[Clinical epidemiological survey of primary liver cancer in Yunnan province from 2005 to 2014].

Objective: To investigate the clinical characteristics and changing trends of primary liver cancer in Yunnan province from 2005 to 2014, in order to provide theoretical basis for the prevention and treatment of liver cancer in this region. Methods: A retrospective survey was used to select inpatient cases of liver cancer who were initially diagnosed and treated in our hospital from 2005 to 2014 with simple random sampling. Patients socio-demographic and clinicopathological characteristics were extracted by a unified and standardized questionnaire, and the data were statistically analyzed. Results: A total of 1000 cases with liver cancer were included, aged (53.2±11.2) years, with a male-to-female ratio of 5.99/1.00. There was no significant change in the gender and age composition ratio of patients in the past 10 years. The proportion of patients with lower education level (primary or junior high school) were increased from 21.8% to 23.4%, and the proportion of patients with relatively higher education level were decreased from 58% to 38.2% (P<0.001). Smokers and non-smokers patients were decreased and increased from 58.8% to 44.4%, and 41.2% to 55.6% (P<0.001). The proportion of drinker patients were decreased from 46.4% to 35.2%. The proportion of patients with advanced liver cancer (stage C and D) were increased, while the proportion of patients with stage A and B showed a downward trend (P<0.001). The proportion of HBsAg-positive patients showed an upward trend, that is, rising from 69% in 2005 to 82% in 2014 (P=0.043). The proportion of HBeAg-positive patients showed a steady trend (P=0.008). The use rate of ultrasound examination in patients with liver cancer were decreased from 91.0% to 58.0% (P=0.001), while the use rate of computed tomography (CT), MRI, and PET/CT examinations were increased from 81.0% to 84.0% (P=0.05), 0 to 22% (P<0.001), and 0 to 3% (P=0.026) between 2005 to 2014. The proportion of surgical patients were increased (P=0.005), but the proportion of interventional patients did not change significantly (P=0.590). Surgery and interventional therapy were the most common treatment methods, and the proportion of patients treated with surgery over the past 10 years showed an upward trend (P=0.005), while the proportion of interventional therapy remained at a high level with no significant change (P=0.590). Conclusion: In Yunnan province, the incidence of liver cancer increases with age, and the proportion of male with liver cancer is almost six times that of women. Moreover, the low positive rate of alpha-fetoprotein levels and advanced clinical stage in this region are presently the main challenges against the liver cancer prevention and treatment. The application scope of CT, magnetic resonance imaging, PET-CT and other examination methods has gradually expanded, but the treatment methods are still mainly surgery and interventional therapy.

[Effect and mechanism of adipocyte co-culture on aquaporin-9 expression in HepG2 cells].

Objective: To observe the effect of differentiated mature adipocytes on hepatic steatosis and aquaporin-9 (AQP9) expressions in HepG2 cells and further explore its possible mechanism of action. Methods: Human preadipocytes were cultured and differentiated to full maturity. HepG2 cells were co-cultured with non-differentiated adipocytes and differentiated mature adipocytes for 48 h, and then labeled as control group and experimental group. Oil red O staining and intracellular triglyceride content were performed on co-cultured HepG2 cells and simultaneous changes in phosphatidylinositol 3-kinase (PI3K) - serine/threonine kinase (Akt) signaling pathway, and AQP9 mRNA and protein levels were detected. The experimental group was co-cultured with recombinant human insulin-like growth factor-I (IGF-I), with the addition of 100ng/ml PI3K-Akt pathway agonist, labeled as experimental group + IGF-I group. The activation of PI3K-Akt pathway was verified by Western blotting (WB). The expression of AQP9 was detected by RT-q PCR and WB. The recombinant lentivirus LV-AQP9 or empty-loaded virus LV-PWPI was transfected with HepG2 cells by recombinant lentiviral transfection tecnique, and labeled as HepG2-AQP9 and HepG2-PWPI. The transfection efficiency was assessed by confocal laser scanning microscopy and RT-qPCR and WB detected the change of AQP9 expression level after virus transfection. Afterwards, the stable over-expressed HepG2-AQP9 cells and the empty-loaded HepG2-PWPI cells were co-cultured with differentiated mature adipocytes for 48h, and labeled as HepG2-AQP9 co-culture group, and then intracellular triglyceride content were detected with Oil red O staining. Finally, IGF-I was added to the HepG2-AQP9 co-culture group, which was recorded as HepG2-AQP9 co-culture + IGF-I group. Intracellular triglyceride content was detected with Oil red O staining, and WB verified PI3K-Akt signaling pathway activation and changes in AQP9 mRNA and protein levels. A t-test was used to compare the two independent samples. Results: The intracellular lipid droplets and triglyceride content (0.052 ± 0.005) in the experimental group was increased significantly than the control group (0.033 ± 0.003) (t= 5.225,P= 0.006), suggesting that adipocyte co-culture had induced steatosis in HepG2 cells. RT-qPCR and WB results indicated that the expression levels of AQP9 mRNA (3.615 ± 0.330) and protein levels (0.072 ± 0.005) in the experimental group were significantly higher than the control group (t= 13.708, 11.225,P= 0.005, < 0.001). WB results showed that the expression level of phosphorylated Akt (p-Akt) protein (0.116±0.003) in the experimental group was significantly lower than the control group (0.202 ± 0.003) (t= 27.136,P< 0.001). The total Akt protein was constant, and the p-Akt/total Akt (0.182 ± 0.017)was significantly lower than the control group (0.327 ± 0.019) (t= 2.431,P= 0.001), suggesting that adipocyte co-culture had inhibited PI3K- Akt signaling pathway in HepG2 cells and up-regulated the expression level of AQP9. WB results indicated that the expression level of p-Akt protein (0.194 ± 0.021) in the experimental group + IGF-I group was significantly higher than the experimental group (0.132 ± 0.003) (t= 5.082,P= 0.007). The total Akt protein was constant, and the p-Akt/total Akt (0.281 ± 0.009) was significantly higher than the control group (0.184 ± 0.132) (t= 10.311,P< 0.001). Simultaneously, RT-qPCR and WB results indicated that the expression levels of AQP9 mRNA (0.327 ± 0.347) and protein levels (0.042 ± 0.004) in the experimental group + IGF-I group were significantly lower than the experimental group (t= 33.573, 5.598,P< 0.001, 0.005), suggesting that adipocyte co-culture had possibility to regulate the expression level of AQP9 through the PI3K-Akt pathway. Confocal laser microscopy analysis showed that the transfection efficiency was more than 90%. RT-q PCR and WB results indicated that the expression levels of AQP9 mRNA and protein levels (0.373 ± 0.221) in HepG2-AQP9 group were significantly higher than HepG2-PWPI group (t=14.953, 28.931,P= 0.002 and 0.000), suggesting that the stable overexpression of AQP9 cell line was successfully constructed. The intracellular lipid droplets and triglyceride content in HepG2-AQP9 co-culture group was significantly increased (t= 5.478, 5.369,P= 0.005) than HepG2-PWPI co-culture group and HepG2-AQP9 co-culture+ IGF-I group, suggesting that the increased expression of AQP9 had promoted HepG2 steatosis in co-cultured adipocytes. WB results showed the expression levels of p-Akt protein (0.168 ± 0.006) and p-Akt/total Akt (0.265±0.009) in HepG2-AQP9 co-culture + IGF-1 group was significantly increased (t= 16.311, 8.769,P< 0.001) than HepG2-AQP9 co-culture group, while the expression levels of AQP9 mRNA (0.327 ± 0.034) and protein (0.375 ± 0.025) was significantly decreased (t= 33.573, 9.146,P< 0.001 and 0.001). Conclusion: Adipocytes co-culture can induce steatosis in HepG2 cells, and may participate in inhibiting PI3K-Akt signaling pathway to upregulate the expression of AQP9 in steatotic HepG2 cells.

[Application of multi-slice spiral CT to assess vascular volume for the diagnosis and treatment of gastric varices].

Objective: To differentiate the inflow and outflow channels of gastric varices in cirrhotic portal hypertension patients using multi-slice spiral CT (MSCT), and to assess the relationship between calculable CT volume of gastric varices and the amount of tissue adhesive. Methods: 97 cases with cirrhotic gastric varices who were admitted from November 2013 to August 2017 were selected. The type and shape of gastric varices were observed before tissue glue injection treatment by MSCT. The correlation between CT volume of gastric varices and the amount of tissue adhesive was evaluated by Spearman rank correlation coefficient and Univariate linear regression analysis. Results: MSCT showed that Le, g type had the highest proportion (54.6%), followed by Le, g, Lg (20.6%). Le, Lg and Lgf type accounted for 17.5%, and 5.2%, respectively, while Lgf+b accounted for 2.1%. On MSCT, varices of the gastric fundus were in the direction from bottom to top, and 75% of the fundus had a large curved side varices combined with gastric and renal shunt. Under the gastroscopy, varices in the small curved side of the gastric fundus from near to far were formless. In addition, varices in the large curved side of the gastric fundus when observed from different angles to the direction of blood flow (reverse gastroscope) were 72.7% (near and far) or 20.5 % (far and near). There was a positive correlation between CT volume (R = 0.97, P < 0.001) and the amount of tissue adhesive (Y(1) = 0.35 + 0.65X1, Univariate linear regression equation; ρ = 0.89, P < 0.001, Spearman correlation analysis). Conclusion: MSCT can recognize the vascular shape and inflow and outflow channels of gastric varices. A positive correlation between CT volume and the amount of tissue adhesive, suggested that the CT volume measurement before treatment could be used as one of the method to predict the amount of tissue adhesive.

Oleic acid-induced hepatic steatosis is coupled with downregulation of aquaporin 3 and upregulation of aquaporin 9 via activation of p38 signaling.

Excess lipid deposition in hepatocytes is a hallmark feature of nonalcoholic fatty liver disease (NAFLD). The present study was designed to explore the expression and regulation of aquaporin (AQP) 3 and AQP9 in oleic acid-induced hepatic steatosis. HepG2 cells were incubated with oleic acid at different concentrations and time points. Oil-Red-O staining and triglyceride content measurement were done to assess the extent of hepatic steatosis. The expression of AQP3 and AQP9 was assessed using quantitative real-time PCR and Western blot analyses. The mitogen-activated protein kinase (MAPK) pathways involved in the regulation of AQP3 and AQP9 expression were checked. Compared to untreated control cells, oleic acid treatment significantly (p<0.05) induced hepatic steatosis in HepG2 cells in a dose- and time-dependent fashion. Oleic acid-treated cells showed a significant reduction in the AQP3 expression and a concomitant increase in the AQP9 expression. Oleic acid exposure led to enhanced phosphorylation of p38, but not ERK1/2 or JNK MAPK. Pharmacological inhibition of p38 rather than ERK1/2 signaling significantly blocked the regulation of AQP3 and AQP9 expression by oleic acid. Oleic acid-induced hepatic steatosis in HepG2 cells is associated with the coordinated regulation of AQP3 and AQP9 via activation of p38 signaling. These findings warrant functional studies of aquaglyceroporins in NAFLD.