Objective: To investigate the influence of TNF-α on activity of P311/TGF-β1/α-SMA signaling pathway in rabbit bile duct fibroblasts and the interventional effect of tetramethylpyrazine (TMP). Methods: Bile duct fibroblasts of rabbits were isolated and cultured, and then identified. The bile duct fibroblasts were exposed to TNF-α or TNF-α plus different concentrations (0.08, 0.4 and 2.0 mg/mL) of TMP respectively for 48 h, using untreated bile duct fibroblasts as blank control. Afterwards, the cell proliferation was determined by CCK-8 assay, mRNA expressions of P311, TGF-β1 and α-SMA were measured by real-time PCR, and protein expressions of TGF-β1 and α-SMA were examined by Western blot analysis. Results: In bile duct fibroblasts after TNF-α treatment, the cell proliferation significantly accelerated, and the mRNA expressions of P311, TGF-β1 and α-SMA as well as the protein expressions of TGF-β1 and α-SMA were significantly up-regulated compared with blank control cells (all P<0.05); TMP inhibited the above effects of TNF-α with a concentration-dependent tendency, and the inhibitions exerted by TMP at 0.4 and 2.0 mg/mL were significant (all P<0.05). Conclusion: TNF-α can promote proliferation of bile duct fibroblasts possibly via regulating P311/TGF-β1/α-SMA signaling pathway in fibroblasts, and TMP can inhibit the activation of this pathway induced by TNF-α, so it may have preventive and therapeutic effect on benign biliary stricture.

Objective: To investigate the role of Toll-like receptor 4 (TLR4) in the repairing process of bile duct injury. Methods: The TLR4 expressions in 12 specimens of bile duct tissue with biliary stricture and 4 specimens of normal bile duct tissue were determined by immunohistochemical staining. Biliary injury and repair model was established in TLR4 deficient (TLR4–/–) and wild-type (TLR4+/+) mice using clamping method, with their corresponding sham-operated mice as control, and the pathological changes and liver function status were observed in each group of mice 48 h after operation. Results: TLR4 was expressed mainly in the endothelial cells of the bile ducts, and its positive expression rate in bile duct wall with benign biliary stricture was significantly higher than that in bile duct wall of normal tissue (83.33% vs. 25.00%, P<0.01). No pathological changes were noted in the liver and bile duct tissues in the two groups of sham-operated mice, while obvious liver and bile duct injuries were seen in model groups of both TLR4–/– and TLR4+/+ mice, but the injuries were markedly milder in the former than those in the latter; compared with corresponding sham-operated control, the serum levels of alanine transaminase, and total or direct bilirubin in model groups of both TLR4–/– and TLR4+/+ mice were significantly increased (all P<0.05), but the increasing amplitudes of these parameters in the former were significantly slighter than those in the latter (all P<0.05). Conclusion: TLR4 contributes critically to benign bile duct stricture probably via participating in the natural immune responses of biliary epithelial cells, initiating the expressions of a range of inflammatory cytokines, and prompting fibroblast proliferation.

Objective: To investigate the expression of paired-related homeobox 1 protein (Prrx-1) in gallbladder cancer tissue and its significance. Methods: The Prrx-1 expressions in 45 specimens of gallbladder cancer tissues and 35 specimens of gallbladder tissues with benign diseases were determined by immunohistochemical staining, and the relations of Prrx-1 expression with clinicopathologic profiles of gallbladder cancer were analyzed. Results: The expression rate of Prrx-1 was 55.6% (25/45) in gallbladder cancer tissue, and was 5.7% (2/35) in gallbladder tissue with benign diseases, and the difference between them had statistical significance (χ2=21.87, P<0.05). Statistical analysis indicated that the Prrx-1 expression was closely associated with the degree of differentiation and clinical stage of gallbladder cancer (both P<0.05), but irrelevant to the sex and age of the patients and histological type of gallbladder cancer (all P>0.05). Conclusion: Prrx-1 expression is increased in gallbladder cancer and its expression may be closely connected to the occurrence and development of gallbladder cancer.

Objective: To investigate the expressions of annexin A2 and the epithelial-mesenchymal transition (EMT) markers E-cadherin and vimentin in hepatolithiasis-associated intrahepatic cholangiocarcinoma and their significance. Methods: The expressions of annexin A2, E-cadherin and vimentin in tumor tissues from 46 patients with hepatolithiasis-associated intrahepatic cholangiocarcinoma (tumor group), bile duct tissues with chronic inflammation from 50 patients with simple hepatolithiasis (inflammation group) and normal bile duct tissues form 35 patients undergoing surgical resection for hepatic hemangioma or liver injury (normal group) were determined by immunohistochemical staining. The expressions of the three proteins among the groups were compared and their relations with clinicopathologic factors and prognosis of the patients with hepatolithiasis-associated intrahepatic cholangiocarcinoma were analyzed. Results: The positive expression rates of both annexin A2 and vimentin were presented in descending order as tumor group>inflammation group>normal group (69.6% vs. 36.0% vs. 11.4%; 54.3% vs. 28.0% vs. 8.6%, all P<0.05), while the opposite pattern was seen in that of E-cadherin (21.7% vs. 48.0% vs. 71.4%, all P<0.05). In cholangiocarcinoma tissues, there was a positive correlation between the annexin A2 and vimentin expression (r=0.627, P<0.05), and negative correlation between the E-cadherin expression and either the annexin A2 or vimentin expression (r=–0.682; r=–0.575, both P<0.05). The expressions of annexin A2 and vimentin were significantly associated with the degree of tumor differentiation, lymph node metastasis and TNM stage, and E-cadherin expression was significantly associated with the degree of tumor differentiation and lymph node metastasis (all P<0.05). Among the patients with hepatolithiasis-associated intrahepatic cholangiocarcinoma, the survival rate in cases with positive annexin A2 or vimentin expression was significantly lower than that in their corresponding negative ones, and the survival rate in cases with negative E-cadherin expression was significantly lower than that in those with its positive expression (all P<0.05). Conclusion: The annexin A2/EMT pathway may probably play an important role in the development and progression of hepatolithiasis-associated intrahepatic cholangiocarcinoma. The expressions of annexin A2, E-cadherin and vimentin are closely related to the degree of malignancy of the tumor and prognosis of the patients.

Objective: To investigate the Snail expression in intrahepatic cholangiocarcinoma (ICC) and its relations with clinicopathologic features and prognosis of the patients. Methods: The clinical and follow-up data of 55 ICC patients undergoing surgical treatment between December 1999 and January 2010 were retrospectively analyzed. The Snail expressions in specimens of tumor and adjacent tissues from these patients were determined by immunohistochemical staining, and the relations of Snail expression with the clinicopathologic characteristics and prognosis of the patients were statistically analyzed. Results: In tumor tissues compared with their adjacent tissues, both the expression level (2.764 vs. 0.914) and high expression rate of Snail (48.6% vs. 18.0%) were significantly increased (both P<0.05), and Snail expression was significantly associated with the tumor differentiation (χ2=4.231, P=0.040), TNM stage (χ2=6.631, P=0.010), lymphatic metastasis (χ2=4.134, P=0.042), microvascular invasion (χ2=10.197, P=0.001) and recurrence (χ2=4.610, P=0.032); the postoperative overall survival rate was decreased (P=0.018) and accumulative recurrence rate was increased (P=0.032) in patients with high Snail expression compared with those with low expression. Results of univariate and multivariate Cox regression analysis revealed that microvascular invasion, lymphatic metastasis and Snail expression were independent influential factors for prognosis of the patients (all P<0.05). Conclusion: Snail expression is increased in ICC tissue, and its overexpression is closely related to malignant pathological profiles and dismal prognosis of the ICC patients.

Objective: To investigate the clinical value of contrast-enhanced ultrasonography (CEUS) immediately after radiofrequency ablation (RFA) for malignant hepatic tumors. Methods: Sixty consecutive patients with malignant hepatic tumors (76 lesions) admitted between October 2014 and December 2015 in Xiangya Hospital were randomly designated to study group and control group with 30 cases in each group (41 lesions in study group, 35 lesions in control group). All patients underwent percutaneous RFA under real time ultrasound guidance. In patients in study group, CEUS was performed immediately (within 5-10 min) after RFA, and supplementary ablation was conducted immediately for the tumor residues until no residue was detected by contrast examination, while immediate CEUS was not performed in those in control group. Patients in study group underwent CEUS again at 36-48 h after RFA, and patients in both groups underwent CEUS together with contrast-enhanced CT or MRI as well as detection of liver function parameters and tumor markers at 1 month after the RFA. Results: A total of 5 tumor residues were found by immediate CEUS in 5 patients of study group , and supplementary treatments were performed. By review examinations for the two groups at 1 month after RFA, one tumor residual (1/41) was found in study group and 7 tumor residuals (7/35) were found in control group, and the complete ablation rates between study group and control group were statistically different (96.7% vs. 80.0%, P<0.05). In study group, the thickness of the hematoma or edema around the ablation zone detected by CEUS in arterial phase examination immediately after RFA and 36–48h later was (6.89±2.73) mm and (4.68±2.05) mm respectively, and the difference had statistically significant (P<0.05). Conclusion: CEUS immediately after RFA can timely detect the tumor residuals and direct supplementary therapy, and effectively increase the complete ablation rate.

Hepatolithiasis is one of the common hepatobiliary diseases. It has been confirmed that biliary infection, cholestasis and epithelial mesenchymal transformation are important factors for the occurrence and the development of this condition, and recent studies found that inflammation mediated by LPS/TLR4 pathway plays a crucial role in the pathogenic processes of these factors. Here, the authors address the potential actions and relevant mechanisms of LPS/TLR4 signaling pathway in hepatolithasis, so as to provide new paths to the mechanism research as well as prevention and treatment of hepatolithiasis.