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This study investigated whether a standard calorie diet that is high in glucose and deficient in dietary fibre (described as HGD [high glucose diet]) induces hepatic fat accumulation in mice. We evaluated hepatic steatosis at 7 days and 14 days after the commencement of the HGD. Hepatic triglycerides and areas of oil droplets increased in the HGD group both at day 7 and day 14, whereas weight gain, weight of epididymal fat, and plasma levels of triglycerides were unaffected by HGD consumption. A microarray analysis of the livers revealed that the expression of lipogenesis-related genes was the most affected by HGD consumption. Furthermore, HGD consumption induced the expression of hepatic proteins of fatty acid synthetase, acetyl-CoA carboxylase alpha, and stearoyl-CoA desaturase 1, which are known to be involved in the synthesis of triglyceride. These results indicate that HGD consumption causes fat accumulation in the liver, with an increase in enzymes that are involved in de novo lipogenesis without an accompanying weight or obesity phenotype. Our new findings suggest that HGD consumption could serve as a breeding ground for liver steatosis., Competing Interests: The authors have no conflicts of interest to declare., (© 2024 The Authors.)

Background: Specific bacterial species in the oral cavity contribute to cerebral hemorrhage and microbleeds. The relationship between oral bacterial flora and asymptomatic brain lesions (ABL) remains unclear. This study aimed to investigate this relationship in a healthy Japanese cohort., Methods: This cross-sectional study included participants who underwent health examinations at our Brain Dock facility between October 2020 and March 2021. The oral microbiomes of participants with and without ABL were compared using magnetic resonance imaging. To extensively assess the oral bacterial flora, the differences in genes and species compositions between the ABL and noBL (without brain lesions) groups were statistically evaluated via extensive analysis using 16S rRNA gene-based cloning., Results: Among 143 patients, 48.3 % had ABL. In the univariate analyses, Fusobacterium and Leptotrichia were associated with ABL (P = 0.017 and P < 0.001, respectively). In the adjusted models, Fusobacterium was associated with ABL (P = 0.006). In an intergroup comparison of seven Fusobacterium species, F. nucleatum , F. naviforme , and F. canifelinum were associated with ABL (P < 0.001, P = 0.002, P < 0.001)., Conclusions: The elevation of Fusobacterium in the ABL indicates the importance of the microbiome in the oral cavity as a factor in inducing cerebral small-vessel disease in healthy individuals, whose preventive approach might have an impact on therapeutic applications., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Background: Cholesterol levels and bile acid metabolism are important drivers of metabolic dysfunction-associated steatohepatitis (MASH) progression. Using a mouse model, we investigated the mechanism by which cholesterol exacerbates MASH and the effect of colestyramine (a bile acid adsorption resin) and elobixibat (an apical sodium-dependent bile acid transporter inhibitor) concomitant administration on bile acid adsorption and MASH status., Methods: Mice were fed a high-fat high-fructose diet with varying concentrations of cholesterol to determine changes in fatty liver according to liver status, water intake, defecation status, insulin resistance, bile acid levels, intestinal permeability, atherosclerosis (in apolipoprotein E knockout mice), and carcinogenesis (in diethylnitrosamine mice). Using small interfering ribonucleic acid (siRNA), we evaluated the effect of sterol regulatory element binding protein 1c (SREBP1c) knockdown on triglyceride synthesis and fatty liver status following the administration of elobixibat (group E), colestyramine (group C), or both (group EC)., Results: We found greater reductions in serum alanine aminotransferase levels, serum lipid parameters, serum primary bile acid concentrations, hepatic lipid levels, and fibrosis area in EC group than in the monotherapy groups. Increased intestinal permeability and watery diarrhea caused by elobixibat were completely ameliorated in group EC. Group EC showed reduced plaque formation rates in the entire aorta and aortic valve of the atherosclerosis model, and reduced tumor counts and tumor burden in the carcinogenesis model., Conclusions: Excessive free cholesterol in the liver can promote fatty liver disease. Herein, combination therapy with EC effectively reduced free cholesterol levels in MASH model mice. Our study provides strong evidence for combination therapy as an effective treatment for MASH., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.)

Introduction: Periodontal disease is associated with nonalcoholic fatty liver disease (NAFLD). We evaluated periodontal treatment efficacy in patients with NAFLD and periodontal disease., Methods: This multicenter, 2-arm, randomized study recruited adult patients with NAFLD and periodontitis, alanine aminotransferase levels ≥40 U/L, and equivalent steatosis grade ≥1. Forty eligible patients (18 men and 22 women) were randomly assigned to 2 groups (scaling and root planning [SRP; n = 20] and tooth brushing [n = 20] groups) stratified by age and sex. The primary and secondary endpoints were changes in alanine aminotransferase levels and serum Porphyromonas gingivalis IgG antibody titers from baseline to 12 weeks, respectively. Efficacy analysis was performed using an intention-to-treat approach ( t test). This trial was registered with the University Hospital Medical Information Network Clinical Trials Registry (UMIN000022079)., Results: We observed a significantly higher decrease in absolute alanine aminotransferase levels and P. gingivalis IgG antibody titers in the SRP group than in the tooth brushing group (-12 vs 1 U/L; mean difference [δ], -12; 95% confidence interval [CI], -20 to -5; P = 0.002). The decrease in P. gingivalis IgG antibody titer was significantly higher in the SRP group than in the tooth brushing group (FDC381, -1.6 [2.5]; δ, -1.6; 95% CI, -2.7 to -0.4; P = 0.0092; SU63, -1.7 [2.0]; δ, -1.7; 95% CI, -2.7 to -0.7). No life-threatening events or treatment-related deaths occurred., Discussion: Periodontal treatment induced significant short-term and mid-term reductions in liver enzyme levels and antibody titers. Further research is warranted to clearly define SRP efficacy and tolerability in patients with NAFLD and periodontitis., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

The risk factors for non-alcoholic fatty liver disease (NAFLD) progression are not completely known. Porphyromonas gingivalis infection is a risk factor for systemic diseases. We investigated the association of P. gingivalis infection with the risk of non-alcoholic steatohepatitis progression. Here, hematological tests, periodontal examination, and saliva collection were performed for 164 patients with NAFLD. P. gingivalis was identified in saliva using polymerase chain reaction. Hepatic steatosis and stiffness were evaluated using vibration-controlled transient elastography (VCTE) and magnetic resonance imaging. In patients with NAFLD, P. gingivalis positivity (P. gingivalis ratio ≥ 0.01%) in saliva correlated with liver stiffness determined using magnetic resonance elastography (MRE; p < 0.0001). A P. gingivalis ratio of 0.01% corresponds to 100,000 cells/mL and indicates the proportion of P. gingivalis in the total number of bacteria in the oral cavity. Patients with NAFLD and advanced fibrosis on MRE showed significantly elevated endotoxin activity; those who had > 10 periodontal pockets with depths ≥ 4 mm had significantly increased hepatic stiffness on both VCTE and MRE., (© 2022. The Author(s).)

Introduction: No reports on both blood and fecal bile acids (BAs) in patients with nonalcoholic fatty liver disease (NAFLD) exist. We simultaneously assessed the serum and fecal BA patterns in healthy participants and those with NAFLD., Methods: We collected stool samples from 287 participants from 5 hospitals in Japan (healthy control [HC]: n = 88; mild fibrosis: n = 104; and advanced fibrosis group: n = 95). Blood samples were collected and analyzed for serum BAs and 7α-hydroxy-4-cholesten-3-one (C4)-a surrogate marker for BA synthesis ability-from 141 patients. Concentrations of BAs, including cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid, ursodeoxycholic acid, and lithocholic acid (LCA), were measured using liquid chromatography-mass spectrometry., Results: The total fecal BA concentration was significantly higher in the NAFLD group with worsening of fibrosis than in the HC group. Most of the fecal BAs were secondary and unconjugated. In the fecal BA fraction, CA, DCA, chenodeoxycholic acid, ursodeoxycholic acid, and LCA were significantly higher in the NAFLD than in the HC group. The total serum BA concentration was higher in the NAFLD group with worsening of fibrosis than in the HC group. In the serum BA fraction, CA, LCA, and C4 concentrations were significantly higher in the NAFLD than in the HC group., Discussion: Fecal and serum BA and C4 concentrations were high in patients with NAFLD with worsening of fibrosis, suggesting involvement of abnormal BA metabolism in NAFLD with fibrosis progression. Abnormalities in BA metabolism may be a therapeutic target in NAFLD with fibrosis., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

Fusobacterium nucleatum is associated with the progression of colorectal cancer. Thus, the possibility of preventing colorectal cancer or its progression by targeting F. nucleatum has been explored. As F. nucleatum is associated with periodontitis, we analysed whether treating periodontitis could influence F. nucleatum abundance in the colon. Patients with colorectal tumours who underwent colonoscopy were recruited. Patients diagnosed with periodontitis by a dentist were treated for approximately 3 months. Endoscopic resection of colorectal tumours was performed after periodontitis treatment, and resected tumours were pathologically classified as high-(HGD) or low-grade dysplasia (LGD). Saliva and stool samples were collected before and after the treatment. Of the 58 patients with colorectal tumours, 31 were included in the study, 16 showed improvement in periodontitis, and 11 showed no improvement. Stool F. nucleatum levels before treatment were significantly lower in the LGD group than in the HGD group. A significant decrease in faecal F. nucleatum levels was observed in patients who underwent successful treatment but not in those whose treatment failed. Salivary F. nucleatum levels were not altered in patients despite periodontal treatment. Thus, successful periodontitis treatment reduces stool F. nucleatum levels and may aid research on periodontitis and suppression of colorectal cancer development., (© 2021. The Author(s).)

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide. It occurs with a prevalence of up to 25%, of which 10-20% cases progress to nonalcoholic steatohepatitis (NASH), cirrhosis, and liver cancer. The histopathology of NASH is characterized by neutrophilic infiltration, and endotoxins from gram-negative rods have been postulated as a contributing factor. Elevations in endotoxin levels in the blood can be classified as intestinal and hepatic factors. In recent years, leaky gut syndrome, which is characterized by impaired intestinal barrier function, has become a significant issue. A leaky gut may prompt intestinal bacteria dysbiosis and increase the amount of endotoxin that enters the liver from the portal vein. These contribute to persistent chronic inflammation and progressive liver damage. In addition, hepatic factors suggest that liver damage can be induced by low-dose endotoxins, which does not occur in healthy individuals. In particular, increased expression of CD14, an endotoxin co-receptor in the liver, may result in leptin-induced endotoxin hyper-responsiveness in obese individuals. Thus, elevated blood endotoxin levels contribute to the progression of NASH. The current therapeutic targets for NASH treat steatosis and liver inflammation and fibrosis. While many clinical trials are underway, no studies have been performed on therapeutic agents that target the intestinal barrier. Recently, a randomized placebo-controlled trial examined the role of the intestinal barrier in patients with NAFLD. To our knowledge, this study was the first of its kind and study suggested that the intestinal barrier may be a novel target in the future treatment of NAFLD., Competing Interests: ANa receives grants and research support from Gilead, Mylan EPD, EA Pharma, Kowa, Taisho, and Biofermin. ANa is also a consultant for Gilead, Boehringer Ingelheim, Bristol Myers Squibb, Kowa, Astellas, EA Pharma, and Mylan EPD. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kessoku, Kobayashi, Imajo, Tanaka, Yamamoto, Takahashi, Kasai, Ozaki, Iwaki, Nogami, Honda, Ogawa, Kato, Higurashi, Hosono, Yoneda, Okamoto, Usuda, Wada, Kobayashi, Saito and Nakajima.)

Cardiovascular diseases including blood vessel disorders represent a major cause of death globally. The essential roles played by local and systemic vascular inflammation in the pathogenesis of cardiovascular diseases have been increasingly recognized. Vascular inflammation triggers the aberrant activation of endothelial cells, which leads to the functional and structural abnormalities in vascular vessels. In addition to humoral mediators such as pro-inflammatory cytokines and prostaglandins, the alteration of physical and mechanical microenvironment - including vascular stiffness and shear stress - modify the gene expression profiles and metabolic profiles of endothelial cells via mechano-transduction pathways, thereby contributing to the pathogenesis of vessel disorders. Notably, connexins and integrins crosstalk each other in response to the mechanical stress, and, thereby, play an important role in regulating the mechano-transduction of endothelial cells. Here, we provide an overview on how the inter-play between connexins and integrins in endothelial cells unfold during the mechano-transduction in vascular inflammation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder. Traumatic stress during adolescence increases the risk of IBS in adults. The aim of this study was to characterize the juvenile social defeat stress (SDS)-associated IBS model in mice. Juvenile mice were exposed to an aggressor mouse for 10 min once daily for 10 consecutive days. Behavioral tests, visceral sensitivity, immune responses, and fecal bacteria in the colon were evaluated in 5 weeks after SDS exposure. Social avoidance, anxiety- and depression-like behavior, and visceral hypersensitivity were observed. Juvenile SDS exposure significantly increased the number of 5-HT-containing cells and calcitonin gene-related peptide-positive neurons in the colon. The gut microbiota was largely similar between the control and juvenile SDS groups. The alterations in fecal pellet output, bead expulsion time, plasma corticosterone concentration, and colonic 5-HT content in response to restraint stress were exacerbated in the juvenile SDS group compared with the control group. The combination of juvenile SDS and restraint stress increased the noradrenaline metabolite 3-Methoxy-4-hydroxyphenylglycol (MHPG) content and MHPG/noradrenaline ratio in the amygdala when compared with restraint stress in control mice. These results suggest that juvenile SDS exposure results in later onset of IBS-like symptoms., (© 2021. The Author(s).)

Background and Aim: Gut microbiota composition is associated with the pathogenesis of non-alcoholic fatty liver disease. However, the association between gut microbiota composition and non-alcoholic fatty liver disease in non-obese patients remains unclear. We compared clinical parameters and gut microbiota profiles of healthy controls and non-obese and obese patients with non-alcoholic fatty liver disease., Methods: We examined the clinical parameters and gut microbiota profiles by 16S rRNA sequences and short-chain fatty acid levels in fecal samples from 51 non-obese patients with non-alcoholic fatty liver disease (body mass index <25 kg/m 2 ) and 51 obese patients with non-alcoholic fatty liver disease (body mass index ≥30 kg/m 2 ) who underwent pathological examination and 87 controls at five hospitals in Japan., Results: Although no significant differences between the non-obese and other groups were observed in alpha diversity, a significant difference was found in beta diversity. We observed a significant decrease in serum alanine aminotransferase levels, Eubacterium population, and butyric acid levels in non-obese patients with non-alcoholic fatty liver disease compared with those in obese patients with non-alcoholic fatty liver disease. A significant negative correlation was found between the stage of hepatic fibrosis and Eubacterium abundance in non-obese patients with non-alcoholic fatty liver disease., Conclusions: The decrease in the abundance of Eubacterium that produces butyric acid may play an important role in the development of non-alcoholic fatty liver disease in non-obese individuals. This study was registered at the University Hospital Medical Information Network clinical trial registration system (UMIN000020917)., (© 2021 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

The liver directly accepts blood from the gut and is, therefore, exposed to intestinal bacteria. Recent studies have demonstrated a relationship between gut bacteria and nonalcoholic fatty liver disease (NAFLD). Approximately 10-20% of NAFLD patients develop nonalcoholic steatohepatitis (NASH), and endotoxins produced by Gram-negative bacilli may be involved in NAFLD pathogenesis. NAFLD hyperendotoxicemia has intestinal and hepatic factors. The intestinal factors include impaired intestinal barrier function (leaky gut syndrome) and dysbiosis due to increased abundance of ethanol-producing bacteria, which can change endogenous alcohol concentrations. The hepatic factors include hyperleptinemia, which is associated with an excessive response to endotoxins, leading to intrahepatic inflammation and fibrosis. Clinically, the relationship between gut bacteria and NAFLD has been targeted in some randomized controlled trials of probiotics and other agents, but the results have been inconsistent. A recent randomized, placebo-controlled study explored the utility of lubiprostone, a treatment for constipation, in restoring intestinal barrier function and improving the outcomes of NAFLD patients, marking a new phase in the development of novel therapies targeting the intestinal barrier. This review summarizes recent data from studies in animal models and randomized clinical trials on the role of the gut-liver axis in NAFLD pathogenesis and progression.

Intestinal tract is the boundary that prevents harmful molecules from invading into the mucosal tissue, followed by systemic circulation. Intestinal permeability is an index for intestinal barrier integrity. Intestinal permeability has been shown to increase in various diseases-not only intestinal inflammatory diseases, but also systemic diseases, including diabetes, chronic kidney dysfunction, cancer, and cardiovascular diseases. Chronic increase of intestinal permeability is termed 'leaky gut' which is observed in the patients and animal models of these diseases. This state often correlates with the disease state. In addition, recent studies have revealed that gut microbiota affects intestinal and systemic heath conditions via their metabolite, especially short-chain fatty acids and lipopolysaccharides, which can trigger leaky gut. The etiology of leaky gut is still unknown; however, recent studies have uncovered exogenous factors that can modulate intestinal permeability. Nutrients are closely related to intestinal health and permeability that are actively investigated as a hot topic of scientific research. Here, we will review the effect of nutrients on intestinal permeability and microbiome for a better understanding of leaky gut and a possible mechanism of increase in intestinal permeability.

Chronic constipation is a functional disorder that decreases a patient's quality of life (QOL). Because dysbiosis has been associated with constipation, we aimed to investigate the efficacy of Bifidobacterium bifidum G9-1 (BBG9-1) in improving QOL in patients with constipation. This was a prospective, single-center, non-blinded, single-arm feasibility trial. A total of 31 patients with constipation and decreased QOL received BBG9-1 treatment for 8 weeks, followed by a 2-week washout period. The primary endpoint was change in the overall Japanese version of the patient assessment of constipation of QOL (JPAC-QOL) score after probiotic administration relative to that at baseline. Secondary endpoints included changes in gut microbiota, stool consistency, frequency of bowel movement, degree of straining, sensation of incomplete evacuation, and frequency of rescue drug use. The overall JPAC-QOL scores and frequency of bowel movement significantly improved after BBG9-1 administration from those at baseline (p<0.01 and p<0.01, respectively). There were no statistically significant changes in other clinical symptoms. Subset analysis revealed that patients with initial Bristol Stool Form Scale stool types of <4 had improvements in stool consistency, a significant increase in the frequency of bowel movements, and a significant alleviation in the degree of straining, following BBG9-1 administration. At the genus and species levels, Sarcina and Sarcina maxima were significantly increased. Functional analysis showed that butanoate metabolism increased significantly, whereas methane metabolism decreased significantly. We concluded that BBG9-1 is safe and improves QOL in patients with constipation. The underlying improvements may be due to changes in stool consistency., (©2021 BMFH Press.)

Background: The laxative drug lubiprostone improves intestinal permeability in healthy volunteers. We aimed to assess efficacy and safety of lubiprostone in patients with non-alcoholic fatty liver disease (NAFLD) with constipation via attenuation of intestinal permeability., Methods: This randomised, double-blind, placebo-controlled, phase 2a study in Yokohama City University Hospital, Japan, recruited patients (aged 20-85 years) with NAFLD and constipation, alanine aminotransferase (ALT) at least 40 U/L, liver stiffness (≤6·7 kPa), and hepatic fat fraction at least 5·2% when assessed by MRI-proton density fat fraction. Eligible patients were randomly assigned (11:10:9) by a computer-based system and stratified by age and sex to receive 24 μg lubiprostone, 12 μg lubiprostone, or placebo, orally, once per day for 12 weeks. The primary endpoint was the absolute changes in ALT at 12 weeks. Efficacy analysis was done by intention to treat. Safety was assessed in all treated patients. This trial was registered with University Hospital Medical Information Network Clinical Trials Registry (UMIN000026635)., Findings: Between March 24, 2017, and April 3, 2018, we screened 288 patients, of whom 150 (52%) were randomly assigned to treatment: 55 patients were assigned to receive 24 μg lubiprostone, 50 to receive 12 μg lubiprostone, and 45 to receive placebo. A greater decrease in the absolute ALT levels from baseline to 12 weeks was seen in the 24 μg lubiprostone group (mean -13 U/L [SD 19]) than in the placebo group (1 U/L [24]; mean difference -15 U/L [95% CI -23 to -6], p=0·0007) and in the 12 μg lubiprostone group (-12 U/L [21]) than in the placebo group (mean difference -13 U/L [-22 to -5], p=0·0023). 18 (33%) of 55 patients in the 24 μg group had at least one adverse event, as did three (6%) of 47 patients in the 12 μg group and three (7%) of 43 in the placebo group. The most common adverse event was diarrhoea (17 [31%] of patients in the 24 μg group, three [6%] in the 12 μg group, none in the placebo group). No life-threatening events or treatment-related deaths occurred., Interpretation: Lubiprostone was well tolerated and reduced the levels of liver enzymes in patients with NAFLD and constipation. Further studies are necessary to better define the efficacy and tolerability of lubiprostone in patients with NAFLD without constipation., Funding: Mylan EPD G.K., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Introduction: Non-alcoholic fatty liver disease (NAFLD) pathogenesis involves abnormal metabolism of cholesterol and hepatic accumulation of toxic free-cholesterol. Elobixibat (EXB) inhibits the ileal bile acid (BA) transporter. EXB and cholestyramine (CTM) facilitate the removal of free cholesterol from the liver by decreasing BA recirculation to the liver, thereby stimulating novel BA synthesis from cholesterol. In this randomised, double-blind, placebo-controlled, parallel-group, phase IIa study, we aim to provide a proof-of-concept assessment by evaluating the efficacy and safety of EXB in combination with CTM in patients with NAFLD., Methods and Analysis: A total of 100 adult patients with NAFLD, diagnosed based on low-density lipoprotein cholesterol (LDL-C) level of >120 mg/dL and liver fat content of ≥8% by MRI-based proton density fat fraction (MRI-PDFF), who meet the inclusion/exclusion criteria will be enrolled. The patients will be randomly assigned to receive the combination therapy of 10 mg EXB and 9 g CTM powder (4 g CTM), 10 mg EXB monotherapy, 9 g CTM powder monotherapy or a placebo treatment (n=25 per group). Blood tests and MRIs will be performed 16 weeks following treatment initiation. The primary study endpoint will be the absolute LDL-C level change at week 16 after treatment initiation. The exploratory endpoint will include absolute changes in the liver fat fraction as measured by MRI-PDFF. This proof-of-concept study will determine whether the combination therapy of EXB and CTM is effective and safe for patients with NAFLD., Ethics and Dissemination: Ethics approval was obtained from the Ethics Committee of Yokohama City University Hospital before participant enrolment. The results of this study will be submitted for publication in international peer-reviewed journals and the key findings will be presented at international scientific conferences., Trial Registration Number: NCT04235205., Competing Interests: Competing interests: Data will be retained in accordance with the Japanese ethical guidelines for clinical research. Participants will be allocated a unique identification (ID) number at entry. The master list linking participant personal information and ID number will be maintained in a separate locked cabinet and password-protected hard drive. Data will be analysed by ID number only. Records will be retained for 5 years after study completion, and then destroyed by the data center. AN reports grants and research support from Gilead, Mylan EPD, EA Pharma, Kowa, Taisho, Biofermin; is a consulting adviser for Gilead, Boehringer Ingelheim, BMS, Kowa, Astellas, EA Pharma, Mylan EPD. Other authors declare no competing interests., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Elobixibat, a novel inhibitor of apical sodium-dependent bile acid transporter for treating chronic constipation, increases colonic bile acid concentrations, stimulating bowel function. However, it is not clear which bile acids are altered, or whether altered gut microbiota are associated with functional effects that may alter bowel function., Aims: To investigate the effects of elobixibat on changes in the faecal concentrations of total and individual bile acids and in faecal microbiota., Methods: This was a prospective, single-centre study. After baseline period, patients received 10 mg daily of elobixibat for 2 weeks. We evaluated the effects on bowel function, changes in faecal bile acid concentrations and composition of gut bacteria, before and after elobixibat administration., Results: In the 30 patients analysed, the frequency of pre- and post-treatment bowel movements per fortnight was 7 and 10 (P < 0.001), respectively. The pre-treatment faecal bile acid concentration increased significantly from 10.9 to 15.0 µg/g stool post-treatment (P = 0.030), with a significant increase in faecal deoxycholic acid (pre-treatment 3.94 µg/g stool to post-treatment 5.02 µg/g stool, P = 0.036) and in glycine-conjugated deoxycholic and chenodeoxycholic acids. Shannon index was significantly decreased, but there were no significant changes at the genus and phylum levels., Conclusions: Short term treatment with elobixibat increased the concentrations of total bile acids and deoxycholic acid and decreased the diversity of faecal microbiota. The biological effects of elobixibat are associated with its effects on secretory bile acids, rather than the structural changes of an altered faecal microbiota., (© 2020 John Wiley & Sons Ltd.)

Endothelial cellular stiffening has been observed not only in inflamed cultured endothelial cells but also in the endothelium of atherosclerotic regions, which is an underlying cause of monocyte adhesion and accumulation. Although recombinant soluble thrombomodulin (rsTM) has been reported to suppress the inflammatory response of endothelial cells, its role in regulating endothelial cellular stiffness remains unclear. The purpose of this study was to investigate the impact of anticoagulant rsTM on lipopolysaccharide (LPS)-induced endothelial cellular stiffening. We show that LPS increases endothelial cellular stiffness by using atomic force microscopy and that rsTM reduces LPS-induced cellular stiffening not only through the attenuation of actin fiber and focal adhesion formation but also via the improvement of gap junction functionality. Moreover, post-administration of rsTM, after LPS stimulation, attenuated LPS-induced cellular stiffening. We also found that endothelial cells regulate leukocyte adhesion in a substrate- and cellular stiffness-dependent manner. Our result show that LPS-induced cellular stiffening enhances monocytic THP-1 cell line adhesion, whereas rsTM suppresses THP-1 cell adhesion to inflamed endothelial cells by reducing cellular stiffness. Endothelial cells increase cellular stiffness in reaction to inflammation, thereby promoting monocyte adhesion. Treatment of rsTM reduced LPS-induced cellular stiffening and suppressed monocyte adhesion in a cellular stiffness-dependent manner.

Background: We report the first protocol for a multicenter, randomized comparison study to compare the efficacies of periodontal scaling and root-planing treatment against that of tooth-brushing treatment for nonalcoholic fatty liver disease (NAFLD) (PERION: PERIOdontal treatment for NAFLD). Nonalcoholic steatohepatitis (NASH) is an advanced form of NAFLD, which can progress to cirrhosis and hepatocellular carcinoma. Increased endotoxemia is associated with the progression of NAFLD. Periodontal bacteria possess endotoxins; Porphyromonas gingivalis is well-known as a major pathogenic bacterium in periodontitis, and serum antibody levels for P. gingivalis are high in patients with periodontitis. Several reports have indicated that P. gingivalis is related to NAFLD. This study aims to investigate the effect of periodontal treatment for liver damage, P. gingivalis infection, and endotoxemia on patients with NAFLD., Methods: We will include adult patients (20-85 years old) with NAFLD, alanine aminotransferase (ALT) ≥ 40 IU/L, and equivalent steatosis grade ≥ 1 (target sample size, n = 40 patients; planned number of patients with outcome data, n = 32). Participants will be randomly assigned to one of two groups: a scaling and root-planing group or tooth-brushing as the usual group. The primary outcome will be the change in ALT levels from baseline to 12 weeks; the key secondary outcome will be the change in the serum immunoglobulin G (IgG) antibody titer for P. gingivalis at 12 weeks., Discussion: This study should determine whether periodontal treatment decreases liver damage, P. gingivalis infection, and endotoxemia in patients with NAFLD., Trial Registration: University Hospital Medical Information Network (UMIN) Clinical Trials Registry, ID: UMIN000022079.

Nonalcoholic fatty liver disease (NAFLD) is characterized by a spectrum of liver pathologies, from simple steatosis to steatohepatitis. Recent studies have increasingly noted the aberrant expression of microRNAs closely related to NAFLD pathologies. We have previously shown the presence of increased levels of microRNA-27b (miR-27b) in patients with NAFLD. In this study, we investigated the role of miR-27b in NAFLD by examining the impact of up-regulated miR-27b on the differentiation of preadipocytes into mature adipocytes. We found that miR-27b-3p remarkably enhances the adipocyte differentiation of 3T3-L1 cells associated with lipid accumulation and intracellular triglyceride contents. Furthermore, we have demonstrated not only that miR-27b-3p induces acyl-CoA thioesterase 2 (ACOT2) expression in 3T3-L1 cells, but also that the knockdown of ACOT2 suppresses lipid accumulation and adipocyte differentiation in both the presence and absence of miR-27b-3p treatment. Our data strongly suggest that the miR-27b-ACOT2 axis is an important pathway in adipocyte differentiation and may play a role in the pathogenesis of NAFLD., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2019 Yuka Murata et al.)

Angiogenesis-the sprouting and growth of new blood vessels from the existing vasculature-is an important contributor to tumor development, since it facilitates the supply of oxygen and nutrients to cancer cells. Endothelial cells are critically affected during the angiogenic process as their proliferation, motility, and morphology are modulated by pro-angiogenic and environmental factors associated with tumor tissues and cancer cells. Recent in vivo and in vitro studies have revealed that the gap junctions of endothelial cells also participate in the promotion of angiogenesis. Pro-angiogenic factors modulate gap junction function and connexin expression in endothelial cells, whereas endothelial connexins are involved in angiogenic tube formation and in the cell migration of endothelial cells. Several mechanisms, including gap junction function-dependent or -independent pathways, have been proposed. In particular, connexins might have the potential to regulate cell mechanics such as cell morphology, cell migration, and cellular stiffness that are dynamically changed during the angiogenic processes. Here, we review the implication for endothelial gap junctions and cellular mechanics in vascular angiogenesis.

The increased stiffness of the extracellular microenvironment observed in cancer and atherosclerosis is thought to regulate the activation of tissue-resident immune cells. However, it remains to be determined whether such substrate stiffness affects macrophage activation phenotypes. Here, we have studied the impact of substrate stiffness on in vitro activation phenotypes of the human monocyte cell line THP-1. THP-1 cells were activated while being cultured on 1%, 4%, 10% agarose gel (soft substrate) or on a plastic plate (stiff substrate). We have shown that a soft, versus a stiff, substrate attenuates the pro-inflammatory activity of M1 promoting-activated THP-1 cells. In addition, we have found that M1-related marker expression and phagocytic activity was lower in THP-1 cells activated on a soft substrate compared to cells on stiff substrates. THP-1 cells alternatively activated on soft substrates showed enhanced M2-like phenotypes. We have found that peroxisome proliferator-activated receptor γ (PPARγ) expression was up-regulated in THP-1 cells activated on a soft substrate. We have shown that the PPARγ antagonist GW9662 partially suppresses M2-like activation of THP-1 cells activated on a soft substrate. Substrate stiffness is, therefore, an important factor in regulating the balance of the pro-inflammatory M1 and anti-inflammatory M2 activation phenotypes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: This paper reports the protocol of a randomised, double-blind, placebo-controlled study to test the efficacy, safety, and tolerability of lubiprostone (LUB) vs. placebo on suppressing gut permeability in non-alcoholic fatty liver disease (NAFLD) patients with constipation. NAFLD, including non-alcoholic steatohepatitis (NASH), is a common chronic liver disorder. Progression is associated with increased gut permeability and gut-derived endotoxins. Most NAFLD/NASH clinical trial drugs aim to improve liver function or systemic metabolism. LUB is a type 2 chloride channel activator used as a laxative for the treatment of patients with constipation. LUB suppresses gut permeability induced by non-steroidal anti-inflammatory drugs in healthy volunteers and lowers blood endotoxin levels. There have been no clinical studies of LUB for NAFLD/NASH patients., Methods: The study plans to enrol adult patients (20-85 years, planned enrolment, n = 150; planned sample size, n = 120) with NAFLD and constipation, alanine aminotransferase ≥40 IU/L, equivalent steatosis grade ≥1, and equivalent fibrosis stage <4 measured using non-invasive vibration-controlled transient elastography and magnetic resonance imaging. Participants will be randomly allocated into three groups: LUB 12 μg, LUB 24 μg, and a placebo group., Results: The primary endpoint will be changes in alanine aminotransferase from baseline at 12 weeks. The main secondary endpoint will be changes in intestinal permeability from baseline at 12 weeks using the lactulose mannitol ratio., Conclusions: This study will determine whether LUB improves gut permeability in NAFLD patients with constipation., Trial Registration: This trial is registered in the University Hospital Medical Information Network (UMIN) Clinical Trials Registry (UMIN000026635)., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: The aim of this study was to investigate the therapeutic effects of a Sasa veitchii leaf extract (SE) on acetaminophen (APAP)-induced hepatotoxicity., Methods: Seven-week-old male ddY mice were orally administered SE or saline (0.2 mL) once a day for a week. Twenty-four hours after the last pretreatment, the mice were intraperitoneally injected with 550 mg/kg APAP or saline under fasting conditions. The mice from each group were euthanized and bled for plasma analysis 2, 6, 24, and 72 h after the injection., Results: We found that pretreatment with SE significantly decreased hepatic injury markers (i.e., alanine aminotransferase and aspartate aminotransferase), oxidative stress (malondialdehyde and glutathione level), inflammatory cytokines, histological damage, c-jun N-terminal kinase activation, and receptor-interacting protein-1 activation. Further, SE pretreatment decreased Cyp2e1 expression and increased total antioxidant capacity in the liver., Conclusion: Our findings demonstrate that prophylactic SE treatment protects mice from APAP-induced hepatotoxicity through modulation of Cyp2e1 expression and antioxidant capacity.

The aim of this study was to determine whether calcium potentiates acute carbon tetrachloride (CCl4) -induced toxicity. Elevated calcium levels were induced in mice by pre-treatment with cholecalciferol (vitamin D3; V.D3), a compound that has previously been shown to induce hypercalcemia in human and animal models. As seen previously, mice injected with CCl4 exhibited increased plasma levels of alanine aminotransferase, aspartate aminotransferase, and creatinine; transient body weight loss; and increased lipid peroxidation along with decreased total antioxidant power, glutathione, ATP, and NADPH. Pre-treatment of these animals with V.D3 caused further elevation of the values of these liver functional markers without altering kidney functional markers; continued weight loss; a lower lethal threshold dose of CCl4; and enhanced effects on lipid peroxidation and total antioxidant power. In contrast, exposure to V.D3 alone had no effect on plasma markers of liver or kidney damage or on total antioxidant power or lipid peroxidation. The potentiating effect of V.D3 was positively correlated with elevation of hepatic calcium levels. Furthermore, direct injection of CaCl2 also enhanced CCl4-induced hepatic injury. Since CaCl2 induced hypercalcemia transiently (within 3 h of injection), our results suggest that calcium enhances the CCl4-induced hepatotoxicity at an early stage via potentiation of oxidative stress.

Background and Aims: The barrier function of the small intestinal mucosa prevents the introduction of undesired pathogens into the body. Breakdown of this barrier function increases intestinal permeability. This has been proposed to induce not only gastrointestinal diseases, including inflammatory bowel disease and irritable bowel syndrome, but also various other diseases, including allergies, diabetes mellitus, liver diseases, and collagen diseases, which are associated with this so called "leaky gut syndrome." As such, a method to prevent leaky gut syndrome would have substantial clinical value. However, no drugs have been demonstrated to improve disturbed intestinal permeability in humans to date. Therefore, we investigated whether a drug used to treat chronic constipation, lubiprostone, was effective for this purpose., Methods: Healthy male volunteers were treated with lubiprostone (24 μg/day) for 28 days. Intestinal permeability was evaluated by measuring the lactulose-mannitol ratio (LMR) after administration of diclofenac and compared with an untreated group. The examination was conducted three times in total, i.e., at baseline before diclofenac administration and after 14 and 28 days of lubiprostone treatment. Blood endotoxin activity was also evaluated at the same time points., Results: The final analysis was conducted on 28 subjects (14 in the lubiprostone group and 14 in the untreated group). The LMR after 28 days of treatment was significantly lower in the lubiprostone group than that in the untreated group (0.017 vs. 0.028, respectively; 95% confidence interval, -0.022--0.0001; p = 0.049). Blood endotoxin activity exhibited almost no change over time in the lubiprostone and untreated groups and displayed no significant differences at any time point of examination., Conclusions: This study is the first to report an improvement in leaky gut using an available drug in humans. The result suggests that lubiprostone may prevent and ameliorate "leaky gut syndrome". However, a pivotal trial is needed to confirm our finding.

Fibrates, which are widely used lipidaemic-modulating drugs, are emerging environmental pollutants. However, fibrate concentrations in the environment have not been thoroughly surveyed. Here, we determined concentrations of the most commonly used fibrates and their metabolites in source water and drinking water samples from ten drinking water treatment plants in Shanghai and Zhejiang, China, using solid-phase extraction and liquid chromatography-tandem mass spectrometry. All the target compounds were detected in at least some of the source water samples, at concentrations ranging from 0.04 ng/L (fenofibrate) to 1.53 ng/L (gemfibrozil). All the compounds except fenofibrate were also detected in at least some of the drinking water samples, at recoveries ranging from 35.5% to 91.7%, suggesting that these compounds are poorly removed by typical drinking water treatment processes. In a peroxisome proliferator-activated receptor α agonistic activity assay, the target compounds showed no significant activity at nanogram per litre concentrations; therefore, our results suggest that the fibrate concentrations in drinking water in Shanghai and Zhejiang, China do not significantly affect human health. However, because of the increasing westernization of the Chinese diet, fibrate use may increase, and thus monitoring fibrate concentrations in aquatic environments and drinking water in China will become increasingly important.

Carbon tetrachloride (CCl4) is commonly used as a chemical inducer of experimental liver injury. Several compounds have been demonstrated to attenuate the hepatic damage caused by sublethal doses of CCl4. However, rescue from lethal toxicity of CCl4 has not been reported. In the present study, we evaluated the protective effect of metallothionein (MT), an endogenous scavenger of free radicals, on CCl4-induced lethal toxicity of mice. To induce MT production in male ddY mice, we administered Zn (as ZnSO4) at 50 mg/kg as a once-daily subcutaneous injection for 3 days prior to a single intraperitoneal administration of 4 g/kg CCl4. Animals were observed for mortality every 3 hr for 24 hr after CCl4 injection. Liver damage was assessed by determining (in a subset of these mice) blood levels of alanine aminotransferase (ALT; a marker of liver injury) and liver histopathology at 6 hr after CCl4 injection. Our results showed that three times pretreatment with Zn yielded > 40-fold induction of hepatic MT protein levels compared to control group. Zn pretreatment completely abolished the CCl4-induced mortality of mice. We also found that pretreatment of mice with Zn significantly decreased the ALT levels and reduced the histological liver damage as assessed at 6 hr post-CCl4. These findings suggest that prophylaxis with Zn protects mice from CCl4-induced acute hepatic toxicity and mortality, presumably by induction of radical-scavenging MT.

Carbon tetrachloride (CCl4) is commonly used as a chemical inducer of experimental liver injury. In addition, many studies showed that CCl4 can induce kidney damage. In the current study, we evaluated the protective effect of zinc (Zn) against CCl4-induced nephrotoxicity. We hypothesized that this protective effect would result from the ability of Zn to serve as an inducer of metallothionein (MT), a known endogenous scavenger of free radicals. We administered Zn (as ZnSO4) 50 mg/kg subcutaneously once daily for 3 successive days prior to a single intraperitoneal administration of CCl4 4 g/kg in male ddY mice. Our results showed that Zn pretreatment significantly decreased creatinine and blood urea nitrogen levels and reduced renal histopathological damage at 6 h post-CCl4 injection, observations consistent with enhanced antioxidative activity in the kidney. Moreover, kidney MT levels in the Zn+CCl4-treated group decreased by greater than 70% compared with levels in the Zn-alone group, implying that MT was consumed by CCl4-induced radicals. These findings suggest that prophylaxis with Zn protects mice from CCl4-induced acute nephrotoxicity, presumably by induction of MT, which in turn scavenges radicals induced by CCl4 exposure.

Serotonin (5-hydroxytryptamine; 5-HT) is known to be activated during ischemia-reperfusion and triggers contractile dysfunction and pathological apoptosis. Here, the beneficial effects of the selective serotonin reuptake inhibitor (SSRI) fluvoxamine was demonstrated on ischemia-reperfusion injury in guinea-pig hearts perfused using the Langendorff technique. The recovery (%) of left ventricular developed pressure (LVDP) by fluvoxamine (5×10(-8) M) was 95.4% (control: 32%), which was consistent with the inhibition of mitochondrial Ca(2+)([Ca(2+)]m) uptake induced by changes in the Ca(2+) content and acidification of the perfusate, and similar to reperfusion following global ischemia in Langendorff-perfused hearts. Fluvoxamine inhibited the increase in [Ca(2+)]m induced by changes in the Ca(2+) content of the perfusate in perfused preparations of mitochondria, which was similar to the results obtained with the mitochondrial permeability transition pore (MPTP) opener atractyroside. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive cells were significantly less in fluvoxamine-treated hearts than in control hearts, with decreases in caspase-3 activity. These results suggest that SSRI inhibits opening of the MPTP by preventing [Ca(2+)]m overload-induced apoptosis related to the endogenous accumulation of 5-HT in ischemia-reperfusion hearts.

Formaldehyde (FA) is irritating to the skin and is the main cause of sick building syndrome. However, the cutaneous reaction induced by long-term FA exposure has not been fully investigated. In our previous study, we demonstrated that repeated painting of 2% - 10% FA on mouse ears caused marked ear swelling and increased mRNA expression of transient receptor potential vanilloid 1 (TRPV1) and neurotrophins in the ear. TRPV1 is reported to be involved in neurogenic inflammation; therefore, in the present study, we investigated the role of TRPV1 in FA-induced skin inflammation using TRPV1 gene-knockout mice. Mice were painted with 5% FA once a week for 5 weeks, and ear swelling and mRNA expression were investigated. Ear swelling and increased expression of neurotrophins mRNA by FA provocation in wild-type mice were attenuated by disruption of the TRPV1 gene. Furthermore, painting with a threshold dose of capsaicin, which does not induce ear swelling in intact mice, caused marked ear swelling after painting the ear 5 times with FA, indicating that inflamed tissues after FA application are hypersensitive to various ligands of TRPV1 in mice. These results demonstrated that neurogenic inflammation via TRPV1 and neurotrophins could be involved in FA-induced dermatitis.

Volatile organic compounds (VOCs) are considered the main cause of sick building syndrome and they are likely to irritate the skin, eyes, and mucous membrane; however, the toxic threshold and the mechanisms of cutaneous reaction induced by long-time VOC exposure have not been clarified. In the present study, we investigated the effect of repeated painting of VOCs onto mouse skin. Various concentrations of toluene, xylene, and formaldehyde (FA) were applied once a week for 5 weeks. While FA solution (2-10%) induced remarkable ear swelling and caused evident infiltration of inflammatory cells, high concentrations of toluene and xylene (50 or 100%) evoked mild ear swelling and marginal inflammatory cell invasion. In addition, FA exposure markedly increased the expression of interleukin-4 (IL-4), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and transient receptor potential vanilloid-1 (TRPV-1) mRNAs in the ears and IL-4 and NT-3 mRNAs in the cervical lymph nodes. Furthermore, capsazepine, a TRPV-1 antagonist, significantly suppressed ear swelling caused by repeated painting of 5% FA. These findings demonstrate that FA has more potent irritancy against skin than toluene or xylene and suggest that the Th2 response, neurotrophins and TRPV-1 play important roles in FA-induced skin inflammation., (Copyright © 2009 Wiley Periodicals, Inc.)