Flagellar brake protein YcgR interacts with motor proteins MotA and FliG to regulate the flagellar rotation speed and direction.

In E. coli and related species, flagellar brake protein YcgR responds to the elevated intracellular c-di-GMP, decreases the flagellar rotation speed, causes a CCW rotation bias, and regulates bacterial swimming. Boehm et al. suggested that c-di-GMP-activated YcgR directly interacted with the motor protein MotA to curb flagellar motor output. Paul et al. proposed that YcgR disrupted the organization of the FliG C-terminal domain to bias the flagellar rotation. The target proteins are controversial, and the role of motor proteins remains unclear in flagellar rotation speed and direction regulation by YcgR. Here we assayed the motor proteins' affinity via a modified FRET biosensor and accessed the role of those key residue via bead assays. We found that YcgR could interact with both MotA and FliG, and the affinities could be enhanced upon c-di-GMP binding. Furthermore, residue D54 of YcgR-N was needed for FliG binding. The mutation of the FliG binding residue D54 or the MotA binding ones, F117 and E232, restored flagellar rotation speed in wild-type cells and cells lacking chemotaxis response regulator CheY that switched the flagellar rotation direction and decreased the CCW ratio in wild-type cells. We propose that c-di-GMP-activated YcgR regulated the flagellar rotation speed and direction via its interaction with motor proteins MotA and FliG. Our work suggest the role of YcgR-motor proteins interaction in bacterial swimming regulation.

The effects of bariatric surgery on cognition in patients with obesity: a systematic review and meta-analysis.

Obesity impairs cognition. Bariatric surgery can result in substantial weight loss in patients with severe obesity; however, the impact of bariatric surgery on cognitive function remains controversial. To quantify the effect of bariatric surgery on cognition in patients with severe obesity, we performed a meta-analysis of 20 studies retrieved from PubMed, Cochrane, and Embase. Of these, 6 cohort studies found that Roux-en-Y gastric bypass leads to better performance for immediate verbal memory function (standardized mean difference [SMD] = .56; 95% confidence interval [CI]: .30-.82, P < .0001; I2 = 0%) and delayed memory function (SMD = .64; 95% CI: .38-.90, P < .00001; I2 = 0%) during in the short term. Similarly, positive impacts on immediate verbal memory function (SMD = .46; 95% CI: .09-.83, P < .00001) and delayed memory function (SMD = .84; 95% CI: .46-1.22, P < .0001) were identified during a long-term follow-up. The Roux-en-Y gastric bypass group showed no improvements in attention, cognitive speed, and executive function compared with the control obese group. In 14 longitudinal studies (12 single-arm pre-post comparison studies and 2 cohort studies whose control group had no follow-up cognitive data), patients performed better postoperatively than preoperatively in all cognitive domains during repeated assessments. The analysis for the 20 operative groups showed that individuals treated with bariatric surgery had higher scores after repeated assessment of most neuropsychological tests except for animal fluency and letter fluency than baseline scores. These findings suggest that patients with severe obesity may obtain immediate verbal and delayed memory function benefits from Roux-en-Y gastric bypass.

Pre-colonoscopy special guidance and education on intestinal cleaning and examination in older adult patients with constipation.

BACKGROUND: The prevalence of constipation in the Chinese population over 60 years of age is 11.5%, and this prevalence increases with age, which seriously affects the quality of life in older adults. Therefore, reducing the incidence of constipation in older adults is necessary to promote a healthy lifestyle as well as biochemical health. AIM: To explore the value of preoperative guidance and education to improve the effects of bowel cleaning in older adult patients undergoing colonoscopy. METHODS: In this study, 160 older adult patients with constipation requiring colonoscopy at Shandong Provincial Hospital between January 2019 and March 2021 were selected and randomly divided into a study group and a control group, with 80 patients in each group. The study group received medication guidance and targeted educational guidance before the operation, while the control group received only medication and dietary guidance. The baseline data, colonoscopy duration, bowel preparation compliance, Boston bowel preparation (BBPS) assessment score, intestinal bubble score, the incidence of adverse reactions during bowel preparation, and nursing appointment satisfaction were compared between the two groups. RESULTS: The colonoscopy duration times and intestinal bubble scores of the study group were shorter than those of the control group, with statistically significant differences. The BBPS scores for the right, left, and interrupted colon in the study group were also higher than those in the control group, and the difference was statistically significant. Additionally, the study group had a higher rate of liquid diet one day before the examination, higher rate of correct bowel-clearing agent dilution method, higher rate of accurate time of ingesting the bowel-clearing agent, and a higher proportion of patients ingesting bowel-clearing agent at the specified time than the control group, with statistically significant differences. The incidence of nausea and vomiting during bowel clearance in the study group was significantly lower than that in the control group. The incidence of abdominal pain, abdominal distension, dizziness, and fatigue was compared between the two groups, but the difference was not statistically significant. The scores of service attitude, detailed notification of dietary precautions, clear and easy-to-understand health educational content, and receiving care and comfort in the study group were significantly higher than those in the control group. CONCLUSION: Preoperative special guidance and education were shown to significantly improve bowel clearance and compliance and reduce the incidence of adverse reactions in older adult patients with constipation undergoing colonoscopy. These factors are beneficial for improving patient satisfaction with nursing services.

Combination of neutrophil gelatinase-associated lipocalin and matrix metalloproteinase-9 are biomarkers for the detection of colon tubular adenocarcinoma.

BACKGROUND: Tubular adenocarcinoma of the colon, which originates from the epithelium of the glands, is a major health concern worldwide. However, it is difficult to detect at an early stage. The lack of biomarkers is a main barrier to the diagnosis and treatment of tubular adenocarcinoma. Neutrophil gelatinase-associated lipocalin (NGAL) is a secreted protein that induces the expression of matrix metalloproteinase-9 (MMP-9) and is involved in various tumors. NGAL and MMP-9 have been reported to be associated with tumorigenesis and development. They may have potential as biomarkers for diagnosis of tubular adenocarcinoma of the colon. AIM: To determine whether NGAL and MMP-9 can be used as potential biomarkers to indicate the progression of tubular adenocarcinoma of the colon. METHODS: Samples were collected from surgically excised tissue from various patients. The content of pro-gastrin-releasing peptide (pro-GRP) in the serum was measured by an electrochemiluminescence immunoassay. The expression patterns of NGAL and MMP-9 and the relationship between NGAL and MMP-9 were examined by quantitative real-time PCR, Western blotting and immunohistochemical analysis. RESULTS: In this study, we found that NGAL and MMP-9 can be used as biomarkers for the detection of tubular adenocarcinoma of the colon and that their combination improved diagnostic accuracy. By analyzing the expression of NGAL in tubular adenocarcinoma at different levels, we found that NGAL expression was significantly upregulated in primary tubular adenocarcinoma tissues compared with normal tissues. The upregulation of NGAL expression was strongly correlated with both the degree of differentiation and the disease stage (I-III), indicating that NGAL could serve as a diagnostic biomarker for tubular adenocarcinoma. When using NGAL as a biomarker for diagnosis, the accuracy was similar to that achieved with the widely used biomarker pro-GRP, suggesting that NGAL is reliable. Moreover, the expression of MMP-9 was also strongly correlated with the differentiation stage, demonstrating that MMP-9 could be used as a biomarker to indicate the progression of tubular adenocarcinoma of the colon. More importantly, the combination of NGAL and MMP-9 produced a more accurate diagnosis of tubular adenocarcinoma, and these results were further confirmed by immunohistochemical analysis of tissue sections. CONCLUSION: Our study demonstrated that both NGAL and MMP-9 can be used as biomarkers for the diagnosis of colon tubular adenocarcinoma and that the results could be further improved by combining them.

The Effects of Hyperbaric Oxygen Therapy on Pelvic Radiation Induced Gastrointestinal Complications (Rectal Bleeding, Diarrhea, and Pain): A Meta-Analysis.

Background: Radiotherapy is a routine treatment for pelvic cancer patients. While it had been proven effective, gastrointestinal side effects remain a concern, impairing the quality of life. A few studies focused on the effects of hyperbaric oxygen (HBO) treatment to alleviate radiation-induced gastrointestinal complications. This meta-analysis aimed to critically review and summarize existing literature, assessing the effectiveness of HBO therapy for the treatment of radiation-induced gastrointestinal side effects. Methods: Medical literature search was performed with PubMed, Cochrane Library, and EMBASE up to March 14, 2019. Literatures about HBO treatment upon patients undergoing pelvic cancer (endometrial, cervix, rectum, or prostate cancers) radiotherapy were collected, and the effects of HBO treatment on radiotherapy-induced gastrointestinal complications were evaluated. A random-effects model was used to calculate the pooled effect size. Subgroup analyses were performed to search for sources of heterogeneity. Publication bias was detected with Funnel plots and Egger's test. Results: Three different radiotherapy-related gastrointestinal complications, including rectal bleeding, diarrhea, and pain, were analyzed after screening. It was revealed that the improvement rates were considerable in rectal bleeding (0.81, 95% CI: 0.74-0.89) and diarrhea (0.75, 95% CI: 0.61-0.90) and slightly in pain (0.58, 95% CI: 0.38-0.79). Subgroup analysis revealed factors that significantly influenced the heterogeneity of rectal bleeding, diarrhea, and pain (evaluation criteria, follow-up time, and scoring system, respectively). No significant publication bias was detected. Conclusion: HBO treatment might have the potential to alleviate radiotherapy-related gastrointestinal complications, including rectal bleeding, diarrhea, and pain, but more data are needed for further conclusions. Other symptoms were not further analyzed, as the number of studies was insufficient. More large-scale and prospective studies are needed for better evaluation of HBO's therapeutic values.

Risk factors and prognostic analysis of acute-on-chronic liver failure of chronic hepatitis B after cessation of nucleos(t)ide analogs.

BACKGROUND: To explore the risk factors and prognostic factors related to the acute-on-chronic liver failure (ACLF) occurrence and adverse outcome after withdrawal of nucleos(t)ide analogs (NAs) in chronic hepatitis B (CHB) patients. METHODS: Hospitalized CHB patients with relapse after NAs withdrawal at our medical center were retrospectively included in the present study from January 2011 to May 2018. Logistic regression, Cox regression analysis, Kaplan-Meier log-rank test, and area under the receiver operating characteristic curves (AUROC) were used. RESULTS: A total of 389 CHB patients (including 46 ACLF patients) were included. Their median age was 48.0 years; 315 patients were male and 74 were female. The age ≥30 years and HBVDNA ≤1000 copies at admission in logistic regression were the independent risk factors for ACLF after NAs withdrawal in CHB patients. In patients who developed ACLF, only the model of end-stage liver disease combining serum natrium concentration (MELD-Na) score and relapse after Lamivudine (LAM) cessation in the Cox multivariate regression analysis were independent predictors for 12-week mortality. The artificial liver support system (ALSS) showed no improvement in the 12-week survival of ACLF patients. We further defined 22.35 as the optimal cutoff value of MELD-Na score to predict 12-week mortality for ACLF patients, with the AUROC of 0.817, a sensitivity of 76.5%, and a specificity of 75.9%. CONCLUSION: The age ≥30 years and HBVDNA ≤1000 copies at admission strongly correlate with occurrence of ACLF, and higher MELD-Na score and relapse after LAM withdrawal are closely related with 12-week mortality among patients with ACLF after NAs withdrawal.

Relation of Circulating Resistin to Insulin Resistance in Type 2 Diabetes and Obesity: A Systematic Review and Meta-Analysis.

Background: Resistin, a cysteine-rich polypeptide encoded by the RETN gene, which plays an important role in many mechanisms in rodent studies, including lipid metabolism, inflammation and insulin resistance. Nevertheless, the relationship between resistin and insulin resistance in humans is under debate. The present study was designed to clarify the correlation between resistin and insulin resistance. Methods: A systematic literature search was performed using PubMed, Embase and Cochrane Library until March 3, 2019 with the keywords "resistin" and "insulin resistance." Funnel plots and Egger's test were used to detect publication bias. A random-effects model was used to calculate the pooled effect size. Subgroup analysis and meta regression was performed to identify the sources of heterogeneity. Results: Fifteen studies were included in our systematic review. Among them, 10 studies with Pearson coefficients were used for meta-analysis. We found resistin levels were weakly correlated with insulin resistance in those with T2DM and obesity (r = 0.21, 95% CI: 0.06-0.35, I 2 = 59.7%, P = 0.003). Nevertheless, subgroup analysis suggested that circulating resistin levels were significantly positively correlated with insulin resistance in individuals with hyperresistinemia (≥14.8 ng/ml) (r = 0.52, 95% CI: 0.35-0.68, I 2 = 0.0%, P = 0.513). And there was no relationship between circulating resistin and insulin resistance in those with normal circulating resistin levels (<14.8 ng/ml) (r = 0.08, 95% CI: -0.01-0.18, I 2 = 0.0%, P = 0.455). Publication bias was insignificant (Egger's test P = 0.592). Conclusion: In T2DM and obese individuals, resistin levels were positively correlated with insulin resistance in those with hyperresistinemia, but not in those with normal circulating resistin levels.

Recombinant betatrophin (Angptl­8/lipasin) ameliorates streptozotocin­induced hyperglycemia and ß­cell destruction in neonatal rats.

Betatrophin [also known as lipasin, angiopoietin­like 8 (ANGPTL8), refeeding induced in fat and liver (RIFL), or hepatocellular carcinoma­associated gene TD26], a 22­kDa protein in the angiopoietin­like family, is a liver­derived hormone that promotes pancreatic ß­cell proliferation and lipid metabolism. The aim of the present study was to investigate the effect of recombinant betatrophin on ß­cell regeneration in a neonatal streptozotocin (STZ)­induced diabetic rat model. One­day­old Wistar rats were injected with STZ (100 mg/kg), followed by intraperitoneal administration of betatrophin to the STZ­injected rats for 6 days. Plasma glucose and body weight were monitored. On days 4 and 7, expression levels of pancreatic duodenal homeobox gene­1 (PDX­1), the Bax/B­cell lymphoma­2 (Bcl­2) ratio and plasma insulin were assessed, and the ß­cell proliferation rate was determined. Pancreatic islet area and number were determined at 10 weeks. It was found that betatrophin treatment alleviated STZ­induced hyperglycemia, elevated pancreatic expression levels of Bcl­2, PDX­1, plasma insulin levels and the ß­cell proliferation rate on days 4 and 7. Long­term betatrophin treatment improved glucose tolerance, associated with improved plasma insulin levels and ß­cell mass. These results suggest that early administration of betatrophin promotes ß­cell proliferation in STZ­induced diabetic neonates and prevents the development of diabetes in adults.

Effects of Lifestyle Modification and Anti-diabetic Medicine on Prediabetes Progress: A Systematic Review and Meta-Analysis.

Background: Pre-diabetes is a risk factor for full-blown diabetes; it presents opportunities to prevent the actual diseases. It is therefore essential to identify effective preventive strategies, and to clarify the direction of future research. Methods: PubMed, Embase and Cochrane Central Register of Controlled Trials were searched using key terms (Supplementary Table 1). We applied network meta-analysis to multiple comparisons among various diabetic preventive strategies, including lifestyle and pharmacological interventions; traditional meta-analysis for the synthesis of basal metabolic changes after interventions; and trial sequential analysis for determinations as to whether analysis conclusions meet expectations. Results: We included 32 randomized controlled trials comprising 43,669 patients and 14 interventions in the meta-analysis. Both lifestyle modifications and anti-diabetic medications improved physical conditions, including weight loss, blood glucose, and blood pressure. Network meta-analysis suggested that the progression of diabetes could be delayed to varying degrees by lifestyle and pharmacological interventions, except for angiotensin-converting enzyme inhibitors, statins, sulfonylureas and vitamin D. The risk ratios (RR) [95% credible interval (CrI)] compared with control were: GLP-1RAs 0.28 (0.15, 0.50), Orlistat 0.33 (0.18, 0.55), TZM 0.33 (0.16, 0.63), TZD 0.39 (0.27, 0.53), LST 0.54 (0.32, 0.88), lifestyle 0.58 (0.49, 0.67), LSM 0.62 (0.45, 0.80), GI 0.66 (0.46, 0.88), SU 0.67 (0.40, 1.00), Vitamin D 0.91 (0.59, 1.40), ACEI 0.93 (0.62, 1.40), statins 1.20 (0.84, 1.60). Conclusions: In adults with pre-diabetes, firm evidence supports the notion that lifestyle modifications and metformin reduces the incidence of diabetes with an average of 20% relative risk reduction, while statins increase the relative risk 20%. We found that lifestyle modifications, promising long-term strategies involving three factors (nutrition, exercise, and weight loss) contribute to health by reducing BMI, body weight, waist and hip circumference, systolic and diastolic pressure, fasting, and 2-h postprandial blood glucose, total cholesterol and by increasing HDL. We made this determination using TSA, avoiding further waste of experimental resources.

Correction to: Systematic Review and Meta-Analysis of the Change in Ghrelin Levels After Roux-en-Y Gastric Bypass.

This article was initially published with incorrect copyright information. Upon publication of this correction, the copyright of this article changed to "The Author(s)." The original article has been corrected.

Systematic Review and Meta-analysis of the Change in Ghrelin Levels After Roux-en-Y Gastric Bypass.

BACKGROUND: Roux-en-Y gastric bypass (RYGB) is considered effective for weight loss and for treatment of many obesity-related metabolic diseases. Ghrelin is an essential orexigenic peptide that plays an indispensable role in controlling body weight and energy homeostasis of post-operative patients. This systematic review and meta-analysis aimed to investigate changes in the level of fasting total ghrelin following RYGB. METHODS: A systematic literature search of PubMed, EMBASE, and the Cochrane Library until April 2018 with keywords "ghrelin" and "gastric bypass" was performed in accordance with the MOOSE guidelines and PRISMA statement. Three reviewers independently selected the studies and extracted data. Quality assessment of the included studies was undergone. A random effects model was employed to calculate overall effect sizes. Subgroup analyses and meta-regression were subsequently performed. RESULTS: Sixteen studies with 325 patients were included. We found ghrelin levels had an increasing tendency (SMD = 0.30; 95% CI = 0.04 to 0.57) despite moderate heterogeneity (I2 = 58%). Subsequent subgroup analysis indicated that ghrelin levels decreased (SMD = - 0.49; 95% CI = - 0.98 to 0.00) in the short term (≤ 3 months) and increased (SMD = 0.46; 95% CI = 0.22 to 0.69) in the long term (> 3 months) after RYGB. Meta-regression showed that gastric pouch volume, alimentary limb length and biliopancreatic limb length were not associated with changes in ghrelin levels. CONCLUSION: Fasting total ghrelin levels decreased in the short term (≤ 3 months) and increased in the long term (> 3 months) after RYGB.

Ghrelin Receptor Is Required for the Effect of Nesfatin-1 on Glucose Metabolism.

Studies of nesfatin-1 in glucose metabolism have become a topic of interest recently, however, the specific receptor for nesfatin-1 has not yet been identified. Some studies hinted at a connection between nesfatin-1 and the ghrelin receptor, growth hormone secretagogue receptor. Therefore, we aimed to study the role of GHSR in the glycemic effects of nesfatin-1 as well as its downstream pathways. We employed C57/BL6 mice (wild type and GHSR knockout mice) eating a normal chow diet and a high fat diet in this study, and the experimental technique included western blot, real-time PCR, immunofluorescence and ELISA. We found that in mice fed a normal chow diet (NCD), nesfatin-1 improved glucose tolerance, up-regulated AKT kinase (AKT) mRNA levels and phosphorylation and GLUT4 membrane translocation in skeletal muscle. These effects were blocked by co-injection of GHSR antagonist [D-Lys3]-GHRP-6 and were attenuated in GHSR knockout mice. In mice fed high-fat diet (HFD), nesfatin-1 not only exerted the effects observed in NCD mice, but also suppressed appetite and raised AKT levels in liver tissues that also required GHSR. Peripheral nesfatin-1 suppressed c-fos expression of GHSR immunoreactive neurons induced by fasting in hypothalamic nuclei, indicating that nesfatin-1 inhibited the activation of central GHSR. We concluded that the effects of nesfatin-1 on food intake and glucose metabolism were GHSR-dependent, and that the glycemic effect was associated with AKT and GLUT4. This study should stimulate further exploration of the nesfatin-1 receptor.

The Correlation Between Circulating Ghrelin and Insulin Resistance in Obesity: A Meta-Analysis.

Background: Ghrelin, a peptide mainly produced by stomach X-A cells. It plays a pivotal role in the regulation of food intake and energy metabolism, including glucose metabolism and insulin sensitivity. However, the correlation between circulating ghrelin levels and insulin resistance in obesity remained uncertain. This meta-analysis aimed to clarify the association between ghrelin and IR in obesity. Methods: A systematic literature search was performed using PubMed, EMBASE, Cochrane Library and Web of Science until April 18, 2018 with the keywords "ghrelin" and "insulin resistance." Two independent reviewers selected studies and assessed data. Subgroup analyses were performed to search for sources of heterogeneity. Funnel plots and Egger's test were used to detect publication bias. A random-effects model was used to calculate the pooled effect size. Results: Ten studies with 546 participants were included in this meta-analysis. We found that ghrelin levels were negatively correlated with IR in obese individuals. (r = -0.31; 95% CI: -0.45 to -0.18). Subgroup analysis revealed that circulating ghrelin levels were significantly negatively correlated with IR in people with normal fasting blood glucose (FBG) (<6.9 mmol/dl) (r = -0.28; 95% CI: -0.47 to -0.09, I 2 = 39.5%), while there was no relationship between circulating ghrelin levels and IR in the high FBG group (>6.9 mmol/dl) (r = -0.15; 95% CI: -0.33 to 0.03, I 2 = 0.0%). Publication bias was insignificant (Egger's test: P = 0.425). Conclusion: In obesity, circulating ghrelin levels were significantly negative correlated with insulin resistance in individuals with normal fasting blood glucose.

Nesfatin-1 in the Lateral Parabrachial Nucleus Inhibits Food Intake, Modulates Excitability of Glucosensing Neurons, and Enhances UCP1 Expression in Brown Adipose Tissue.

Nesfatin-1, an 82-amino acid neuropeptide, has been shown to induce anorexia and energy expenditure. Food intake is decreased in ad libitum-fed rats following injections of nesfatin-1 into the lateral, third, or fourth ventricles of the brain. Although the lateral parabrachial nucleus (LPBN) is a key regulator of feeding behavior and thermogenesis, the role of nesfatin-1 in this structure has not yet been delineated. We found that intra-LPBN microinjections of nesfatin-1 significantly reduced nocturnal cumulative food intake and average meal sizes without affecting meal numbers in rats. Because glucose sensitive neurons are involved in glucoprivic feeding and glucose homeostasis, we examined the effect of nesfatin-1 on the excitability of LPBN glucosensing neurons. In vivo electrophysiological recordings from LPBN glucose sensitive neurons showed that nesfatin-1 (1.5 × 10-8 M) excited most of the glucose-inhibited neurons. Chronic administration of nesfatin-1 into the LPBN of rats reduced body weight gain and enhanced the expression of uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) over a 10-day period. Furthermore, the effects of nesfatin-1 on food intake, body weight, and BAT were attenuated by treatment with the melanocortin antagonist SHU9119. These results demonstrate that nesfatin-1 in LPBN inhibited food intake, modulated excitability of glucosensing neurons and enhanced UCP1 expression in BAT via the melanocortin system.

Modulation of NRF2 and UGT1A expression by epigallocatechin-3-gallate in colon cancer cells and BALB/c mice.

BACKGROUND: Green tea is an important source of flavonoids in human diets and epidemiological data correlate green tea consumption with a reduced cancer risk. Given its complicated properties at effective concentrations, we put epigallocatechin-3-gallate (EGCG) that previously reported on its anti-proliferative activities against several cancer cell lines on our research agenda to further examine the mechanism of its chemopreventive potential. METHODS: RNA interference (RNAi) expression vector pSilencer 3.1-H1 was used to construct recombinant nuclear factor erythroid 2 related factor 2 (Nrf2)-targeting RNAi plasmids. EGCG (5 microg/ml) was added into the culture fluid of cells before and after transfection. RT-PCR and Western blotting were used to detect the expression of uridine 5'-diphosphate-glucuronosyltransferase (UGT) 1A in cells. Forty male BALB/c mice were assigned to four groups: a normal unexposed control and three groups treated with varying doses of EGCG. Four weeks later, the mice were sacrificed, and their colon tissues were subjected to mRNA and protein expression of Nrf2 and UGT1A via RT-PCR and Western blotting analysis. RESULTS: EGCG up-regulated the expression of Nrf2 and increased the level of UGT1A in cells. The blockade of Nrf2 activity via RNA intervention largely attenuated the induction of UGT1A expression by EGCG. In mice, the mRNA and protein levels of Nrf2 and UGT1A detected by RT-PCR and Western blotting increased (both P < 0.05 compared with the control). This increase in Nrf2 expression also had a positive correlation with an increased UGT1A expression. CONCLUSIONS: EGCG mediated its effect in part by inducing the NRF2 signaling pathway and increasing UGT1A expression. Both in vitro and in vivo studies demonstrated the role of NRF2 and UGT1A expression in the potential use of EGCG as a possible chemopreventive agent and supported further study of EGCG for cancer treatment.

Effect of ursodeoxycholic acid on TGF beta1/Smad signaling pathway in rat hepatic stellate cells.

BACKGROUND: Hepatic fibrosis is the key stage of the pathological progress from hepatic injury to cirrhosis. Ursodeoxycholic acid (UDCA) has been known as having significant clinical therapeutic effects on chronic liver diseases. Our research aimed to study the effect of UDCA on the signaling pathway of transforming growth factor beta1 (TGFbeta1)/Smad and discuss its possible molecular mechanisms of inhibiting hepatic fibrosis. METHODS: Rat hepatic stellate cells were cultured in vitro and randomly assigned to 4 groups. Group A was control group, with only DMEM culture medium applied, and groups B, C, D were experimental groups, with different doses of UDCA (1.0 mmol/L, 0.5 mmol/L and 0.25 mmol/L respectively) added into their DMEM culture medium for further culture of 24 hours and 48 hours. The protein expressions of TGFbeta1, TGF type I receptor, Smad3, Smad4 and Smad7 were measured by Western blotting, as well as the expressions of TGFbeta1, Smad3, Smad7 and cAMP response element (CREB) binding protein (CBP) mRNA by real-time PCR. SPSS 11.5 statistical package was adopted for data analyses. RESULTS: Compared with control group, the mRNA expressions of TGFbeta1 in the high and middle UDCA dose groups for 24 hours and 48 hours significantly decreased (P < 0.05), the protein expressions of TGFbeta1 in the two above groups for 48 hours and in the high dose group for 24 hours significantly decreased (P < 0.05). The protein and mRNA expressions of Smad3 in each UDCA dose group for 24 hours and 48 hours significantly decreased, with significant difference among different UDCA dose groups and between that of 24 hours and 48 hours observed (P < 0.05). The protein and mRNA expressions of Smad7 in the high and middle UDCA dose groups for 24 hours and 48 hours significantly increased. The CBP mRNA expression in each UDCA dose group for 24 hours and 48 hours significantly decreased (P < 0.05), with significant difference among different UDCA dose groups observed (P < 0.05). CONCLUSION: UDCA could curb the development of hepatic fibrosis through affecting the signaling pathway of TGFbeta1/Smad by inhibiting the expressions of TGFbeta1, Smad3 and CBP and increasing the expression of Smad7.

Protective effects of epigallocatechin gallate on colon preneoplastic lesions induced by 2-amino-3-methylimidazo[4,5-f ] quinoline in mice.

Epigallocatechin gallate (EGCG), a key active ingredient in green tea, has multiple anticarcinogenic effects. The aim of the present study was to investigate if EGCG could prevent the formation of colon aberrant crypt foci (ACF) induced by 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ) and to explore possible mechanisms for resultant effects. Sixty male BALB/cA nude, immunodeficient mice were divided into six groups including a normal unexposed control, mice induced with IQ alone, three groups treated with varying doses of EGCG post-IQ induction, and a EGCG-treated control population. Six weeks later, the mice were killed, and tissues subjected to hematoxylin-eosin (H&E) and 0.2% methylene blue staining to observe histopathological alterations of colon mucus and the formation of ACF, respectively. Protein expression of NF-E2-related factor 2 (Nrf2) was assessed via immunohistochemistry (IHC) and Western analysis, and mRNA levels of Nrf2 and uridine 5'-diphosphate-glucuronosyltransferase (UGT)1A10 were determined in colon tissues. Our results demonstrate that, compared with IQ-induced controls, the degree of atypical hyperplasia decreased and the number of total ACF and total AC also decreased significantly (P < 0.05 and P < 0.01, respectively) in mice belonging to all EGCG dosing groups. At the same time, the protein levels of Nrf2 detected by IHC and Western blotting increased (both P < 0.01 compared with IQ group), and the mRNA levels of Nrf2 and UGT1A10 increased (both P < 0.01 compared with IQ group). In conclusion, EGCG had preventive effects on preneoplastic lesions induced by IQ. Our observations suggest that this effect may be the result of activation of the Nrf2-UGT1A10 signaling pathway.

Inhibition of epigallocatechin gallate on orthotopic colon cancer by upregulating the Nrf2-UGT1A signal pathway in nude mice.

Epigallocatechin gallate (EGCG), a key active ingredient in green tea, has many anti-carcinogenic activities. The aim of the present study was to investigate whether EGCG could prevent the occurrence or metastases of orthotopic colon cancer and probe the underlined mechanisms. We observed the inhibition of EGCG on growth and metastases of colon tumor implanted orthotopically in the cecum of nude mice. Immunohistochemistry and Western-blotting analysis were used to detect NF-E2-related factor 2 (Nrf2) protein expressions. RT-PCR was also applied to detect the mRNA levels of Nrf2, uridine 5'-diphosphate-glucuronosyltransferase (UGT) 1A, UGT1A8 and UGT1A10 in colon tumors. As a result, the inhibition rates on tumor growth in the 3 EGCG groups were significantly different (all p < 0.001) compared with the control group. In addition, different doses of EGCG were able to inhibit liver and pulmonary metastases to varying degrees. The protein level of Nrf2 and the mRNA levels of Nrf2, UGT1A, UGT1A8 and UGT1A10 significantly increased in EGCG-treated mice in comparison with the control group (all p < 0.01). The results demonstrated that EGCG has a preventive effect on the growth and liver and pulmonary metastases of orthotopic colon cancer in nude mice, and this anticancer effect could be partly caused by activating the Nrf2-UGT1A signal pathway.

[The role of NF-E2-related factor 2 in the induction of uridine 5'-diphosphate-glucuronosyltransferase 1A and its isoforms by epigallocatechin gallate in colon cancer cells].

OBJECTIVE: To investigate the role of human transcription factor NF-E2-related factor 2 (Nrf2) in the induction of the gene expression of uridine 5'-diphosphate-glucuronosyltransferase (UGT) 1A and its isoforms by epigallocatechin gallate (EGCG). METHODS: (1) Human colon carcinoma cells Caco-2 and HT-29 were cultured. Immunocytochemistry, western blotting and confocal laser microscopy were used to detect the protein expression of Nrf2. Twenty samples of colon carcinoma with surrounding normal tissues were collected during endoscopic course. (2) RNA interference expression vector pSilencer 3.1-H1 was used to construct four Nrf2-trageting plasmids: pSilence-Nrf2-A, B, C, and D and a control pSilence-CON. Cells were transfected with pSilence-Nrf2 for 48 hours to observe the effects of transient transfection. Cells were stably transfected with pSilence-Nrf2-B for 4 weeks and re-named as Caco-2-siNrf2 and HT-29-siNrf2 (siNrf2 cells), and others stably transfected with blank plasmid pSilencer 3.1-H1 were used as controls. (3) EGCG was added into the culture fluid of cells before and after the stably transfection. RT-PCR was used to detect the mRNA expression of Nrf2, UGT1A, UGT1A8 and UGT1A10 in cells and the samples of human colon cancer tissue. RESULTS: (1) The expressions of UGT1A8 and UGT1A10 mRNA were significantly lower than that in the surrounding healthy mucosa. (2) The mRNA expression of Nrf2, UGT1A8, and UGT1A10 increased by 1.8-9.2 times after the addition of EGCG (all P < 0.05). Immunocytochemistry, western blotting and immunofluorescence demonstrated a significant increase of Nrf2 protein expression in the nucleus after treatment with EGCG. (3) SalIenzyme digestion and DNA sequencing confirmed that pSilence-Nrf2-A, B, C, and D and pSilence-CON were all successfully constructed. The inhibition rate of Nrf2 gene expression was above 80% 48 h after transfection with pSilence-Nrf2-B, and that was no significant difference after transfection with pSilence-CON (P > 0.05). There was specific inhibition of Nrf2 in Caco-2-siNrf2, HT-29-siNrf2 cells (both P < 0.01). (4) The basal levels of UGT1A8 and UGT1A10 mRNA expression in the Caco-2-siNrf2 and HT-29-siNrf2 cells were lower by 15%-65% in comparison with those in control, and the induction of genes by EGCG was largely attenuated in them (all P > 0.05). CONCLUSION: Nrf2 is localized in the cytoplasm of non-stimulated cells, and EGCG triggered its rapid nuclear accumulation. Suppression of Nrf2 gene expression results in down-regulation of the constructive expression of UGT genes and their induction by EGCG. EGCG induces the expression of UGT1A, UGT1A8 and UGT1A10 genes via a Nrf2-dependent mechanism.