Pancreatobiliary reflux increases macrophage-secreted IL-8 and activates the PI3K/NFκB pathway to promote cholangiocarcinoma progression.

Tingting Wu: Writing – original draft, Formal analysis. Ruiqian Gao: Writing – original draft, Data curation. Xiaowei Wang: Writing – review & editing, Formal analysis. Dong Guo: Writing – review & editing, Formal analysis. Yuwei Xie: Formal analysis, Methodology, Supervision. Bingzi Dong: Formal analysis, Software, Writing – review & editing. Xiwei Hao: Supervision, Validation, Writing – review & editing. Chengzhan Zhu: Funding acquisition, Supervision, Validation.

In the investigation of the prevalence of PBR in patients diagnosed with biliary disorders, we conducted a quantitative assessment of biliary amylase concentration within the resected gallbladders of 227 subjects. The findings indicated that in 65.2 % of cases (148/227), the biliary amylase levels surpassed the upper threshold established by the serum amylase criteria of our institution. This observation aligns with the diagnostic criteria for PBR. For the purpose of our study, individuals whose biliary amylase levels exceeded the upper limit (135 IU/L) were classified as the high amylase bile population, while those below were enrolled as low amylase patients. Table 1 delineates the distribution of pancreaticobiliary reflux across various biliary diseases. Notably, the top three conditions exhibiting a higher prevalence of pancreaticobiliary reflux were adenomyosis of the gallbladder (81.5 %), gallbladder stones (68.9 %), and the co-occurrence of gallbladder stones with either adenomyosis of the gallbladder or gallbladder polyps (68.4 %). Further exploration of clinical factors associated with pancreaticobiliary reflux is presented in Table 2 . The statistical analysis unveiled a significant association ( p 45 U/L). Examination of the data elucidates a notably higher incidence of pancreaticobiliary reflux among female patients compared to their male counterparts. Table 1 The proportion of pancreaticobiliary reflux in different biliary diseases Table 1 Diseases BA < 135 U/L(N) BA ≥ 135 U/L(N) BA ≥ 1000 U/L(N) Cholecystolithiasis 32 (31.1 %) 71 (68.9 %) 11 Gallbladder polyps (GP) 19 (55.9 %) 15 (44.1 %) 4 Gallbladder adenomyosis (GA) 5 (18.5 %) 22 (81.5 %) 1 Cholecystolithiasis with GP or GA 6(35.3 %) 11(64.7 %) 0 Cholecystolithiasis with Choledocholithiasis 12(31.6 %) 26(68.4 %) 8 Gallbladder carcinoma (GC) 5(62.5 %) 3(37.5 %) 0 Total 79(34.8 %) 148(65.2 %) 24 Table 2 The clinical factors correlated with pancreaticobiliary reflux Table 2 Variables BA 60 years) 32(42.1 %) 65(44.2 %) 0.763 Symptom  Abdominal pain 52(67.1 %) 114(77.6 %) 0.092  Nausea 15(19.7 %) 41(27.9 %) 0.183  Pyrexia 8(10.5 %) 11(7.5 %) 0.440 Gallbladder wall thickness (>0.3 cm) 32(42.1 %) 93(63.3 %) 0.003 BMI (>24 kg/m 2 ) 37(52.1 %) 73(56.6 %) 0.543 Total bilirubin (>22 μmol/L) 19(25.0 %) 54(36.7 %) 0.087 GGT (>45 U/L) 13(18.3 %) 45(34.9%) 0.013 The proportion of pancreaticobiliary reflux in different biliary diseases The clinical factors correlated with pancreaticobiliary reflux To investigate the effect of high-amylase bile on bile duct epithelial cell, we treated human intrahepatic biliary epithelial cell (HIBEpiC) with different amylase concentration bile. The γH2AX expression levels were elevated after the cells were treated with high-amylase bile compared to low-amylase bile ( Fig. 1 B), indicating the DNA damage was induced high-amylase bile. Then the gallbladder mucosa was performed RNA-sequencing to investigate the transcriptome change in PBR patients. The enrichment analysis of the differentially expressed genes indicated cytokine-cytokine receptor interactions was significant ( Fig. 1 C). Then we measured the concentration of tumor-related inflammatory factor in the bile from 28 patients with Elisa. The IL-8, IL-17A, and IL-13 levels were significantly higher in the high-amylase bile than in the low-amylase bile ( p < 0.0001; Fig. 1 D). The IL-4 levels were slightly lower in subjects with high-amylase bile than in those with low-amylase bile ( p < 0.05; Fig. 1 D). Fig. 1 Pancreaticobiliary reflux incidence and high-amylase bile induced DNA damage and inflammatory changes in gallbladder tissues and cells. Fig 1 (A) Bile amylase concentration in 200 patients with biliary disease. (B) High-amylase bile treatment increases γ-H2AX immunofluorescence intensity. (IF images are 20× magnification). (C) Volcano plot and KEGG enrichment analysis showed the differentially expressed genes and enriched pathways comparing high- with low-amylase bile. (D) The concentration of IL-4, IL-17A, IL-13 and IL-8 levels in high- and low-amylase bile. (E) and (F) Effects of different concentrations of amylase bile on cell viability of HUCCT1 and QBC939. Results are expressed as mean ± SD ( n = 3). Statistically significant differences are as follows: * p < 0.05; ** p < 0,01; *** p < 0.001; Student's t-test. Pancreaticobiliary reflux incidence and high-amylase bile induced DNA damage and inflammatory changes in gallbladder tissues and cells. To investigate the biological effect of high-amylase bile on cancer cells, we selected HUCCT and QBC939 cell lines for further study. Cell activity changes as the bile concentration increases. In low-amylase bile, cell activity was not significantly altered. In high-amylase bile (>16U/L), cell activity gradually decreases with the increase of bile concentration ( Fig. 1 E, F). The final concentration of high-amylase bile was selected 8 U/L, and the corresponding concentration of low-amylase bile was 0.1 U/L at the same dilution of bile ( Fig. 1 E, F). In CCK-8 assay and colony formation, cholangiocarcinoma cells showed significantly higher cell viability ( Fig. 2 A) and more colony formation in the high-amylase bile group compared with the low-amylase bile treatment ( Fig. 2 B). The high-amylase bile also promoted cellular migration and invasion in wound healing and Transwell experiment compared to low-amylase bile treatment group ( Fig. 2 C, D). Fig. 2 High-amylase bile promoted the cellular progression of cholangiocarcinoma cells Fig. 2 (A) Effect of high-amylase bile on HUCCT1 and QBC939 cells proliferation determined by CCK-8 assay. (B) Effect of high-amylase bile on cholangiocarcinoma cellular colony formation. (C) Transwell assay indicates high-amylase bile promoted of cells migration and invasion (Images are 10× magnification). (D) Wound healing assays examined the effects of high and low-amylase bile on the migration of HUCCT1 and QBC939 cells (Images are 10× magnification). Results are expressed as mean ± SD ( n = 3). Statistically significant differences are as follows: * p < 0.05; ** p < 0,01; *** p < 0.001; Student's t-test. High-amylase bile promoted the cellular progression of cholangiocarcinoma cells To validate the expression and investigate the origin of up-regulated inflammatory factors, we performed RT-PCR to confirm the expression in different cells including human umbilical vein endothelial cells (HUVEC), bile duct epithelial cell (HIBEpiC) and macrophage (M1) differentiated from monocytes (THP-1). The high-amylase bile showed no effect on HUVEC ( Fig. 3 A). IL-8 was significantly increased in HIBEpiC cells after treatment of high-amylase bile ( Fig. 3 B). Immunofluorescence staining showed that high-amylase bile promoted IL-8 expression in HIBEpiC cells ( Fig. 3 C). It was found that high amylase bile caused an elevation of the reactive oxygen species level in THP-1 ( Fig. 3 D). And the high-amylase bile promoted cellular expression of IL-8, IL-13 and IL-17A in M1 cell ( Fig. 4 B) which was differentiated from THP-1 cells. The M1 phonotype was verified by CD14, IL-6 and CD80 expression ( Fig. 4 A). Immunofluorescence results demonstrated that high amylase bile promoted IL-8 expression in M1 cells ( Fig. 4 C). Heightened levels of IL-8 are induced in response to oxidative stress, a phenomenon initiated by polarized macrophages. This mechanism is orchestrated through the activation of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), phosphorylated p38 (p-p38), and phosphorylated c-Jun N-terminal kinase (p-JNK) proteins. Notably, these proteins are phosphorylated and thus clearly distinguishable from their unphosphorylated counterparts (ERK1/2, p38, and JNK) ( Fig. 4 D). The IL-8 concentration in cell supernatant of HUVEC, HIBEpiC and M1cells was verified by ELISA ( Fig. 4 E). Fig. 3 The expression and cellular origin of the inflammatory factors in HUVEC and HIBEpiC. Fig. 3 (A) and (B) qRT-PCR results of IL-8, IL-13 and IL-17A mRNA expression in HUVEC and HIBEpiC cells after high- or low- amylase bile treatment. Results are expressed as mean ± SD ( n = 3). (C) Immunofluorescence staining with CY3-labeled secondary antibodies was used to analyze the expression of IL-8 in HIBEpiC cells (IF images are 20× magnification). (D) Detection of intracellular reactive oxygen species after high and low amylase bile treatment of THP-1 (IF images are 20× magnification). Results are expressed as mean ± SD ( n = 3). Statistically significant differences are as follows: * p < 0.05; ** p < 0,01; *** p < 0.001; Student's t-test. Fig. 4 The expression IL-8 in macrophage differentiated from THP1 cell. Fig. 4 (A) mRNA expression of M1 macrophage markers after inducing differentiation. (B) IL-8 mRNA expression in M1 macrophages after treatment with high- or low-amylase bile. (C) Immunofluorescence staining using CY3-labeled secondary antibodies was used to detect IL-8 in M1 macrophage cells (IF images are 20× magnification). (D) Detection of protein expression of p-Erk1/2, p-JNK, p-P38 and IL-8 under the effects of high and low amylase. (E) IL-8 secretion in the culture medium was measured by ELISA after treatment with bile. Results are expressed as mean ± SD ( n = 3). Statistically significant differences are as follows: * p < 0.05; ** p < 0,01; *** p < 0.001; Student's t-test. The expression and cellular origin of the inflammatory factors in HUVEC and HIBEpiC. The expression IL-8 in macrophage differentiated from THP1 cell. To further study whether IL-8 enhances cell progression, we treated cholangiocarcinoma cells with IL-8. Through concentration gradient screening, 50 ng/mL was finally selected as the experimental drug concentration ( Fig. 5 A). The IL-8 promoted cellular proliferation and colony formation of HUCCT and QBC939 cells through CCK8 assay ( Fig. 5 B) and colony formation assay ( Fig. 5 C). And IL-8 also promoted cellular migration and invasion of HUCCT and QBC939 cells, proved by wound healing assay ( Fig. 5 D) and Transwell experiment ( Fig. 5 E). To investigate the mechanism by which high amylase bile regulates cells progression and migration, we explored whether high amylase bile could affect the expression of EMT-related proteins. The high amylase-treated group promoted the expression of N-calmodulin and Vimentin proteins and inhibited the expression of E-cadherin in HIBEpiC, HUCCT1 and QBC939 cells ( Fig. 5 F). In the following study, PI3K phosphorylation was discovered to be increased by IL-8. The elevated phosphorylation level of NFκB is also further activated by AKT to IκBα ( Fig. 5 G). Fig. 5 The IL-8 promoted cholangiocarcinoma cell progression. Fig. 5 (A) Using CCK8 Assay to measure the cell viability of cholangiocarcinoma cells after treated with IL-8 at different concentrations. (B) The effect of IL-8 on HUCCT1 and QBC939 cells proliferation was detected by CCK-8 assay. (C) Effect of IL-8 on cellular colony formation. (D) IL-8 promotes migration and invasion of cells in Transwell assay (Images are 10× magnification). (E) Wound healing assay examining the effect of IL-8 on cells migration (Images are 10× magnification). (F) IL-8 promoted the expression of N-cadherin and Vimetin proteins and decreased the expression of E-cadherin. (G) The protein expression of p-PI3K, p-AKT, p-IκBαand p-NFκB was detected by western blot. The IL-8 promoted cholangiocarcinoma cell progression.

Two hundred patients who underwent surgical treatment for biliary diseases at our hospital between August 2022 and October 2023 participated in this study. The exclusion criteria were as follows: 1) history of percutaneous transhepatic gallbladder drainage, cholangiojejunostomy, or sphincterotomy; 2) gallstone in Hartmann's pouch, acute pancreatitis, suppurative cholecystitis, or gallbladder perforation; and 3) bile that was too thick or too small in volume to be tested. This study was approved by the Ethics Committee of the Affiliated Hospital of Qingdao University. Informed consent was obtained from all patients. The clinicopathological variables included symptoms; age; sex; BMI; total bilirubin, gamma-glutamyl transferase (GGT), and BA levels; pathological diagnosis; and gallbladder wall thickness. Bile specimens were obtained from the gallbladder during surgery and were testes at a clinical laboratory for amylase concentration. Continuous variables were converted into categorical variables using the upper limit of the normal (ULN) as the cutoff value. PBR was diagnosed when the BA concentration was >135 IU/L (ULN serum amylase). Two human cholangiocarcinoma cell lines (HUCCT1 and QBC939) were purchased from the Cell Bank of the Shanghai Institute for Biological Sciences (Chinese Academy of Sciences, Shanghai, China). Human intrahepatic biliary epithelial cells (HIBEpiCs) were purchased from ScienCell (ScienCell, USA). The cell lines were cultured in high glucose-DMEM (Procell, China) supplemented with 10 % fetal bovine serum (FBS; Biological Industries, Israel) and 1 % Penicillin-Streptomycin-Gentamicin Solution (Solarbio, China). Human monocytic THP-1 (Procell) cells were cultured in Roswell Park Memorial Institute medium (Procell) culture medium with 10 % of heat inactivated FBS (Procell) and supplemented with 0.05 mM β-mercaptoethanol and 1 % Penicillin-Streptomycin Solution (Procell). THP-1 monocytes were differentiated into macrophages by incubation with 150 nM phorbol 12-myristate 13-acetate (Sigma, Germany) for 24 h, followed by incubation in the original medium for 24 h. Then it was incubated with 20 ng/mL IFN-γ (R&D Systems, USA) and 10 pg/mL LPS (Solarbio) to polarize it from M0–M1. They were cultured together in a humidified incubator at 37 °C with 5 % CO 2 . Cells were treated with filtered bile containing varying concentrations of amylase or bile containing varying concentrations of IL-8. HIBEpiC were seeded at a density of 6 × 10 4 cells/well in 24-well plates. The cells were washed three times with phosphate-buffered saline (PBS), fixed with 5 % paraformaldehyde (Beyotime, China) for 20 min in cell immunostaining fixative (Beyotime), and then washed three times with staining and washing solution (Beyotime). Add γ-H2AX rabbit monoclonal antibody (Beyotime) and incubate at room temperature for 1 h. We washed with the washing liquid three times; then, anti-rabbit 488 (Beyotime) was added, incubated at room temperature for 1 h, and washed three times with washing liquid. Subsequently, 100 μL of nuclear staining solution (DAPI) was added and stained at room temperature for about 5 min. The culture medium was discarded. The cells were washed with PBS and observed under a fluorescence microscope (Nikon ECLIPSE Ts2R; Shanghai, China). Quantification of the proinflammatory cytokine IL-8 in the bile and culture media of high-amylase or low-amylase-bile treated THP-1 cells using a human IL-8 (CXCL8) ELISA kit (Ruixin Biotech, China). The absorbance was measured at 450 nm using an enzyme label analyzer (Rayto RT-6100). IL-4, IL-13, and IL-17A in the cell culture medium were measured using the corresponding human IL-4, IL-17 and IL-13 ELISA kits (Ruixin Biotech) [16] . To judge cell viability after treatment with amylase bile and IL-8 at different concentrations, Cell Counting Kit 8 (CCK-8) (Beyotime) was used. Cholangiocarcinoma cells were incubated with high- or low-amylase bile or IL-8 for 24 h. The cells were then evenly spread in a 96-well plate at a density of 5 × 103 cells/well. At the designated time, CCK-8 reagent (10 μL) was added to each well and reacted at 37 °C for 1 h. The corresponding absorbance (450 nm) was measured using a multiwell plate spectrometer. After incubating HUCCT1 and QBC939 cells with bile and IL-8 for 24 h, the cells were trypsinized and dispersed in fresh medium to a final concentration of 1 × 105 cells in 1 mL. Subsequently, 100 μL/well of cell suspension was added evenly into the 96-well plate. There were six subholes in each group. Humidity was maintained and cells were cultured at 37 °C, 5 % CO 2 for 24, 48, 72, 96, and 120 h. Then, add 100 μL master mix (10 μL CCK-8 reagent + 90 μL DMEM). The cells were added to each well, counted, and incubated for 2 h. The absorbance of each well was measured at 450 nm using a microplate reader. Overall, 1000 HUCCT1 or QBC939 cells were cultured in complete medium, maintained at this cell density, added to each well of a six-well plate and cultured for 14 d. The cell colonies were fixed with paraformaldehyde then stained with crystal violet. Finally, count the number of colonies in the clusters after the six-well cells were propagated to determine the colony-forming ability of the cells. The invasive ability of cholangiocarcinoma cells was determined using 24-well Millicell plates. Eight micrometer pore Millicell chambers were coated with 30 μg of Matrigel (BD Biosciences, NJ, USA). A cell suspension of 300 μL (1.0 × 105) in serum-free medium (DMEM) was added to the upper chamber, and 500 μL of high-glucose DMEM containing 10 % fetal calf serum was added to the lower chamber as an attractant. After incubation in a cell box for 24 h, the stably adherent cells were fixed with methanol and stained with 0.5 % crystal violet. Residual stained components were removed from the upper chamber by cleaning and wiping. Three areas were randomly selected to photograph the migrating cells. Migration experiments were performed as described above without Matrigel in a Millicell chamber. Cholangiocarcinoma cells were trypsinized and resuspended in fresh media after treatment with diluted bile containing different concentrations of amylase or IL-8. The final cell concentration was 1 × 10 6 cells/ml. Then add the cell suspension (1 mL/well) into a 6 mm petri dish mixed with 4 mL of serum-free DMEM at 37 °C. After 12 h, the dish was scratched with a pipette tip and the width was recorded. The cells were then cultured for 24 h. The widths of the wound gaps were recorded and photographed. Total RNAs was extracted from treated cells using RNA-easy isolation reagent. Then, cDNA was synthesized using the HiScript® II Q RT SuperMix for qPCR (+gDNA wiper) Kit (Vazyme, Nanjing, China), the reaction system was described in our previous study [17] . Upon completion, mRNA expression was quantified using the SYBR Green/ROX qPCR Master Mix on a Roche 480 system. Cells were co-cultured with different treatments for 12 h, and ROS levels were detected using an ROS Assay Kit (Solarbio). Cells were washed three times with PBS then incubated with a ROS fluorescent probe (2,7-dichlorodihydrofluorescein diacetate,) for 1 h, and the ROS signal was observed by fluorescence microscopy (Nikon ECLIPSE Ts2R). After the treated cells were washed three times with PBS, the protein was extracted using a premix of RIPA (Beyotime) and PMSF (Solarbio), and a BCA protein assay kit (Beyotime) was used to determine the protein concentration of the sample. After electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes (Merck Millipore, MA, USA) membrane by transfer, then blocked with 5 % skim milk powder at room temperature for 2 h. Subsequently, the membrane was incubated with primary antibody for 16 h at 4 °C, and then used with HRP-labeled secondary antibody (1:10,000) at room temperature for 2 h. After washing three times with PBST, band matrix development was observed using ECL Western blotting (KF005; Affinity, MA, USA) [18] . THP-1 monocytes were evenly seeded into 24-well plates containing coverslips at a density of 6 × 104 cells/well and subjected to differentiation induction and various treatments. For immunofluorescence experiments, cells were fixed with pre-chilled 4 % paraformaldehyde for 10 min, then permeabilized by reacting with PBS (containing 0.1–0.25 % Triton X-100) for 10 min. The cells were then washed three times with 2 % PBS-BSA (containing bovine serum albumin) for 10 s each time and incubated with PBST containing 1 % BSA, 22.52 mg/mL glycine (PBS + 0.1 % Tween 20) reagent for a total of 30 min to block non-specific binding of various antibodies to cells. The primary antibody was then diluted 1:100 in 2 % PBS-BSA: anti-IL-8 from Proteintech (17038-1-AP, China) and incubated at 4 °C for 16 h. The cells were washed with 2 % PBS-BSA and then incubated with Cy3-labeled fluorescent secondary antibody diluted 1/500 (Beyotime, A0516) for 1 h in the dark. The cells were then washed three times with PBS, observed, and photographed under a fluorescence microscope (Nikon ECLIPSE Ts2R). Categorical variables are expressed as proportions, and continuous variables are expressed as means, standard deviations, and ranges. Comparative analysis was performed by analysis of variance, where the χ test was used to compare each categorical variable, Bonferroni test was used for multiple comparisons, and Wilcoxon non-parametric test was used to compare amylase means. Statistical significance was set at p < 0.05. Statistical analyses were performed using SPSS 22.0 for Windows (SPSS, Chicago, IL, USA).

Pancreaticobiliary reflux (PBR) is one of the common causes of benign and malignant biliary diseases. Here we found that about 64.5 % of patients with benign and malignant gallbladder diseases occurred PBR. The high-amylase bile from PBR patient induced DNA damage of bile duct epithelial cells and promoted cholangiocarcinoma cell progression. Activation of the MAPK pathway through oxidative stress activated macrophage expression of IL-8. Cytokine IL-8, derived from macrophage and bile duct epithelial cells, maybe one of the key regulators that promoted cancer cell progression. IL-8 influenced the expression of proteins associated with the epithelial mesenchymal transition, and also activates phosphorylation of PI3K/ NFκB pathway proteins, which triggers tumor cell value-addition and survival. A schematic diagram of PBR and mechanisms of PBR promoting tumor progression is shown in Fig. 6 . Fig. 6 Mechanism of high-amylase bile increased macrophage-original IL-8 promoting cancer progression in pancreaticobiliary reflux patients. Fig. 6 Mechanism of high-amylase bile increased macrophage-original IL-8 promoting cancer progression in pancreaticobiliary reflux patients. Pancreaticobiliary reflux often occurs in individuals with long common channels [ 19 , 20 ] or in patients with Oddi sphincter dysfunction [21] , [22] , [23] . OPBR indicates the presence of high-amylase bile without PBM, which manifests as high BA. PBR may cause precancerous lesions and carcinogenesis of the bile duct or gallbladder [24] . The main malignant lesions induced by PBM are gallbladder cancer, 62.3 % in patients with PBM with congenital biliary dilatation and 88.1 % in patients with PBM without biliary dilatation [9] . Patients with PBM without bile duct dilation should undergo preventive cholecystectomy. Whether extrahepatic bile duct resection should be performed in patients with PBM without bile duct dilation is controversial. Some surgeons believe that bile ducts are at risk of cancer and that extrahepatic bile duct resection should be performed [25] . In contrast, some surgeons believe that preventive extrahepatic bile duct and biliary shunting are unnecessary. Long-term follow-up after cholecystectomy alone in PBM without bile duct dilatation noted that the 10- and 15-year survival rates were 100 and 89.7 %, respectively [26] . ERCP is a relatively safe treatment with the advantages of less trauma, fewer complications, and better reproducibility [27] . The overall effective rate of ERCP treatment was 60.7 %, which significantly reduced the elevated levels of transaminases and bilirubin, and the symptoms of abdominal pain also significantly decreased. ERCP is used as a bridge treatment to stabilize PBM symptoms prior to radical surgery [28] . The mechanism by which PBR induces carcinoma in the bile duct remains unknown. PBR are associated with bile duct mucosal hyperplasia and intestinal metaplasia [ 5 , 6 ]. The activation conditions for pancreatic enzymes in refluxed bile were studied. Trypsin, elastase, and proteolytic enzymes were activated in the bile of a dog model of PBM [29] . The concentrations of lysolecithin in phospholipids, deoxycholic acid, lithocholic acid, and unconjugated bile acids in the patient's bile were increased, which may be a risk factor for biliary cancer [ 30 , 31 ]. Gene mutations have been found in the gallbladder and bile duct mucosa of PBM, including p53 and KRAS [32] . Additionally, MSI is closely associated with bile duct cancer [33] . PBM-related gallbladder cancer has a high frequency of allelic loss and may contain tumor suppressor genes at two new common LOH loci [34] . PBM may increase TGFα expression and induce cell proliferation through autocrine and/or paracrine mechanisms, which promotes the incidence of gallbladder cancer [35] . COX2 is expressed to varying degrees in normal bile duct mucosa and cholangiocarcinoma of patients with PBM without bile duct dilation and may be associated with cholangiocarcinoma incidence [36] . The expression of tight junction proteins, including occludin, claudin-1, and myosin light chain kinase, in the common bile duct epithelium of patients were significantly higher than that in patients without PBM [37] . We confirmed that DNA damage occurred after the bile duct epithelial cells were treated with high-amylase bile. High-amylase bile promotes proliferation and invasion of cholangiocarcinoma cells. Long-term chronic inflammatory response triggers changes in DNA. Thus, PBR-induced carcinogenesis may be a process of inflammatory cancer transformation. Cytokine shuttles play important roles in cancer-related validation. It has been demonstrated that the antitumor response of natural killer (NK) cells can be influenced by positively or negatively regulating their functions through exerting different cytokines. For example, IL-12 and type I interferon positively regulate NK cells, however TGBβ, IL-6 and IL-10 are inhibitors of NK cell function [38] . In myelofibrosis, serum levels of tumor necrosis factor-a are higher than those of TGF-b, thereby influencing acute myeloid leukemia progression [39] . Tumor-related inflammatory factors expression, such as IL-8, IL-13, and IL-17A, were increased in the refluxed bile. Previous studies have found that tissue expression of IL-33 mRNA in PBM-related gallbladder cancer is significantly higher than that in non-PBM gallbladder cancer. IL-33 regulates the microenvironment under cancer-promoting conditions. IL-33 is produced by the endothelium, cancer cells, and non-neoplastic biliary epithelium [40] . Inspired by research focusing on cytokines associated to tumourigenesis, we investigated the elevated expression of IL-8 produced by cells after experimental treatment with high amylase bile. IL-8 activates PI3K/AKT signaling through CXCR1/2 receptors in many cancer types and has been shown to promote cell invasion and tumor growth in intrahepatic cholangiocarcinomas [41] . We observed high levels of IL-8 expression in cholangiocarcinoma after high amylase bile treatment. IL-8 promotes cellular proliferation and invasion in cholangiocarcinoma similar to bile from PBR. The subsequent role of IL-8 belongs to the elastin-like recombinant + CXC chemokine family and is produced by macrophages, epithelial cells, airway smooth muscle cells, and endothelial cells [ 42 , 43 ]. We found that macrophages may be the primary source of IL-8 and that the bile duct epithelium secretes a small amount of it. By conducting mechanistic investigations, we delineated the subcellular localization of the ERK, JNK, and P38 signaling pathways. Under normal conditions, the ERK pathway is typically localized in the cytoplasm. Upon activation, ERK promptly translocates across the nuclear membrane, activating transcription factors such as P70 nucleosome S6 kinase. Through phosphorylation, ERK regulates the activity of specific transcription factors including EIk2l, c2Myc, STATs, Jun, Fos, ATF2, and Max [44] . These transcription factors modulate the transcription of their respective target genes, leading to alterations in the expression and activity of specific proteins. This cascade of events ultimately governs cellular function and metabolism and plays a pivotal role in cell growth, differentiation, and gene expression [ 45 , 46 ]. We investigated the phosphorylation level of JNK and observed that the phosphorylation of p38 elucidated the process of THP-1 (M1) inflammatory factor secretion following polarization. Our findings substantiate the inflammatory factor secretion process based on the aforementioned molecular pathways [ 47 , 48 ]. Pancreatic juice reflux may stimulate macrophages to produce IL-8 and promote cholangiocarcinoma progression. Upon stimulation of the inflammatory microenvironment within the tumor, increased levels of IL-8 contribute to tumor development. The PI3K/NFκB pathway constitutes a pivotal signaling cascade integral to various cellular processes, and its aberrations have been implicated in tumorigenesis [ 49 , 50 ]. This pathway promoted cell survival and proliferation. PI3K activation initiates the production of phosphatidylinositol-3,4,5-trisphosphate, which activates the AKT pathway which promotes cell survival and cell cycle progression. Downstream of PI3K, NFκB activation further contributes to the regulation of genes associated with cell survival. This pathway exerts anti-apoptotic effects by inhibiting pro-apoptotic proteins and enhancing the expression of anti-apoptotic proteins, enabling cancer cells to circumvent programmed cell death, fortifying their survival [ 51 , 52 ]. The activation of the PI3K/NFκB pathway is intricately linked to the promotion of angiogenesis—a pivotal process fostering new blood vessels formation, is indispensable for tumor growth and the provision of nutrients and oxygen. Beyond its role in angiogenesis, the PI3K/NFκB pathway significantly influences cell migration and invasion, thereby contributing to the metastatic potential of cancer cells. This involves the regulation of genes associated with EMT and cell motility [53] . Dysregulation of this pathway was implicated in genomic instability induction, a recognized hallmark of cancer. Although PBR is a common cause of biliary system tumors, it has not received sufficient attention, and many problems remain to be solved. Clinically, ERCP is necessary for diagnosing PBM without bile duct dilation, and there is no effective method for diagnosing OPBR in advance. The mechanisms of PBR-induced DNA damage and carcinogenesis remain unknown. The indirect impact of PBR extract on macrophages and other inflammatory cells, and the direct effects of PBR extract on epithelial cells require further study. Further studies should be conducted to explore these issues. In conclusion, we found that high-amylase bile from patients with PBR induced DNA damage in bile duct epithelial cells. High-amylase bile may promote cholangiocarcinoma progression through IL-8, which is derived from macrophages and bile duct epithelial cells. Further studies are required to investigate the mechanisms underlying high amylase bile-induced carcinogenesis.

Pancreaticobiliary reflux (PBR) is the reflux of pancreatic juice into the bile ducts in patients with or without pancreaticobiliary maljunction (PBM). Occult pancreaticobiliary reflux (OPBR), diagnosed by high biliary amylase, was found in 15.97–62.5 % patients with benign or malignant biliary diseases [ 1 , 2 ]. OPBR leads to precancerous lesions, including dysplasia and metaplasia, in the gallbladder mucosa. Intestinal metaplasia was observed in 16.8 % benign gallbladder diseases [ 3 , 4 ]. In patients with metaplasia of the gallbladder mucosa, the rate of high biliary amylase levels is significantly higher than that in patients with low biliary amylase levels [ 4 , 5 ]. In biliary cancer, OPBR was found in 26.7 and 62,5 % of cholangiocarcinomas and gallbladder cancer, respectively [2] . Although the clinical significance of OPBR has not been seriously considered, long-term follow-up is required [6] . PBR is a primary cause of benign and malignant biliary diseases [ 2 , 7 ]. Biliary amylase levels are high in patients with chronic cholecystitis and gallbladder carcinoma [7] . Compared to benign biliary diseases, biliary amylase and lipase levels are higher in patients with biliary cancer [8] . In patients with biliary amylase levels >10,000 IU/L, 38 % had gallbladder cancer and 46 % had precancerous changes [9] . The Ki-67 (molecular immunology borstel antibody 1) labeling index was significantly higher in the gallbladder mucosa of high-amylase patients than in that of low-amylase patients [10] . The pathological changes in OPBR were consistent with those in PBM owing to persistent pancreatic juice reflux. PBR can lead to inflammatory changes in the biliary epithelium [11] . Proinflammatory cytokines play a key role in the initiation, growth, progression, and metastasis of cholangiocarcinoma [12] . Interleukin-8 (IL-8) is a chemokine with autocrine and/or paracrine tumor-promoting metastatic effects via binding to its receptors CXCR1 and CXCR2 [ 13 , 14 ]. Furthermore, reflux of pancreatic juice into the bile duct leads to changes in the biochemical components of bile, which leads to pathophysiological processes such as hyperplasia or dysplasia of the biliary mucosal epithelium. Hyperplasia-dysplasia-carcinoma progression is a possible mechanism involved in biliary carcinogenesis [9] . Reflux of pancreatic juice leads to DNA mutations and abnormal gene expression [15] . Previous studies have confirmed that PBR leads to genetic mutations in the tumor protein P53 (TP53) and kirsten rat sarcoma viral oncogene homolog (KRAS) genes. PBR also leads to abnormal expression of genes such as cyclooxygenase-2 (COX2), transforming growth factor alpha (TGFα), occluding and claudin-1 [10] . However, the mechanism by which PBR leads to tumor development and progression remains to be elucidated. Herein, we measured bile amylase (BA) concentrations to determine the proportion of pancreaticobiliary reflux in patients with benign and malignant biliary diseases. We then preliminarily explored DNA damage in bile duct epithelial cells and cholangiocarcinoma cell function induced by high-amylase bile or major tumor-associated inflammatory factors. Our findings suggest that high-amylase bile promotes tumor cell progression by affecting epithelial mesenchymal transition. Additionally, high-amylase bile promotes IL-8 production by stimulating the production of reactive oxygen species (ROS), which triggers the activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 signaling pathways. Subsequently, IL-8 promoted the progression of HUCCT1 (human intrahepatic cholangiocarcinoma) and QBC939 (human extrahepatic cholangiocarcinoma) cells by regulating epithelial-mesenchymal transition-related protein expression and activating the PI3K/NF-κB pathway.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.