra high-performance liquid chromatography-mass spectrometry. Organic acid concentrations in cecal contents were analyzed by using ultra high-performance liquid chromatography with post-column pH buffered electric conductivity method. RESULTS: A positive correlation of aortic 12αOH BA concentration was observed with energy intake and visceral adipose tissue weight. We found that an increase of 12αOH BAs in enterohepatic circulation, intestinal contents and feces in the HFS-fed rats compared to those in control rats regardless of no significant increase of total BA concentration in the feces in the test period. Fecal 12αOH BA concentration was positively correlated with maximal insulin level in OGTT and area under curve of insulin in IPGTT. There was a positive correlation between aortic 12αOH BAs concentration and changes in plasma glucose level in both OGTT and IPGTT. In contrast, a decrease in the concentration of organic acids was observed in the cecal contents of the HFS-fed rats. Multiple linear regression analysis in the IPGTT revealed that the concentrations of aortic 12αOH BA and cecal acetic acid were the predictors of insulin secretion. Moreover, there was a positive correlation between concentration of portal 12αOH BAs and change in insulin concentration of peripheral blood in the IPGTT. CONCLUSION: The distribution analysis of BA compositions accompanied by GTTs revealed a close relationship between 12αOH BA metabolism and insulin secretion in GTTs in rats.

Thermal stability and bioavailability of inclusion complexes of perilla oil with γ-cyclodextrin.

Perilla oil is abundant in α-linolenic acid, which is metabolized to long-chain n-3 fatty acids. This study aimed to determine thermal stability and bioavailability of perilla oil that was powdered by inclusion complexation with γ-cyclodextrin. Fatty acid analysis revealed that the relative abundance of α-linolenic and linoleic acids in the complexes was not affected by heating at 40 °C for six days but decreased after heating at 60 °C for three days. No adverse events occurred in rats fed with an experimental diet containing the complexes for two weeks. Plasma α-linolenic and eicosapentaenoic acids in rats fed with diets containing complexes and liquid perilla oil were equally high, indicating the preserved bioavailability of perilla oil in the complexes. Plasma arachidonic acid decreased only in rats fed with a diet containing the complexes. Results suggest that the complexes have potential as a useful source of α-linolenic acid to increase plasma n-3 fatty acids.

Ingestion of difructose anhydride III partially suppresses the deconjugation and 7α-dehydroxylation of bile acids in rats fed with a cholic acid-supplemented diet.

Difructose anhydride III (DFAIII) is a prebiotic involved in the reduction of secondary bile acids (BAs). We investigated whether DFAIII modulates BA metabolism, including enterohepatic circulation, in the rats fed with a diet supplemented with cholic acid (CA), one of the 12α-hydroxylated BAs. After acclimation, the rats were fed with a control diet or a diet supplemented with DFAIII. After 2 weeks, each group was further divided into two groups and was fed diet with or without CA supplementation at 0.5 g/kg diet. BA levels were analyzed in aortic and portal plasma, liver, intestinal content, and feces. As a result, DFAIII ingestion reduced the fecal deoxycholic acid level via the partial suppression of deconjugation and 7α-dehydroxylation of BAs following CA supplementation. These results suggest that DFAIII suppresses production of deoxycholic acid in conditions of high concentrations of 12α-hydroxylated BAs in enterohepatic circulation, such as obesity or excess energy intake. Abbreviation: BA: bile acid; BSH: bile salt hydrolase; CA: cholic acid; DCA: deoxycholic acid; DFAIII: difructose anhydride III; MCA: muricholic acid; MS: mass spectrometry; NCDs: non-communicable diseases; LC: liquid chromatography; SCFA: short-chain fatty acid; TCA: taurocholic acid; TCDCA: taurochenodeoxycholic acid; TDCA: taurodeoxycholic acid; TUDCA: tauroursodeoxychlic acid; TαMCA: tauro-α-muricholic acid; TßMCA: tauro-ß-muricholic acid; TωMCA: tauro-ω-muricholic acid.

Skatole regulates intestinal epithelial cellular functions through activating aryl hydrocarbon receptors and p38.

Intestinal bacteria produce skatole (3-methylindole) from tryptophan in dietary proteins and ingesting large quantities of animal protein is associated with increased fecal skatole concentrations. Although possibly associated with disrupted intestinal homeostasis, the influence of skatole on intestinal epithelial cellular function has not been characterized in detail. The present study aimed to determine whether skatole induces intestinal epithelial cell (IEC) dysfunction. We found that skatole dose-dependently caused IEC death and time-dependently induced IEC apoptosis. Since skatole directly interacts with aryl hydrocarbon receptors (AhR), we investigated whether these receptors influence the skatole-induced death of IEC. In addition to increased AhR transcriptional activity induced by skatole, the AhR antagonist CH223191 partially suppressed of skatole-induced IEC death. Extracellular signal-related kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) are mitogen-activated protein kinases (MAPK) induced by skatole. None of them were repressed by CH223191, whereas the p38 inhibitor SB203580 promoted skatole-induced IEC death. These findings together indicated that skatole induces both AhR-dependent activation pathways and the AhR-independent activation of p38, consequently regulating the amount of IEC death. Accumulating evidence indicates that consuming large amounts of animal protein is associated with the pathogenesis and progression of inflammatory bowel diseases (IBD). Thus, intestinal skatole production induced by large amounts of dietary animal protein might be associated via IEC death with intestinal pathologies such as IBD.