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ObjectiveTo investigate the difference in the level of biliary calprotectin between patients with cholangiocarcinoma and those with choledocholithiasis. MethodsClinical data and bile samples were collected from 34 patients with cholangiocarcinoma and 78 patients with choledocholithiasis who were diagnosed and treated with endoscopic retrograde cholangiopancreatography in The First Affiliated Hospital of Anhui Medical University from May 2021 to September 2022. Fluorescence lateral flow immunoassay was used to measure the levels of calprotectin， hemoglobin， and lactoferrin in bile. The Mann-Whitney U test was used for comparison of continuous data between two groups， and the chi-square test was used for comparison of categorical data between two groups； the Spearman correlation test was used for correlation analysis； the DeLong test was used for comparison of the area under the ROC curve （AUC）. ResultsCompared with the choledocholithiasis group， the cholangiocarcinoma group had significant increases in the levels of calprotectin ［4 795.50 （2 286.79 — 20 179.73） ng/mL vs 411.16 （67.03 — 1 991.88） ng/mL， Z=5.572， P<0.001］ and fluoride ［115.70 （109.10 — 125.50） mmol/L vs 106.60 （98.60 — 114.40） mmol/L， Z=2.702， P=0.007］. The patients with cholangiocarcinoma were further divided into high cholangiocarcinoma group and low cholangiocarcinoma group， and there was no significant difference between the two groups in the level of calprotectin ［3 867.71 （2 235.66 — 26 407.40） ng/mL vs 4 795.50 （2 361.15 — 13 070.53） ng/mL， Z=0.129， P>0.05］. Biliary calprotectin level was correlated with white blood cell count， hemoglobin concentration， and lactoferrin concentration in bile （r=0.316， 0.353， and 0.464， all P<0.05）. The ROC curve analysis showed that biliary calprotectin （with a sensitivity of 79.4% and a specificity of 75.6%）， blood CA19-9 （with a sensitivity of 82.4% and a specificity of 78.2%）， and their combination （with a sensitivity of 88.2% and a specificity of 73.1%） had good sensitivity and specificity in the diagnosis of cholangiocarcinoma. ConclusionThere is an increase in the level of biliary calprotectin in patients with cholangiocarcinoma， and therefore， it might become a biomarker for the diagnosis of cholangiocarcinoma.
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Objective:To investigate the activation level of neutrophil extracellular trap (NET) in the bile of patients with choledocholithiasis and its clinical significance.Methods:From May 2021 to October 2022, 130 patients underwent endoscopic retrograde cholangiopancreatography (ERCP) at the Department of Gastroenterology, the First Affiliated Hospital of Anhui Medical University were enrolled. The patients were divided into choledocholithiasis group (90 cases) and non-choledocholithiasis group (40 cases), and the choledocholithiasis group was further divided into large stone group (maximum diameter >1 cm, 36 cases) and small stone group (maximum diameter≤1 cm, 54 cases). The bile samples were collected from 130 patients during operation and 16 choledocholithiasis patients with nasobiliary drainage at 24 h after operation.The levels of myeloperoxidase(MPO), neutrophilelastase(NE), and citrullinated histone H3(CitH3) in bile were detected by enzyme-linked immunosorbent assay.The levels of MPO, NE, and CitH3 were compared between choledocholithiasis group and non-choledocholithiasis group, between large stone group and small stone group, as well as between choledocholithiasis patients before ERCP and after ERCP. Mann-Whitney U test and Wilcoxon signed rank test were used for statistical analysis. Results:The levels of MPO, NE and CitH3 in the bile of choledocholithiasis group were 32.6 U/L(28.5 U/L), 30.6 ng/L(35.2 ng/L) and 0.37 μg/L(0.73 μg/L), respectively, which were all higher than those of non-choledocholithiasis group (19.9 U/L(36.4 U/L), 18.2 ng/L(27.4 ng/L), and 0.10 μg/L(0.25 μg/L)), and the differences were statistically significant ( Z=2.91, 3.20 and 3.34; P=0.004, 0.001 and 0.001). The levels of MPO, NE and CitH3 of large stone group were 47.0 U/L(49.4 U/L), 48.4 ng/L(39.5 ng/L) and 0.83 μg/L(1.08 μg/L), respectively, which were all higher than those of small stone group (29.3 U/L(17.5 U/L), 24.0 ng/L(25.8 ng/L), and 0.27 μg/L(0.45 μg/L)), and the differences were statistically significant ( Z=2.01, 3.58 and 3.63; P=0.044, <0.001 and <0.001). The levels of MPO, NE and CitH3 in the bile of choledocholithiasis patients after ERCP significantly decreased compare with those before ERCP (19.4 U/L(19.8 U/L) vs. 33.6 U/L(36.7 U/L), 12.7 ng/L(15.1 ng/L) vs. 22.7 ng/L(25.9 ng/L), 0.05 μg/L(0.12 μg/L) vs. 0.14 μg/L(0.27 μg/L)), and the differences were statistically significant ( Z=3.52, 3.30 and 3.18; all P<0.001). Conclusion:The activation level of NET in the bile of patients with choledocholithiasis increase, while the activation level of NET decrease after ERCP, which indicate that NET may be involved in the formation of choledocholithiasis.
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Objective To investigate the endoscopic ultrasound (EUS) features of distal biliary stricture (DBS), and to provide a clinical basis for the evaluation of DBS by EUS. Methods Related clinical data were collected from 175 patients with DBS who underwent EUS examination in The First Affiliated Hospital of Anhui Medical University from April 2016 to March 2020 to analyze their clinical manifestation, laboratory examination results, imaging findings, and EUS findings, and the patients were followed up to summarize the EUS features of DBS. The chi-square test was used for comparison of categorical data between groups, and the t -test was used for comparison of continuous data between groups. Results Among the 175 patients with DBS, 85(48.57%) had benign DBS and 90(51.43%) had malignant DBS. Compared with the patients with benign DBS, the patients with malignant DBS had a significantly longer length of stricture on EUS (14.1±3.0 mm vs 7.9±3.0 mm, t =13.358, P < 0.001) and significantly higher incidence rates of the characteristic changes on EUS such as hypoechoic space-occupying lesions in lumen (57.8% vs 34.1%, χ 2 =9.843, P =0.002), peripheral lymph node enlargement (26.7% vs 12.9%, χ 2 =5.147, P =0.023), and pancreatic duct dilatation (51.1% vs 28.2%, χ 2 =9.532, P =0.002). EUS combined with magnetic resonance cholangiopancreatography had a sensitivity of 70.6% in the diagnosis of benign DBS and a sensitivity of 92.2% in the diagnosis of malignant DBS. Conclusion The characteristic EUS features of DBS, such as long length of stricture, hypoechoic lesion, peripheral lymph node enlargement, and pancreatic duct dilatation, may help with the differential diagnosis of DBS in clinical practice.
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ObjectiveTo investigate the effect of different cytopathological grading standards on the efficiency of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) in the diagnosis of pancreatic cancer. MethodsRelated clinical data and pancreatic cytopathological results were collected from 256 patients with pancreatic space-occupying lesions who underwent EUS-FNA in The First Affiliated Hospital of Anhui Medical University from May 2011 to March 2019, and the influencing factors for the diagnostic efficiency of EUS-FNA were analyzed based on surgical pathology and follow-up results. The independent samples t-test or the Mann-Whitney U test was used for comparison of continuous data between two groups, and the chi-square test was used for comparison of categorical data between two groups. The receiver operating characteristic (ROC) curve was used to evaluate the value of different cytopathological grading standards in the diagnosis of pancreatic cancer. ResultsA total of 67 patients who were lost to follow-up were excluded, and a total of 189 patients were included in the study. According to the Papanicolaou cytopathological standard, there were 47 cases of heterotypic cells, 25 cases of suspected cancer cells, 20 cases of cancer cells, and 97 cases without tumor cells based on EUS-FNA. A total of 133 patients were confirmed to have pancreatic cancer by postoperative pathology and follow-up results, among whom 52 had no tumor cells, 36 had heterotypic cells, 25 had suspected cancer cells, and 20 had cancer cells based on cytopathological results. EUS-FNA had a true positive rate of 6090% (81 patients) and a false negative rate of 39.10% (52 patients) in the diagnosis of pancreatic cancer; for the 56 patients without pancreatic cancer, EUS-FNA had a false positive rate of 19.64% (11 patients) and a true negative rate of 80.36% (45 patients). EUS-FNA had an area under the ROC curve of 0.643 (95% confidence interval: 0.561-0.724) in the diagnosis of pancreatic cancer. In combination with different cytopathological grading standards and with the diagnostic criteria of “the identification of heterotypic cells or suspected cancer cells or cancer cells was considered positive”, “the identification of suspected cancer cells or cancer cells was considered positive”, and “the identification of cancer cells was considered positive”, the results showed that the diagnostic criteria of “the identification of heterotypic cells or suspected cancer cells or cancer cells was considered positive” improved the efficiency of EUS-FNA in the diagnosis of pancreatic cancer, with a sensitivity of 50.38% and a specificity of 75.00%. Among the 189 patients, 13 (6.88%) experienced complications after EUS-FNA, which included hyperamylasemia and abdominal pain. ConclusionThe combination of different cytopathological grading standards can help improve the efficiency of EUS-FNA in the diagnosis of pancreatic cancer.
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To observe the effect of indomethacin suppository 100 mg before endoscopic retrograde cholangiopancreatography (ERCP) on the level of platelet microparticles (PMPs) in patients with post-ERCP pancreatitis (PEP). A total of 191 patients receiving ERCP were collected from June 2019 to October 2020 in the First Affiliated Hospital of Anhui Medical University and were randomly divided into the indometacin group ( n=96) and the control group ( n=95) by random number table method. The indometacin group received 100 mg indometacin suppositories before ERCP and the control group received placebo of equal quality. Levels of PMPs before operation, 3 hours and 24 hours after operation were measured by flow cytometry. The levels of IL-1, IL-6 and TNF-α in the plasma before ERCP, 3 hours and 24 hours after ERCP were also detected. The incidence of PEP in the indometacin group was 5.21% (5/96), which was significantly lower than that in the control group [13.68% (13/95), P=0.044]. The preoperative PMPs level in the indometacin group (1 910.01/μL) was slightly lower than that in the control group (2 351.87/μL) with no significant difference ( P>0.05). The PMPs levels in the indometacin group 3 hours and 24 hours after ERCP (1 671.47 /μL, 862.74/μL) were significantly lower than those of the control group (2 443.75/μL, 2 536.76/μL, both P<0.05). Inflammatory cytokines including IL-1, IL-6 and TNF-α showed the same tendency. Indometacin can reduce the incidence of PEP, for the reason that indometacin may decrease the levels of PMPs.
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Objective@#To investigate the effects of resolvin D1 on autophagy in the prevention of acute pancreatitis (AP) in mice. @*Methods@#Thirty C57BL/6 mice were divided into control group, AP group and resolvin D1 group. AP model was established by intraperitoneal injection of cerulein at 50 μg·kg-1·h-1. Resolvin D1 was intraperitoneally given at 50 μg/kg one hour before and four hours after modeling. The mice of control group were intraperitoneally injected the same volume of 0.9% sodium chloride solution. The serum levels of amylase and lipase were measured by colorimetric method. The pathological injury of the lung and pancreatitis were observed under optical microscope. Autophagic vacuoles in acinar cells of pancreas of mice were evaluated by transmission electron microscope. And the expressions of autophagy related markers Beclin-1, p62 and LC3-Ⅱ at the mRNA and protein levels in pancreas of mice were detected by real time quantitative polymerase chain reaction (RT-qPCR) and Western blotting method. One-way analysis of variance and SNK-q were performed for statistical analysis. @*Results@#There were statistically significant differences in serum amylase and lipase levels between control group, AP group and resolvin D1 group (F=62.99 and 149.69, both P<0.01). The serum amylase and lipase levels of mice in resolvin D1 group were lower than those of AP group ((525.08±41.12) U/L vs. (752.62±42.03) U/L, (758.24±134.77) U/L vs. (1 201.06±112.53) U/L), and the differences were both statistically significant (both SNK-q test and P<0.01). In addition, there were statistically significant differences in the ratio of the pancreas and lung wet mass to body mass between control group, AP group and resolvin D1 group (F=11.36 and 18.51, both P<0.05). Pathological injury scores of pancreas and lung of resolvin D1 group were both lower than those of AP group (3.3±0.6 vs. 5.6±0.6, 5.4±0.5 vs. 8.8±0.4), and the differences were statistically significant (both SNK-q test and P<0.05). The results of transmission electron microscopy observation revealed that the number of autophagic vacuole of resolvin D1 group was less than that of AP group, and the size was smaller. Moreover, there were statistically significant differences in Beclin-1, p62 and LC3-Ⅱ mRNA between control group, AP group and resolvin D1 group (F=270.95, 151.83 and 124.77, all P<0.05). The relative expression mRNA levels of Beclin-1, p62 and LC3-Ⅱ of resolvin D1 group were 1.59±0.12, 2.75±0.27 and 1.34±0.14, respectively, which were lower than those of AP group (2.68±0.13, 3.32±0.30 and 3.37±0.26, respectively), and the differences were statistically significant (all SNK-q test and P<0.05). There were statistically significant differences in the expressions of Beclin-1, p62 and LC3-Ⅱat the protein level between control group, AP group and resolvin D1 group (F=116.63, 384.40 and 192.45, all P<0.05). The expressions of Beclin-1, p62 and LC3-Ⅱ at protein level of resolvin D1 group were 0.98±0.03, 0.57±0.04 and 0.31±0.03, respectively, which were lower than those of AP group (1.34±0.07, 1.02±0.03 and 0.48±0.04, respectively), and the differences were statistically significant (all SNK-q test and P<0.05). @*Conclusion@#Resolvin D1 ameliorates the severity of AP by attenuating the impaired autophagy and restoring autophagic flux in AP mice.
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Objective To determine the pathway of macrophage metalloelastase (MME)generate active angiostatin by decomposing plasminogen and its effect on inhibiting growth of tumor and microvessel density (MVD) in vivo in mouse models. Methods The recombined plasmid pEGFPC1-MME was constructed. Thirty mice were subcutaneously inoculated with CT-26 cells that were stably transfected with pEGFP-C1-MME (MME-transfected group), 30 with CT-26 cells transfected with empty vector pEGFP-C1 (vector-transfected group) and 30 with CT-26 cells (non-transfected group). Radioiodination and radioisotope tracer were used to explore the pathway of angiostatin generation in vivo. Results SDS-PAGE electrophoresis analysis revealed that, in the PAGE gel contained the protein with molecular weights of 35 000 and 38 000, radioactivity in MME-transfected group was significantly higher than vector-transfected and non-transfected groups (P = 0. 00).Western blotting analysis demonstrated two bands containing 35 000 and 38 000 fragments in three groups. Quantification of the protein signals by image analysis revealed that the levels of 35 000 and 38 000 fragments were obviously increased in MME-transfected group (9.32±1.52 and 5.61±2.24,respectively) than those in vector-transfected (2.47 ± 0.23 and 0. 67 ± 0. 12, respectively) and nontransfected (1.21±0. 69 and 0. 86 ± 0.44, respectively) groups (P= 0.00). The average value of MVD and fluorescent express of vascular endothelial growth factor (VEGF) were lower in MMEtransfected group when compared with those in vector-transfected and non-transfected groups (P =0.00). The average tumor size in MME-transfected group was small in comparison with vectortransfected and non-transfected groups (P= 0.00). Conclusions MME is demonstrated to be one of matrix metalloproteinase that closely related with angiostatin production and has inhibitory effect on tumor growth in tumor-bearing mice.
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Objective To determine the correlation between mouse maerophage metalloelastase (MME)and vascular endothelial growth factor(VEGF)expression involved in angiogenesis of colon cancer.Methods A eDNA fragment coding for domainsⅠandⅡof MME was transfected into murine CT-26 colon cancer cells that were MME deficient.The enzymatic activity of recombinant MME was confirmed by cleavage of native substrate in vitro.An orthotopic implantation model was established by using MME-transfected cells and control cells.Tumor samples were subjected to in situ hybridization (ISH)and immunohistochemical staining(IHC)to detect expressions of MME and VEGF.The microvessel counting was used to assess angiogenesis of murine colon tumors.Results It was demon- strated that the tumor growth was significantly inhibited in MME-transfected group compared with pcDNA3.1 transfected and nontransfected groups(P＜0.001).It was also found that,compared with pcDNA3.1-transfected and nontransfected groups,the microvessel formation in MME transfected group was significantly reduced(P＜0.001).The expression of VEGF mRNA and protein was significantly lower in MME-transfected group than those in the controls,as demonstrated by ISH(MME-transfected group versus pcDNAa.1-transfected group,P=0.028;and versus nontransfected group,P=0.003) and by IHC(MME-transfected group versus pcDNA3.1-transfected group,P=0.025;and versus non- transfected group,P=0.008).Conclusions The MME gene transfected into murine colon cancer cells can effectively suppress the growth of orthotopic tumors by inhibition of vaseularity.Both MME and VEGF gene expression is highly associated with the vascularity of tumors,which may depend on a hal- ance between MME and VEGF expression.