[Effect of Qingyi Decoction on gene expression profiles of severe acute pancreatitis rats by gene chip technique].

OBJECTIVE: To investigate the effect of Qingyi Decoction (QYD) on pancreatic gene expression profiles in rats with severe acute pancreatitis (SAP). METHODS: Totally 60 Sprague-Dawley (SD) rats were randomly divided into the sham-operation group (SO group), the SAP group, and the QYD group, 20 in each group. SAP model was replicated by the pancreatic duct retrograde injection with 4% sodium taurocholate. Rats in the QYD group was intragastrically intervened by QYD (0.75 mL/100 g) for 3 times. Pancreatic RNA expression was analyzed using Illuminate whole genome expression profiles. Changes of mRNA and protein in specific genes [heat shock proteins a8 (Hspa8) and heat shock proteins b1 (Hspb1)] were verified by real-time quantitative PCR and Western blot analysis. RESULTS: Compared with the SAP group, 575 differential genes were screened in the QYD group, including 92 up-regulated genes and 483 down-regulated genes. Gene Ontology (GO) categories indicated the genes are associated with negative regulation of transcription regulator activity, oxidoreductase activity and enzyme inhibitor activity. Effects of QYD on the SAP rats were major related to mitogen-activated protein kinase (MAPK), NOD like receptors (NLR) receptor-like signaling pathway, cell cycle, metabolic pathways, oxidoreductase activity. Protein and mRNA changes of Hspa8 and Hspb1 in microarray were verified [relative mRNA expression for Hspa8 and Hspb1 was increased by (13.24 +/- 1.22) times and (7.55 +/- 1.09) times respectively, P < 0.01]. CONCLUSION: QYD was effective in treating experimental SAP involved the MAPK and NLR signaling pathways, cell cycle, metabolic pathways, and oxide reductase activities.

Predicting the risk of variceal rehemorrhage in cirrhotic patients with portal vein thrombosis: A two-center retrospective study.

OBJECTIVES: Although portal vein thrombosis (PVT) was thought to deteriorate portal hypertension and contribute to poor prognosis, risk stratification remains unclear. This study aimed to evaluate its effect on the risk of variceal rehemorrhage and to develop a competitive risk model in cirrhotic patients with PVT. METHODS: Cirrhotic patients with and without PVT admitted for acute variceal hemorrhage were retrospectively included after matching (1:1) for age, gender and etiology of cirrhosis from two tertiary centers with 1-year follow-up. Those with PVT were subsequently divided into the training and validation cohorts. Cox regression analysis was performed to identify risk factors and develop a competitive risk model, of which the predictive performance and optimal decision threshold were evaluated by C-index, competitive risk curves, calibration curves and decision curve analysis. RESULTS: Among 398 patients, PVT significantly increased the variceal rehemorrhage risk. Multivariate Cox regression analysis identified that the Child-Turcotte-Pugh score (P = 0.013), chronic PVT (P = 0.025), C-reactive protein (P < 0.001), and aspartate aminotransferase (P = 0.039) were independently associated with variceal rehemorrhage, which were incorporated into the competitive risk model, with high C-index (0.804 and 0.742 of the training and validation cohorts, respectively), risk stratification ability, and consistency. The optimal decision range of the threshold probability was 0.2-1.0. CONCLUSION: We confirmed the adverse effect of PVT on variceal rehemorrhage and developed a competitive risk model for variceal rehemorrhage in cirrhotic patients with PVT, which might be conveniently used for clinical decision-making.

Effects of n-3 polyunsaturated fatty acids from seal oils on nonalcoholic fatty liver disease associated with hyperlipidemia.

AIM: To investigate the efficacy and safety of n-3 polyunsaturated fatty acids (PUFA) from seal oils for patients with nonalcoholic fatty liver disease (NAFLD) associated with hyperlipidemia. METHODS: One hundred and forty-four patients with NAFLD associated with hyperlipidemia were included in the 24-wk, randomized, controlled trial. The patients were randomized into two groups. Group A (n=72) received recommended diet and 2 g n-3 PUFA from seal oils, three times a day. Group B (n=72) received recommended diet and 2 g placebo, three times a day. Primary endpoints were fatty liver assessed by symptom scores, liver alanine aminotransferase (ALT) and serum lipid levels after 8, 12, 16, and 24 wk. Hepatic fat infiltration was detected by ultrasonography at weeks 12 and 24 after treatment. RESULTS: A total of 134 patients (66 in group A, 68 in group B) were included in the study except for 10 patients who were excluded from the study. After 24 wk of treatment, no change was observed in body weight, fasting blood glucose (FBG), renal function and blood cells of these patients. Total symptom scores, ALT and triglyceride (TG) levels decreased more significantly in group A than in group B (P<0.05). As expected, there was a tendency toward improvement in aspartate aminotransferase (AST), gamma-glutamyltranspeptidase (GGT), and total cholesterol (TCHO) and high-density lipoprotein (HDL) cholesterol levels (P<0.05) after administration in the two groups. However, no significant differences were found between the two groups. The values of low-density lipoprotein (LDL) were significantly improved in group A (P<0.05), but no significant change was found in group B at different time points and after a 24-wk treatment. After treatment, complete fatty liver regression was observed in 19.70% (13/66) of the patients, and an overall reduction was found in 53.03% (35/66) of the patients in group A. In contrast, in group B, only five patients (7.35%, 5/68) achieved complete fatty liver regression (P=0.04), whereas 24 patients (35.29%, 24/68) had a certain improvement in fatty liver (P=0.04). No serious adverse events occurred in all the patients who completed the treatment. CONCLUSION: Our results indicate that n-3 PUFA from seal oils is safe and efficacious for patients with NAFLD associated with hyperlipidemia and can improve their total symptom scores, ALT, serum lipid levels and normalization of ultrasonographic evidence. Further study is needed to confirm these results.

[Antitumor effects of specific cyclooxygenase inhibitors combined with chemotherapeutic agents on gastric cancer cells in vitro].

OBJECTIVE: To study the effects of two specific cyclooxygenase inhibitors (SCI), rofecoxib and celecoxib, combined with chemotherapeutic drugs 5-Fu, DDP and VP-16 on gastric cancer cell line BGC-823, and to evaluate whether specific cyclooxygenase inhibitors can be used as a synergetic agent in chemotherapy. METHODS: The gastric cancer cell line BGC-823 cells were incubated for 48 hours with rofecoxib and celecoxib, 5-Fu, DDP and VP-16 (concentration gradient of 5-Fu, DDP and VP-16:1 microg/ml, 10 microg/ml and 100 microg/ml), or in combination, respectively. MTT working solution was added to each culture and calculated the survival rates of gastric cancer cells. Median-effect principle and Professor Jin's evaluation methods were applied to detect the interaction between the specific cyclooxygenase inhibitors and chemotherapeutic agents. RESULTS: The inhibition rates of gastric cancer cells were 42.63% +/- 1.26% and 50.67% +/- 2.35% by treatment with 0.1 micromol/L rofecoxib and 50 micromol/L celecoxib, respectively. The inhibition rates of gastric cancer cells by treatment with 5-Fu, DDP and VP-16 at different concentrations (1 microg/ml, 10 microg/ml and 100 microg/ml) were 39.75% +/- 3.14%, 49.96% +/- 2.08%, 87.93% +/- 3.66%; 48.28% +/- 2.08%, 59.46% +/- 1.69%, 88.23% +/- 4.81%; and 29.23% +/- 3.27%, 49.34% +/- 3.75%, 79.24% +/- 2.44%, respectively. However, the inhibition rates showed a synergetic role while combined the two SCI (0.1 micromol/L rofecoxib and 50 micromol/L celecoxib) with chemotherapeutic agent at different concentrations (P <0.05). CONCLUSION: Both rofecoxib and celecoxib have an ability to suppress gastric cancer cells in vitro, and the synergetic role becomes evident when rofecoxib and celecoxib are combined with chemotherapeutic agents at different concentrations, which indicate that the two specific cyclooxygenase inhibitors may be used as a chemotherapeutic sensitizer.

[A study of extraesophageal presentations in gastroesophageal reflux disease].

OBJECTIVE: The aim of this prospective multi-center study was to evaluate the clinical characteristics of extraesophageal reflux disorders (EED) in gastroesophageal reflux disease (GERD) patients and the therapeutic effect of proton pump inhibitor (PPI) on EED. METHODS: We investigated GERD patients in 4 hospitals in Shanghai in a same time period. These patients were diagnosed as GERD by finding reflux esophagitis (RE) on endoscopy or with abnormal reflux during 24 h esophageal pH monitoring. Typical GERD symptoms and EED symptoms were evaluated by questionnaire. Patients with EED symptoms underwent videolaryngoscopy and abnormalities were recorded. RESULTS: Totally 200 subjects were enrolled in this study. Among them 95 patients complained of EED. The RE cases were 134 in number and EED occurred in 65 of the RE patients. The commonest presenting symptom of EED was globus or foreign body feeling in the throat (27%), followed by cough, soar throat and hoarseness. Asthma was a rare symptom, the occurrence being 21%, 16%, 11% and 3% respectively. The rate of typical GERD symptoms existing in EED group was 56%. The severity of EED symptoms showed no significant difference between RE and NERD patients. Abnormalities were found in 58% of subjects with EED on laryngoscopy, the occurrence of arytenoids medial wall erythema/edema was 25%, vocal cord erythema/edema was 32%, posterior pharyngeal wall cobble stoning was 20%, and 42% of the patients showed no abnormalities on laryngoscopy. Higher dosage PPI therapy showed effects on the relief of EED, and the relief rate was 95% after 8 weeks of treatment. CONCLUSIONS: Our results suggest that a significant part of GERD patients suffered from EED, and value of laryngoscopy and 24 h pH monitoring is limited for the diagnosis of EED. Higher dosage of PPI was effective for the treatment of EED.

Detection of micrometastasis of gastric carcinoma in peripheral blood circulation.

AIM: To detect the micrometastasis of gastric carcinoma in peripheral blood circulation using immunomagnetic beads sorting technique and RT-PCR technique, and to discuss its significance and the difference between the two methods. METHODS: Density gradient centrifugation was used to isolate mononuclear cells from peripheral blood, immunomagnetic beads sorting technique and RT-PCR technique were used to detect the disseminated carcinoma cells. HE, immunocytochemical and immunofluorescence staining were also used to identify the characteristics of the cells separated with immunomagnetic beads sorting technique. RESULTS: Cells expressing cytokeratin were separated and enriched from the peripheral blood specimens of patients suffering from gastric carcinoma or chronic gastritis. After HE staining, two kinds of cells with little cytoplasm were found. Majority of these cells had small and round nuclei, even chromatins and the thickness of nuclear membrane was normal. Immunohistochemical staining indicated that there were CD34 and CD45 expression on the cell membrane of this kind of cells and these cells also showed expressed human telomerase reverse transcriptase by immunofluorescence staining, but the expression of carcinoembryonic antigen was absent. So, these cells might hematopoiesis precursors. Another kind of cells had larger and abnormal nuclei with thicker nuclear membranes. Massed chromatins and poly-nucleoli were found in the nuclei. These cells expressed human telomerase reverse transcriptase and carcinoembryonic antigen, but CD34 and CD45 were not found on the cell membrane. So, these cells were considered as gastric carcinoma cells escaping from the original focuses and existing in the peripheral blood circulation. Carcinoma cells were found in 25 of 60(41.7%) specimens of peripheral blood from patients with gastric carcinoma, while there were no such cells separated from the blood specimens of chronic gastritis patients. The difference of positive rates of disseminated carcinoma cells between two groups was markedly significant (P<0.005). The expressions of CK20 mRNA in peripheral blood specimens were examinated with RT-PCR. CK20 mRNA was detected from 32 of 60(53.3%) peripheral blood specimens in the group of gastric carcinoma patients, while none of the specimens from patients suffering from chronic gastritis had CK20 mRNA. Significant difference was also found between two groups (P<0.005). Statistic analyses also showed that there was a significant difference between the positive rates of two methods in detecting the disseminated carcinoma cells from the peripheral blood circulation of gastric carcinoma patients (P<0.05). CONCLUSION: The results demonstrated that there were disseminated carcinoma cells in the peripheral blood circulation of some patients with gastric carcinoma. Disseminated carcinoma cells can be detected from the peripheral blood samples with immunomagnetic beads sorting technique and RT-PCR technique. The positive rate of RT-PCR technique is higher than that of immunomagnetic beads sorting technique in detecting micrometastasis.

Pioglitazone ameliorates nonalcoholic steatohepatitis by down-regulating hepatic nuclear factor-kappa B and cyclooxygenases-2 expression in rats.

BACKGROUND: Pioglitazone is effective in nonalcoholic steatohepatitis (NASH), but the mechanisms of action are not completely understood. This study was designed to investigate the effects of pioglitazone on hepatic nuclear factor-kappa B (NF-κB) and cyclooxygenases-2 (COX-2) expression in NASH rats. METHODS: Thirty Sprague-Dawley male rats were randomly assigned to a control group (n = 10), NASH group (n = 10), and pioglitazone treatment group (n = 10). Liver tissues were processed for histology by hematoxylin & eosin and Masson stained. Serum alanine aminotransferase (ALT), cholesterol, triglyceride, fasting blood glucose (FBG), fasting insulin (FINS) levels and biochemical parameters of antioxidant enzyme activities, tumor necrosis factor alpha (TNF-α), prostaglandin E(2) (PGE(2)) levels in serum and liver were measured. The mRNA and protein expression of peroxisome proliferator-activated receptor gamma (PPARγ), NF-κB and COX-2 were determined by real-time polymerase chain reaction, Western blotting and immunohistochemistry. One-way analysis of variance (ANOVA) and Wilcoxon's signed-rank test was used for the statistical analysis. RESULTS: There were severe steatosis, moderate inflammatory cellular infiltration and fibrosis in NASH rats. After pioglitazone treatment, steatosis, inflammation and fibrosis were significantly improved compared with the NASH group (χ(2) = 20.40, P < 0.001; χ(2) = 20.17, P < 0.001; χ(2) = 13.98, P = 0.002). Serum ALT, cholesterol, triglyceride, FBG, FINS levels were significantly elevated in the NASH group (P < 0.05). In the NASH group, total anti-oxidation competence (T-AOC), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX) and malondialdehyde (MDA) levels in serum and liver were conspicuous disordered than those parameters in the control group. Meanwhile, TNF-α and PGE(2) levels in serum and liver were significantly increased compared with the control group. Immunohistochemistry showed NF-κB and COX-2 expression in liver was significantly elevated. However, PPAR? level was decreased in the NASH group. Real-time PCR and Western blotting revealed mRNA and protein expression of COX-2 were increased in the NASH group compared with the control group (0.57 ± 0.08 vs. 2.83 ± 0.24; 0.38 ± 0.03 vs. 1.00 ± 0.03, P < 0.001 and P = 0.004, respectively). After pioglitazone intervention, all of those parameters markedly improved (P < 0.05 or P < 0.01). CONCLUSION: Down-regulating hepatic NF-κB and COX-2 expression, at least in part, is one of the possible therapeutic mechanisms of pioglitazone in NASH rats.

Rofecoxib augments anticancer effects by reversing intrinsic multidrug resistance gene expression in BGC-823 gastric cancer cells.

OBJECTIVE: To investigate combined chemotherapeutic effects of rofecoxib in combination with 5-fluorouracil (5-FU), cisplatin (DDP) and etoposide (VP-16) in vitro, and to explore the potential mechanisms in modulating multidrug resistance (MDR) expression. METHODS: The BGC-823 gastric cancer cell line was incubated for 48 h with 0.1 micromol/L rofecoxib, 5-FU, DDP and VP-16 (1 microg/mL, 10 microg/mL and 100 microg/mL) alone, and combined with rofecoxib, respectively. Methyl-thiazolyl-tetrazolium and the terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-yriphosphate nick-end labeling assays were performed to calculate inhibitory rates and apoptotic index. Middle effects principles (CI values) were used to determine the interaction between rofecoxib and chemotherapeutic agents. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were employed to determine expression of MDR1, multidrug resistance-associated protein 1 (MRP1), glutathione S-tranferase-pi (GST-pi) mRNA and protein in gastric cancer cells administered by rofecoxib, respectively. RESULTS: Both anticancer drugs such as 5-FU, DDP and VP-16 and rofecoxib inhibited the cells' proliferation and induced apoptosis in a dose-dependent manner, and a more significant inhibition was achieved when the cells were co-treated with anticancer drugs and rofecoxib. There was a synergetic role when different concentrations of chemotherapeutic agents were combined with rofecoxib (all CI < 1, P < 0.01 or 0.05). RT-PCR analyses of MDR gene families in BGC-823 gastric cancer cells revealed a strong expression in MRP1 and GST-pi mRNA, but MDR1 mRNA was undetectable. After administration with different concentrations of rofecoxib (0.1, 1.0, 10 micromol/L), significant downregulation of MRP1 and GST-pi mRNA was observed (MRP1: from 0.984 +/- 0.093-0.513 +/- 0.098; GST-pi: from 1.078 +/- 0.201-0.472 +/- 0.084, P < 0.01 or 0.05). In addition, MRP1 and GST-pi protein expression induced by rofecoxib were also reduced (P < 0.01 or 0.05). CONCLUSION: Rofecoxib, a specific cyclooxygenase-2 inhibitor, plays a chemotherapeutic sensitizer role in various anticancer agents on the BGC-823 gastric cancer cell line, which could be partly explained by its ability to reverse the intrinsic MRP1 and GST-piin vitro.