Tetrahydroxylated bile acids prevents malignant progression of Barret esophagus in vitro by inhibiting the interleukin-1β-nuclear factor kappa-B pathway

Abstract

BACKGROUND

Barrett esophagus (BE), a metaplastic adaptive process to gastrointestinal reflux, is associated with a higher risk of developing esophageal adenocarcinoma. However, the factors and mechanism that drive the malignant progression of BE is not well understood.

AIM

To investigate the role of bile acids, a component of the reflux fluid, in the malignant progression of BE.

METHODS

Using engineered green fluorescent protein- labeled adult tissue-resident stem cells isolated from BE clinical biopsies (BE-ASCs) as the target, we studied the effect of hydrophobic deoxycholic acid (DCA) and hydrophilic tetrahydroxylated bile acids (THBA) on cell viability by fluorescence intensity analysis, mucin production by dark density measurement, tissue structure by pathology analysis, expression of different pro-inflammatory factors gene by quantitative polymerase chain reaction and proteins by Western blot.

RESULTS

We found that hydrophobic DCA has cytotoxic and proinflammatory effects through activation of interleukin-1β (IL-1β)-nuclear factor kappa-B (NF-κB) inflammatory pathway on BE-ASCs. This action results in impaired cell viability, tissue intactness, reduced mucin production, and increased transition to disorganized atypical cells without intestinal features. In contrast, co-culture with hydrophilic THBA inhibited the IL-1β-NF-κB inflammatory pathway with maintenance of mature intestinal type cellular and histomorphology.

CONCLUSION

Our data indicates that the hydrophilic bile acid THBA can counteract the cytotoxic and proinflammatory effect of hydrophobic DCA and prevent the malignant progression of BE by inhibiting the IL-1β-NF-κB pathway.

Key Words: Barrett esophagus; Inflammation; Metaplasia; Mucin; Cell viability; Histology; Tetrahydroxylated bile acids; Deoxycholic bile acid; Pathway

Core Tip: Barrett’s esophagus is a pathological precancerous condition. Hydrophobic bile acids could stimulate processes of malignant transition of Barrett esophagus tissue via activation proinflammatory signaling pathways. This study demonstrated that hydrophilic tetrahydroxylated bile acid could inhibit the cytotoxic and proinflammatory action of hydrophobic deoxycholic acid in adult stem cells isolated from Barrett esophagus. Co-treatment with hydrophilic tetrahydroxylated bile acids maintained the metaplastic intestinal-type cells in their differentiated state with preserved histology and mucin production. Thus, tetrahydroxylated bile acids have a potential protective role against the malignant progression of Barrett esophagus.

INTRODUCTION

Gastroesophageal reflux disease (GERD) is caused by retrograde flow from the stomach into the esophagus. Refluxate contains an admixture of gastric and intestinal contents including a set of different bile acids. It is well known that long-term exposure to reflux leads to chronic inflammation of esophageal tissue and replacement of squamous esophageal epithelium with a mature intestine-like columnar epithelium, an adaptive process first described by Dr. Barrett[1] in 1950 and subsequent named “Barrett esophagus (BE)”. BE is considered a precancerous lesion and is closely related to the occurrence of esophageal adenocarcinoma[2-12]. However, it is still not clear what drives the progression from the adaptive metaplastic process to a malignant dysplastic process in the setting of BE.

Histologically dysplasia is characterized by loss of cell polarity, abnormal tissue architecture, and nuclear atypia[13]. Studies showed that gastric acid in the reflux content was not the primary cause of malignant progression of BE as inhibition of gastric acid secretion using proton pump inhibitors was unable to significantly reduce the incidence of esophageal carcinoma of BE patients[14-16]. On the other hand, hydrophobic bile acids were found to be one of the most important promoters of esophageal cancer initiation[17-21].

Recent studies showed that hydrophilic bile acid can counteract the effect of hydrophobic bile acid[17-20]. Previous investigation by our team and others showed that a hydrophilic bile acid, tetrahydroxylated bile acids (THBA), can mitigate liver damage and reduced formation of hepatocellular carcinoma in Mdr2-deficient mice that has high levels of toxic bile acids[22,23]. However, the potential role of THBA in the setting of BE has not been reported. Besides, THBA are the most potent hydrophilic bile acids characterized in mammals. THBA are significantly more hydrophilic comparatively to ursodeoxycholic acid (UDCA)[24], which is one of the most investigated hydrophilic bile acids and approved for clinical use by the United States Food and Drug Administration[25]. Thus, there was an increased interest in investigating THBA efficiency in contexts of increased hydrophilicity of this bile acid towards the clinically approved UDCA. In the study, we investigated whether the hydrophilic THBA can counteract the toxic hydrophobic deoxycholic bile acid in the gastroesophageal refluxate and protect the esophageal tissue from dysplasia, cancer initiation and progression.

MATERIALS AND METHODS

Cells culture and cytotoxicity study

This study was conducted using engineered green fluorescent protein-labeled (GFP) adult tissue-resident stem cells isolated from BE clinical biopsies (BE-ASCs) following approval by the institutional research ethics board (No. B22-0164). The study abides to the principles of the declaration of Helsinki. BE-ASCs were isolated from the clinical biopsies tissue using a modified green protocol, by digestion in dispase, collagenase type I, trypsin, and DNAse with ROCK inhibitor Y-27632 (Selleckchem) at 37 °C for 1 hour with agitation[26-29]. Cells were cultivated on Matrigel (Corning) and irradiated mouse embryonic 3T3 fibroblasts (Kerafast) feeder layer in optimized expansion culture Chinese Family Assessment Device media contained: 2.5 mmol/L Rock-inhibitor (Calbiochem, United States), 10 mmol/L nicotinamide (Sigma-Aldrich, United States), 1 mmol/L Jagged-1 (AnaSpec Inc, United States), 125 ng/mLR-spondin1 (RD systems, United States), 100 ng/mL human Noggin (Peprotech, United States) and 2 mmol/L SB431542 (Cayman Chemical, United States). Bile acids cytotoxicity was assessed in three dimensional conditions (in 6.5 mm, pore size of 0.4 μm transwell; Corning) according to an optimized experimental scheme (Figure 1). 1 × 10⁶ BE-ASCs were seeded into 12-transwells coated with 20% v/v Matrigel in cold phosphate-buffered saline and irradiated 3T3 mouse embryonic fibroblasts (1 × 10⁵ fibroblasts per transwell). Briefly, BE-ASCs were cultured in an expansion cell medium for 4 days, then induced by air liquid interface (ALI) for 1 day and cultured in differentiation medium contained the same compounds excluding nicotinamide (Figure 1). Under these conditions BE-ASCs can form structures recapitulating Barrettes esophagus tissue (fragments of columnar well-polarized cells and interspersed goblet-like cells).

Figure 1 Scheme of cultivation of adult tissue-resident stem cells isolated from Barrett esophagus clinical biopsies and cytotoxicity investigation using different concentrations of deoxycholic acid, tetrahydroxylated bile acids and their combinations. BE-ASCs: Barrett esophagus clinical biopsies; DCA: Deoxycholic acid; THBA: Tetrahydroxylated bile acids.

On day 6, the cells were treated with different concentration of DCA in differentiating medium (10, 20, 30, 40, 50, 100, 150, 200, 400 μM) and synthetic THBA (200-5000 μM) to determine the optimal concentrations for treatment. Viability was measured after 2 weeks of cultivation. To evaluate viability, the levels of GFP fluorescence were measured and analyzed by using ImageJ software.

Mucin production

BE-ASCs were cultured in the same manner as described above. Levels of mucin production were measured on day 15 of BE-ASCs cultivation either without addition of bile acids (control) or treated with DCA, THBA and their combinations. Measurement was performed using ImageJ software.

Histological analysis

Inserts of membranes with BE-ASC ALI tissues were taken for fixation in 4% paraformaldehyde (15 minutes). All procedures [dehydration, paraffin-embedding, sectioning of ALI tissue, rehydration and hematoxylin and eosin (HE) staining of ALI tissue sections] were performed according to the protocol of Manna and Caradonna[30].

Proinflammatory gene analysis

Quantitative polymerase chain reaction (qPCR) was used to study the expression of proinflammatory genes [interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-1α, cyclooxygenase-2 (COX-2)], and MUC-2 (intestinal marker) in BE-ASCs under the influence of DCA, THBA and their combination. BE-ASCs were cultured using optimized media with or w/o 100 μM DCA, 2000 μM THBA and their combination (2000 μM THBA/100 μM DCA). Total cellular RNA was isolated after 48 hours cultivation treatment using RNeasy mini kit (QIAGEN) according to the manufacturer’s protocol. The RNA was quantified on the Nano Drop 1000 where the quality of message RNA was evaluated by the optical density (OD)₂₆₀/OD₂₈₀ ratio. For reverse transcription reaction, 1 μg of total RNA (high capacity completely DNA Archive kit) was used and 10 ng of transcribed DNA was spent for each qPCR reaction. As target probes, TaqMan IL-6, IL-1β, TNF-1α, COX-2 were used along with a housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Proinflammatory protein analysis

Detection of cleaved (active form) of IL-1β and nuclear factor kappa-B (NF-κB) was conducted using Western blotting. Briefly, BE-ASCs were cultivated with optimized media with or w/o DCA 100 μM, THBA 2000 μM and their combination. Cellular lysates were obtained after 48 hours of cultivation using radioimmunoprecipitation assay lysis buffer (Millipore Sigma) and Halt^TM Protease Phosphatase Inhibitor Cocktail (Thermo Scientific) according to the manufacturer’s protocol. The total protein concentration in the BE-ASCs lysates was detected in a 96-well plate using a Pierce™ bicinchoninic acid protein assay kit (Thermo Scientific) according to the manufacturer’s protocol. Western blot was performed using the protocol of Mahmood and Yang[31]. The following rabbit primary antibodies were used: IL-1β, NF-κB, GAPDH, β-actin and horseradish peroxidase-labeled goat anti-rabbit secondary antibody (Cell Signaling). Incubation membranes with antibodies were conducted according to the manufacturer’s protocol.

Statistical analysis

The results were represented as mean ± SD, and calculated with a two-tailed Student’s t test. Statistical significance was defined as P value of < 0.05, < 0.01, < 0.001 and < 0.0001. All experiments were performed using at least three biological replicates.

RESULTS

THBA protects the viability of DCA-treated BE-ASCs cells

To determine if THBA can protect BE-ASCs from the cellular damage caused by DCA, we tested the dose-dependent effect of each bile acid on these cells (Figure 2). As can be seen in Figure 2A and D, there was no decrease in viability in BE-ASCs at concentrations of up to 50 μM of DCA but there was visible cytotoxicity starting at 100 μM and a significant loss of viability (22-fold) when cells were treated with 150 μM or higher. Since the goal was to avoid extreme toxicity and possible non-specific effects, we chose to use 100 μM as the effective concentration of DCA to be used for this study.

Figure 2 Effect of deoxycholic acid, tetrahydroxylated bile acids and their combination on adult tissue-resident stem cells isolated from Barrett esophagus clinical biopsies cytotoxicity. Representative green fluorescent protein-labeled (GFP) fluorescent images of Barrett esophagus clinical biopsies (BE-ASCs) in transwell inserts after 15 days in culture with various deoxycholic acid (DCA) and hydrophilic tetrahydroxylated bile acids (THBA) concentrations. 1 × 10⁶ BE-ASCs were plated in 12 Matrigel coated transwell inserts on the feeder layer of irradiated 3T3 mouse embryonic fibroblasts (1 × 10⁵ fibroblasts per transwell). A: BE-ASCs cultured with 100 μM DCA showed slight cytotoxicity and significant decreasing of viability (22-fold) after treatment with 150 μM DCA or higher; B: BE-ASCs treated with 3000 μM THBA or higher demonstrated significant decreases in viability; C: BE-ASCs co-treated with 2000 μM THBA/150 μM DCA showed 17-fold higher viability in comparison to cells treated with DCA alone; D-F: Viability levels of BE-ASCs treated with DCA (D), THBA (E), their combination (F), were correlated with GFP fluorescence intensity measured using ImageJ software. Averages from triplicates are presented as mean ± SD (n = 3). ^cP < 0.001. ^dP < 0.0001. DCA: Deoxycholic acid; THBA: Tetrahydroxylated bile acids.

In a similar manner, we determined the highest non-toxic concentration of THBA that can be used in this system. As can be seen in Figure 2B and E no significant cytotoxicity was observed in BE-ASCs treated with THBA using concentrations ranging from 200-2000 μM; however, significant cytotoxicity was observed at 3000 μM and increasing to greater than 20 fold at 4000 and 5000 μM of THBA. We therefore chose 2000 μM of THBA to test the potential protective effect of this bile acid.

We next tested if the presence of THBA can protect EB-ASCs from the cellular damage caused by DCA using these two elected concentrations of bile acids (Figure 2C and F). We observed that co-treatment with 2000 μM THBA/150 μM DCA resulted in significantly higher cell viability (17-fold) compared with treatment with DCA alone.

THBA counteracted the inhibitory effect of DCA on BE-ASCs mucin production

Mucin production is an important gastro-intestinal functional indicator of the mucosa tissue and plays an important role in tissue protection. We investigated whether THBA could preserve mucin secretion in BE-ASCs exposed to DCA. As demonstrated (Figure 3), 150 μM DCA inhibits mucin secretion in BE-ASCs. The same cells co-treated with THBA (2000 μM) restored mucin production (the level of secretion was 17-fold higher in comparison to the cells treated with 150 μM DCA alone).

Figure 3 Effects of deoxycholic acid and tetrahydroxylated bile acids co-exposure on adult tissue-resident stem cells isolated from Barrett esophagus clinical biopsies mucin production. Representative bright field images of Barrett esophagus clinical biopsies (BE-ASCs) in transwell inserts. A: BE-ASCs treated with 2000 μM tetrahydroxylated bile acids restored mucin production (the level of secretion was 17-fold higher in comparison to the cells treated with 150 μM deoxycholic acid alone); B: Mucin levels were correlated with dark density measured by ImageJ software. Data are presented as mean ± SD (n = 3). ^dP < 0.0001. DCA: Deoxycholic acid; THBA: Tetrahydroxylated bile acids.

THBA supports intestinal differentiation in DCA-treated BE-ASCs

Inflammation, cellular disarray, tissue fragmentation and loss of goblet cells are early histological signs of progression from metaplasia to dysplasia. We investigated the effects of DCA and THBA on tissue histology using a model of ALI with transwell inserts. HE-stained tissue sections revealed a high level of differentiation in samples treated with THBA and control. There were interspersed goblet-like cells between well polarized columnar intestine-like cells resembling mature intestinal mucosa. Samples treated with DCA showed attenuation of the cell layer and reduced number of goblet-like cells, which becomes more severe with increasing DCA concentration. BE-ASCs treated with combination DCA/THBA showed high levels of intestinal differentiation. (Figure 4). Further, we examined the expression of MUC2, a molecular marker of intestinal differentiation. As shown in Figure 5, DCA treatment decreased MUC2 expression by 2 to 2.3-fold compared to untreated BE-ASCs, while THBA co-treatment was able to significantly restore MUC2 expression in DCA treated cells to over 50 percent of control. These data indicate that THBA can counteract the damaging effect of DCA by preserving intestinal differentiation in the metaplastic mucosa.

Figure 4 Effects of deoxycholic acid and tetrahydroxylated bile acids co-exposure on adult tissue-resident stem cells isolated from Barrett esophagus clinical biopsies intestinal differentiation. Representative pictures of hematoxylin and eosin stained air liquid interface Barrett esophagus clinical biopsies tissue sections (× 20 magnification). Samples treated with 2000 μM tetrahydroxylated bile acids (THBA), co-treated with THBA and deoxycholic acid (DCA) (2000 μM THBA/150 μM DCA, 2000 μM THBA/100 μM DCA) and control demonstrated high level intestinal differentiation with distinct interspersed goblet-like cells between well polarized columnar intestine-like cells resembling mature intestinal mucosa. Samples treated with DCA alone showed attenuation of the cell layer, reduced number of goblet dells and nuclear atypia. (¹goblet-like cells, ²columnar polarized intestine-like cells). DCA: Deoxycholic acid; THBA: Tetrahydroxylated bile acids; Scale bar = 250 μm.

Figure 5 Effects of deoxycholic acid and tetrahydroxylated bile acids co-exposure on MUC2 gene expression in adult tissue-resident stem cells isolated from Barrett esophagus clinical biopsies. Barrett esophagus clinical biopsies cells treated with deoxycholic acid (DCA) showed decreased level of MUC2 expression by 2.3-fold in comparison to control cells, while tetrahydroxylated bile acids co-treatment significantly restored MUC2 expression in DCA treated cells to over 50 percent of control. Data are presented as mean ± SD (n = 3). ^bP < 0.01. ^cP < 0.001. DCA: Deoxycholic acid; THBA: Tetrahydroxylated bile acids.

THBA inhibits IL-1β-NF-κB pathway in DCA-treated BE-ASCs

Since one of the most important actions of hydrophobic bile acids is inflammatory stimulation[32-35], we studied the influence of DCA on gene expression of the most important inflammatory markers (IL-1β, IL-6, COX-2, TNF-α) and effect of THBA in combined treatment of BE-ASCs. We did not observe significant differences in the gene expression IL-6, COX-2, TNF-α in the cells treated with DCA, THBA, their combination and control (Figure 6). However, we found significant 2-fold increase in IL-1β gene expression after 48 hours exposure with 100 μM DCA (Figure 6).

Figure 6 Effects of deoxycholic acid and tetrahydroxylated bile acids co-exposure on proinflammatory (interleukin-6, interleukin-1β, tumor necrosis factor-α, cyclooxygenase-2) genes expression in adult tissue-resident stem cells isolated from Barrett esophagus clinical biopsies. Barrett esophagus clinical biopsies treated with deoxycholic acid, tetrahydroxylated bile acids, their combination did not demonstrate significant difference in the gene expression interleukin-6, cyclooxygenase-2, and tumor necrosis factor-α. At the same time, these cells demonstrated significant 2-fold increase in interleukin-1β gene expression after cultivation with 100 μM deoxycholic acid. Data are presented as mean ± SD (n = 3). ^aP < 0.05. ^bP < 0.01. IL: Interleukin; TNF-α: Tumor necrosis factor-α; COX2: Cyclooxygenase-2; DCA: Deoxycholic acid; THBA: Tetrahydroxylated bile acids.

Western blot analysis demonstrated that IL-1β is expressed in its bioactive form (17 kDa) in BE-ASCs (Figure 7). Treatment with 100 μM DCA increases IL-1β protein expression in BE-ASCs by 2-fold versus non-treated cells. THBA plus DCA co-exposure resulted in significant decrease of IL-1β expression vs DCA alone (Figure 7A and B).

Figure 7 Effect of deoxycholic acid and tetrahydroxylated bile acids co-exposure on interleukin-1β-nuclear factor kappa-B pathway activity in adult tissue-resident stem cells isolated from Barrett esophagus clinical biopsies. Representative immunoblots demonstrating effects of deoxycholic acid (DCA) and tetrahydroxylated bile acids (THBA) co-exposure on interleukin-1β (IL-1β)-nuclear factor kappa-B (NF-κB) pathway activity in Barrett esophagus clinical biopsies (BE-ASCs). A: Cells treated with 100 μM DCA showed significant 2-fold increase of IL-1β protein expression in comparison to untreated cells. THBA and DCA co-exposure resulted in significant decrease of IL-1β expression vs DCA alone; B: Expression levels of IL-1β in BE-ASCs treated with 100 μM DCA, 2000 μM THBA and their combination-2000 μM THBA/100 μM DCA, were correlated with density levels measured by using ImageJ software; C: BE-ASCs treated with DCA demonstrated significant higher level of NF-κB phosphorylation in comparison to untreated cells and treated with combination THBA/DCA, THBA; D: Expression levels of NF-κB in BE-ASCs treated with 100 μM DCA, 2000 μM THBA and their combination-2000 μM THBA/100 μM DCA, were correlated with density levels measured by using ImageJ software. Data are presented as mean ± SD (n = 3). ^aP < 0.05. ^bP < 0.01. IL: Interleukin; DCA: Deoxycholic acid; THBA: Tetrahydroxylated bile acids; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; NF-κB: Nuclear factor kappa-B.

IL-1β inflammatory factor is one of the important participants of the NF-κB pathway which in turn is a central regulator of inflammation. NF-κB signaling promotes the development of different cancers including esophageal adenocarcinoma[36-39]. Using Western blot analysis, BE-ASCs exposed with DCA showed around 1.5 folds higher level of phosphorylated (active form p-p65) NF-κB comparatively to the cells treated with combination THBA/DCA, THBA and untreated cells (Figure 7C and D). These results suggest THBA may prevent dysplasia and esophageal cancer development through inhibiting the NF-κB signaling pathway.

DISCUSSION

BE is a process of trans differentiation from squamous cells into goblet-cell containing mature intestinal mucosa cells. The stem cells of the distal esophagus and gastro-esophageal junction are thought to be the origin of BE and may be affected by biliary reflux[40]. BE is considered an aberrant wound healing process that protects the damage from the reflux as the intestinal mucosa is supposedly more resistant to digestion and toxic effect of intestinal contents in the reflux[41]. However, the mechanisms that regulate wound healing have been shown to promote transformation and growth of malignant cells. As a matter of fact, chronic inflammation has been associated with malignant transformation in many tissues[42]. Timely blockage of the inflammatory process in the metaplastic mucosa has the potential to maintain tissue homeostasis and prevent the malignant progression in the setting of BE. In this study, using cell viability, mucin production, MUC2 expression, and histological analysis, we found that hydrophilic bile acid THBA could counteract the toxic effect of hydrophobic bile acid DCA on BE-ASCs. Further analysis using PCR and Western blot indicate that the protective effect of THBA is at least in part mediated through the inhibition of IL-1β-NF-κB inflammatory pathway. Our study provided experimental evidence that hydrophilic bile acid THBA has the potential to prevent malignant progression of BE through inhibition of the inflammatory process caused by continuous reflux and exposure to toxic hydrophobic bile acid in the reflux content.

Progression of BE to adenocarcinoma is strongly influenced by bile acid exposure[21]. The hydrophobic bile acid DCA, in particular, has been shown to be genotoxic to esophageal cells through inducing the release of reactive oxygen species within the cytoplasm of exposed cells[43-47]. In turn, DNA damage caused by DCA increase the possibility of oncogenic mutations. In addition, hydrophobic bile acids have the ability to induce damage in the cell membrane through lipid peroxidation, and induction of apoptosis through different pathways[45,48,49]. The mechanism of cytoprotective action of THBA needs further investigation. We assume that THBA can be involved in similar antioxidant mechanisms as another hydrophilic acid UDCA. It has been demonstrated that hepatocytes pretreated with UDCA increased level of thiol-containing proteins which effectively neutralized hydroxyl radicals[45,50].

The present investigation demonstrated the ability of THBA to commit BE-ASCs in intestinal state, with saving functional and morphological properties of intestinal (metaplastic) tissue. We have shown that THBA treatment restored in 17 times mucin production in DCA treated BE-ASCs, which was further confirmed at the molecular level using qPCR analysis of MUC2 gene expression. MUC2 expression was significantly higher in BE-ASCs treated with a combination of THBA/DCA compared to cells treated with DCA alone (Figure 5). Moreover, THBA treatment alone increased MUC2 gene expression compared to untreated BE-ASCs. These results are consistent with previous investigation demonstrating that DCA decreased mucin levels in differentiated enterocyte-like cells, which can be prevented by co-incubation with another hydrophilic tauroursodeoxycholic acid[44]. We speculate that stimulation of mucin production can prevent damage to the mucosal layer and protect underlying cells susceptible to toxins or carcinogens. Thus, THBA can attenuate the transition from metaplasia to dysplasia and cancer initiation induced by hydrophobic bile acid.

Further, the present investigation revealed that THBA could suppress the activity of IL-1β-NF-κB proinflammatory pathway induced by DCA. Similar phenomenon has been observed in previous investigations with using another hydrophilic acid UDCA which inhibited IL-1β and DCA induced activation of NF-κB in human colon cancer cells[49,51]. In addition, it has been shown that UDCA inhibits expression of the pro- inflammatory factors IL-1β and nitric oxide and suppressed NF-κB activation in lipopolysaccharides-exposed (inflammatory inducer) rat, a model of neuroinflammation associated with neurodegeneration[52,53]. Miura et al[54] revealed that UDCA can activate glucocorticoid receptor (GR) with consecutive suppression of NF-κB-dependent transcription through the intervention of interaction between GR and phosphorylated unit of NF-κB p65. This study suggests that THBA may exploit similar molecular mechanisms of inactivation of IL-1β-NF-κB pathways as UDCA.

One of the limitations of our study is its sample size. Although the experiment was repeated at least three times, further research with expanded sample size could help to improve the generalization of the results. Secondly, in vivo studies using gastroesophageal reflux-BE animal models are required to further validate these in vitro results. Thirdly, more in-depth molecular studies using advanced target discovery technologies such as proteomics and proteolysis-targeting chimeras probe can be applied to further explore the underlying specific molecular mechanism and targets of THBA[55]. Lastly, the safety and efficacy of THBA needs to be well studied in both small and large animals before introducing it into clinic trials. If proved safe and effective for preventing gastroesophageal cancer in the clinical setting, we will further explore the role of THBA in other types of cancer such as cancer of the gastric antrum or other diseases that affects the metabolism and secretion of bile acids.

CONCLUSION

In summary, we have demonstrated that the hydrophilic bile acid THBA could counteract the toxic effect of hydrophobic bile acid DCA, maintain BE tissue in metaplastic intestinal-like state and hamper transition to “oncogenic” phase: dysplasia and adenocarcinoma initiation. Our data indicate that the protective effect of THBA is at least in part mediated through the inhibition of IL-1β-NF-κB inflammatory pathway. If validated in preclinical trials, THBA could be a viable preventive regimen for patients with BE that has ongoing gastroesophageal reflux.