Anti-inflammatory effect of polyherbal composition with hepatoprotective and choleretic properties on LPS-stimulated murine macrophages.

OBJECTIVES: A polyherbal formulation with hepatoprotective and choleretic properties combining pharmacological potential of eight medicinal plants was developed in Nargiz Medical center (Republic of Azerbaijan) for the use as herbal tea. To explore the effect of polyherbal composition on the metabolism of LPS-stimulated macrophages in vitro. METHODS: The qualitative and quantitative phytochemical analysis was conducted using specific color reactions and gas chromatography-mass spectrometry (GC-MS). Nitric oxide (NO) assay was determined using the Griess reaction. Reactive oxygen species (ROS) generation was measured using ROS-sensitive fluorescence indicator, H2DCFDA, by flow cytometry. Arginase activity was examined by colorimetric method. RESULTS: The studied polyherbal formulation exerted anti-inflammatory activity in LPS-stimulated macrophages which was evidenced by dose-dependent decrease of ROS generation and by shift of arginine metabolism to the increase of arginase activity and decrease of NO release. CONCLUSIONS: Our findings suggest that the herbal tea reduces macrophage inflammatory activity, that provide an important rationale to utilize it for the attenuation of chronic inflammation typical of hepatobiliary disorders.

Dietary Flavone Baicalein Combinate with Genipin Attenuates Inflammation Stimulated by Lipopolysaccharide in RAW264.7 Cells or Pseudomonas aeruginosa in Mice via Regulating the Expression and Phosphorylation of AKT.

Mounting evidence has shown that single-targeted therapy might be inadequate to achieve satisfactory effects. Thus, drug combinations are gaining attention as they can regulate multiple targets to obtain more beneficial effects. Heat shock protein 90 (HSP90) is a molecular chaperone that assists the protein assembly and folding of client proteins and maintains their stability. Interfering with the interaction between HSP90 and its client proteins by inhibiting the latter's activity may offer a new approach toward combination therapy. The HSP90 client protein AKT plays an important role in the inflammatory response syndrome caused by infections. In this study, the dietary flavone baicalein was identified as a novel inhibitor of HSP90 that targeted the N-terminal ATP binding pocket of HSP90 and hindered the chaperone cycle, resulting in AKT degradation. Combining baicalein with genipin, which was extracted from Gardenia jasminoides, could inhibit the pleckstrin homology domain of AKT, significantly increasing the anti-inflammatory effects both in vitro and in vivo. This synergistic effect was attributed to the reduction in AKT expression and phosphorylation. Thus, elucidating the mechanism underlying this effect will provide a new avenue for the clinical application and development of synergistic anti-inflammatory drugs.

Oral Ursodeoxycholic Acid Crosses the Blood Retinal Barrier in Patients with Retinal Detachment and Protects Against Retinal Degeneration in an Ex Vivo Model.

Rhegmatogenous retinal detachment (RD) is a threatening visual condition and a human disease model for retinal degenerations. Despite successful reattachment surgery, vision does not fully recover, due to subretinal fluid accumulation and subsequent photoreceptor cell death, through mechanisms that recapitulate those of retinal degenerative diseases. Hydrophilic bile acids are neuroprotective in animal models, but whether they can be used orally for retinal diseases is unknown. Ursodeoxycholic acid (UDCA) being approved for clinical use (e.g., in cholestasis), we have evaluated the ocular bioavailability of oral UDCA, administered to patients before RD surgery. The level of UDCA in ocular media correlated with the extent of blood retinal barrier disruption, evaluated by the extent of detachment and the albumin concentration in subretinal fluid. UDCA, at levels measured in ocular media, protected photoreceptors from apoptosis and necrosis in rat retinal explants, an ex vivo model of RD. The subretinal fluid from UDCA-treated patients, collected during surgery, significantly protected rat retinal explants from cell death, when compared to subretinal fluid from control patients. Pan-transcriptomic analysis of the retina showed that UDCA upregulated anti-apoptotic, anti-oxidant, and anti-inflammatory genes. Oral UDCA is a potential neuroprotective adjuvant therapy in RD and other retinal degenerative diseases and should be further evaluated in a clinical trial.

Regulation of autophagy by bile acids and in cholestasis - CholestoPHAGY or CholeSTOPagy.

Autophagy is a lysosomal degradation pathway in which the cell self-digests its own components to provide nutrients in harsh environmental conditions. It also represents an opportunity to rid the cell of superfluous and damaged organelles, misfolded proteins or invaded microorganisms. Liver autophagy contributes to basic hepatic functions such as lipid, glycogen and protein turnover. Deregulated hepatic autophagy has been linked to many liver diseases including alpha-1-antitrypsin deficiency, alcoholic and non-alcoholic fatty liver diseases, hepatitis B and C infections, liver fibrosis as well as liver cancer. Recently, bile acids and the bile acid receptor FXR have been implicated in the regulation of hepatic autophagy, which implies a role of autophagy also for cholestatic liver diseases. This review summarizes the current evidence of bile acid mediated effects on autophagy and how this affects cholestatic liver diseases. Although detailed studies are lacking, we suggest a concept that the activity of autophagy in cholestasis depends on the disease stage, where autophagy may be induced at early stages ("cholestophagy") but may be impaired in prolonged cholestatic states ("cholestopagy").

Inhibiting P2Y12 in Macrophages Induces Endoplasmic Reticulum Stress and Promotes an Anti-Tumoral Phenotype.

The P2Y12 receptor is an adenosine diphosphate responsive G protein-coupled receptor expressed on the surface of platelets and is the pharmacologic target of several anti-thrombotic agents. In this study, we use liver samples from mice with cirrhosis and hepatocellular carcinoma to show that P2Y12 is expressed by macrophages in the liver. Using in vitro methods, we show that inhibition of P2Y12 with ticagrelor enhances tumor cell phagocytosis by macrophages and induces an anti-tumoral phenotype. Treatment with ticagrelor also increases the expression of several actors of the endoplasmic reticulum (ER) stress pathways, suggesting activation of the unfolded protein response (UPR). Inhibiting the UPR with tauroursodeoxycholic acid (Tudca) diminishes the pro-phagocytotic effect of ticagrelor, thereby indicating that P2Y12 mediates macrophage function through activation of ER stress pathways. This could be relevant in the pathogenesis of chronic liver disease and cancer, as macrophages are considered key players in these inflammation-driven pathologies.

Impact of Deoxycholic Acid on Oesophageal Adenocarcinoma Invasion: Effect on Matrix Metalloproteinases.

Bile acids (BAs) have been implicated in the development of oesophagitis, Barrett's oesophagus and oesophageal adenocarcinoma (OAC). However, whether BAs promote cancer invasiveness has not been elucidated. We evaluated the role of BAs, in particular deoxycholic acid (DCA), in OAC invasion. Migration and invasiveness in untreated and BA-treated oesophageal SKGT-4 cancer cells were evaluated. Activity and expression of different matrix metalloproteinases (MMPs) were determined by zymography, ELISA, PCR and Western blot. Finally, human OAC tissues were stained for MMP-10 by immunohistochemistry. It was found that SKGT-4 cells incubated with low concentrations of DCA had a significant increase in invasion. In addition, MMP-10 mRNA and protein expression were also increased in the presence of DCA. MMP-10 was found to be highly expressed both in-vitro and in-vivo in neoplastic OAC cells relative to non-neoplastic squamous epithelial cells. Our results show that DCA promotes OAC invasion and MMP-10 overexpression. This study will advance our understanding of the pathophysiological mechanisms involved in human OAC and shows promise for the development of new therapeutic strategies.

Genipin suppression of growth and metastasis in hepatocellular carcinoma through blocking activation of STAT-3.

BACKGROUND: The signal transducer and activator of transcription-3 (STAT-3) can facilitate cancer progression and metastasis by being constitutively active via various signaling. Abundant evidence has indicated that STAT-3 may be a promising molecular target for cancer treatment. METHODS: In this study, a dual-luciferase assay-based screening of 537 compounds for STAT-3 inhibitors of hepatocellular carcinoma (HCC) cells was conducted, leading to the identification of genipin. Effects of genipin on HCC were assessed in a patient-derived xenograft nude mice model. Western blotting assay, chromatin immunoprecipitation (ChIP) assay, molecular docking study, tube formation assay, three-dimensional top culture assay, histological examination, and immunofluorescence were utilized to evaluate the regulatory signaling pathway. RESULTS: Our research demonstrated that genipin suppresses STAT-3 phosphorylation and nuclear translocation, which may be attributed to the binding capacity of this compound to the Src homology-2 (SH2) domain of STAT-3. In addition, the therapeutic effects of genipin in a patient-derived HCC xenograft nude mice model were also demonstrated. CONCLUSIONS: In conclusion, genipin showed therapeutic potential for HCC treatment by interacting with the SH2-STAT-3 domain and suppressing the activity of STAT-3. In the future, further research is planned to explore the potential role of genipin in combination with chemotherapy or radiotherapy for HCC.

Therapeutic potential of genipin in various acute liver injury, fulminant hepatitis, NAFLD and other non-cancer liver diseases: More friend than foe.

Genipin is an aglycone derived from the geniposide, the most abundant iridoid glucoside constituent of Gardenia jasminoides Ellis. For decades, genipin is the focus of studies as a versatile compound in the treatment of various pathogenic conditions. In particularly, Gardenia jasminoides Ellis has long been used in traditional Chinese medicine for the prevention and treatment of liver disease. Mounting experimental data has proved genipin possesses therapeutic potential for cholestatic, septic, ischemia/reperfusion-triggered acute liver injury, fulminant hepatitis and NAFLD. This critical review is a reflection on the valuable lessons from decades of research regarding pharmacological activities of genipin. Of note, genipin represents choleretic effect by potentiating bilirubin disposal and enhancement of genes in charge of the efflux of a number of organic anions. The anti-inflammatory capability of genipin is mediated by suppression of the production and function of pro-inflammatory cytokines and inflammasome. Moreover, genipin modulates various transcription factor and signal transduction pathway. Genipin appears to trigger the upregulation of several key genes encoding antioxidant and xenobiotic-metabolizing enzymes. Furthermore, the medicinal impact of genipin extends to modulation of regulated cell death, including autophagic cell death, apoptosis, necroptosis and pyroptosis, and modulation of quality of cellular organelle. Another crucial effect of genipin appears to be linked to dual role in targeting uncoupling protein 2 (UCP2). As a typical UCP2-inhibiting compound, genipin could inhibit AMP-activated protein kinase or NF-κB in circumstance. On the contrary, reactive oxygen species production and cellular lipid deposits mediated by genipin through the upregulation of UCP2 is observed in liver steatosis, suggesting the precise role of genipin is disease-specific. Collectively, we comprehensively summarize the mechanisms and pathways associated with the hepatoprotective activity of genipin and discuss potential toxic impact. Notably, our focus is the direct medicinal effect of genipin itself, whereas its utility as a crosslinking agent in tissue engineering is out of scope for the current review. Further studies are therefore required to disentangle these complicated pharmacological properties to confer this natural agent a far greater potency.

The endoplasmic reticulum stress-autophagy pathway controls hypothalamic development and energy balance regulation in leptin-deficient neonates.

Obesity is associated with the activation of cellular responses, such as endoplasmic reticulum (ER) stress. Here, we show that leptin-deficient ob/ob mice display elevated hypothalamic ER stress as early as postnatal day 10, i.e., prior to the development of obesity in this mouse model. Neonatal treatment of ob/ob mice with the ER stress-relieving drug tauroursodeoxycholic acid (TUDCA) causes long-term amelioration of body weight, food intake, glucose homeostasis, and pro-opiomelanocortin (POMC) projections. Cells exposed to ER stress often activate autophagy. Accordingly, we report that in vitro induction of ER stress and neonatal leptin deficiency in vivo activate hypothalamic autophagy-related genes. Furthermore, genetic deletion of autophagy in pro-opiomelanocortin neurons of ob/ob mice worsens their glucose homeostasis, adiposity, hyperphagia, and POMC neuronal projections, all of which are ameliorated with neonatal TUDCA treatment. Together, our data highlight the importance of early life ER stress-autophagy pathway in influencing hypothalamic circuits and metabolic regulation.

Evidence for functional selectivity in TUDC- and norUDCA-induced signal transduction via α5ß1 integrin towards choleresis.

Functional selectivity is the ligand-specific activation of certain signal transduction pathways at a receptor and has been described for G protein-coupled receptors. However, it has not yet been described for ligands interacting with integrins without αI domain. Here, we show by molecular dynamics simulations that four side chain-modified derivatives of tauroursodeoxycholic acid (TUDC), an agonist of α5ß1 integrin, differentially shift the conformational equilibrium of α5ß1 integrin towards the active state, in line with the extent of ß1 integrin activation from immunostaining. Unlike TUDC, 24-nor-ursodeoxycholic acid (norUDCA)-induced ß1 integrin activation triggered only transient activation of extracellular signal-regulated kinases and p38 mitogen-activated protein kinase and, consequently, only transient insertion of the bile acid transporter Bsep into the canalicular membrane, and did not involve activation of epidermal growth factor receptor. These results provide evidence that TUDC and norUDCA exert a functional selectivity at α5ß1 integrin and may provide a rationale for differential therapeutic use of UDCA and norUDCA.

Mesna Alleviates Cerulein-Induced Acute Pancreatitis by Inhibiting the Inflammatory Response and Oxidative Stress in Experimental Rats.

BACKGROUND: Acute pancreatitis (AP) is a sudden inflammation of the pancreas that may be life-threatening disease with high mortality rates, particularly in the presence of systemic inflammatory response and multiple organ failure. Oxidative stress has been shown to be involved in the pathophysiology of acute pancreatitis. AIM: This study is designed to investigate the possible effect of mesna on an experimental model of cerulein-induced acute pancreatitis. METHODS: Animals were divided into five groups: Group 1 served as a control group given the saline; group II (mesna group) received mesna at a dose of (100 mg/kg per dose, i.p.) four times; group III (acute pancreatitis group) received cerulein at a dose of (20 µg/kg/dose, s.c.) four times with 1-h intervals; group VI, cerulein + mesna, was treated with mesna at a dose of (100 mg/kg, i.p.) 15 min before each cerulein injection. RESULTS: Animals with acute pancreatitis showed elevated serum amylase and lipase levels. Biochemical parameters showed increased pancreatic tumor necrosis factors-α (TNF-α) and interleukin-1ß (IL-1ß) levels. A disturbance in oxidative stress markers was evident by elevated pancreatic lipid peroxides (TBARS) and decline in pancreatic antioxidants' concentrations including reduced glutathione (GSH); superoxide dismutase (SOD); and glutathione peroxidase (GSH-Px). Histological examination confirmed pancreatic injury. Pre-treatment with mesna was able to abolish the changes in pancreatic enzymes, oxidative stress markers (TBARS, SOD, GSH and GSH-Px), pancreatic inflammatory markers (TNF-α, IL-1ß) as well as histological changes. CONCLUSIONS: Mesna mitigates AP by alleviating pancreatic oxidative stress damage and inhibiting inflammation.

Scoparone improves hepatic inflammation and autophagy in mice with nonalcoholic steatohepatitis by regulating the ROS/P38/Nrf2 axis and PI3K/AKT/mTOR pathway in macrophages.

BACKGROUND AND AIMS: Scoparone has been shown to ameliorate many forms of liver disease, and several underlying molecular mechanisms involved have been previously revealed. However, the potential role of scoparone in autophagy, which is dysregulated in nonalcoholic fatty liver disease-nonalcoholic steatohepatitis (NAFLD-NASH), has not been evaluated. In the current study, we investigated the effect and potential mechanisms of scoparone in hepatic autophagy in mice with NASH. METHODS: In vivo, mice were fed a methionine-choline deficient (MCD) diet to establish a NASH model and then subjected to treatment with or without scoparone for 4 weeks. In vitro, scoparone was applied in a hepatocellular lipid overload model in AML12 cells challenged with palmitic acid (PA) and in lipopolysaccharide (LPS)-induced RAW264.7 cells. RESULTS: Scoparone improved impaired autophagy and several key features of NASH in mice fed an MCD diet. In vitro, scoparone had an effect on the autophagy of macrophages but not hepatocytes. In RAW264.7 cells, scoparone reduced the LPS-induced accumulation of autophagosomes and autophagy substrates, the production of reactive oxygen species (ROS) and the inflammatory response. Scoparone inhibited the upregulation of p62 transcription, which is mediated by the ROS/P38/Nrf2 axis. Chloroquine (CQ), an inhibitor of autophagic flux, significantly inhibited scoparone-mediated protection against inflammation. In addition, scoparone suppressed activation of the PI3K/AKT/mTOR pathway, and MHY1485 (an mTOR activator that inhibits autophagy) inhibited the anti-inflammatory effect of scoparone. CONCLUSIONS: In LPS-induced macrophages, scoparone regulates autophagy and further suppresses inflammation by inhibiting the ROS/P38/Nrf2 axis and PI3K/AKT/mTOR pathway and enhancing autophagic flux. Scoparone may improve hepatic autophagy and NASH partly through enhancing autophagy in macrophages but not hepatocytes. Scoparone is expected to become a novel therapeutic drug for NASH or diseases associated with dysregulated autophagy in macrophages.

Oncomir MicroRNA-346 Is Upregulated in Colons of Patients With Primary Sclerosing Cholangitis.

INTRODUCTION: Primary sclerosing cholangitis (PSC) is a cholestatic liver disorder that is frequently associated with ulcerative colitis (UC). Patients with PSC and UC (PSC-UC) have a higher risk of colorectal neoplasia compared with patients with UC. The oncogenic properties of microRNA-346 (miR-346) have been recently reported. We investigated the expression of miR-346 and its 2 target genes, the receptor of vitamin D (VDR), and the tumor necrosis factor-α (TNF-α), which are known to modulate carcinogenesis. METHODS: Ascending and sigmoid colon biopsies were obtained from patients with PSC, PSC and UC (PSC-UC), UC, and healthy controls (n = 10 in each group). Expressions of VDR, TNF-α, 18S RNA, p27, miR-346, and reference microRNA, miR-191, were evaluated by real-time PCR using human TaqMan Gene Expression and TaqMan MicroRNA Assays. Functional studies with miR-346 mimic and inhibitor were conducted in HepG2 and Caco-2 cells. The effect of ursodeoxycholic acid on miR-346 expression was examined in Caco-2 cells. RESULTS: An increased expression of miR-346 in the ascending colon of PSC-UC was observed (P < 0.001 vs all groups). In patients with UC, an exceptionally low colonic expression of miRNA-346 was accompanied by the extensive upregulation of VDR and TNF-α genes. A functional in vitro analysis demonstrated that inhibition of miR-346 resulted in the upregulation of VDR and TNF-α, whereas the induction of miR-346 activity suppressed VDR, TNF-α, and p27. DISCUSSION: The upregulation of miRNA-346 in the colon of patients with PSC may be responsible for the disturbance of VDR and TNF-α signaling pathway, which could result in an inadequate suppression of neoplasia.

Chemical chaperones improve the functional recovery of stunned myocardium by attenuating the endoplasmic reticulum stress.

AIM: Myocardial ischaemia/reperfusion (I/R) produces structural and functional alterations depending on the duration of ischaemia. Brief ischaemia followed by reperfusion causes reversible contractile dysfunction (stunned heart) but long-lasting ischaemia followed by reperfusion can result in irreversible injury with cell death. Events during I/R can alter endoplasmic reticulum (ER) function leading to the accumulation of unfolded/misfolded proteins. The resulting ER stress induces activation of several signal transduction pathways, known as unfolded protein response (UPR). Experimental evidence shows that UPR contributes to cell death in irreversible I/R injury; however, there is still uncertainty for its occurrence in the stunned myocardium. This study investigated the ER stress response and its functional impact on the post-ischaemic cardiac performance of the stunned heart. METHODS: Perfused rat hearts were subjected to 20 minutes of ischaemia followed by 30 minutes of reperfusion. UPR markers were evaluated by qRT-PCR and western blot. Post-ischaemic mechanical recovery was measured in absence and presence of two chemical chaperones: tauroursodeoxycholic acid (TUDCA) and 4-phenylbutyric acid (4-PBA). RESULTS: Analysis of mRNA and protein levels of various ER stress effectors demonstrated that different UPR signalling cascades, involving both pro-survival and pro-apoptotic pathways, are activated. Inhibition of the UPR with chemical chaperones improved the post-ischaemic recovery of cardiac mechanical function without affecting the I/R-induced increase in oxidative stress. CONCLUSION: Our results suggest that prevention of ER stress by chemical chaperones could be a therapeutic tool to limit deterioration of the contractile function in clinical settings in which the phenomenon of myocardial stunning is present.

Histological Analysis of the Effect of ATX-101 (Deoxycholic Acid Injection) on Subcutaneous Fat: Results From a Phase 1 Open-Label Study.

BACKGROUND: ATX-101 is approved for submental fat reduction. OBJECTIVE: To characterize the histological effect of ATX-101 injection into subcutaneous fat. METHODS: This Phase 1 open-label study enrolled 14 adults to receive injections of ATX-101 into abdominal fat at varying concentrations (0.5%, 1.0%, 2.0%, or 4.0%), volumes (0.2 or 0.4 mL), spacing (0.7, 1.0, or 1.5 cm), and time points before scheduled abdominoplasty (1, 3, 7, or 28 days). During abdominoplasty, tissue was excised and preserved for histology. RESULTS: All injection paradigms resulted in histological changes confined to the subcutaneous layer, which were more prominent at higher concentrations and independent of volume and spacing. Key features at Day 1 after injection were adipocytolysis, blood vessel injury, neutrophilic inflammation, and lysis of locally present neutrophils. At Day 3, inflammation was reduced versus Day 1, and hemorrhage and lipid lake formation (at higher concentrations) were observed. Day 7 samples exhibited prominent adipocytolysis, mild inflammation, lipid-laden macrophages in the septae, and repair of vascular injury. At Day 28, inflammation was largely resolved and prominent features were septal thickening, neovascularization, and atrophy of fat lobules. CONCLUSION: Subcutaneous injection of ATX-101 induces adipocytolysis and local inflammation with septal thickening and resolution of inflammation by 28 days after injection.

Enhancing Retinal Endothelial Glycolysis by Inhibiting UCP2 Promotes Physiologic Retinal Vascular Development in a Model of Retinopathy of Prematurity.

Purpose: We address the hypothesis that uncoupling protein 2 (UCP2), a cellular glucose regulator, delays physiologic retinal vascular development (PRVD) by interfering with glucose uptake through glucose transporter 1 (Glut1). Methods: In the rat 50/10 oxygen-induced retinopathy (OIR) model, retinal Glut1 and UCP2 were measured and compared to room air (RA)-raised pups at postnatal day 14 (p14). Pups in OIR and RA received intraperitoneal genipin, an UCP2 inhibitor, or control every other day from p3 until p13. Analyses at p14 included avascular/total retinal area (AVA), Western blots of retinal UCP2 and Glut1, and immunostaining of Glut1 in retinal cryosections. Intravitreal neovascular/total retinal area (IVNV) was analyzed at p18, and electroretinograms were performed at p26. Glut1 and phosphorylated VEGFR2 (p-VEGFR2), glucose uptake, adenosine triphosphate (ATP) production, and cell proliferation were measured in human retinal microvascular endothelial cells (hRMVECs) pretreated with genipin or transfected with UCP2siRNA, Glut1siRNA, or control siRNA when incubated with VEGF or PBS. Results: At p14, OIR pups had increased AVA with decreased Glut1 and increased UCP2 in the retina compared to RA retinas. Intraperitoneal genipin increased retinal Glut1 and reduced AVA. Compared to control, treatment with genipin or knockdown of UCP2 significantly increased Glut1, glucose uptake, ATP production, VEGF-induced p-VEGFR2 and cell proliferation in hRMVECs. Knockdown of Glut1 inhibited VEGF-induced p-VEGFR2. Genipin-treated OIR pups with decreased AVA at p14 had reduced IVNV at p18 and increased amplitudes in a- and b-waves at p26. Conclusions: Extending PRVD by increasing retinal endothelial glucose uptake may represent a strategy to prevent severe retinopathy of prematurity and vision loss.

Ursodeoxycholic acid ameliorates hepatic lipid metabolism in LO2 cells by regulating the AKT/mTOR/SREBP-1 signaling pathway.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD), the most common chronic liver disease, can progress into nonalcoholic steatohepatitis (NASH), cirrhosis, and even hepatocellular carcinoma. Bile acids such as ursodeoxycholic acid (UDCA) play an essential role in the pathogenesis of NAFLD by regulating the level of sterol regulatory element-binding protein (SREBP) 1c, but the underlying regulatory mechanism remains elusive. Increased evidence indicates that the AKT/mTOR/SREBP-1 signaling pathway is a key pathway to regulate hepatic cellular lipid metabolism. UDCA may regulate the AKT/mTOR/SREBP-1 signaling pathway to ameliorate hepatic lipid metabolism. AIM: To investigate the functional mechanism of UDCA in an oleic acid (OA)-induced cellular model of NAFLD. METHODS: The cellular model of NAFLD was established using OA and treated with UDCA. First, the best concentration of UDCA was selected. For the best time-dependent assay, cells were stimulated with OA only or co-treated with OA and 2 mmol/L UDCA for 24 h, 48 h, and 72 h. Oil red O staining was used to observe the accumulation of intracellular lipids, while the intracellular contents of triglyceride, alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (GGT), and aspartate aminotransferase (AST) were detected by enzymatic methods. Meanwhile, the expression levels of AKT/mTOR/SREBP-1 signaling pathway-related proteins were detected by real-time PCR and Western blot. RESULTS: In the NAFLD cell model established with LO2 cells induced using OA, lipid accumulation was obvious. UDCA significantly inhibited lipid accumulation at different concentrations (especially 2 mmol/L) and decreased cell growth ability at different time points. The biochemical parameters like ALT, AST, and GGT were significant improved by UDCA. UDCA treatment vividly repressed the activation of AKT, mTOR, and CRTC2 and the expression of nSREBP-1 in LO2 cells induced with OA. CONCLUSION: Our findings demonstrate the effect of UDCA in improving NAFLD. UDCA attenuates OA-induced hepatic steatosis mainly by regulation of AKT/mTOR/SREBP-1 signal transduction.

Systemic bile acids induce insulin resistance in a TGR5-independent manner.

Bile acids are involved in the emulsification and absorption of dietary fats, as well as acting as signaling molecules. Recently, bile acid signaling through farnesoid X receptor and G protein-coupled bile acid receptor (TGR5) has been reported to elicit changes in not only bile acid synthesis but also metabolic processes, including the alteration of gluconeogenic gene expression and energy expenditure. A role for bile acids in glucose metabolism is also supported by a correlation between changes in the metabolic state of patients (i.e., obesity or postbariatric surgery) and altered serum bile acid levels. However, despite evidence for a role for bile acids during metabolically challenging settings, the direct effect of elevated bile acids on insulin action in the absence of metabolic disease has yet to be investigated. The present study examines the impact of acutely elevated plasma bile acid levels on insulin sensitivity using hyperinsulinemic-euglycemic clamps. In wild-type mice, elevated bile acids impair hepatic insulin sensitivity by blunting the insulin suppression of hepatic glucose production. The impaired hepatic insulin sensitivity could not be attributed to TGR5 signaling, as TGR5 knockout mice exhibited a similar inhibition of insulin suppression of hepatic glucose production. Canonical insulin signaling pathways, such as hepatic PKB (or Akt) activation, were not perturbed in these animals. Interestingly, bile acid infusion directly into the portal vein did not result in an impairment in hepatic insulin sensitivity. Overall, the data indicate that acute increases in circulating bile acids in lean mice impair hepatic insulin sensitivity via an indirect mechanism.

Vitamin A can ameliorate fibrosis of liver in an established rat model of biliary atresia and Kasai portoenterostomy.

BACKGROUND: Antifibrosis therapy may prevent progressive liver fibrosis after successful Kasai portoenterostomy (KPE) in biliary atresia (BA) patients. The aim of this study is to evaluate the efficacy of antifibrosis therapy in a rat model of BA and KPE. METHODS: BA model was created on three-week-old Sprague-Dawley rats by intrabiliary alcohol injection as previously described, and KPE was performed at postoperative week (POW) 5 by cystoenterostomy. Liver biopsies were performed at the time of BA creation, during KPE, POW 9, and at sacrifice (POW 17). Prednisolone (0.1 mg/100 g/day, group 1, n = 20), Vitamin A (0.5 mg/100 g/day, group 2, n = 20), and ursodeoxycholic acid (UDCA, 1.5 mg/100 g/day, group 3, n = 20) were respectively given to three groups after KPE and continued daily until sacrifice. Histological evaluation of fibrosis and immunohistochemistry stains for 8 fibrosis markers were compared to the control group (without medication, n = 10). RESULTS: Among the four markers, namely ɑ-smooth muscle actin (ɑ-SMA), glial fibrillary acidic protein (GFAP), tumor growth factor ß1 (TGFß1) receptors 1 and 2, which showed persistently high expression after successful KPE in the examined 8 markers, only the expression of ɑ-SMA was significantly reduced in all treatment groups at POW17. However, the fibrosis grade at POW 17 was only significantly reduced in group 2 in comparison with the control group (Vitamin A vs. control group, Ishak score 3 vs. 1.8, p < 0.05). CONCLUSION: In our rat model of BA with KPE, Vitamin A was effective in reducing liver fibrosis, and the mechanisms deserve further study. LEVEL OF EVIDENCE: Basic science.

Peroxiredoxin I maintains luteal function by regulating unfolded protein response.

BACKGROUND: Mounting evidence shows that ROS regulation by various antioxidants is essential for the expression of enzymes involved in steroidogenesis and maintenance of progesterone production by the corpus luteum (CL). However, the underlying mechanisms of peroxiredoxin 1 (PRDX1), an antioxidant enzyme, in luteal function for progesterone production in mice have not been reported. The aim of this study was to evaluate the functional link between PRDX1 and progesterone production in the CL of Prdx1 knockout (K/O) mice in the functional stage of CL. METHODS: The expression pattern of the unfolded protein response (UPR) signaling pathways, endoplasmic reticulum (ER) stress-induced apoptosis related genes and peroxiredoxins 1 (PRDX1) were investigated by western blotting analysis in CL tissue of 10 weeks mice during functional stage of CL. The protein levels of these genes after ER-stress inducer tunicamycin (Tm), ER-stress inhibitor tauroursodeoxycholic acid (TUDCA) and ROS scavenger, N-acetylcysteine (NAC) stimulation by intraperitoneal (i.p) injection were also investigated in CL tissue of wild type (WT) mice. Finally, we examined progesterone production and UPR signaling related gene expression in CL tissue of Prdx1 K/O mice. RESULTS: We demonstrated that PRDX1 deficiency in the functional stage activates the UPR signaling pathways in response to ER stress-induced apoptosis. Interestingly, CL number, serum progesterone levels, and steroidogenic enzyme expression in Prdx1 K/O mice decreased significantly, compared to those in wild type mice. Levels of UPR signaling pathway markers (GRP78/BIP, P50ATF6, and phosphorylated (p)-eIF2) and ER-stress associated apoptotic factors (CHOP, p-JNK, and cleaved caspase-3) were dramatically increased in the CL tissue of Prdx1 K/O mice. In addition, administration of the NAC, reduced progesterone production and activated ER-stress-induced UPR signaling in the CL tissue obtained from the ovary of Prdx1 K/O mice. Taken together, these results indicated that reduction in serum progesterone levels and activation of ER-stress-induced UPR signaling are restored by NAC injection in the CL of Prdx1 K/O mice. CONCLUSION: These observations provide the first evidence regarding the basic mechanisms connecting PRDX1 and progesterone production in the functional stage of CL.