Lycium barbarum polysaccharide alleviates DSS-induced chronic ulcerative colitis by restoring intestinal barrier function and modulating gut microbiota.

PURPOSE: This study examined the protective effects and mechanism of Lycium barbarum polysaccharides (LBP) in the context of intestinal barrier function and intestinal microbiota in mice with dextran sulfate sodium (DSS)-induced chronic ulcerative colitis (UC). METHODS: C57BL/6J male mice were assigned to a standard normal diet without DSS (control group), a normal diet with DSS (DSS group, 2% DSS given discontinuously for 3 weeks) or a normal diet supplemented with LBP (1% dry feed weight, LBP group, 2% DSS given discontinuously for 3 weeks) for a total of 8 weeks, at which point colonic tissues and caecal contents were collected. RESULTS: LBP exerted a significant effect against colitis by increasing body weight, colon length, DAI and histopathological scores. LBP inhibited proinflammatory cytokines (IL-1ß, IL-6, iNOS and TNF-α) expression, improved anti-inflammatory cytokine (IL-10) expression, promoted the expression of tight junction proteins (Occludin and ZO-1) via nuclear factor erythroid 2-related factor 2 (Nrf2) activation and decreased Claudin-2 expression to maintain the intestinal mucosal barrier. In addition, the abundances of some probiotics (Ruminococcaceae, Lactobacillus, Butyricicoccus, and Akkermansia) were decreased with DSS treatment but increased obviously with LBP treatment. And LBP reduced the abundance of conditional pathogens associated with UC (Mucispirillum and Sutterella). Furthermore, LBP improved the production of short-chain fatty acids (SCFAs), including acetic acid, propionic acid, butyric acid and isobutyric acid. CONCLUSION: LBP can alleviate DSS-induced UC by regulating inflammatory cytokines and tight junction proteins. Moreover, LBP promotes probiotics, suppresses conditional pathogens and increases SCFAs production, showing a strong prebiotic effect.

Development and validation of a risk prediction score for the severity of acute hypertriglyceridemic pancreatitis in Chinese patients.

BACKGROUND: The frequency of acute hypertriglyceridemic pancreatitis (AHTGP) is increasing worldwide. AHTGP may be associated with a more severe clinical course and greater mortality than pancreatitis caused by other causes. Early identification of patients with severe inclination is essential for clinical decision-making and improving prognosis. Therefore, we first developed and validated a risk prediction score for the severity of AHTGP in Chinese patients. AIM: To develop and validate a risk prediction score for the severity of AHTGP in Chinese patients. METHODS: We performed a retrospective study including 243 patients with AHTGP. Patients were randomly divided into a development cohort (n = 170) and a validation cohort (n = 73). Least absolute shrinkage and selection operator and logistic regression were used to screen 42 potential predictive variables to construct a risk score for the severity of AHTGP. We evaluated the performance of the nomogram and compared it with existing scoring systems. Last, we used the best cutoff value (88.16) for severe acute pancreatitis (SAP) to determine the risk stratification classification. RESULTS: Age, the reduction in apolipoprotein A1 and the presence of pleural effusion were independent risk factors for SAP and were used to construct the nomogram (risk prediction score referred to as AAP). The concordance index of the nomogram in the development and validation groups was 0.930 and 0.928, respectively. Calibration plots demonstrate excellent agreement between the predicted and actual probabilities in SAP patients. The area under the curve of the nomogram (0.929) was better than those of the Bedside Index of Severity in AP (BISAP), Ranson, Acute Physiology and Chronic Health Evaluation (APACHE II), modified computed tomography severity index (MCTSI), and early achievable severity index scores (0.852, 0.825, 0.807, 0.831 and 0.807, respectively). In comparison with these scores, the integrated discrimination improvement and decision curve analysis showed improved accuracy in predicting SAP and better net benefits for clinical decisions. Receiver operating characteristic curve analysis was used to determine risk stratification classification for AHTGP by dividing patients into high-risk and low-risk groups according to the best cutoff value (88.16). The high-risk group (> 88.16) was closely related to the appearance of local and systemic complications, Ranson score ≥ 3, BISAP score ≥ 3, MCTSI score ≥ 4, APACHE II score ≥ 8, C-reactive protein level ≥ 190, and length of hospital stay. CONCLUSION: The nomogram could help identify AHTGP patients who are likely to develop SAP at an early stage, which is of great value in guiding clinical decisions.

New traction method (traction ring) for colorectal endoscopic submucosal dissection.

OBJECTIVES: Colorectal endoscopic submucosal dissection (ESD) is challenging because of the difficulty in adequately visualizing the submucosal layer. Many traction methods have been developed to facilitate submucosal dissection; however, they are not widely applied. Therefore, we designed a new traction device, a traction ring, and conducted this pilot study to evaluate its feasibility and safety for colorectal ESD. METHODS: Twenty patients with colorectal lesions who underwent traction ring-assisted ESD were retrospectively included. The main outcomes included en bloc resection rate, R0 resection rate, procedure time, resection time, and intraoperative and postoperative complications. RESULTS: The median procedure time was 74.5 min (range 35-269 min). The median resection time was 55 min (range 25-209 min). Application of the traction system accounted for only 2.7% of the entire procedure time. The en bloc resection rate was 95.0% (19/20), whereas the R0 resection rate was 90.0% (18/20). All traction rings were successfully set and retrieved. Significant intraoperative bleeding was not observed. One patient experienced perforation after treatment, but no further intervention was required. No delayed complications were observed within 1 month post-ESD. CONCLUSION: Traction ring is an effective and safe method for colorectal ESD and can be used at any location in the colorectum.

The effect of quality of segmental bowel preparation on adenoma detection rate.

BACKGROUND: The effectiveness in surveillance colonoscopy largely depends on the quality of bowel preparation. We aimed to investigate the quality of bowel preparation segmentally and its effect on Adenoma Detection Rate (ADR) and Advanced Adenoma Detection Rate (AADR) at corresponding bowel segments. METHODS: This is a single-centered and cross-sectional study. A consecutive of 5798 patients who underwent colonoscopy examination were included. Bowel preparation was evaluated based on Bowel Bubble Scale (BBS) in general and Boston Bowel Preparation Scale (BBPS) in each segment (right side, transverse and left side of colon) and total BBPS scores. The quality of bowel preparation was correlated with ADR and AADR. RESULTS: Four thousand nine hundred forty colonoscopies (14,820 bowel segments) were included in the final analysis. In which 30.9% scored 3, 57.5% scored 2, 11.2% scored 1 and 0.4% scored 0 on basis of BBPS. For each score, ADR were 10.8, 7.7, 4.9 and 3.2%, respectively; whereas AADR were 4.5, 2.8,1.8 and 1.6% (P < 0.05). 36.9% of the colonoscopies showed presence of minimal bubbles and 34.3% with no bubble. For bowels without bubbles and with a large amount of bubbles, ADR were 28.3 and 20.0% respectively; and AADR were 13.3 and 7.1% respectively. CONCLUSIONS: Segmental bowels' cleanliness and the amount of bubbles in bowels significantly affect ADR and AADR. The better the bowel preparation at each segment is and the less bubbles in the bowel there are, the higher ADR and AADR we got. We suggest repeating colonoscopy if any segment of the bowel preparation is poor, or if there is more bubbles, even if the total score of BBPS indicates good or fair bowel preparation.

Efficacy and safety of using premedication with simethicone/Pronase during upper gastrointestinal endoscopy examination with sedation: A single center, prospective, single blinded, randomized controlled trial.

BACKGROUND AND AIM: To investigate the efficacy and safety of premedication with simethicone/Pronase during esophagogastroduodenoscopy (EGD) with sedation. METHODS: Six hundred and ten patients were randomly allocated to two groups based on type of premedication given. Premedication used in the control group was 10 mL lidocaine hydrochloride mucilage (LHM, N = 314) and premedication used in the intervention group was 80 mL simethicone/Pronase solution plus 10 mL lidocaine hydrochloride mucilage (SP/LHM, N = 296). EGD was done under sedation. Visibility scores, number of mucosal areas that needed cleansing, water consumption for cleansing, time taken for examination, diminutive lesions, pathological diagnosis, patients' gag reflex and oxygenation (pulse oximetry) were recorded. RESULTS: SP/LHM has significantly lower total visibility score than LHM (7.978 ± 1.526 vs 6.348 ± 1.097, P < 0.01). During the procedure, number of intragastric areas that needed cleansing and amount of water consumed were significantly less in the SP/LHM than in the LHM group (P < 0.01). In SP/LHM (P = 0.01), endoscopy procedure duration was significantly longer. Although there was no significant difference in rate of detection of diminutive lesions between LHM and SP/LHM, the endoscopist carried out more biopsies in SP/LHM. This led to a higher rate of diagnosis of atrophic gastritis (P = 0.014) and intestinal metaplasia (P = 0.024). There was no significant difference in gag reflex (P = 0.604) and oxygenation during the endoscopy procedure for either group of patients. CONCLUSION: Routine use of premedication with simethicone/Pronase should be recommended during EGD with sedation.

CacyBP/SIP nuclear translocation regulates p27Kip1 stability in gastric cancer cells.

AIM: To investigate the mechanism of calcyclin binding protein/Siah-1 interacting protein (CacyBP/SIP) nuclear translocation in promoting the proliferation of gastric cancer (GC) cells. METHODS: The effect of CacyBP/SIP nuclear translocation on cell cycle was investigated by cell cycle analysis. Western blot analysis was used to assess the change in expression of cell cycle regulatory proteins and proteasome-mediated degradation of p27Kip1. Co-immunoprecipitation (co-IP) analysis was performed to examine the binding of CacyBP/SIP with Skp1. A CacyBP/SIP truncation mutant which lacked the Skp1 binding site was constructed and fused to a fluorescent protein. Subsequently, the effect on Skp1 binding with the fusion protein was examined by co-IP, while localization of fluorescent fusion protein observed by confocal laser microscopy, and change in p27Kip1 protein expression assessed by Western blot analysis. RESULTS: CacyBP/SIP nuclear translocation induced by gastrin promoted progression of GC cells from G1 phase. However, while CacyBP/SIP nuclear translocation was inhibited using siRNA to suppress CacyBP/SIP expression, cell cycle was clearly inhibited. CacyBP/SIP nuclear translocation significantly decreased the level of cell cycle inhibitor p27Kip1, increased Cyclin E protein expression whereas the levels of Skp1, Skp2, and CDK2 were not affected. Upon inhibition of CacyBP/SIP nuclear translocation, there were no changes in protein levels of p27Kip1 and Cyclin E, while p27Kip1 decrease could be prevented by the proteasome inhibitor MG132. Moreover, CacyBP/SIP was found to bind to Skp1 by immunoprecipitation, an event that was abolished by mutant CacyBP/SIP, which also failed to stimulate p27Kip1 degradation, even though the mutant could still translocate into the nucleus. CONCLUSION: CacyBP/SIP nuclear translocation contributes to the proliferation of GC cells, and CacyBP/SIP exerts this effect, at least in part, by stimulating ubiquitin-mediated degradation of p27Kip1.

CacyBP/SIP nuclear translocation induced by gastrin promotes gastric cancer cell proliferation.

AIM: To investigate the role of nuclear translocation of calcyclin binding protein, also called Siah-1 interacting protein (CacyBP/SIP), in gastric carcinogenesis. METHODS: The expression of CacyBP/SIP protein in gastric cancer cell lines was detected by Western blot. Immunofluorescence experiments were performed on gastric cancer cell lines that had been either unstimulated or stimulated with gastrin. To confirm the immunofluorescence findings, the relative abundance of CacyBP/SIP in nuclear and cytoplasmic compartments was assessed by Western blot. The effect of nuclear translocation of CacyBP/SIP on cell proliferation was examined using MTT assay. The colony formation assay was used to measure clonogenic cell survival. The effect of CacyBP/SIP nuclear translocation on cell cycle progression was investigated. Two CacyBP/SIP-specific siRNA vectors were designed and constructed to inhibit CacyBP/SIP expression in order to reduce the nuclear translocation of CacyBP/SIP, and the expression of CacyBP/SIP in stably transfected cells was determined by Western blot. The effect of inhibiting CacyBP/SIP nuclear translocation on cell proliferation was then assessed. RESULTS: CacyBP/SIP protein was present in most of gastric cancer cell lines. In unstimulated cells, CacyBP/SIP was distributed throughout the cytoplasm; while in stimulated cells, CacyBP/SIP was found mainly in the perinuclear region. CacyBP/SIP nuclear translocation generated a growth-stimulatory effect on cells. The number of colonies in the CacyBP/SIP nuclear translocation group was significantly higher than that in the control group. The percentage of stimulated cells in G1 phase was significantly lower than that of control cells (69.70% ± 0.46% and 65.80% ± 0.60%, control cells and gastrin-treated SGC7901 cells, P = 0.008; 72.99% ± 0.46% and 69.36% ± 0.51%, control cells and gastrin-treated MKN45 cells, P = 0.022). CacyBP/SIPsi1 effectively down-regulated the expression of CacyBP/SIP, and cells stably transfected by CacyBP/SIPsi1 were then chosen for further cellular assays. In CacyBP/SIPsi1 stably transfected cells, CacyBP/SIP was shown to be distributed throughout the cytoplasm, irregardless of whether they were stimulated or not. After CacyBP/SIP nuclear translocation was reduced, there had no major effect on cell proliferation, as shown by MTT assay. There had no enhanced anchorage-dependent growth upon stimulation, as indicated by colony formation in flat plates. No changes appeared in the percentage of cells in G0-G1 phase in either cell line (71.09% ± 0.16% and 70.86% ± 0.25%, control cells and gastrin-treated SGC7901-CacyBP/SIPsi1 cells, P = 0.101; 74.17% ± 1.04% and 73.07% ± 1.00%, control cells and gastrin-treated MKN45-CacyBP/SIPsi1 cells, P = 0.225). CONCLUSION: CacyBP/SIP nuclear translocation promotes the proliferation and cell cycle progression of gastric cancer cells.

Potent inhibition of human gastric cancer by HER2-directed induction of apoptosis with anti-HER2 antibody and caspase-3 fusion protein.

BACKGROUND AND AIMS: HER2, an oncogene, has been found to be over-expressed in 10-40% of human gastric carcinomas. The aims of this study were to investigate if a fusion protein consisting of anti-HER2 sFv and constitutively active caspase-3 was capable of inducing apoptosis in HER2-expressing human gastric cancer cells and blocking the growth of human gastric cancer xenografts in nude mice. METHODS: NIH3T3 cells stably transduced with the pcDNA3.1-HER-PE-CP3 recombinant plasmid containing a secretion signal, a single-chain anti-HER2 monoclonal antibody fragment, a Pseudomonas exotoxin A translocation domain and a constitutively active caspase-3 molecule were used to induce apoptosis in human gastric cancer cells both in vitro and in vivo. Immunofluorescence staining and western blotting were used to examine the expression of the recombinant protein HER-PE-CP3. Apoptosis was determined by flow cytometry and TUNEL assay. RESULTS: Co-cultivation of HER-PE-CP3/ NIH3T3 with human gastric cancer cells led to internalisation of HER-PE-CP3 and apoptosis in HER2-expressing human gastric cancer cells but not in HER2-negative cancer cells. Inoculation of HER-PE-CP3/NIH3T3 in nude mice resulted in potent inhibition of human gastric cancer xenografts and much prolonged survival time of the tumour-bearing mice compared with the control. Significantly more apoptotic cells were detected in xenografts in mice receiving HER-PE-CP3/NIH3T3 than in control mice. CONCLUSIONS: The HER-PE-CP3 chimeric molecule could induce selective apoptosis and potent growth inhibition of HER2-positive human gastric cancer cells and might represent a novel HER2-directed treatment option for human gastric cancer.

[Prokaryotic expression and purification of RPS13 and preparation of polyclonal antibody against RPS13].

AIM: To construct prokaryotic expression plasmid of RPS13, express and purify the protein for the preparation of polyclonal antibody. METHODS: RPS13 gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) from the highly expressed cell of gastric multidrug cell SGC7901/VCR. After sequenced, the gene was cloned into the expression vector pET-28a(+) to construct RPS13 expression plasmid pET-28a(+)-RPS13. The expression plasmid was transformed into the E.coli BL21 and screened by ampiline, and then the E.coli BL21 containing the expression plasmid was induced by IPTG and the protein was purifed by Histag column. BALB/c mice were immunized by the immunogen His-RPS13. The titer was measured by ELISA and the polyclonal antibody was obtained. RESULTS: The expression plasmid pET-28a(+)-RPS13 was constructed. The 19 kDa recombined protein was successfully expressed in the E.coli BL21 by IPTG for 3 hours, purified by Histag column and confirmed by SDS-PAGE and Western blot. The polyclonal anti-His-RPS13 antibody was obtained by immunizing the mice with RPS13 protein. RPS13 could be specifically recognized by the antibody based on Western blot. CONCLUSION: RPS13 has bene expressed and purified successfully and polyclonal anti-His-RPS13 antibody has been prepared. Our study can be used for the preparation of monoclonal anti-His-RPS13 antibody and for further research of the function of the RPS13 protein in tumor.

Potential involvement of the cyclooxygenase-2 pathway in hepatocellular carcinoma-associated angiogenesis.

Angiogenesis plays a crucial role in tumor development and growth. The present study was carried out to investigate the potential involvement of the cyclooxygenase-2 (Cox-2) pathway in the regulation of angiogenesis in hepatocellular carcinoma (HCC). We inhibited Cox-2 expression in HCC cell line HuH-7 by selective Cox-2 inhibitor (SC-58635) or Cox-2 siRNA. Conditioned media (CMs) from HuH-7 cells were used in angiogenic assays in vitro and in vivo. Compared with CMs from untreated and negative siRNA treated HuH-7 cells, CMs from SC-58635 and Cox-2 siRNA treated HuH-7 dramatically suppressed the proliferation, migration, and differentiation of human umbilical vein endothelial cells (HUVECs) in vitro and neovascularization in vivo. These inhibitory effects could be partially reversed by the addition of exogenous PGE2 to CMs. Furthermore, Cox-2 inhibition by SC-58635 resulted in PGE2 reduction accompanied by the down-regulation of four PGE2 receptor (EP receptor) subtypes. Treatment with SC-58635 led to the down-expression of proangiogenic factors such as VEGF, HGF, FGF2, ANGPT1 and ANGPT2 in HCC. An approximately 78% reduction of VEGF level has been found in the CM from SC-58635 treated HuH-7. Our results suggest an involvement of Cox-2 in the control of HCC-associated angiogenesis. PGE2 as a vital angiogenic factor may act directly on endothelial cells to promote HuH-7-stimulated angiogenic process. Moreover, Cox-2/PGE2/EP/VEGF pathway possibly also contributes to tumor angiogenesis in HCC. This study provides the rationale for clinical studies of Cox-2 inhibitors on the treatment or chemoprevention of HCC.

Temporal expression of estrogen receptor alpha in rat bone marrow mesenchymal stem cells.

Estrogen responsiveness of bone formation is mediated by the estrogen receptor alpha (ERalpha) in osteoblastic lineage. As osteoblasts arise from the multipotent bone marrow stromal (mesenchymal) cells, this study was undertaken to observe the ERalpha in primary female adult rat bone marrow mesenchymal stem cells (BMSCs). The ERalpha was localized using immunocytochemical analysis in identified primary BMSCs. Then, using real-time PCR analysis, we measured the expression of ERalpha messenger RNA (mRNA) in BMSCs. ERalpha transcripts showed different trends between untreated cultures (control group) and osteogenic-induced cultures (treated group). In the control group, ERalpha mRNA climbed at peak levels at a confluence stage and decreased until day 20, whereas, in the treated group, the ERalpha mRNA kept climbing from a low level until day 20. Thus, the observed developmental expression of ERalpha mRNA correlates with progressive BMSCs growth and osteogenic differentiation and BMSCs may be a primary target cell for estrogen in maintaining bone formation.

[Screening and identification of phage-displayed polypeptides specifically binding to human gastric cancer with high metastatic potential to peritoneum].

OBJECTIVE: By means of phage-display technique, to screen polypeptides that specifically bind to human gastric cancer with high metastatic potential to peritoneum. METHODS: Two human gastric cancer cell lines were used: GC9811-P with high metastatic potential to peritoneum and its wild type parental GC9811, to carry out subtractive screening with a phage display-12 peptide library. RESULTS: After three rounds of screening, 40 phage clones bond to GC9811-P cells were randomly selected. When injected into the peritoneal cavity of nude mice, 6 of the 40 clones did not bind to mouse peritoneum as examined by immunohistochemical staining. They were considered to be capable of binding specifically to GC9811-P cells. Sequence analysis revealed two different exogenous peptides: TLNINRLILPRT and SMSI(X)SPYI(XXX). CONCLUSION: Two peptides have been obtained that specifically bind to a gastric cancer cell variant GC9811-P, which easily disseminates to the peritoneum. Whether or not they could block GC9811-P metastasis to peritoneum in vivo remains to be determined.

Apoptosis-inducing effect of recombinant Caspase-3 expressed by constructed eukaryotic vector on gastric cancer cell line SGC7901.

AIM: To investigate the apoptosis-inducing effect of Caspases-3 expressed by constructed eukaryotic vector on gastric cancer cell line SGC7901. METHODS: PCR was employed to amplify the sequences of both small and large subunits of Caspases-3. Its products were separately cloned into the Sma I site of pBluescript KS(+) to generate both plasmids pBS/SS and pBS/LS. The small subunit fragment was excised from plasmid pBS/SS with BamH I and then inserted into the BamH I site of plasmid pBS/LS preceding that of the large subunit to yield plasmid pBS/Rev-Caspase-3. Rev-Caspase-3 cDNA was excised with Kpn I+Xba I and then subcloned into plasmid pcDNA3.1 (+) to construct Rev-Caspase-3 eukaryotic expression vector pcDNA/Rev-Caspase-3, which was used to transiently transfect SGC7901 cell line. Cell count, MTT assay and electron microscopy were used to confirm the antiproliferation and apoptosis-inducing effect of Rev-Caspase-3 expression on gastric cancer cells. RESULTS: Plasmid pBS/Rev-Caspase-3 and eukaryotic expression vector pcDNA/Rev-Caspase-3 were successfully constructed. SGC7901 cells were transiently transfected by either pcDNA/Rev-Caspase-3 or pcDNA3.1 (+) for 24, 48, 72, and 96 h respectively. Cell growth was measured by cell count and MTT assay. In cell count assay, the cell numbers were 1.8 X 10(6), 1.55 X 10(6), 2.0 X 10(6), and 3.1 X 10(6) in the experimental group and 2.5 X 10(6), 3.1 X 10(6), 4.0 X 10(6), and 5.7 X 10(6) in the control group at 24, 48, 72 and 96 h respectively. The growth of SGC7901 cells was suppressed by Rev-Caspase-3 in a time-dependent manner (P<0.05). The results of MTT assay were similar to that of cell count (P<0.05). The characteristics of apoptosis such as chromatin condensation, crescent formation and margination were seen and more obvious with time in the given-experimental period in the experimental group, but not easily observed in the control group. CONCLUSION: The expression of Rev-Caspase-3 by the constructed eukaryotic vector can significantly induce apoptosis of gastric cancer cell line SGC7901, which may exhibit a potential way in gastric cancer gene therapy.