Characterization of the intestinal digesta and mucosal microbiome of the grass carp (Ctenopharyngodon idella).

The intestinal microbiome plays a pivotal role in the nutritional digestion and metabolism of the grass carp (Ctenopharyngodon idella). Here, we characterized the digesta and mucosal microbiome of the anterior, middle, and posterior intestine of the grass carp, using 16S rRNA next-generation sequencing. Based on 16S rRNA amplicon data, Proteobacteria, Firmicutes and Bacteroides were the dominant phyla in the intestine of grass carp. Our results also showed that microbial communities of the middle intestine exhibited higher alpha diversity indices compared with the anterior and posterior intestine. The clustering of microbial communities that had either colonized in the digesta or were attached to the mucosa, were significantly tighter in the posterior intestine, based on average unweighted Unifrac distances (P < 0.05). The digesta or mucosa of the anterior and middle intestines were similar in microbial composition, but were significantly different to the posterior intestine (P < 0.05). In digesta and mucosa samples from the posterior intestine, we observed a significantly increased abundance of cellulose-degrading microbiomes, such as Bacteroides, Clostridiales and Spirochaetia (P < 0.05). Our results suggested that the microbiomes of the posterior intestine, either attached to the mucosa or colonized in the digesta, were distinct from the microbiomes of the anterior and middle intestine in grass carp.

The Diagnosis Performance of the TCM Syndromes of Irritable Bowel Syndrome by Gastroenterologists Based on Modified Simple Criteria Compared to TCM Practitioners: A Prospective, Multicenter Preliminary Study.

PURPOSE: Traditional Chinese medicine (TCM) including Chinese patent medicine has been widely used to treat irritable bowel syndrome (IBS). Syndrome differentiation is the essence of TCM. However, the diagnostic ability of gastroenterologists to detect TCM syndromes in IBS in China remains unknown. The aim of this study was to investigate the ability of gastroenterologists to diagnose the TCM syndromes of IBS based on modified simple criteria compared with TCM practitioners. METHODS: Patients meeting the Rome III criteria for IBS-D or IBS-C were recruited from six tertiary referral centers between January 2016 and December 2017. After learning the diagnosis criteria of the TCM syndromes in IBS, gastroenterologists first diagnosed the syndromes of the enrolled patients. Subsequently, the patients were diagnosed by TCM practitioners. The rate of agreement between the gastroenterologists and TCM practitioners was analyzed. In addition, demographic data and the distribution of TCM syndrome types in IBS were also analyzed. RESULTS: A total of 178 patients (93 males and 85 females), including 131 patients with IBS-D and 47 patients with IBS-C, were enrolled in this study. The rate of agreement of the syndrome diagnosis between the gastroenterologists and TCM practitioners was 84.3%. The diagnosis consistency rates among IBS-D patients and IBS-C patients were 87.0% and 76.5%, respectively. The most common TCM syndrome type in IBS-D patients was liver depression and spleen deficiency syndrome (27.5%), followed by spleen-yang deficiency syndrome (19.8%). Dryness and heat in intestine syndrome was the most common TCM syndrome in IBS-C patients (57.4%). CONCLUSIONS: Gastroenterologists had good diagnostic agreement with TCM practitioners for diagnosing TCM syndrome types in IBS after learning the diagnostic criteria. This knowledge can aid gastroenterologists in selecting suitable Chinese patent medicine to treat IBS.

Identification, characterization, and immunological analysis of complement component 4 from grass carp (Ctenopharyngodon idella).

Complement component 4 (C4) has critical immunological functions in vertebrates. In the current study, a C4 homolog (gcC4) was identified in grass carp (Ctenopharyngodon idella). The full-length 5458 bp gcC4 cDNA contained a 5148 bp open reading frame (ORF) encoding a protein of 1715 amino acids with a signal peptide and eight conservative domains. The gcC4 protein has a high level of identity with other fish C4 counterparts and is phylogenetically clustered with cyprinid fish C4. The gcC4 transcript shows wide tissue distribution and is inducible by Aeromonas hydrophila in vivo and in vitro. Furthermore, its expression also fluctuates upon lipopolysaccharide or flagellin stimulation in vitro. During infection, the gcC4 protein level decreases or increases to varying degrees, and the intrahepatic C4 expression location changes. With gcC4 overexpression, interleukin 1 beta, tumor necrosis factor alpha, and interferon transcripts are all upregulated by A. hydrophila infection. Meanwhile, overexpression of gcC4 reduces bacterial invasion or proliferation. Moreover, gcC4 may activate the NF-κB signaling pathway. These findings demonstrate the vital role of gcC4 in the innate immunity of grass carp.

16S rRNA sequencing analysis of the correlation between the intestinal microbiota and body-mass of grass carp (Ctenopharyngodon idella).

There appears to be a close correlation between intestinal microbiotas and obesity. Still, our understanding of the relationship between the intestinal microbiota and body-mass in grass carp (Ctenopharyngodon idella) remains limited. Herein, we explored this association in the anterior, middle, and posterior intestine of cohabitating grass carp by using next-generation sequencing of the 16S rRNA gene. The results showed that alpha diversity indices of the low-weight-gain (LWG) groups were higher than that of the high-weight-gain (HWG) groups. HWG groups possessed the decreased ratio of Bacteroidetes to Firmicutes compared with that in the LWG groups. Principal coordinate analysis (PCoA) and analysis of similarities (ANOSIM) revealed that there were significant differences between the HWG and LWG groups. Furthermore, linear discriminant analysis (LDA) coupled with effect size (LEfSe) showed that the order Clostridiales were significantly abundant in the HWG groups. Phylogenetic molecular ecology networks (pMENs) showed a lower average path distance (GD), higher average clustering coefficient (avgCC), and higher average degree (avgK) in the HWG group. Our results suggested that there appeared to be a tight correlation between the intestinal microbiota and body-mass in grass carp. The study provides a referable resource for establishing the relationship between intestinal microbiotas and economic traits, which also lays a foundation for the progress of new fish probiotic in the future.

Agreement of gastroenterologists in traditional Chinese medical differential diagnosis of functional dyspepsia compared with traditional Chinese medical practitioners: A prospective, multicenter study.

OBJECTIVE: To compare the performance of gastroenterologists major in western medicine in diagnosing traditional Chinese medicine (TCM) syndrome types of functional dyspepsia (FD), postprandial distress (PDS) and epigastric pain syndromes (EPS) based on the main symptoms, with that of traditional TCM practitioners in outpatient services. METHODS: Patients with PDS or EPS were enrolled in the study from six tertiary referral centers between January 2016 and December 2017. Their symptoms were first diagnosed by medical doctors, and then by the TCM practitioners. The diagnostic agreement between the gastroenterologists and the TCM practitioners was calculated. The patients' data and their types of FD syndrome were collected and analyzed. RESULTS: In total 160 patients, including 81 with PDS and 79 with EPS were enrolled. The total diagnostic consistency rate between the gastroenterologists and TCM practitioners was 86.3%, while that of PDS and EPS was 85.2% and 87.3%, respectively. The most common type of PDS diagnosed by TCM practitioners was liver-stomach disharmony syndrome (33.3%), spleen deficiency and qi-stagnation syndrome (33.3%), while that for EPS was liver-stomach disharmony syndrome (36.7%). CONCLUSIONS: Gastroenterologists had a high diagnostic agreement about the types of FD syndromes based on differential diagnosis of the main symptoms, compared with TCM practitioners. This may aid gastroenterologists in selecting Chinese medicine for FD-based on syndrome differentiation.

A Randomized, Double-Blind, Multicenter, Placebo-Controlled Trial of Qi-Zhi-Wei-Tong Granules on Postprandial Distress Syndrome-Predominant Functional Dyspepsia.

BACKGROUND: Functional dyspepsia (FD) is a common upper gastrointestinal disorder worldwide, but the current treatments for FD are still unsatisfactory. The aims of this study were to investigate the efficacy and safety of Qi-Zhi-Wei-Tong granules in patients with postprandial distress syndrome (PDS)-predominant FD. METHODS: The study was conducted as a randomized, double-blinded, multicenter, placebo-controlled design in 197 patients with PDS. All participants received placebo treatment for 1 week. Patients whose total symptom score decreased by <50% after the placebo treatment were recruited into the 4-week treatment period, in which they were randomly assigned to be treated with either Qi-Zhi-Wei-Tong granules or placebo. The patients were then followed for 2 weeks without any treatment. Dyspeptic symptoms were scored at weeks 2 and 4 during the random treatment period and 2 weeks after the treatment. Anxiety and depression symptoms were also scored and compared. RESULTS: (1) The total effective rates in the Qi-Zhi-Wei-Tong granules group at weeks 2 and 4 during the random treatment period and 2 weeks after treatment were all significantly higher than those in the placebo group (38.82% vs. 8.75%, P < 0.001; 69.14% vs. 16.25%, P < 0.001; 77.65% vs. 21.25%, P < 0.001). (2) The total dyspeptic symptoms scores in the Qi-Zhi-Wei-Tong granules group at weeks 2 and 4 and 2 weeks after treatment were significantly lower than those in the placebo group. (3) The severity and frequency of each dyspeptic symptom at weeks 2 and 4 and the follow-up period were all significantly lower than those in the placebo group. (4) The anxiety scores in the Qi-Zhi-Wei-Tong granules group were significantly lower than those in the placebo group. (5) Qi-Zhi-Wei-Tong granules did not have more adverse effects than the placebo. CONCLUSION: Qi-Zhi-Wei-Tong granules offer significant symptomatic improvement in PDS with no more adverse effects than placebo. TRIAL REGISTRATION: https://clinicaltrials.gov/, NCT02460601.

Biological parameters, immune enzymes, and histological alterations in the livers of grass carp infected with Aeromonas hydrophila.

Aeromonas hydrophila is the causative agent of bacterial septicemia that is frequently observed in grass carp, Ctenopharyngodon idellus. In this study, we evaluated the biological parameters and immune enzymes in the liver of grass carp following A. hydrophila infection and quantified the alterations in liver histology using a semi-quantitative system. For the biological parameters, we found that the liver somatic index (LSI) was more sensitive than Fulton's condition factor (CF) and was significantly decreased at three days post-injection (DPI). At the immune enzyme level, the level of peroxidase (POD) in the liver significantly increased at 1 and 3 DPI. The activity of alkaline phosphatase (ALP) significantly increased at 3 DPI. Similarly, acid phosphatase (ACP) activity significantly increased at 1, 3, and 5 DPI. Histologically, the results indicated that the liver index at 3, 5, and 7 DPI was significantly higher than that of control groups. The regressive alterations as the highly variable reactions patterns and its index at 5 DPI was significantly higher than that of 1, 21 DPI, and the control groups. Based on our results, we suggest that grass carp resist A. hydrophila infection via an innate immune mechanism in the liver. The findings of this study will help elucidate the underlying mechanisms of resistance to A. hydrophila infection.

Comparison of efficacy and safety of hyoscine butylbromide versus anisodamine for acute gastric or intestinal spasm-like pain: A randomized, double-blinded, multicenter Phase III trial.

OBJECTIVE: Acute gastric or intestinal spasm-like pain is common in clinical setting. Hyoscine butylbromide (HBB), an anti-cholinergic agent, relieves pain in stomach and bowel cramps by inhibiting smooth muscle contractility. In this study, we aimed to compare the efficacy and safety of parenteral HBB and anisodamine for treating acute gastric or intestinal pain. METHODS: In this randomized, controlled, double-blind, parallel-group, multicenter non-inferiority trial, 299 Chinese patients were randomly assigned to HBB or anisodamine in a ratio of 1:1. They were administrated a single dose of either HBB 20 mg or anisodamine 10 mg, and a second dose was given when needed. The primary end-point was the difference in pain intensity (PID) from the pre-dose baseline at 20 min after the first injection. RESULTS: Altogether 295 patients completed the protocol (153 in the HBB and 142 in the anisodamine group). For the primary end-point, the PID was -4.09 (95% confidence interval [CI]: -4.41, -3.76) for the HBB group and -3.66 (95% CI: -4.02, -3.31) for the anisodamine group (P < 0.0001 for non-inferiority). The percentage of patients with at least one adverse event was lower in the HBB group than in the anisodamine group (13.1% vs 17.6%), but there was no statistical significance (P = 0.279). The most frequent adverse events were thirst (7.8%) and dry mouth (2.6%) in the HBB group, and thirst (7.0%), dry mouth (3.5%) and nodal arrhythmia (2.1%) in the anisodamine group. CONCLUSIONS: HBB 20 mg was not inferior to anisodamine 10 mg in pain relief of patients with acute gastric or intestinal spasm-like pain. Both drugs were safe and well tolerated.

Mannan-binding lectin-associated serine protease-1 (MASP-1) mediates immune responses against Aeromonas hydrophila in vitro and in vivo in grass carp.

The mannan-binding lectin-associated serine protease-1 (MASP-1) gene is a crucial component of the lectin pathway in the complement and coagulation cascade. Although MASP-1 has been found in the immune system of teleosts, its immune functions in response to bacterial infection are unclear. In this study, we identified a MASP-1 homolog (gcMASP-1) in the grass carp (Ctenopharyngodon idella). The full-length 3308-bp gcMASP-1 cDNA includes a 2160-bp open reading frame encoding a protein composed of 719 amino acids with epidermal growth factor-like, complement control protein, and trypsin-like domains. gcMASP-1 shares a high similarity with MASP-1 counterparts in other species, and it is most closely related to Cyprinus carpio MASP-1 and Sinocyclocheilus anshuiensis MASP-1. Transcription of gcMASP-1 was widely distributed in different tissues and induced by Aeromonas hydrophila in vivo and in vitro. Expression of gcMASP-1 was also affected by lipopolysaccharide and flagellin stimulation in vitro. In cells over-expressing gcMASP-1, transcript levels of almost all components, except gcMBL and gcC5, were significantly enhanced, and gcIL1ß, gcTNF-α, gcIFN, gcCD59, gcC5aR1, and gcITGß-2 were significantly upregulated after exposure to A. hydrophila; gcMASP-1 interference downregulated the transcript levels after A. hydrophila challenge. In addition, gcMASP-1 activated NF-κB signaling. These findings indicate the vital role of gcMASP-1 in innate immunity in C. idella.

Intravenous Esomeprazole for Prevention of Peptic Ulcer Rebleeding: A Randomized Trial in Chinese Patients.

INTRODUCTION: High-dose intravenous esomeprazole is the only approved pharmacological treatment for the prevention of peptic ulcer rebleeding (currently approved in over 100 countries worldwide), but has not yet been approved in China. This study aimed to evaluate a high-dose esomeprazole intravenous regimen vs. an active control (cimetidine) for the prevention of rebleeding in Chinese patients with a high risk of peptic ulcer rebleeding who had undergone primary endoscopic hemostatic treatment. METHODS: This was a parallel-group study conducted at 20 centers in China. The study comprised a randomized, double-blind, intravenous treatment phase of 72 h in which 215 patients received either high-dose esomeprazole (80 mg + 8 mg/h) or cimetidine (200 mg + 60 mg/h), followed by an open-label oral treatment phase in which all patients received esomeprazole 40 mg tablets once daily for 27 days. The primary outcome was the rate of clinically significant rebleeding within the first 72 h after initial endoscopic hemostatic therapy. Secondary outcomes included the rates of clinically significant rebleeding within 7 and 30 days; proportions of patients who had endoscopic retreatment and other surgery due to rebleeding; and number of blood units transfused. RESULTS: The rate of clinically significant rebleeding within 72 h was low overall (3.3%) and numerically lower in patients treated with esomeprazole compared with cimetidine (0.9% vs. 5.6%). Overall, the results of the secondary outcomes also showed a numerical trend towards superiority of esomeprazole over cimetidine. All treatments were well tolerated. CONCLUSION: In this phase 3, multicenter, randomized trial conducted in China, esomeprazole showed a numerical trend towards superior clinical benefit over cimetidine in the prevention of rebleeding in patients who had successfully undergone initial hemostatic therapy of a bleeding peptic ulcer, with a similar safety and tolerability profile. These findings suggest that esomeprazole may be an alternative treatment option to cimetidine for this indication in China. FUNDING: AstraZeneca. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT01757275.

Interleukin-18 Down-Regulates Multidrug Resistance-Associated Protein 2 Expression through Farnesoid X Receptor Associated with Nuclear Factor Kappa B and Yin Yang 1 in Human Hepatoma HepG2 Cells.

Multidrug resistance-associated protein 2 (MRP2) plays an important role in bile acid metabolism by transporting toxic organic anion conjugates, including conjugated bilirubin, glutathione, sulfate, and multifarious drugs. MRP2 expression is reduced in cholestatic patients and rodents. However, the molecular mechanism of MRP2 down-regulation remains elusive. In this report, we treated human hepatoma HepG2 cells with interleukin-18 (IL-18) and measured the expression of MRP2, nuclear factor kappa B (NF-κB), farnesoid X receptor (FXR), and the transcription factor Yin Yang 1 (YY1) by quantitative real-time quantitative polymerase chain reaction (PCR) and western blotting. We found that expression of MRP2 was repressed by IL-18 at both the mRNA and protein levels in a dose- and time-dependent manner. Furthermore, the activated NF-κB pathway increased YY1 and reduced FXR. These changes were all attenuated in HepG2 cells with knockdown of the NF-κB subunit, p65. The reduced expression of FXR and MRP2 in HepG2 cells that had been caused by IL-18 treatment was also attenuated by YY1 knockdown. We further observed significantly elevated IL-18, NF-κB, and YY1 expression and decreased FXR and MRP2 expression in bile duct-ligated Sprague Dawley rat livers. Chromatin immunoprecipitation assays also showed that FXR bound to the promoter region in MRP2 was less abundant in liver extracts from bile duct-ligated rats than sham-operated rats. Our findings indicate that IL-18 down-regulates MRP2 expression through the nuclear receptor FXR in HepG2 cells, and may be mediated by NF-κB and YY1.

Peptic ulcer bleeding in China: a multicenter endoscopic survey of 1006 patients.

OBJECTIVE: We aimed to describe the clinical picture, management and outcomes of Chinese patients with peptic ulcer bleeding (PUB), especially in those with high risks. METHODS: A multicenter endoscopic survey was conducted. All consecutive patients with endoscopy confirmed PUB from October 2010 to June 2011 were enrolled. Data including patients' gender, age, symptoms and endoscopic findings, Forrest classification, and endoscopic and medical treatment were documented. High-risk ulcer was defined as Forrest grades Ia to IIb upon endoscopy. Rates of rebleeding, surgery and mortality were recorded. RESULTS: In all, 1006 patients were included. Of these 437 (43.4%) were categorized with high-risk PUB, among whom 110 (25.2%) received endoscopic treatment, and the success rate was 99.1%. Rebleeding rates 1-3 days, 4-5 days and 6-30 days after treatment in high-risk patients who did and did not receive endoscopic treatment were 10.9% versus 10.4%, 3.6% versus 3.7% and 0.9% versus 1.5%, respectively. The surgery rates of high-risk patients with or without endoscopic treatment were 1.8% (2/110) versus 1.8% (6/327). During the 9-month study period, two patients with high-risk PUB died, therefore, the overall mortality rate of high-risk PUB was 0.5% (2/437). CONCLUSION: The study suggests that the proportions of high-risk PUB in China is 43.4%, while rebleeding and surgery rate after endoscopic treatment as well as the mortality rate of high-risk PUB in China are 15.6%, 1.8% and 0.5%, respectively.

[Genetic variation analysis of wild populations of grass carp (Ctenopharyngodon idella) using microsatellite markers].

Twelve microsatellites were used to analyze the genetic diversity and genetic structure of eight wild populations of grass carp, among which six populations from Yangtze River (Hanjiang, Wujian, Jiujiang, Shishou, Mudong, and Wanzhou), one population from Pearl River and Heilongjiang River for each, Zhaoqing, and Nenjiang, respectively. Twelve markers showed highly polymorphic and all the eight populations contained high genetic variations. The variations of six populations of Yangtze River and Zhaoqing population of Pearl River were higher than Nenjiang population of Heilongjiang River. Bottleneck analysis revealed that four populations (Zhaoqing, Nenjiang, Mudong, and Wangzhou) had experienced a recent genetic bottleneck, and the effective population size was reduced. Pairwise FST and AMOVA analysis detected significant genetic difference among populations. The pairwise population genetic distances and the UPGMA tree demonstrated that the genetic distances between six populations of Yangtze River and Zhaoqing population were closer and clustered together earlier, as compared to those populations with Nenjiang population. The genetic structure simulation analysis suggested that there were five logic populations of all individuals. The genetic structures of Zhaoqing and Nenjiang populations were shown with independent separation, but the genetic structures of populations from Yangtze River were shown with fuzzy distribution. The high diversity was found in the wild grass carp from three major watersheds in China, which would supply a basis for future genetic improvement. However, the bottleneck effect of some populations should be taken into account in the practical breeding programs.

MiR-26a regulates cell cycle and anoikis of human esophageal adenocarcinoma cells through Rb1-E2F1 signaling pathway.

Resistance to anoikis, the subtype of apoptosis induced by lack of matrix adhesion, contributes to malignant transformation and development of metastasis. MicroRNAs play key regulatory roles in tumorigenesis and metastasis. In this study, we described that miR-26a, which is usually downregulated in tumor cells, is involved in the acquisition of anoikis-resistance of human esophageal adenocarcinoma (EA) cells. Results of qRT-PCR in clinical samples showed that downregulated miR-26a expression is related to tumorigenesis and metastasis of EA. In vitro experiments determined that miR-26a directly participates in the regulation of cell cycle and anoikis of human EA OE33 cells. Further, we identified that Rb1 is the direct functional target of miR-26a, and revealed that the reduction of miR-26a expression leads to increased Rb1 protein level and thus inhibits the function of E2F1, by which it influences the phenotypes of cell cycle and anoikis. The findings we reported here presented the evidence that miR-26a may be involved in regulation of anoikis-resistance of EA cells. Targeting miR-26a may provide a novel strategy to inhibit metastasis.

CpG oligodeoxynucleotides discriminately enhance binding capacity of human naïve B cells to Hepatitis B virus epitopes.

CpG oligodeoxynucleotides (CpG ODN) have the potential to enhance the antigen-presenting cells function of human naïve B cells. In this study, we aim to define the effect of CpG ODNs on the binding capacity of human naïve B cells for different Hepatitis B virus (HBV) epitopes. Three HLA-A2 restricted epitopes were selected to incubate with CpG ODN-primed human naïve B cells. Binding capacity for each epitope and expression of CD80, CD86, class I major histocompatibility complex (MHC), and class II MHC of naïve B cells was tested, respectively, by flow cytometry. CpG ODNs, especially ODN 2216, enhanced the binding capacity of human naïve B cells for HBV epitopes (p < 0.01), and induced markedly higher expression of CD80, CD86, class I MHC, and class II MHC. The binding capacity of CpG-treated naive B cells for each epitope was significantly different. In all the 3 subjects, CpG ODN 2216-primed naïve B cells showed the highest binding ability for Env172-180 compared with the other epitopes with a high expression of co-stimulatory and MHC molecules. CpG ODN showed the potential to selectively enhance the binding capacity of human naïve B cells for HBV epitopes. These results suggest new strategies for development of vaccine design.

SLCO1B1 *15 haplotype is associated with rifampin-induced liver injury.

The organic anion transporting polypeptide 1B1 (OATP1B1, encoded by SLCO1B1) plays an important role in the transport of endogenous and xenobiotic compounds, such as bile acids and rifampin. In this study, the association between OATP1B1 polymorphisms and rifampin hepatotoxicity was investigated using integrated population genetic analysis and functional studies. A total of 273 unrelated patients treated with rifampin were recruited. The allele frequencies were examined in patients with drug (rifampin)-induced liver injury (DILI) (n = 118) and without (non-DILI) (n = 155). Functional analyses were conducted to determine whether the inhibition of bile acids by rifampin was associated with OATP1B1 variants. In the present study, 24 single nucleotide polymorphisms (SNPs) in OATP1B1 were detected in a Chinese population, with two of them causing an amino acid change (rs2306283 and rs4149056). The haplotypes constructed by these two SNPs were OATP1B1 *1a, *1b, *5 and *15, with their respective frequencies being 23.44, 66.30, 0.73 and 9.52% in a total of 273 individuals. The logistic regression analysis indicated that the *15 haplotype was associated with susceptibility to DILI (p = 0.03, OR = 2.04, 95% CI 1.05-3.96). The frequency of the *15 haplotype in DILI patients was significantly higher than that in non-DILI patients (p = 0.03). In the subgroup analysis, the *15 haplotype was associated with susceptibility to cholestatic/mixed injury (p = 0.03, OR = 2.31, 95% CI 1.06-5.02). Functional assessment of the OATP1B1 *15 haplotype revealed that the activity of bile acid uptake was markedly reduced compared to the three other haplotypes. In the inhibition study, the inhibition by rifampin in the *15 haplotype was greater compared to that in the other haplotypes. These results suggest that the OATP1B1 *15 haplotype is an important predisposing factor for rifampin-induced liver injury.

Silencing of the hPOT1 gene by RNA inference promotes apoptosis and inhibits proliferation and aggressive phenotype of gastric cancer cells, likely through up-regulating PinX1 expression.

BACKGROUND: The human protection of telomeres 1 (hPOT1) protein, a single-strand telomeric DNA binding protein, plays an important role in telomere protection and telomere length regulation. However, its effect on invasion of gastric cancer remains unclear. AIMS: To explore the role of hPOT1 in the proliferation and invasion of gastric cancer cells. METHODS: The gastric expression of hPOT1 was examined in normal gastric mucosa (n=25), intestinal metaplasia (n=20), gastric dysplasia (n=20) and gastric cancer (n=150) by immunohistochemistry. The mean optical density (MOD) of the immunostaining was determined by semi-quantitative image analysis. The role of hPOT1 in the cell proliferation, apoptosis and invasion of gastric cancer 7901 cells was determined by means of the RNA interference (RNAi) of hPOT1 mRNA. The effects of hPOT1 RNAi on the expression of hPinX1 and hTERT were detected with western blotting. RESULTS: The hPOT1 MOD was progressively increased from the normal mucosa to intestinal metaplasia, dysplasia, and gastric cancer. An increased hPOT1 expression significantly correlated with tumour serosal invasion, node metastasis and advanced stage. Transfection of hPOT1 siRNA into SGC-7901 cells led to a decrease in cell proliferation, colony formation and invasion, and also an increase of apoptosis. An up-regulation of hPinX1 and down-regulation of hTERT were found in gastric cancer cells with hPOT1 siRNA. CONCLUSIONS: Increased hPOT1 expression is associated with an advanced tumour stage. hPOT1 RNAi inhibits proliferation and invasion, and induces apoptosis of gastric cancer cells. The effects of hPOT1 RNAi seem to be functionally linked to up-regulation of PinX1 and down-regulation of hTERT.

CIAPIN1 confers multidrug resistance through up-regulation of MDR-1 and Bcl-L in LoVo/Adr cells and is independent of p53.

Recent investigations discovered that CIAPIN1 might be another drug resistance-associated molecule in cancer cells. However, the underlying mechanisms of CIAPIN1-related multidrug resistance (MDR) remain elusive. In the present study, we investigated the role and possible mechanisms of CIAPIN1 in MDR of human colon carcinoma LoVo/Adr cells which express the wild-type p53 gene. By using small interference RNA and gene transfection techniques, we found that knockdown of CIAPIN1 expression re-sensitized LoVo/Adr cells to anti-cancer drugs and up-regulation of CIAPIN1 in sensitive LoVo cells resulted in a distinct MDR phenotype. We further revealed that CIAPIN1 conferred the MDR phenotype in LoVo/Adr cells through up-regulating expression of MDR-1 (P-gp) and Bcl-xL. Finally, by analyzing the effect of inactivation of wild-type p53 on CIAPIN1-induced up-regulation of P-gp and Bcl-xL, we determined that CIAPIN1 could exhibit its MDR-related function independently of the p53 signaling pathway. Overall, the results presented here further suggest that over-expression of CIAPIN1 is an important mechanism of drug resistance in human cancers, even if not the sole one.

Programmed cell death 4 (PDCD4) enhances the sensitivity of gastric cancer cells to TRAIL-induced apoptosis by inhibiting the PI3K/Akt signaling pathway.

OBJECTIVE: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is thought to be a promising anti-neoplastic agent because of its ability to selectively induce apoptosis in cancer cells. However, some cancer cells are resistant to TRAIL. The mechanisms underlying this resistance are unclear. The aim of this study was to explore the role of programmed cell death 4 (PDCD4) in regulating TRAIL sensitivity in gastric cancer cells. METHODS: PDCD4 complementary DNA and PDCD4-specific short-hairpin RNA (shRNA) fragments were transfected into TRAIL-sensitive and -resistant gastric cancer cells. Expression of PDCD4 and Akt was detected via western blot. Cell survival and apoptosis were measured using 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry (FCM) assays. RESULTS: We found that upregulation of PDCD4 enhanced TRAIL sensitivity in gastric cancer cells. Downregulation of PDCD4 decreased TRAIL sensitivity. Inhibition of Akt by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 induced PDCD4 activity and enhanced TRAIL sensitivity in TRAIL-resistant gastric cancer cells. CONCLUSION: We demonstrated that PDCD4 regulates TRAIL sensitivity in gastric cancer cells by inhibiting the PI3K/Akt signaling pathway.

RNA interference-mediated hTERT inhibition enhances TRAIL-induced apoptosis in resistant hepatocellular carcinoma cells.

TRAIL has been reported to induce apoptosis in a variety of tumor cell types including hepato-cellular carcinoma (HCC) cell lines. However, considerable numbers of HCC cells, especially some highly malignant tumors, show resistance to TRAIL-induced apoptosis. The molecular mechanisms that regulate sensitivity versus resistance of tumor cells to TRAIL-induced apoptosis remain poorly defined. It has been shown that human telomerase catalytic subunit (hTERT) is overexpressed in human HCCs. In this study, we investigated the effects and the mechanisms of hTERT RNAi on the TRAIL-induced apoptosis of HCC cells that exhibit resistance to TRAIL. Our results indicate that hTERT RNAi sensitizes TRAIL-resistant HCC cells to TRAIL-induced apoptosis. hTERT RNAi-mediated sensitization to TRAIL-induced apoptosis is accompanied up-regulation of procaspases-8 and -9, inhibition of telomerase activity and loss of telomere length. Our results suggest that hTERT RNAi overcame the resistance of the HCC cells against TRAIL, at least in part, via the mitochondrial type II apoptosis pathway and telomerase-dependent pathway.