Allocholic acid protects against α-naphthylisothiocyanate-induced cholestasis in mice by ameliorating disordered bile acid homeostasis.

Cholestasis is a pathological condition characterized by disruptions in bile flow, leading to the accumulation of bile acids (BAs) in hepatocytes. Allocholic acid (ACA), a unique fetal BA known for its potent choleretic effects, reappears during liver regeneration and carcinogenesis. In this research, we investigated the protective effects and underlying mechanisms of ACA against mice with cholestasis brought on by α-naphthylisothiocyanate (ANIT). To achieve this, we combined network pharmacology, targeted BA metabolomics, and molecular biology approaches. The results demonstrated that ACA treatment effectively reduced levels of serum AST, ALP, and DBIL, and ameliorated the pathological injury caused by cholestasis. Network pharmacology analysis suggested that ACA primarily regulated BA and salt transport, along with the signaling pathway associated with bile secretion, to improve cholestasis. Subsequently, we examined changes in BA metabolism using UPLC-MS/MS. The findings indicated that ACA pretreatment induced alterations in the size, distribution, and composition of the liver BA pool. Specifically, it reduced the excessive accumulation of BAs, especially cholic acid (CA), taurocholic acid (TCA), and ß-muricholic acid (ß-MCA), facilitating the restoration of BA homeostasis. Furthermore, ACA pretreatment significantly downregulated the expression of hepatic BA synthase Cyp8b1, while enhancing the expression of hepatic efflux transporter Mrp4, as well as the renal efflux transporters Mdr1 and Mrp2. These changes collectively contributed to improved BA efflux from the liver and enhanced renal elimination of BAs. In conclusion, ACA demonstrated its potential to ameliorate ANIT-induced liver damage by inhibiting BA synthesis and promoting both BA efflux and renal elimination pathways, thus, restoring BA homeostasis.

Dual Agonist of Farnesoid X Receptor and Takeda G Protein-Coupled Receptor 5 Inhibits Hepatitis B Virus Infection In Vitro and In Vivo.

BACKGROUND AND AIMS: Chronic HBV infection is a major health problem worldwide. Currently, the first-line treatment for HBV is nucleos(t)ide analogs or interferons; however, efficient therapeutic approaches that enable cure are lacking. Therefore, anti-HBV agents with mechanisms distinct from those of current drugs are needed. Sodium taurocholate cotransporting polypeptide (NTCP) was previously identified as an HBV receptor that is inhibited by several compounds. Farnesoid X receptor (FXR) activation also inhibits NTCP function. APPROACH AND RESULTS: In this study, we investigated the inhibitory effect of bile acid (BA) derivatives-namely obeticholic acid (OCA), 6α-ethyl-24-nor-5ß-cholane-3α,7α,23-triol-23 sulfate sodium salt (INT-767; a dual agonist of FXR and Takeda G protein-coupled receptor [TGR5]), and 6α-ethyl-23(S)-methyl-cholic acid (INT-777; a TGR5 agonist)-3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole (GW4064; a FXR agonist), cyclosporin A, and irbesartan. OCA and INT-777 suppressed HBV infection in HepG2-human NTCP-C4 cells. Interestingly, INT-767 showed potent inhibition by attaching to HBV particles rather than binding to NTCP. As an entry inhibitor, INT-767 was stronger than various natural BAs. Furthermore, in chimeric mice with humanized liver, INT-767 markedly delayed the initial rise of HBsAg, HBeAg, and HBV DNA and reduced covalently closed circular DNA. The strong inhibitory effect of INT-767 may be due to the cumulative effect of its ability to inhibit the entry of HBV and to stimulate FXR downstream signaling, which affects the postentry step. CONCLUSIONS: Our results suggest that BA derivatives, particularly INT-767, are prospective candidate anti-HBV agents. Clarifying the underlying mechanisms of BA derivatives would facilitate the development of anti-HBV agents.

New Drugs for NASH and HIV Infection: Great Expectations for a Great Need.

In recent years, there has been an increasing number of clinical trials for the treatment of nonalcoholic steatohepatitis (NASH). People living with human immunodeficiency virus (PLWH) are commonly excluded from these studies, usually due to concerns over drug-drug interactions associated with antiretroviral therapy. The Steatohepatitis in HIV Emerging Research Network, a group of international experts in hepatology and infectious diseases, discusses our current understanding on the interaction between human immunodeficiency virus and NASH, and the issues related to the inclusion of PLWH in NASH clinical trials. Recent trials addressing NASH treatment in PLWH are discussed. The risk of drug-drug interactions between antiretroviral therapy and aramchol, cenicriviroc, elafibranor, obeticholic acid and resmetirom (MGL-3196), which are currently in phase 3 trials for the treatment of NASH, are reviewed. A model for trial design to include PLWH is proposed, strongly advocating for the scientific community to include this group as a subpopulation within studies.

Muricholic Acids Promote Resistance to Hypercholesterolemia in Cholesterol-Fed Mice.

BACKGROUND AND AIMS: Hypercholesterolemia is a major risk factor for atherosclerosis and cardiovascular diseases. Although resistant to hypercholesterolemia, the mouse is a prominent model in cardiovascular research. To assess the contribution of bile acids to this protective phenotype, we explored the impact of a 2-week-long dietary cholesterol overload on cholesterol and bile acid metabolism in mice. METHODS: Bile acid, oxysterol, and cholesterol metabolism and transport were assessed by quantitative real-time PCR, western blotting, GC-MS/MS, or enzymatic assays in the liver, the gut, the kidney, as well as in the feces, the blood, and the urine. RESULTS: Plasma triglycerides and cholesterol levels were unchanged in mice fed a cholesterol-rich diet that contained 100-fold more cholesterol than the standard diet. In the liver, oxysterol-mediated LXR activation stimulated the synthesis of bile acids and in particular increased the levels of hydrophilic muricholic acids, which in turn reduced FXR signaling, as assessed in vivo with Fxr reporter mice. Consequently, biliary and basolateral excretions of bile acids and cholesterol were increased, whereas portal uptake was reduced. Furthermore, we observed a reduction in intestinal and renal bile acid absorption. CONCLUSIONS: These coordinated events are mediated by increased muricholic acid levels which inhibit FXR signaling in favor of LXR and SREBP2 signaling to promote efficient fecal and urinary elimination of cholesterol and neo-synthesized bile acids. Therefore, our data suggest that enhancement of the hydrophilic bile acid pool following a cholesterol overload may contribute to the resistance to hypercholesterolemia in mice. This work paves the way for new therapeutic opportunities using hydrophilic bile acid supplementation to mitigate hypercholesterolemia.

MRI Assessment of Treatment Response in HIV-associated NAFLD: A Randomized Trial of a Stearoyl-Coenzyme-A-Desaturase-1 Inhibitor (ARRIVE Trial).

Aramchol, an oral stearoyl-coenzyme-A-desaturase-1 inhibitor, has been shown to reduce hepatic fat content in patients with primary nonalcoholic fatty liver disease (NAFLD); however, its effect in patients with human immunodeficiency virus (HIV)-associated NAFLD is unknown. The aramchol for HIV-associated NAFLD and lipodystrophy (ARRIVE) trial was a double-blind, randomized, investigator-initiated, placebo-controlled trial to test the efficacy of 12 weeks of treatment with aramchol versus placebo in HIV-associated NAFLD. Fifty patients with HIV-associated NAFLD, defined by magnetic resonance imaging (MRI)-proton density fat fraction (PDFF) ≥5%, were randomized to receive either aramchol 600 mg daily (n = 25) or placebo (n = 25) for 12 weeks. The primary endpoint was a change in hepatic fat as measured by MRI-PDFF in colocalized regions of interest. Secondary endpoints included changes in liver stiffness using magnetic resonance elastography (MRE) and vibration-controlled transient elastography (VCTE), and exploratory endpoints included changes in total-body fat and muscle depots on dual-energy X-ray absorptiometry (DXA), whole-body MRI, and cardiac MRI. The mean (± standard deviation) of age and body mass index were 48.2 ± 10.3 years and 30.7 ± 4.6 kg/m2 , respectively. There was no difference in the reduction in mean MRI-PDFF between the aramchol group at -1.3% (baseline MRI-PDFF 15.6% versus end-of-treatment MRI-PDFF 14.4%, P = 0.24) and the placebo group at -1.4% (baseline MRI-PDFF 13.3% versus end-of-treatment MRI-PDFF 11.9%, P = 0.26). There was no difference in the relative decline in mean MRI-PDFF between the aramchol and placebo groups (6.8% versus 1.1%, P = 0.68). There were no differences in MRE-derived and VCTE-derived liver stiffness and whole-body (fat and muscle) composition analysis by MRI or DXA. Compared to baseline, end-of-treatment aminotransferases were lower in the aramchol group but not in the placebo arm. There were no significant adverse events. Conclusion: Aramchol, over a 12-week period, did not reduce hepatic fat or change body fat and muscle composition by using MRI-based assessment in patients with HIV-associated NAFLD (clinicaltrials.gov ID:NCT02684591).

G Protein-Coupled Bile Acid Receptor TGR5 Activation Inhibits Kidney Disease in Obesity and Diabetes.

Obesity and diabetes mellitus are the leading causes of renal disease. In this study, we determined the regulation and role of the G protein-coupled bile acid receptor TGR5, previously shown to be regulated by high glucose and/or fatty acids, in obesity-related glomerulopathy (ORG) and diabetic nephropathy (DN). Treatment of diabetic db/db mice with the selective TGR5 agonist INT-777 decreased proteinuria, podocyte injury, mesangial expansion, fibrosis, and CD68 macrophage infiltration in the kidney. INT-777 also induced renal expression of master regulators of mitochondrial biogenesis, inhibitors of oxidative stress, and inducers of fatty acid ß-oxidation, including sirtuin 1 (SIRT1), sirtuin 3 (SIRT3), and Nrf-1. Increased activity of SIRT3 was evidenced by normalization of the increased acetylation of mitochondrial superoxide dismutase 2 (SOD2) and isocitrate dehydrogenase 2 (IDH2) observed in untreated db/db mice. Accordingly, INT-777 decreased mitochondrial H2O2 generation and increased the activity of SOD2, which associated with decreased urinary levels of H2O2 and thiobarbituric acid reactive substances. Furthermore, INT-777 decreased renal lipid accumulation. INT-777 also prevented kidney disease in mice with diet-induced obesity. In human podocytes cultured with high glucose, INT-777 induced mitochondrial biogenesis, decreased oxidative stress, and increased fatty acid ß-oxidation. Compared with normal kidney biopsy specimens, kidney specimens from patients with established ORG or DN expressed significantly less TGR5 mRNA, and levels inversely correlated with disease progression. Our results indicate that TGR5 activation induces mitochondrial biogenesis and prevents renal oxidative stress and lipid accumulation, establishing a role for TGR5 in inhibiting kidney disease in obesity and diabetes.

The fatty acid-bile acid conjugate Aramchol reduces liver fat content in patients with nonalcoholic fatty liver disease.

BACKGROUND & AIMS: We investigated the effects of the fatty acid-bile acid conjugate 3ß-arachidyl-amido, 7α-12α-dihydroxy, 5ß-cholan-24-oic acid (Aramchol; Trima Israel Pharmaceutical Products Ltd, Maabarot, Israel) in a phase 2 trial of patients with nonalcoholic fatty liver disease (NAFLD). METHODS: We performed a randomized, double-blind, placebo-controlled trial of 60 patients with biopsy-confirmed NAFLD (6 with nonalcoholic steatohepatitis) at 10 centers in Israel. Patients were given Aramchol (100 or 300 mg) or placebo once daily for 3 months (n = 20/group). The main end point was the difference between groups in the change in liver fat content according to magnetic resonance spectroscopy. The secondary end points focused on the differences between groups in alterations of liver enzyme levels, levels of adiponectin, homeostasis model assessment scores, and endothelial function. RESULTS: No serious or drug-related adverse events were observed in the 58 patients who completed the study. Over 3 months, liver fat content decreased by 12.57% ± 22.14% in patients given 300 mg/day Aramchol, but increased by 6.39% ± 36.27% in the placebo group (P = .02 for the difference between groups, adjusted for age, sex, and body mass index). Liver fat content decreased in the 100-mg Aramchol group, by 2.89% ± 28.22%, but this change was nonsignificant (P = .35), indicating a dose-response relationship (P for trend = .01). Groups given Aramchol had nonsignificant improvements over time in endothelial function and levels of alanine aminotransferase and adiponectin, but homeostasis model assessment scores did not change. The appropriateness of a single daily dose was confirmed by pharmacokinetic analysis. CONCLUSIONS: Three months' administration of the fatty acid-bile acid conjugate Aramchol is safe, tolerable, and significantly reduces liver fat content in patients with NAFLD. The reduction in liver fat content occurred in a dose-dependent manner and was associated with a trend of metabolic improvements, indicating that Aramchol might be used for the treatment of fatty liver disease. ClinicalTrials.gov number: NCT01094158.

Ursocholanic acid rescues mitochondrial function in common forms of familial Parkinson's disease.

Previous drug screens aiming to identify disease-modifying compounds for Parkinson's disease have typically been based on toxin-induced in vitro and in vivo models of this neurodegenerative condition. All these compounds have failed to have a reliable disease-modifying effect in subsequent clinical trials. We have now established a novel approach, namely to screen an entire compound library directly in patient tissue to identify compounds with a rescue effect on mitochondrial dysfunction as a crucial pathogenic mechanism in Parkinson's disease. The chosen Microsource Compound library contains 2000 compounds, including 1040 licensed drugs and 580 naturally occurring compounds. All 2000 compounds were tested in a step-wise approach for their rescue effect on mitochondrial dysfunction in parkin (PARK2) mutant fibroblasts. Of 2000 compounds, 60 improved the mitochondrial membrane potential by at least two standard deviations. Subsequently, these 60 compounds were assessed for their toxicity and drug-like dose-response. The remaining 49 compounds were tested in a secondary screen for their rescue effect on intracellular ATP levels. Of 49 compounds, 29 normalized ATP levels and displayed drug-like dose response curves. The mitochondrial rescue effect was confirmed for 15 of these 29 compounds in parkin-mutant fibroblasts from additional patients not included in the initial screen. Of 15 compounds, two were chosen for subsequent functional studies, namely ursocholanic acid and the related compound dehydro(11,12)ursolic acid lactone. Both compounds markedly increased the activity of all four complexes of the mitochondrial respiratory chain. The naturally occurring compound ursolic acid and the licensed drug ursodeoxycholic acid are chemically closely related to ursocholanic acid and dehydro(11,12)ursolic acid lactone. All four substances rescue mitochondrial function to a similar extent in parkin-mutant fibroblasts, suggesting a class effect. The mitochondrial rescue effect depends on activation of the glucocorticoid receptor with increased phosphorylation of Akt and was confirmed for both ursocholanic acid and ursodeoxycholic acid in a Parkin-deficient neuronal model system. Of note, both ursocholanic acid and ursodeoxycholic acid also rescued mitochondrial function in LRRK2(G2019S) mutant fibroblasts. Our study demonstrates the feasibility of undertaking drug screens in Parkinson's disease patients' tissue and has identified a group of chemically-related compounds with marked mitochondrial rescue effect. Drug repositioning is considered to be a time- and cost-saving strategy to assess drugs already licensed for a different condition for their neuroprotective effect. We therefore propose both ursolic acid as a naturally occurring compound, and ursodeoxycholic acid as an already licensed drug as promising compounds for future neuroprotective trials in Parkinson's disease.

The human UGT1A3 enzyme conjugates norursodeoxycholic acid into a C23-ester glucuronide in the liver.

Norursodeoxycholic acid (norUDCA) exhibits efficient anti-cholestatic properties in an animal model of sclerosing cholangitis. norUDCA is eliminated as a C(23)-ester glucuronide (norUDCA-23G) in humans. The present study aimed at identifying the human UDP-glucuronosyltransferase (UGT) enzyme(s) involved in hepatic norUDCA glucuronidation and at evaluating the consequences of single nucleotide polymorphisms in the coding region of UGT genes on norUDCA-23G formation. The effects of norUDCA on the formation of the cholestatic lithocholic acid-glucuronide derivative and of rifampicin on hepatic norUDCA glucuronidation were also explored. In vitro glucuronidation assays were performed with microsomes from human tissues (liver and intestine) and HEK293 cells expressing human UGT enzymes and variant allozymes. UGT1A3 was identified as the major hepatic UGT enzyme catalyzing the formation of norUDCA-23G. Correlation studies using samples from a human liver bank (n = 16) indicated that the level of UGT1A3 protein is a strong determinant of in vitro norUDCA glucuronidation. Analyses of the norUDCA-conjugating activity by 11 UGT1A3 variant allozymes identified three phenotypes with high, low, and intermediate capacity. norUDCA is also identified as a competitive inhibitor for the hepatic formation of the pro-cholestatic lithocholic acid-glucuronide derivative, whereas norUDCA glucuronidation is weakly stimulated by rifampicin. This study identifies human UGT1A3 as the major enzyme for the hepatic norUDCA glucuronidation and supports that some coding polymorphisms affecting the conjugating activity of UGT1A3 in vitro may alter the pharmacokinetic properties of norUDCA in cholestasis treatment.

INT-777 prevents cognitive impairment by activating Takeda G protein-coupled receptor 5 (TGR5) and attenuating neuroinflammation via cAMP/ PKA/ CREB signaling axis in a rat model of sepsis.

BACKGROUND: Survivors of sepsis must often endure significant cognitive and behavioral impairments after discharge, but research on the relevant mechanisms and interventions remains lacking. TGR5, a member of the class A GPCR family, plays an important role in many physiological processes, and recent studies have shown that agonists of TGR5 show neuroprotective effects in a variety of neurological disorders. To date, no studies have assessed the effects of TGR5 on neuroinflammatory, cognitive, or behavioral changes in sepsis models. METHODS: A total of 267 eight-week-old male Sprague-Dawley rats were used in this study. Sepsis was induced via cecal ligation and puncture (CLP). All animals received volume resuscitation. The rats were given TGR5 CRISPR oligonucleotide intracerebroventricularly 48 h before CLP surgery. INT-777 was administered intranasally 1 h after CLP, and the cAMP inhibitor, SQ22536, was administered intracerebroventricularly 1 h after CLP. Survival rate, bodyweight change, and clinical scores were assessed, and neurobehavioral tests, western blot, and immunofluorescence staining were performed. The cognitive function of rats was measured using the Morris water maze during 15-20 days after CLP. RESULTS: The expression of TGR5 in the rat hippocampus was upregulated, and peaked at 3 days after CLP. The survival rate of rats after CLP was less than 50%, and the growth rate, in terms of weight, was significantly decreased. While INT-777 treatment did not improve these changes, the treatment did reduce the clinical scores of rats at 24 h after CLP. On day 15 and later, the surviving mice completed a series of behavioral tests. CLP rats showed spatial and memory deficits and anxiety-like behaviors, but INT-777 treatment significantly improved these effects. Mechanistically, immunofluorescence analysis showed that INT-777 treatment reduced the number of microglia in the hippocampus, neutrophilic infiltration, and the expression of inflammatory factors after CLP in rats. Moreover, INT-777 treatment significantly increased the expression of TGR5, cAMP, p-PKA, and p-CREB, but downregulated the expression of IL-1ß, IL-6, and TNF-α. CRISPR-mediated TGR5 knockdown and SQ22536 treatment abolished the neuroprotective effects of TGR5 activation after CLP. CONCLUSION: This study demonstrates that INT-777 treatment reduced neuroinflammation and microglial cell activation, but improved cognitive impairment in the experimental sepsis rats. TGR5 has translational potential as a therapeutic target to improve neurological outcomes in sepsis survivors.

Hyocholic acid species improve glucose homeostasis through a distinct TGR5 and FXR signaling mechanism.

Hyocholic acid (HCA) and its derivatives are found in trace amounts in human blood but constitute approximately 76% of the bile acid (BA) pool in pigs, a species known for its exceptional resistance to type 2 diabetes. Here, we show that BA depletion in pigs suppressed secretion of glucagon-like peptide-1 (GLP-1) and increased blood glucose levels. HCA administration in diabetic mouse models improved serum fasting GLP-1 secretion and glucose homeostasis to a greater extent than tauroursodeoxycholic acid. HCA upregulated GLP-1 production and secretion in enteroendocrine cells via simultaneously activating G-protein-coupled BA receptor, TGR5, and inhibiting farnesoid X receptor (FXR), a unique mechanism that is not found in other BA species. We verified the findings in TGR5 knockout, intestinal FXR activation, and GLP-1 receptor inhibition mouse models. Finally, we confirmed in a clinical cohort, that lower serum concentrations of HCA species were associated with diabetes and closely related to glycemic markers.

Enhancement of intravesical delivery with Syn3 potentiates interferon-alpha2b gene therapy for superficial bladder cancer.

Intravesical administration of interferon alpha-2b protein (IFN) has been successfully used in the treatment of patients with superficial bladder tumors. Local dosing of IFN minimizes well-known systemic side effects of the drug, but exposure to bladder tumors is limited by the duration of instillation and transient concentrations achieved in the urothelium. Intravesical delivery of the gene encoding interferon results in an alternative strategy for IFN-based therapy of the disease, enabling sustained exposure of IFN protein that results from production by tumor and non-tumor cells in the urothelium. Efficient gene delivery and expression of IFN has been achieved using a recombinant adenovirus gene delivery system (rAd-IFN) in conjunction with the novel small molecule excipient Syn3. Studies with rAd-IFN/Syn3 in animal models result in urine concentrations of IFN that persisted for weeks and correlated with potent anti-tumor effects. The objective of this review is to communicate the rationale and preclinical findings that support ongoing clinical investigation of intravesical rAd-IFN/Syn3 in superficial bladder cancer.

An insight into primary biliary cholangitis and its recent advances in treatment: semi-synthetic analogs to combat ursodeoxycholic-acid resistance.

INTRODUCTION: Primary biliary cholangitis (PBC) is a chronic cholestatic liver disease which on progression causes cirrhosis; various studies also suggested that several diseases can co-exist in patients. In existing depiction of disease PBC, apart from entire use of ursodeoxycholic acid (UDCA), several patients need to step forward to liver-transplantation or death due to resistance or non-responder with UDCA monotherapy. AREAS COVERED: To overcome this non-respondent treatment, novel bile acid semi-synthetic analogs have been identified which shows their potency against for farnesoid X receptor and transmembrane G protein-coupled receptor-5 which are identified as target for many developing analogs which have desirable pharmacokinetic profiles. EXPERT OPINION: A range of studies suggests that adding semisynthetic analogs in therapeutic regime improves liver biochemistries in patients with suboptimal response to UDCA. Thus, the aspire of this review is to abridge and compare therapeutic value and current markets affirm of various bile acids semi-synthetic analogs which certainly are having promising effects in PBC monotherapy or in pooled treatment with UDCA for PBC.

Effects of ursodeoxycholic acid in combination with vitamin E on adipokines and apoptosis in patients with nonalcoholic steatohepatitis.

BACKGROUND/AIMS: Adipokines and hepatocellular apoptosis participate in the pathogenesis of nonalcoholic steatohepatitis (NASH). In a randomized trial ursodeoxycholic acid (UDCA) with vitamin E (VitE) improved serum aminotransferases and hepatic histology. The present work evaluates the effect of this combination on adipokines and hepatocellular apoptosis. METHODS: Circulating levels of adiponectin, resistin, leptin, interleukin (IL)-6, IL-8, retinol binding protein-4, monocyte chemoattractant protein-1 and tumour necrosis factor-alpha were measured by enzyme-linked immunoassays at the beginning and after 2 years of treatment with either UDCA+VitE, UDCA+placebo (P) or P+P. Apoptosis was assessed by immunohistochemistry for activated caspase-3 and circulating levels of apoptosis-associated cytokeratin 18 fragments (M30). RESULTS: Levels of adiponectin increased in patients treated with UDCA+VitE, whereas they decreased in the two other groups (P<0.04) and correlated with the improvement of liver steatosis (P<0.04). M30 levels worsened in the P/P group and improved in the other two groups. They correlated with hepatocellular apoptosis (P<0.02) and steatosis (P<0.02) as well as negatively with adiponectin levels (P<0.04). CONCLUSIONS: UDCA+VitE improves not only aminotransferase levels and liver histology of patients with NASH, but also decreases hepatocellular apoptosis and restores circulating levels of adiponectin. These results suggest that the UDCA+VitE combination has metabolic effects in addition to its beneficial cytoprotective properties.

Addictive evaluation of cholic acid-verticinone ester, a potential cough therapeutic agent with agonist action of opioid receptor.

AIM: The purpose of this work was to search for potential drugs with potent antitussive and expectorant activities as well as a low toxicity, but without addictive properties. Cholic acid-verticinone ester (CA-Ver) was synthesized based on the clearly elucidated antitussive and expectorant activities of verticinone in bulbs of Fritillaria and different bile acids in Snake Bile. In our previous study, CA-Ver showed a much more potent activity than codeine phosphate. This study was carried out to investigate the central antitussive mechanism and the addictive evaluation of CA-Ver. METHODS: Testing on a capsaicin-induced cough model of mice pretreated with naloxone, a non-selective opioid receptor antagonist, was performed for the observation of CA-Ver's central antitussive mechanism. We then took naloxone-induced withdrawal tests of mice for the judgment of CA-Ver's addiction. Lastly, we determined the opioid dependence of CA-Ver in the guinea pig ileum. RESULTS: The test on the capsaicin-induced cough model showed that naloxone could block the antitussive effect of CA-Ver, suggesting the antitussive mechanism of CA-Ver was related to the central opioid receptors. The naloxone-urged withdrawal tests of the mice showed that CA-Ver was not addictive, and the test of the opioid dependence in the guinea pig ileum showed that CA-Ver had no withdrawal response. CONCLUSION: These findings suggested that CA-Ver deserved attention for its potent antitussive effects related to the central opioid receptors, but without addiction, and had a good development perspective.

[Study on the effect of in vitro cultured calculus bovis in perioperative period after H-UPPP operation].

Objective:TThe aim of this study is to investigate the effect of in vitro cultured Calculus Bovis on the inflammation of oropharynx and body in patients with OSA during the perioperative period of H-UPPP.Method:Eighty patients with OSA and H-UPPP indications were enrolled. The patients were divided into experimental group and control group by random number table, 40 cases in each group. The experimental group was given in vitro cultured Calculus Bovis, while the control group was not given bovine bezoar in vitro. The postoperative oropharyngeal pain, time to resume normal diet, local edema, concentration of IL-1ß, IL-8 and TNF-α in saliva, and concentration of IL-1ß, IL-8 and TNF-α in blood were compared between the two groups. Result:The pain of oropharynx in the experimental group was lighter than that in the control group on the 3rd, 5th and 7th day after operation (P<0.05), but there was no significant difference in the pain of oropharynx between the two groups on the 1st day after operation(P>0.05); the time of restoring normal diet in the experimental group was shorter than that in the control group (P<0.05); the edema of oropharynx in the experimental group was lighter than that in the control group on the 5th and 7th day after operation (P<0.05).The levels of IL-1ß, IL-8 and TNF-α in saliva were lower than those in control group on the 3rd, 5th and 7th day after operation (P<0.05), and the levels of IL-1ß, IL-8 and TNF-α in blood on the 5th and 7th day after operation were lower than those in control group (P<0.05).Conclusion:In vitro perioperative period of H-UPPP can improve the postoperative sore throat and local edema of oropharynx, shorten the time of normal diet and reduce the expression of related inflammatory factors in oropharynx and blood.

TGR5 agonist INT-777 mitigates inflammatory response in human endometriotic stromal cells: A therapeutic implication for endometriosis.

Endometriosis is a condition characterized by the presence of endometrial tissues outside the uterus. Endometriotic stromal cells (ESCs) are known to undergo regeneration and are linked to the causation of endometriosis. Activation of stromal cells by local inflammatory cytokines is proposed to be one of the mechanisms of endometriosis development. Takeda-G-protein-receptor-5 (TGR5) is a G protein-coupled bile acid receptor that plays multiple roles in various cells and tissues. In this study, we show that activation of TGR5 by its specific agonist, INT-777, protects ESCs from inflammation and oxidative stress induced by tumor necrosis factor-α (TNF-α). TGR5 is fairly expressed in cultured ESCs, and TNF-α treatment suppresses TGR5 expression. Activation of TGR5 by its synthetic agonist, INT-777, dramatically reduces the production of pro-inflammatory cytokines and adhesion molecules by TNF-α, including interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Moreover, INT-777 suppresses TNF-α-induced NADPH oxidase 4 (NOX4) expression and ameliorates cellular oxidative stress. Mechanistically, our findings demonstrate that INT-777 suppresses TNF-α-induced c-Jun N-terminal kinase (JNK) activation via suppression of p-JNK. INT-777 inhibits TNF-α-induced activation of the activator protein-1 (AP-1) pathway owing to its suppression of c-Jun and c-fos as well as transfected AP-1 promoter. INT-777 also inhibits nuclear factor-κB (NF-κB) activation as revealed by its suppression of TNF-α-induced nuclear p65 accumulation and NF-κB promoter. Collectively, our data indicate that activation of TGR5 by its agonist has protective effects against inflammation and reactive oxygen species (ROS) in cytokine-induced activation of ESCs. Therefore, INT-777 may have an implication in the clinical treatment of endometriosis.

Inhibitory effect of INT-777 on lipopolysaccharide-induced cognitive impairment, neuroinflammation, apoptosis, and synaptic dysfunction in mice.

Neuroinflammation plays an important role in the pathophysiology of Alzheimer's disease (AD) and memory impairment. Herein, we evaluated the neuroprotective effects of 6-ethyl-23(S)-methyl-cholic acid (INT-777), a specific G-protein coupled bile acid receptor 1 (TGR5) agonist, in the LPS-treated mouse model of acute neurotoxicity. Single intracerebroventricular (i.c.v.) injection of LPS remarkably induced mouse behavioral impairments in Morris water maze, novel object recognition, and Y-maze avoidance tests, which were ameliorated by INT-777 (1.5 or 3.0 µg/mouse, i.c.v.) treatment. Importantly, INT-777 treatment reversed LPS-induced TGR5 down-regulation, suppressed the increase of nuclear NF-κB p65, and mitigated neuroinflammation, evidenced by lower proinflammatory cytokines, less activation of microglia, and increased the ratio of p-CREB/CREB or mBDNF/proBDNF in the hippocampus and frontal cortex. In addition, INT-777 treatment also suppressed neuronal apoptosis, as indicated by the reduction of TUNEL-positive cells, decreased activation of caspase-3, increased the ratio of Bcl-2/Bax, and ameliorated synaptic dysfunction as evidenced by the upregulation of PSD95 and synaptophysin in the hippocampus and frontal cortex. Taken together, this study showed the potential neuroprotective effects of INT-777 against LPS-induced cognitive impairment, neuroinflammation, apoptosis, and synaptic dysfunction in mice.

Deciphering the Role of Intramolecular Networking in Cholic Acid-Peptide Conjugates on the Lipopolysaccharide Surface in Combating Gram-Negative Bacterial Infections.

The presence of lipopolysaccharide and emergence of drug resistance make the treatment of Gram-negative bacterial infections highly challenging. Herein, we present the synthesis and antibacterial activities of cholic acid-peptide conjugates (CAPs), demonstrating that valine-glycine dipeptide-derived CAP 3 is the most effective antimicrobial. Molecular dynamics simulations and structural analysis revealed that a precise intramolecular network of CAP 3 is maintained in the form of evolving edges, suggesting intramolecular connectivity. Further, we found high conformational rigidity in CAP 3 that confers maximum perturbations in bacterial membranes relative to other small molecules. Interestingly, CAP 3-coated catheters did not allow the formation of biofilms in mice, and treatment of wound infections with CAP 3 was able to clear the bacterial infection. Our results demonstrate that molecular conformation and internal connectivity are critical parameters to describe the antimicrobial nature of compounds, and the analysis presented here may serve as a general principle for the design of future antimicrobials.

Hypocholesterolemic effects of fatty acid bile acid conjugates (FABACs) in mice.

Fatty acid bile acid conjugates (FABACs) prevent and dissolve cholesterol gallstones and prevent diet induced fatty liver, in mice. The present studies aimed to test their hypocholesterolemic effects in mice. Gallstone susceptible (C57L/J) mice, on high fat (HFD) or regular diet (RD), were treated with the conjugate of cholic acid with arachidic acid (FABAC; Aramchol). FABAC reduced the elevated plasma cholesterol levels induced by the HFD. In C57L/J mice, FABAC reduced plasma cholesterol by 50% (p<0.001). In mice fed HFD, hepatic cholesterol synthesis was reduced, whereas CYP7A1 activity and expression were increased by FABAC. The ratio of fecal bile acids/neutral sterols was increased, as was the total fecal sterol excretion. In conclusion, FABACs markedly reduce elevated plasma cholesterol in mice by reducing the hepatic synthesis of cholesterol, in conjunction with an increase of its catabolism and excretion from the body.