Tojapride prevents CaSR‐mediated NLRP3 inflammasome activation in oesophageal epithelium irritated by acidic bile salts

Abstract Impairment of the oesophageal epithelium in patients with reflux oesophagitis (RE) is a cytokine‐mediated injury rather than a chemical burn. The present study was conducted to explore CaSR/NLRP3 inflammasome pathway activation and cytokines IL‐1β and IL‐18 release in oesophageal epithelia injured by refluxates and the effects of Tojapride on that signal regulation. Using a modified RE rat model with Tojapride administration and Tojapride‐pretreated SV40‐immortalized human oesophageal epithelial cells (HET‐1A) exposed to acidic bile salts pretreated with Tojapride, we evaluated the therapeutic effects of Tojapride on oesophageal epithelial barrier function, the expression of CaSR/NLRP3 inflammasome pathway‐related proteins and the release of downstream cytokines in response to acidic bile salt irritation. In vivo, Tojapride treatment ameliorated the general condition and pathological lesions of the oesophageal epithelium in modified RE rats. In addition, Tojapride effectively blocked the CaSR‐mediated NLRP3 inflammasome activation in modified RE rats. In vitro, Tojapride treatment can reverse the harmful effect of acidic bile salts, which reduced transepithelial electrical resistance (TEER), up‐regulated the CaSR‐mediated NLRP3 inflammasome pathway and increased caspase‐1 activity, LDH release and cytokines secretion. Taken together, these data show that Tojapride can prevent CaSR‐mediated NLRP3 inflammasome activation and alleviate oesophageal epithelial injury induced by acidic bile salt exposure.


| INTRODUC TI ON
Reflux oesophagitis (RE) is caused by the excessive backflow of gastric/duodenal contents into the oesophagus, which induces inflammatory lesions and ulceration of the oesophageal mucosa, finally causing problematic symptoms and/or complications. RE is one of the major phenotypic manifestations of gastroesophageal reflux disease (GERD). Epidemiological estimates indicate a 10%-20% prevalence of RE in Western countries, whereas the prevalence is <5% in Asian countries. 1 RE represents a complication of GERD in approximately 30% of cases. 2 In the past, RE was generally regarded as a chemical injury of the oesophageal epithelium caused by hydrochloric acid and pepsin. However, RE develops through a cytokine-mediated injury rather than a chemical injury and progresses to the deep layers of the epithelium. In 2009, a study first suggested this new paradigm of RE pathogenesis. 3 A recent clinical trial further validated this hypothesis by interrupting proton-pump inhibitor (PPI) therapy in 12 patients with RE and examining inflammation in oesophageal biopsies,the inflammation was found to be predominantly associated with T lymphocytes with few infiltrating neutrophils and eosinophils, and notably, none of the biopsies showed a loss of surface cells. 4 Typical oesophageal morphological abnormalities of RE are mainly characterized by basal cell hyperplasia and elongation of connective tissue papillae attributed to acid/alkali-associated injury of the epithelial cells 5 As mentioned above, studying the molecular pathway of RE injury is of fundamental importance because it offers insight into the pathogenesis of oesophageal mucosal inflammatory injury.
Calcium-sensing receptor (CaSR) is a G protein-coupled receptor that is widely expressed in intact polarized epithelia along the entire gastrointestinal tract to stimulate epithelial cell differentiation 6 and regulate gastrointestinal immunity. 7,8 Notably, depending on the cell type, CaSR also participates in the regulation of cell proliferation via activatory or inhibitory effects. [9][10][11] Up-regulation of CaSR in HET-1A cells can accelerate the secretion of the cytokine IL- 8 12 which is in accordance with research showing that after brief exposure to acidic bile salts, human oesophageal cells do not die but secrete specific cytokines. 3 The nucleotide-binding and oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a signalling platform of the immune system. 13 Specifically, aberrant overactivation of the NLRP3 inflammasome has recently been identified in Barrett's epithelial cells. 14 Activation of the NLRP3 inflammasome is thought to consist of priming and assembly. The priming signal is usually induced by the Toll-like receptor (TLR)/ nuclear factor-kappa B (NF-κB) pathway, which up-regulates the expression of NLRP3, procaspase-1 and pro-IL-1β, the levels of which are otherwise relatively low in the cytoplasm. The NLRP3 inflammasome is a multiprotein complex that consists of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and procaspase-1. Caspase-1, which is derived from its precursor protein, is an inflammatory caspase that catalyses the proteolytic cleavage of pro-IL-1β to its mature, active forms and then initiates a inflammatory cell death, that is pyroptosis. 15 A variety of molecular mechanisms have been confirmed in the activation of NLRP3 inflammation. 16 Lipopolysaccharide (LPS) is a gram-negative bacterial cell wall component that functions as a pathogen-associated molecular pattern (PAMP) and up-regulates the expression of TLR4 for inflammation priming. 17 In the classic two signal activation (priming and activation) of NLRP3 inflammasome formation in various cell types, NF-κB activation and nuclear translocation in response to TLR4 signalling are postulated to be the initial event in NLRP3 and pro-IL-1β transcription, 18 [23][24][25][26] Psychological factors have drawn much attention in the pathogenesis of GERD and have a negative impact on quality of life (QoL). 27 A recent epidemiological survey from China has further demonstrated that the incidence of GERD was correlated with anxiety and depression, and the QoL of patients was significantly reduced. 28 According to the theory of TCM, Tojapride is effective in smoothing the liver and regulating gastric functions and be applied to the GERD patients with liver-gastric disharmony syndrome, which is similar to that of GERD patients with psychological problems. In the present study, we adopted a modified RE rat model, in which the oesophagus was directly connected to the duodenum with a well-preserved stomach, in combination with psychological irritation to investigate the expression of CaSR and the NLRP3 inflammasome in the oesophageal epithelium and the pharmacological efficacy of Tojapride. Furthermore, we explored the expression of the CaSR/NLRP3 inflammasome in acidic bile salt-stimulated HET-1A cells and whether Tojapride treatment might inhibit that expression.

| Establishment of the RE rat model
Eight-week-old male Sprague Dawley rats (Huafukang Bioscience Co. Inc) (No. 11401300049714), weighing 220 ± 20 g, were housed under standard laboratory conditions (room temperature, 25 ± 1°C; relative humidity, 65 ± 5%; regular air change; and a 12 hour light/dark cycle) and supplied with tap water and standard diet ad libitum. The rats were allowed to acclimate for 7 days before surgical intervention and were then was subjected to oesophagoduodenostomy. 29 A total of 110 rats were operated on, and 12 out of 120 rats were randomly selected as sham group that undergone a simple celiotomy. Fasting for 24 hours postoperatively, the rats gradually returned to normal diet.
All experimental procedures were approved by the Ethics Review Committee for Animal Experimentation of Xiyuan Hospital, China Academy of Chinese Medical Sciences, PR China.

| Establishment of the modified RE rat model with tail clamp simulation
Two weeks postoperatively, the rats were housed 6 per cage with tail clamp stimulation to induce anger and fighting. The second third of each rat's tail was clamped with an iron clip for 20 minutes, and the clamp position was changed at 5-minute intervals. The duration of daily stimulation was 45 minutes and lasted for 1 week.

| Grouping and drug administration
Three weeks postoperatively, excluding the sham group, the remaining rats were randomized into seven groups according to the model and treatment of each group, namely the RE model group, the modified RE model group, low-, medium-and high-dose Tojapride

| Open-field test
The experiments were performed in a relatively quiet environment without obvious interference. The rats were placed individually in the middle of a wooden open-field apparatus with 100 cm length, 100 cm width and 40 cm height. Twenty-five squares were delineated on the ground, nine squares were in the middle field, and 16 squares were in the peripheral region. The activity of each rat over a 5-minute period was video-recorded by a camera over the centre of the open-field ground. The distance covered, duration each rat spent in the whole region and the number of times the rat crossed between squares were measured. 30 To exclude batch variation, rats treated with distilled water were tested at irregular intervals between drug-treated rats. The openfield apparatus was carefully cleaned with 70% ethanol after each trial.

| Gastric fluid collection and detection
Twelve hours before killing, the rats were underwent a celiotomy, and the pylorus was ligated with a 3-0 silk suture. Gastric juice was collected in a centrifuge tube and centrifuged at 3000 g for 10 minutes at 4°C. The pH value of the supernatant was measured using a pH meter

| Quantitative real-time polymerase chain reaction (qPCR) analysis
Oesophageal strips were placed in TRIzol and sonicated. Total RNA was isolated from the tissues treated with chloroform, precipitated with isopropyl alcohol and resuspended in diethyl pyrocarbonate (DEPC)treated water. cDNA was reverse transcribed from total RNA by a In addition, the primer of forward primer 5' CCATGGAGAAGGCTGGG 3' and reverse primer 5' CAAAGTTGTCATGGATGACC 3' was used for the detection of GAPDH, which was used as a reference gene.

| Western blot analysis
Oesophageal strips or cultured cells were collected to extract the protein with RIPA lysis buffer (Beyotime Biotech), which contained 1 mmol/L final concentration of phenylmethanesulfonyl fluoride (PMSF). After complete lysis, the samples were centrifuged at 10 000 g for 5 minutes at 4°C to precipitate the tissue debris. The supernatants were used to measure the protein concentration by a BCA Protein Assay Kit (Beyotime Biotech). The proteins were electrophoresed in 10% SDS-PAGE gels and then transferred to PVDF membranes. After blocking with 5% skim milk for 1 hour at room temperature, the membranes were incubated with the following primary antibodies: CaSR The membrane was washed with TBST and incubated with secondary antibodies for 2 hours at room temperature. Protein bands were visualized on the membrane with a Gel Imaging System, and the protein bands were quantified with ImageJ software.
Mounted sections were subsequently observed under an optical microscope. In addition, the sections used to detect NLRP3-ASC colocalization through immunofluorescence were incubated with FITC-or Cy3-labelled secondary antibodies (Beyotime Biotech).
Images were acquired by a confocal laser scanning microscope (FV1000, Olympus).

| Cell culture
HET-1A cells obtained from American Type Culture Collection (ATCC) were cultured in bronchial epithelial basal medium (BEBM; Lonza) supplemented with 100 U/mL penicillin-streptomycin solution (Invitrogen). The cells were cultured in a humidified incubator containing 5% carbon dioxide (CO 2 ).

| Acid and bile salt exposure
Cells were exposed to acid, bile salts and acidic bile salt media. The media have been described by Xiaofang Huo. 31,32 When the cells reached 75%-80% confluence, they were treated with the different media for three times per day at 10 minutes per treatment.

| TEER
The TEER of the oesophageal epithelial monolayer was measured using a chamber system. HET-1A cells, which were cultured in tran-

| Measurement of caspase-1 activity
The activity of caspase-1 was detected using a Caspase 1 Assay kit (ab39412, Abcam) per the manufacturer's instructions.
All assays were performed in triplicate in three independent experiments.

| Elisas for IL-1β and IL-18
Supernatants from HET-1A cell cultures were collected and centrifuged to remove cell debris. The concentration of IL-1β and IL-18 in the culture supernatants was determined by using a cytokinespecific ELISA kit (Biomart) per the manufacturer's instructions.
All assays were performed in triplicate in three independent experiments.

| Modified RE rats exhibited anxiety-related behaviour and Tojapride ameliorated the anxiety of modified RE rats
Thirty-eight of 120 rats died by week 6 after surgery (survival rates: 62%). First, to determine whether the modified RE rats were predisposed to anxiety state, we used an open-field test to evaluate anxiety-related and autonomous behaviours. We found that modi-

| Modified RE rats exhibited reflux of gastric and duodenal contents, and the Tojapride decreased the pH value, bile acid and pepsin of the collected gastric fluid of modified RE rats
Oesophageal epithelial exposure to gastric and bile acids or pepsin is the predominant outcome of mixed reflux. 33 We examined the pH value, bile acid concentration and relative pepsin concentration in the collected gastric fluid of rats in each group. Because the lumen of the oesophagus and duodenum were interlinked, the rats suffered from oesophagoduodenostomy and had higher pH values and bile acid concentrations than the sham rats ( Figure 2D,E). In addition, modified RE rats exhibited an increased pepsin to pepsinogen ratio, which was further increased in RE rats; however, there was no significant difference between RE rats and sham rats

| Modified RE rats exhibited severe pathological lesions of the oesophageal epithelium, and Tojapride treatment partially reversed the pathological changes
Basal cell hyperplasia and papillary elongation were observed in the oesophageal epithelium of RE rats, and these pathological changes peaked to a degree in modified RE rats. Compared with the markedly thickened epithelium of modified RE rats, the epithelium of rats treated with both Tojapride and omeprazole displayed reversed basal cell and papillary hyperplasia, but not to the extent observed in the epithelium of the sham rats (Figure2G).

| Modified RE rats exhibited up-regulation of the CaSR-mediated NLRP3 inflammasome signalling pathway, and Tojapride effectively blocked CaSRmediated NLRP3 inflammasome activation
To assess whether the CaSR/NLRP3 inflammasome signalling pathway is up-regulated in the impaired oesophagus, we used qPCR and Western blot techniques to test the expression of pathway-related proteins. We found that CaSR, NLRP3, caspase-1, ASC and IL-1β were significantly up-regulated in the oesophagus of modified RE rats (Figure 3).
Immunolocalization studies showed CaSR to be mainly expressed in the basal and suprabasal layers, and NLRP3-ASC was localized in the basal layer and lamina propria ( Figure 4). Moreover, NLRP3 was the only pattern recognition receptor (PRR) expressed in the oesophagus of both modified RE rats and pure RE rats to a greater extent than that in sham rats at the mRNA level. There was no significant difference in NLRC4, NLRP1A and AIM2 expression between RE rats and sham rats ( Figure 3G).   Figure 5A). Pretreatment with Tojapride-L and MCC950, a specific small-molecule NLRP3 inhibitor, blocked acidic bile saltinduced TEER decline, but not completely ( Figure 5B).

| Exposure of HET-1A cells to acid and bile salts accelerates CaSR-mediated NLRP3 inflammasome activation, and Tojapride down-regulates the CaSRmediated NLRP3 inflammasome signalling pathway induced by acidic bile salts in HET-1A cells
To further confirm that the CaSR/NLRP3 inflammasome signalling pathway is up-regulated in the oesophageal epithelium, which is impaired by mixed refluxate, we investigated the expression of CaSR and NLRP3 in HET-1A cells stimulated by acidic bile salt treatment. We found that exposure to acidic bile salts significantly increased the expression of

| Exposure of HET-1A cells to acidic bile salts activates caspase-1 and causes the secretion of proinflammatory cytokines and release of LDH. Tojapride diminishes the pyroptotic cell lesions through caspase-1-mediated cytokine and LDH release
The activation of the NLRP3 inflammasome always accompanies caspase-1-induced pyroptosis, which promotes pore formation and the release of the cytokines IL-1β and IL-18. To show that proinflammatory cytokine release and pyroptosis is induced by acidic bile salts, we

| D ISCUSS I ON
In this study, our exploration of modified RE rats and acidic bile salt-   37 Although PPI therapy for RE is effective, the side effects of PPI therapy have drawn public attention to this problem.
Moreover, the failure of PPI therapy may increase the psychological burden, establishing a vicious cycle. 38 We found that Tojapride is clinically efficacious against GERD, fulfils unmet needs for PPI ther- cells is inhibited in serum-supplemented media because of terminal differentiation, like most epithelial cells. 39 Even though we removed F I G U R E 7 The inhibitory effects of Tojapride on acidic bile salt-induced caspase-1 activity increase, LDH release and IL-1β and IL-18 secretion. A, Caspase-1 activity assay. B, LDH release detection. Enzyme-linked immunosorbent assays for (C) IL-1β secretion and (D) IL-18 secretion. Bar graphs represent the means ± SD. *P < .05 and**P < .05, compared with the control group; #P < .05 and ##P < .01, compared with the acidic bile salt-treated group; and ○P < .05, compared with Tojapride-L-treated group the proteins from Tojapride serum through acetonitrile and nitrogen blowing, some serum molecules produced as the concentration increased from low to high may have caused increased toxicity after drug biotransformation. We postulate that the protective effect of increased concentrations of the drug serum is surpassed by the cytotoxic effect.
In summary, the current study demonstrates that a formula granule of the TCM Tojapride effectively ameliorates the RE-related inflammatory response of the oesophageal epithelium through the CaSR/NLRP3 inflammasome pathway. The discovery of the molecular mechanism behind the anti-inflammatory effect of Tojapride provides insight into the potential application of Tojapride in RE complementary therapy. Z141100002214012 and Z161100000116046).

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial interests. Tang