Hemoglobin loss method calculates blood loss during pancreaticoduodenectomy and predicts bleeding-related risk factors.

BACKGROUND: The common clinical method to evaluate blood loss during pancreaticoduodenectomy (PD) is visual inspection, but most scholars believe that this method is extremely subjective and inaccurate. Currently, there is no accurate, objective method to evaluate the amount of blood loss in PD patients. AIM: The hemoglobin (Hb) loss method was used to analyze the amount of blood loss during PD, which was compared with the blood loss estimated by traditional visual methods. The risk factors for bleeding were also predicted at the same time. METHODS: We retrospectively analyzed the clinical data of 341 patients who underwent PD in Shandong Provincial Hospital from March 2017 to February 2019. According to different surgical methods, they were divided into an open PD (OPD) group and a laparoscopic PD (LPD) group. The differences and correlations between the intraoperative estimation of blood loss (IEBL) obtained by visual inspection and the intraoperative calculation of blood loss (ICBL) obtained using the Hb loss method were analyzed. ICBL, IEBL and perioperative calculation of blood loss (PCBL) were compared between the two groups, and single-factor regression analysis was performed. RESULTS: There was no statistically significant difference in the preoperative general patient information between the two groups (P > 0.05). PD had an ICBL of 743.2 (393.0, 1173.1) mL and an IEBL of 100.0 (50.0, 300.0) mL (P < 0.001). There was also a certain correlation between the two (r = 0.312, P < 0.001). Single-factor analysis of ICBL showed that a history of diabetes [95% confidence interval (CI): 53.82-549.62; P = 0.017] was an independent risk factor for ICBL. In addition, the single-factor analysis of PCBL showed that body mass index (BMI) (95%CI: 0.62-76.75; P = 0.046) and preoperative total bilirubin > 200 µmol/L (95%CI: 7.09-644.26; P = 0.045) were independent risk factors for PCBL. The ICBLs of the LPD group and OPD group were 767.7 (435.4, 1249.0) mL and 663.8 (347.7, 1138.2) mL, respectively (P > 0.05). The IEBL of the LPD group 200.0 (50.0, 200.0) mL was slightly greater than that of the OPD group 100.0 (50.0, 300.0) mL (P > 0.05). PCBL was greater in the LPD group than the OPD group [1061.6 (612.3, 1632.3) mL vs 806.1 (375.9, 1347.6) mL] (P < 0.05). CONCLUSION: The ICBL in patients who underwent PD was greater than the IEBL, but there is a certain correlation between the two. The Hb loss method can be used to evaluate intraoperative blood loss. A history of diabetes, preoperative bilirubin > 200 µmol/L and high BMI increase the patient's risk of bleeding.

[Corrigendum] Novel crosstalk between KLF4 and ZEB1 regulates gemcitabine resistance in pancreatic ductal adenocarcinoma.

Subsequently to the publication of the above article, an interested reader drew to the attention of the Editorial Office that, in Fig. 1C on p. 1242, the flow cytometric images contained what appeared to be regular and repeating groups of cells. The office consequently asked the authors to provide the raw data for these images, as they would have been generated from the printouts, and the authors were able to demonstrate that these apparent anomalies were not contained in the original data. It is possible that the anomalous appearance of the data in this Figure may have resulted either from low resolution of the images, or the Figure itself may have been compressed. We are reprinting Fig. 1C opposite, highlighting the data of interest in greater detail. We trust that this satisfies the concerns of the reader in this instance, and thank them for their enquiry to the Editorial Office. The authors also requested that, after having provided the raw data of the original image in order to clarify the concerns of the reader, they may republish Fig. 1 featuring alternative data for Fig. 1C. The revised version of Fig. 1 is consequently shown on the next page. In this figure, flow cytometric analysis demonstrated that treatment with 10 µM gemcitabine induced the death of 66.5% of the BxPC­3 cells, 29.54% of the Panc­1 cells, and 34.52% of the MIApaca­2 cells (Fig. 1C). The authors confirm that these data support the main conclusions presented in their paper, and are grateful to the Editor of International Journal of Oncology for allowing them this opportunity to publish a Corrigendum. They also apologise to the readership for any inconvenience caused. [the original article was published in International Journal of Oncology 51: 1239­1248, 2017; DOI: 10.3892/ijo.2017.4099].

miR-4510 acts as a tumor suppressor in gastrointestinal stromal tumor by targeting APOC2.

Dysregulation of microRNAs (miRNAs) expression has been demonstrated in gastrointestinal stromal tumor (GIST). In this study, we aimed to determine the differential miRNAs expression in GISTs and explore the functional mechanism of differential miRNAs in GIST cells. We measured differential miRNAs in six pairs of GIST tissues and matched adjacent tissues through a high-throughput sequencing, which was confirmed in 64 pairs of GIST tissues and adjacent tissues by real-time polymerase chain reaction. We found that miR-4510 expression was significantly downregulated in GIST tissues compared to matched control tissues. Luciferase reporter assay identified apolipoprotein C-II (APOC2) as a direct target of miR-4510. Overexpression of miR-4510 inhibited the mRNA and protein expression of APOC2. In addition, overexpression of miR-4510 suppressed GIST cell proliferation, migration, and invasion. Overexpression of miR-4510 also inhibited the phosphorylation of AKT and ERK1/2, reduced the expression of matrix metallopeptidase 2 (MMP2) and MMP9. APOC2 knockdown mimicked the effect of miR-4510 overexpression. Further investigation confirmed that APOC2 was notably upregulated in GIST tissues compared to adjacent control tissues. These results suggested that miR-4510 downregulation could promote GIST progression, including growth, invasion, and metastasis, through increasing APOC2 expression.

TWIST1 transcriptionally regulates glycolytic genes to promote the Warburg metabolism in pancreatic cancer.

Reprogrammed glucose metabolism is essential for tumor initiation and development, especially for pancreatic ductal adenocarcinoma (PDAC). Most cancer cells rely on aerobic glycolysis, a phenomenon termed "the Warburg effect", to support uncontrolled proliferation and evade apoptosis. However, the direct regulators of the Warburg effect remain areas of active investigation. In this study, we found that the highly conserved transcription factor, TWIST1, is a crucial regulator of aerobic glycolysis in PDAC. Genetic silencing of TWIST1 significantly inhibited the glycolytic phenotypes of PDAC cells as revealed by reduced glucose uptake, lactate production, and extracellular acidification rate, which can be restored by re-expression of siRNA-resistant TWIST1. Moreover, tamoxifen-inducible expression of TWIST1 promoted the Warburg metabolism of PDAC cells. Mechanistically, by luciferase reporter assay and chromatin immunoprecipitation experiment, we showed that TWIST1 can directly increase the expression of several glycolytic genes, including SLC2A1, HK2, ENO1, and PKM2. Of note, the transcriptional regulation by TWIST1 was not dependent on HIF1α or c-Myc. In The Cancer Genome Atlas and Gene Expression Omnibus accession GSE15471, we confirmed that TWIST1 was closely associated with the glycolysis pathway. Collectively, our findings indicate that TWIST1 is likely to act as important regulator of the Warburg effect in PDAC.

As an independent prognostic factor, USP6 promotes the invasion and metastasis of colon cancer.

The lack of efficient tumor invasion and metastatic biomarkers led to high mortality rates in colon cancer patients. Aberrant expression of ubiquitin-specific protease 6 (USP6) was involved in several diseases including cancer, while its role in the progression of colon cancer was still unclear. In this study, USP6 was evaluated at both mRNA and protein levels by using RT-PCR, western blot and immunohistochemistry staining analyses. The results revealed that high USP6 expression predicted poor disease-specific survival and overall survival through Kaplan-Meier analyses with log-rank tests, univariate and multivariate Cox analyses. Furthermore, cell function assay demonstrated that USP6 could promote colon cancer cells' invasion in vitro and liver metastasis in vivo. These findings indicated that high USP6 expression contributed to the progression of colon cancer and USP6 may be a valuable prognostic factor in patients with colon cancer.

Novel crosstalk between KLF4 and ZEB1 regulates gemcitabine resistance in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with broad resistance to chemotherapeutic drugs. Krüppel-like factor 4 (KLF4) is a candidate tumor suppressor in PDAC. However, the precise role of KLF4 in gemcitabine resistance of PDAC remains largely unclear. In this study, we demonstrated that gemcitabine inhibited KLF4 expression. Moreover, gemcitabine also reduced the levels of miR­200b and miR­183, but promoted ZEB1 expression in PDAC cells. KLF4 knockdown blocked the expression of miR­200b and miR­183, and inversely, KLF4 overexpression promoted the expression of miR­200b and miR­183, suggesting that KLF4 positively regulated the expression of miR­200b and miR­183. Moreover, KLF4 knockdown enhanced ZEB1 expression and gemcitabine resistance while KLF4 overexpression induced the opposite effect. ChIP assays verified that KLF4 positively regulated the expression of miR­200b and miR­183 by directly binding to their promoters. Then, miR­200b and miR­183 directly inhibited ZEB1 expression by targeting its 3'UTR region. ZEB1 knockdown attenuated gemcitabine resistance in PDAC cells. KLF4 overexpression promoted gemcitabine sensitivity of PDAC in vivo by negatively regulating ZEB1 expression. Our results revealed that novel crosstalk between KLF4 and ZEB1 regulated gemcitabine resistance in PDAC.

25-hydroxycholesterol promotes RANKL-induced osteoclastogenesis through coordinating NFATc1 and Sp1 complex in the transcription of miR-139-5p.

25-hydroxycholesterol (25-HC) is implicated in many processes, including lipid metabolism and the immune response. However, the role of 25-HC in RANKL-induced osteoclastogenesis remains largely unknown. Our results showed that 25-HC inhibited miR-139-5p expression in mouse bone marrow macrophages (BMMs) cultured in receptor activator of NF-κB ligand (RANKL) and monocyte macrophage colony-stimulating factor (M-CSF). Further investigation suggested that 25-HC promoted the expression of nuclear factor of activated T cell cytoplasmic 1 (NFATc1) and Sp1, especially in the presence of RANKL and M-CSF. Meanwhile, 25-HC induced nuclear translocation of NFATc1, resulting in the interaction between NFATc1 and Sp1 that was confirmed by co-immunoprecipitation. Chromatin immunoprecipitation assay indicated that Sp1 could bind to miR-139-5p promoter, but NFATc1 had no binding capacity. Although forming NFATc1/Sp1 complex increased its binding to miR-139-5p promoter, the complex inhibited the transcriptional activity of Sp1. Inhibition of NFATc1 increase the expression of miR-139-5p, which might be due to the release of free Sp1 that could bind to the promoter of miR-139-5p. Enforced expression of miR-139-5p impaired osteoclastogenesis induced by co-treatment with 25-HC and RANKL. These results suggested that 25-HC induced the interaction between NFATc1 and Sp1, reducing the level of free Sp1 to inhibit miR-139-5p expression and promote osteoclastogenesis.

25-Hydroxycholesterol promotes migration and invasion of lung adenocarcinoma cells.

25-hydroxycholesterol (25-HC) is enzymatically produced by cholesterol 25-hydorxylase in various organs and is involved in many processes, including lipid metabolism, inflammation and the immune response. However, the role of 25-HC in the migration and invasion of lung adenocarcinoma (ADC) cells remains largely unknown. In this study, we demonstrated that 0.1 µM 25-HC promoted ADC cell migration and invasion without affecting cell proliferation, especially after coculture with THP1-derived macrophages. Further investigation showed that 0.1 µM 25-HC significantly stimulated interleukin-1ß (IL-1ß) secretion in a coculture system and increased the expression of LXR and Snail. IL-1ß also mimicked the effect of 25-HC. LXR knockdown notably blocked the 25-HC-induced Snail expression, migration and invasion in both the monoculture system and the coculture system, but it did not impact the effect of IL-1ß, which suggested that IL-1ß functioned in an LXR-independent manner. These results suggested that 25-HC promoted ADC cell migration and invasion in an LXR-dependent manner in the monoculture system but that in the coculture system, the 25-HC-induced IL-1ß secretion enhanced the effect of 25-HC in an LXR-independent manner.

Simvastatin attenuates macrophage-mediated gemcitabine resistance of pancreatic ductal adenocarcinoma by regulating the TGF-ß1/Gfi-1 axis.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with an intrinsic resistance to almost all chemotherapeutic drugs, including gemcitabine. An abundance of tumor-associated macrophages (TAMs), which can promote the resistance of PDAC to gemcitabine, has been observed in the microenvironments of several tumors. In this study, we confirmed that incubation in TAM-conditioned medium (TAM-CM) reduces the gemcitabine-induced apoptosis of PDAC cells. Simvastatin attenuated the TAM-mediated resistance of PDAC cells to gemcitabine. Further investigation found that simvastatin reversed the TAM-mediated down-regulation of Gfi-1 and up-regulation of CTGF and HMGB1. Simvastatin induced Gfi-1 expression, which increased the sensitivity of PDAC cells to gemcitabine by decreasing TGF-ß1 secretion by TAMs. A luciferase reporter assay and ChIP assay revealed that Gfi-1 directly repressed the transcription of CTGF and HMGB1. Simvastatin also reversed the effects of gemcitabine on the expression of TGF-ß1 and Gfi-1 and reduced the resistance of PDAC to gemcitabine in vivo. These results provide the first evidence that simvastatin attenuates the TAM-mediated gemcitabine resistance of PDAC by blocking the TGF-ß1/Gfi-1 axis. These findings suggest the TGF-ß1/Gfi-1 axis as a novel therapeutic target for treating PDAC.

Effects of sphincter of Oddi motility on the formation of cholesterol gallstones.

AIM: To investigate the mechanisms and effects of sphincter of Oddi (SO) motility on cholesterol gallbladder stone formation in guinea pigs. METHODS: Thirty-four adult male Hartley guinea pigs were divided randomly into two groups, the control group (n = 10) and the cholesterol gallstone group (n = 24), which was sequentially divided into four subgroups with six guinea pigs each according to time of sacrifice. The guinea pigs in the cholesterol gallstone group were fed a cholesterol lithogenic diet and sacrificed after 3, 6, 9, and 12 wk. SO manometry and recording of myoelectric activity were obtained by a multifunctional physiograph at each stage. Cholecystokinin-A receptor (CCKAR) expression levels in SO smooth muscle were detected by quantitative real-time PCR (qRT-PCR) and serum vasoactive intestinal peptide (VIP), gastrin, and cholecystokinin octapeptide (CCK-8) were detected by enzyme-linked immunosorbent assay at each stage in the process of cholesterol gallstone formation. RESULTS: The gallstone formation rate was 0%, 0%, 16.7%, and 83.3% in the 3, 6, 9, and 12 wk groups, respectively. The frequency of myoelectric activity in the 9 wk group, the amplitude of myoelectric activity in the 9 and 12 wk groups, and the amplitude and the frequency of SO in the 9 wk group were all significantly decreased compared to the control group. The SO basal pressure and common bile duct pressure increased markedly in the 12 wk group, and the CCKAR expression levels increased in the 6 and 12 wk groups compared to the control group. Serum VIP was elevated significantly in the 9 and 12 wk groups and gastrin decreased significantly in the 3 and 9 wk groups. There was no difference in serum CCK-8 between the groups. CONCLUSION: A cholesterol gallstone-causing diet can induce SO dysfunction. The increasing tension of the SO along with its decreasing activity may play an important role in cholesterol gallstone formation. Expression changes of CCKAR in SO smooth muscle and serum VIP and CCK-8 may be important causes of SO dysfunction.

Knockdown of GPR137 by RNAi inhibits pancreatic cancer cell growth and induces apoptosis.

G-protein-coupled receptors (GPCRs), the largest family of cell-surface molecules involved in a number of biological and pathological processes, have recently emerged as key players in carcinogenesis and cancer progression. Orphan G protein-coupled receptors (oGPCRs) are a group of proteins lacking endogenous ligands. GPR137, one of the novel oGPCR genes, was discovered by homology screening. However, the biological role of GPR137 in cancers has not yet been discussed and is of great therapeutic interest. In this study, we knocked down GPR137 via a lentivirus system in two human pancreatic cancer cell lines BXPC-3 and PANC-1. Knockdown of GPR137 strongly inhibited cell proliferation and colony formation. Flow cytometry showed that cell cycle was arrested in the sub-G1 phase and apoptotic cells were significantly increased after GPR137 knockdown. Western blotting confirmed that GPR137 silencing induced apoptosis due to cleavage of PARP (poly ADP-ribose polymerase) and upregulation of caspase 3. Furthermore, lentivirus-mediated overexpression of GPR137 promoted the proliferation of PANC-1 cells, suggesting GPR137 as a potential oncogene in pancreatic cancer cells. Taken together, our results prove the importance of GPR137 as a crucial regulator in controlling cancer cell growth and apoptosis.

Stanniocalcin 2 expression predicts poor prognosis of hepatocellular carcinoma.

Previous studies have shown that the expression level of stanniocalcin 2 (STC2) is associated with tumor progression. However, to date, the association between STC2 and clinicopathological factors in hepatocellular carcinoma (HCC) has not been investigated. The clinical significance of STC2 was investigated in 30 fresh HCC samples using western blot analysis and in 240 HCC tissues using immunohistochemical analysis. The level of STC2 in cancerous tissue was higher than in the matched non-cancerous tissues. Using immunohistochemistry, the STC2-positive group exhibited a higher incidence of lymph node metastasis and venous invasion compared with the STC2-negative group. Kaplan-Meier survival analysis revealed that the positive expression of STC2 correlated with poor overall survival (OS) and disease-free survival of HCC patients (P<0.01). STC2 expression was observed to be an independent prognostic factor for OS in HCC patients by multivariate analysis (hazard ratio, 2.39; 95% confidence interval, 1.04-5.89; P=0.013). These data suggest that STC2 expression may be a useful indicator of poor prognosis in HCC patients.

Roles of sphincter of Oddi motility and serum vasoactive intestinal peptide, gastrin and cholecystokinin octapeptide.

AIM: To investigate roles of sphincter of Oddi (SO) motility played in pigment gallbladder stone formation in model of guinea pigs. METHODS: Thirty-four adult male Hartley guinea pigs were divided randomly into two groups: the control group and pigment stone group. The pigment stone group was divided into 4 subgroups with 6 guinea pigs each according to time of sacrifice, and were fed a pigment lithogenic diet and sacrificed after 3, 6, 9 and 12 wk. SO manometry and recording of myoelectric activity of the guinea pigs were obtained by multifunctional physiograph at each stage. Serum vasoactive intestinal peptide (VIP), gastrin and cholecystokinin octapeptide (CCK-8) were detected at each stage in the process of pigment gallbladder stone formation by enzyme-linked immunosorbent assay. RESULTS: The incidence of pigment gallstone formation was 0%, 0%, 16.7% and 66.7% in the 3-, 6-, 9- and 12-wk group, respectively. The frequency of myoelectric activity decreased in the 3-wk group. The amplitude of myoelectric activity had a tendency to decrease but not significantly. The frequency of the SO decreased significantly in the 9-wk group. The SO basal pressure and common bile duct pressure increased in the 12-wk group (25.19 ± 7.77 mmHg vs 40.56 ± 11.81 mmHg, 22.35 ± 7.60 mmHg vs 38.51 ± 11.57 mmHg, P < 0.05). Serum VIP was significantly elevated in the 6- and 12-wk groups and serum CCK-8 was decreased significantly in the 12-wk group. CONCLUSION: Pigment gallstone-causing diet may induce SO dysfunction. The tension of the SO increased. The disturbance in SO motility may play a role in pigment gallstone formation, and changes in serum VIP and CCK-8 may be important causes of SO dysfunction.

CCR9 as a prognostic marker and therapeutic target in hepatocellular carcinoma.

The chemokine receptor CCR9 was recently implicated in tumor biology. In the present study, our objective was to evaluate the clinical significance and potential role of CCR9 in hepatocellular carcinoma (HCC). CCR9 expression was detected by immunohistochemistry, quantitative PCR (qPCR) and western blotting in HCC patients. The prognostic significance of CCR9 expression was assessed. The functional roles of CCR9 in HCC were investigated using MTT, BrdU, colony formation assay and flow cytometry. CCR9 was significantly elevated in HCC tissue samples. High CCR9 expression was correlated with multiple tumor nodes, high Edmondson-Steiner grade and vascular invasion. Multivariate analysis showed that CCR9 expression was an independent prognostic factor for the overall survival (OS) of HCC patients. Further investigations revealed that ectopic expression of CCR9 enhanced cell proliferation and tumorigenicity in HCC cells, whereas CCR9 silencing impaired cell proliferation and tumorigenicity, which was mediated through downregulation of the cell cycle regulators p21, p27 as well as upregulation of cyclin D1. These results suggest that CCR9 can act as a novel prognostic marker and therapeutic target for HCC.

The fading role of T-tube in laparoscopic choledochotomy: primary choledochorrhaphy and over pigtail j and endonasobiliary drainage tubes.

BACKGROUND AND AIM: The traditional management of open/laparoscopic choledochotomy after common bile duct (CBD) exploration is accomplished by placement of a T-tube, a procedure historically associated with complications and discomfort. In this study, we share in humble our laparoscopic experience of the use of primary closure of CBD, primary closure over pigtail J, and endonasobiliary drainage (ENBD) tubes as easy and effective alternatives to T-tubes. METHODS: From April 2006 to March 2009, 27 (16 women) patients with CBD stones underwent laparoscopic choledochotomy at our institute and were engaged in this study by means of T-tube-free approach after bile duct exploration: primary closure, pigtail J tube, and ENBD tube groups. On admission, routine laboratory and imaging workups were performed to confirm choledocholithiasis diagnosis. RESULTS: The mean operative time for primary closure, pigtail J tube, and ENBD tube groups were 95, 100, and 97.5 minutes, respectively. There was no conversion to open surgery nor was intraoperative complication experienced in all the groups. No major biliary complications such as bile leakage or bile peritonitis were seen; however, 1 patient from the pigtail J group experienced premature tube dislodgement and 1 patient from the ENBD tube group was found with a singular CBD retained stone. CONCLUSIONS: Laparoscopic primary closure of the CBD and over pigtail J and ENBD tubes are easy and effective alternatives to T-tube placements; these procedures are safe and with great feasibility, they offer faster recovery time for patients and early discharge with lower hospital charge.

Complications analysis with postoperative choledochoscopy for residual bile duct stones.

BACKGROUND: This study was designed to review the experience of this department with the treatment of post-common bile duct exploration residual stones using choledochoscopy and to analyze the complications of choledochoscopy and explore effective methods of prevention. METHODS: A choledochoscope (PENTAX fibercholedochoscope and electronic choledochoscope PENTAX ECN-1530) was used. A total of 2,882 postoperative percutaneous choledochoscopy (POC) sessions were performed on 986 patients with residual bile duct stones from 1980 to 2008 (408 men, 578 women; ages range, 21-82 years). Forty-five of these had undergone laparoscopic common bile duct exploration (LCBDE); the rest had open bile duct exploration. Seventy-six participants had choledochoscopy examination (for diagnosis only), and in 910 patients it was performed for both diagnosis and therapy (calculi extraction). In 68 cases, plasma shock wave lithotripsy (PSWL) was performed for larger stones before choledochoscopy extraction. RESULTS: The mean duration of choledochoscopy was 25 min (range, 10 min to 2 h), with a mean frequency of 2.85 times (range, 1-11). No mortalities occurred. The procedure was unsuccessful in 28 cases in which stones were not accessible because they were embedded in distal hepatic ducts or because they were in proximal ducts that were severely stenosed. Complications resulted in 13 cases and included perforated sinus, biliary peritonitis, sinus hypoplasia, destruction of the T-tube system leading to obstruction, basket incarceration, bleeding, and intestinal fistular. Choledochoscopic stone clearance was achieved in 95.5% of the cases. CONCLUSIONS: Choledochoscopy is an important treatment option for hepatolithus. It has a high efficiency for stone extraction and fewer complications. However, it should be noted that some of its complications are potentially life-threatening.

Western blotting in the diagnosis of duodenal-biliary and pancreaticobiliary refluxes in biliary diseases.

BACKGROUND: Currently adopted diagnostic methods for duodenal-biliary and pancreaticobiliary refluxes carry many flaws, so the incidence of the two refluxes demands further larger sample size studies. This study aimed to evaluate Western blotting for the diagnosis of refluxes in biliary diseases. METHODS: An oral radionuclide 99mTc-DTPA test (radionuclide, RN) was conducted for the observation of duodenal-biliary reflux prior to measuring bile radioactivity and Western blotting for detecting bile enterokinase (EK). Pancreaticobiliary reflux was assessed by biochemical and Western blotting tests for biliary amylase activity and trypsin-1, respectively. In accordance with bile sample origin, our samples were classified into ductal bile and gall bile groups; based on each individual biliary disease, we further classified the ductal bile group into five sub-groups, and the gall bile group into four sub-groups. Western blotting was conducted to assess the two refluxes in biliary diseases. RESULTS: Consistencies were noted between EK and RN tests when diagnosing duodenal-biliary reflux (P<0.001). The amylase and trypsin-1 tests also showed consistency in diagnosing pancreaticobiliary reflux (P<0.001). Amylase and lipase levels within gall and ductal bile were strongly correlated (P<0.05). In the common bile duct pigment stone group, the EK and trypsin-1 positive rates were found to be insignificant (P>0.05); in the common bile duct cyst group, the EK positive rate was significantly lower than the trypsin-1 positive rate (P<0.05). CONCLUSIONS: Western blotting can accurately reflect duodenal-biliary and pancreaticobiliary refluxes. EK has greater sensitivity than RN for duodenal-biliary reflux. The majority of biliary amylase and lipase comes from the pancreas in all biliary diseases; pancreaticobiliary reflux is the predominant source in the common bile duct cyst group and duodenal-biliary reflux is responsible for the ductal pigment stone group.