Yinchenzhufu decoction protects against alpha-naphthylisothiocyanate-induced acute cholestatic liver injury in mice by ameliorating disordered bile acid homeostasis and inhibiting inflammatory responses.

ETHNOPHARMACOLOGICAL RELEVANCE: Intrahepatic cholestasis is a common condition of many liver diseases with few therapies. Yinchenzhufu decoction (YCZFD) is a representative traditional Chinese herbal formula used for treating jaundice and liver disease. AIM OF THE STUDY: To investigate the hepatoprotective effect of YCZFD against cholestatic liver injury and reveal its potential mechanism. MATERIALS AND METHODS: Mice with alpha-naphthyl isothiocyanate (ANIT)-induced intrahepatic cholestasis were orally administered YCZFD at doses of 3, 6, and 12g crude drug/kg for 2 weeks followed by subsequent analyses. A serum metabolomics study was then performed to explore the different metabolites influenced by YCZFD using ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap hybrid mass spectrometry (UPLC-LTQ-Orbitrap-MS/MS).The levels of individual bile acids in the serum, liver, and bile were determined by UPLC-MS/MS. The expression of metabolic enzymes, transporters, inflammatory factors, and cytokeratin-19 (CK-19) was determined by real-time PCR, western blotting, and immunohistochemistry. RESULTS: YCZFD administration decreased the serum biochemical indexes and ameliorated pathological damage, such as hepatic necrosis and inflammatory cell infiltration. Serum metabolomics revealed that the metabolites influenced by YCZFD were mainly associated with bile acid metabolism and inflammation. YCZFD administration effectively ameliorated the disordered bile acid homeostasis. The bile acid transporter, multidrug-resistance associated protein 2 (Mrp2), and the metabolic enzyme, cytochrome P450 2b10 (Cyp2b10), were upregulated in the YCZFD intervention group compared to those in the ANIT-induced group. YCZFD administration also significantly inhibited nuclear factor-κB (NF-κB) and its phosphorylation and decreased the expression of proinflammatory cytokines including tumor necrosis factor-α, interleukin-1ß, and intercellular adhesion molecule-1 in ANIT-induced cholestatic mice. Additionally, the level of CK-19 was lower in the YCZFD intervention group than in the ANIT-induced cholestatic mice. CONCLUSION: YCZFD administration ameliorated disordered bile acid homeostasis, inhibited NF-κB pathway-mediated inflammation, and protected the liver from bile duct injury. Therefore, YCZFD exerted a protective effect against cholestatic liver injury.

Xiaoyan lidan formula ameliorates α-naphthylisothiocyanate-induced intrahepatic cholestatic liver injury in rats as revealed by non-targeted and targeted metabolomics.

Intrahepatic cholestasis is a clinical syndrome of liver damage with systemic circulation and intrahepatic accumulation of excessive toxic bile acids without effective therapeutic methods so far. Xiaoyan Lidan Formula (XYLDF), a traditional Chinese prescription, has long been clinically applied for hepatobiliary disorders due to cholestasis. But its mechanism remains unknown. In this study, a non-targeted metabolomics approach based on UHPLC-Q-TOF-MS/MS combined with a bile acids (BAs) - targeted metabolomics approach based on UHPLC-MS/MS were performed to elucidate the functional mechanisms of XYLDF on α-naphthylisothiocyanate(ANIT)-induced intrahepatic cholestasis rats. The results showed that a total of 39 endogenous metabolites with significant difference (VIP > 1.00, P < 0.05) were identified as biomarkers of ANIT-induced intrahepatic cholestasis in rats. After treatment by XYLDF, 22 biomarkers were reversed to the control-like levels, which involved in primary BA biosynthesis, bile acid metabolism and excretion, steroids metabolism, retinol metabolism, starch and sucrose metabolism, inter conversions between pentose and glucoronate as well as arachidonic acid metabolism. Meanwhile, the results of contents variation of BAs in liver and serum showed that both hydrophilic and hydrophobic BAs were markedly increased in the model rats, while XYLDF treatment could restore the increase induced by ANIT, which suggested that one of the mechanisms of XYLDF on cholestasis referred to regulation of metabolic homeostasis of cholic acid.

Protective Effects of Ginsenosides on 17[Formula: see text]-Ethynyelstradiol-Induced Intrahepatic Cholestasis via Anti-Oxidative and Anti-Inflammatory Mechanisms in Rats.

The present study was designed to assess the effects and potential mechanisms of ginsenosides on 17[Formula: see text]-ethynyelstradiol (EE)-induced intrahepatic cholestasis (IC). Ginsenoside at doses of 30, 100, 300[Formula: see text]mg/kg body weight was intragastrically (i.g.) given to rats for 5 days to examine the effect on EE-induced IC. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bile acid (TBA) were measured. Hepatic malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined. Protein expression of proinflammatory cytokines TNF-[Formula: see text], IL-6 and IL-1[Formula: see text] was analyzed by immunohistochemistry and Western blot. Results indicated that ginsenosides remarkably prevented EE-induced increase in the serum levels of AST, ALT, ALP and TBA. Moreover, the elevation of hepatic MDA content induced by EE was significantly reduced, while hepatic SOD activities were significantly increased when treated with ginsenosides. Histopathology of the liver tissue showed that pathological injuries were relieved after treatment with ginsenosides. In addition, treatment with ginsenosides could significantly downregulate the protein expression of TNF-[Formula: see text], IL-6 and IL-1[Formula: see text] compared with EE group. These findings indicate that ginsenosides exert the hepatoprotective effect on EE-induced intrahepatic cholestasis in rats, and this protection might be attributed to the attenuation of oxidative stress and inflammation.

Yinzhihuang attenuates ANIT-induced intrahepatic cholestasis in rats through upregulation of Mrp2 and Bsep expressions.

BACKGROUND: The treatment of intrahepatic cholestasis has been limited, and development of an effective drug is needed. Clinical studies have shown that Yinzhihuang (YZH), a traditional Chinese decoction, enhances bilirubin clearance. The goal of this study was to determine the protective effect of YZH on experimental intrahepatic cholestasis in young rats and to explore its underlying molecular mechanisms. METHODS: Intrahepatic cholestasis in rats was induced by α-naphthylisothiocyanate (ANIT) on days 1 and 8. The rats received YZH, ursodeoxycholic acid (UDCA), or vehicle for 9 d and were killed on either day 3 or day 10. Serum biomarkers, liver histology, and the distribution of protein and mRNA expression of Mrp2 and Bsep were analyzed. RESULTS: YZH treatment resulted in decreased levels of serum biomarkers except γ-glutamyl transpeptidase, attenuated liver histological injuries, increased protein expressions of Mrp2 and Bsep, and upregulated expressions of Mrp2 and Bsep mRNAs. The effects of YZH on serum biomarkers (aminotransferase, alanine aminotransferase, and direct bilirubin), liver histology, and Mrp2 mRNA expressions were significantly greater and earlier than those of UDCA. CONCLUSION: Our results suggest that YZH has protective effect against ANIT-induced intrahepatic cholestasis in rats, through upregulation of Mrp2 and Bsep expressions.

Omega-3 Fatty Acid-Rich Parenteral Nutrition: Is It a Double-Edged Sword?

Parenteral nutrition (PN) has been strongly associated with intestinal failure-associated liver disease. Cholestasis, liver steatosis, and liver fibrosis are features of this liver injury, which can progress to end stage liver disease. Omega-3 fatty acid rich PN has been shown to alleviate cholestasis and steatosis. There have been reports although suggesting that it may not be able to arrest or reverse the progression to liver fibrosis. In this article, we develop a hypothesis of the mechanism of how Ω-3 fatty acid rich PN may influence the progression of fibrosis.

Impact of new-generation lipid emulsions on cellular mechanisms of parenteral nutrition-associated liver disease.

Parenteral nutrition (PN) is a life-saving nutritional support for a large population of hospitalized infants, and lipids make a substantial contribution to their energy and essential fatty acid (FA) needs. A challenge in the care of these infants is that their metabolic needs require prolonged PN support that increases the risk of PN-associated liver disease (PNALD). In recent years, the emergence of new parenteral lipid emulsions containing different source lipids and FA profiles has created nutritional alternatives to the first-generation, soybean oil-based lipid emulsion Intralipid. The limited U.S. introduction of the new-generation fish-oil emulsion Omegaven has generated promising results in infants with PNALD and spawned a renewed interest in how PN and lipid emulsions, in particular, contribute to this disease. Studies suggest that the lipid load and constituents, such as specific FAs, ratio of n-3 (ω-3) to n-6 (ω-6) long-chain polyunsaturated FAs, phytosterols, and vitamin E content, may be involved. There is an existing literature describing the molecular mechanisms whereby these specific nutrients affect hepatic metabolism and function via lipid and bile acid sensing nuclear receptors, such as peroxisome proliferator-activated receptor α, liver X receptor, and farnesoid X receptor, yet virtually no information as to how they interact and modulate liver function in the context of PN in pediatric patients or animal models. This article will review the recent development of parenteral lipid emulsions and their influence on PNALD and highlight some of the emerging molecular mechanisms that may explain the effects on liver function and disease.

Protective effect of Calculus Bovis Sativus on intrahepatic cholestasis in rats induced by α-naphthylisothiocyanate.

Calculus Bovis Sativus (also referred to as in vitro Cultured Calculus Bovis), an artificial substitute of natural Calculus Bovis (Niuhuang in Chinese, a traditional Chinese medicine), has been widely used to relieve fever, diminish inflammation and normalize gallbladder function in the last decade. This study aims to investigate the effects and possible mechanisms of Calculus Bovis Sativus on α-naphthylisothiocyanate (ANIT)-induced intrahepatic cholestasis in rats. Calculus Bovis Sativus (50, 100 and 200 mg/kg per day) was intragastrically (i.g.) given to experimental rats for seven consecutive days. A single dose of ANIT (100 mg/kg i.g.) was given to rats on the fifth day to induce intrahepatic cholestasis. The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkalinephosphatase (ALP) and total bilirubin (TBIL) were determined through biochemical methods. The bile duct was cannulated with a PE 10 polyethylene tube to collect bile for two hours and bile flow was calculated by the weight of each specimen. Moreover, the mechanism of Calculus Bovis Sativus was elucidated by determining liver malondialdehyde (MDA) content and superoxide dismutase (SOD) activity. The biochemical observations were supplemented by histopathological examinations. Our results showed that Calculus Bovis Sativus (50, 100 and 200 mg/kg) significantly prevented ANIT-induced changes in bile flow and serum levels of ALT, AST, ALP and TBIL. Furthermore, Calculus Bovis Sativus (50, 100 and 200 mg/kg) significantly reduced the elevated hepatic MDA content induced by ANIT and increased the hepatic SOD activity suppressed by ANIT. Accordingly, histopathology of the liver tissue showed that pathological injuries were relieved after Calculus Bovis Sativus (50, 100 and 200 mg/kg) pretreatment. In conclusion, Calculus Bovis Sativus exerted a protective effect on ANIT-induced intrahepatic cholestasis in rats, which may result from the attenuated oxidative damage in liver tissues.

Impact of new-generation parenteral lipid emulsions in pediatric nutrition.

Advancements in the care of premature infants and infants with severe bowel disease have occurred in which long-term use of i.v. nutrition is a cornerstone of successful therapy. Concern about the role of i.v. lipid emulsions in causing severe liver damage to high-risk infants receiving long-term i.v. nutrition has led to a variety of intervention strategies. These have had relatively limited success until the recent introduction of omega-3 (n-3) fatty acid-containing forms of lipid emulsions in place of the current omega-6 fatty acid-predominant lipid emulsions currently exclusively used in the United States. Preliminary data based on nonrandomized trials performed using compassionate-use protocols in the United States suggest very high rates of resolution of cholestasis with the use of an omega-3 fatty acid-predominant lipid emulsion. This result is supported by animal models of liver disease that demonstrate decreased liver damage when animals are provided omega-3 fatty acid-containing lipid emulsions compared with those primarily omega-6 fatty acid based. However, human trials are limited at this time and further research is needed to establish the best approach to preventing liver damage in infants receiving i.v. nutrition and the optimal dose and timing of intervention with novel lipid emulsions.

Combination of retinoic acid and ursodeoxycholic acid attenuates liver injury in bile duct-ligated rats and human hepatic cells.

UNLABELLED: Cholestasis leads to liver cell death, fibrosis, cirrhosis, and eventually liver failure. Despite limited benefits, ursodeoxycholic acid (UDCA) is the only Food and Drug Administration-approved treatment for cholestatic disorders. Retinoic acid (RA) is a ligand for nuclear receptors that modulate bile salt homeostasis. RA also possesses immunomodulatory effects and is used to treat acute promyelocytic leukemia and inflammatory disorders such as psoriasis, acne, and rheumatoid arthritis. To test whether the supplementation of RA with UDCA is superior to UDCA alone for treating cholestasis, male Sprague-Dawley rats underwent common bile duct ligation (BDL) for 14 days and were treated with phosphate-buffered saline (PBS), UDCA, all-trans retinoic acid (atRA), or UDCA and atRA by gavage. Treatment with UDCA and atRA substantially improved animal growth rates, significantly reduced liver fibrosis and bile duct proliferation, and nearly eliminated liver necrosis after BDL. Reductions in the bile salt pool size and liver hydroxyproline content were also seen with treatment with atRA or atRA and UDCA versus PBS and UDCA. Furthermore, atRA and UDCA significantly reduced liver messenger RNA and/or protein expression of transforming growth factor ß1 (Tgf-ß1), collagen 1a1 (Col1A1), matrix metalloproteinase 2 (Mmp2), cytokeratin 19, α-smooth muscle actin (α-SMA), cytochrome P450 7A1 (Cyp7a1), tumor necrosis factor α, and interleukin-ß1. The molecular mechanisms of this treatment were also assessed in human hepatocytes, hepatic stellate cells, and LX-2 cells. atRA alone or in combination with UDCA greatly repressed CYP7A1 expression in human hepatocytes and significantly inhibited COL1A1, MMP2, and α-SMA expression and/or activity in primary human hepatic stellate cells and LX-2 cells. Furthermore, atRA reduced TGF-ß1-induced Smad2 phosphorylation in LX-2 cells. CONCLUSION: Our findings indicate that the addition of RA to UDCA reduces the bile salt pool size and liver fibrosis and might be an effective supplemental therapy with UDCA for cholestatic diseases.

Fish oil containing intravenous lipid emulsions in parenteral nutrition-associated cholestatic liver disease.

PURPOSE OF REVIEW: Lipid emulsions containing fish oils have been proposed to improve parenteral nutrition associated cholestasis (PNAC), a very serious complication of prolonged parenteral nutrition occurring particularly in infants and children with intestinal failure. Here, we summarize current data. RECENT FINDINGS: The cause of PNAC is multifactorial. Prevention is possible by appropriate management, surgical procedures, infection prevention, and optimal parenteral nutrition management. Plausible hypotheses and experimental data support potential benefits of fish oils for treatment and prevention of PNAC. Improvement of PNAC over weeks and months has been reported in observational case studies in part of the children who received parenteral lipids with fish oil, but similar improvements also occurred by withholding or reducing standard lipid emulsions. No controlled trials are available that would allow final conclusions on efficacy and safety of fish oil-based emulsions in PNAC. Concerns exist regarding possible untoward effects and an inadequate supply of n-6 fatty acids with parenteral fish oil only. First data from controlled trials with mixed lipid emulsions containing partly fish oil suggest safety in infants and children and some possible benefits for liver function. SUMMARY: The observed improvement of PNAC on parenteral lipids with fish oil deserves further exploration. No controlled trials are available, nutritional adequacy and safety of 100% fish oil emulsions are not adequately documented, and currently their use cannot be considered standard care. First data on mixed lipid emulsions with some fish oil are encouraging, and their effects in PNAC should also be explored.

Growth factor modulation of hepatic inflammation: a novel approach to the management of total parenteral nutrition-associated liver disease.

PURPOSE: Dependence on total parenteral nutrition in intestinal failure or short bowel syndrome patients can lead to many complications. The most significant complication is progressive liver injury leading to liver failure. This study assesses the potential of hepatocyte growth factor (HGF) in modulating the hepatic response in a rat cholestatic liver injury model. METHODS: Female Sprague-Dawley rats were divided into 3 groups: control (n = 5), chronic liver injury (alpha-naphtylisocyocyanate [ANIT] every 3.5 days at 75 mg/kg; n = 5), and chronic liver injury plus HGF (ANIT + HGF at 250 microg kg(-1) d(-1); n = 5). The rats initially underwent massive (80%) small bowel resections. Seven days later, they were given intraperitoneal injections of saline (control) or ANIT and implantation of an osmotic minipump for continuous intravenous saline or HGF. Intraperitoneal saline or ANIT injections were subsequently administered every 3.5 days to create a chronic cholestatic model. After 14 days, the animals were euthanized, and liver biopsies were obtained. The liver biopsies were evaluated by histology, immunofluorescence staining for interleukin-6 and tumor necrosis factor alpha, and assessment of apoptosis by terminal dUTP-transferase-mediated nick end labeling (TUNEL) technique. RESULTS: In this chronic liver injury model, HGF did not effect the grade of inflammation. However, HGF did induce retention of the ductal structures and avoided ductal proliferation, damage, and evidence of primary sclerosing cholangitis (P < .05). Hepatocyte growth factor induced less interleukin-6 (P < .011) and tumor necrosis factor alpha (P < .01) expression. Apoptotic activity was also significantly less in the HGF group (P < .01). CONCLUSION: Hepatocyte growth factor preserved the hepatic ductal system, modulated the hepatic inflammatory response, and reduced the apoptotic index in this chronic cholestatic liver injury model. It may diminish or prevent liver damage in patients with total parenteral nutrition-induced liver injury.

Reducing stillbirths: prevention and management of medical disorders and infections during pregnancy.

BACKGROUND: An estimated two-thirds of the world's 3.2 million stillbirths occur antenatally, prior to labour, and are often overlooked in policy and programs. Poorly recognised, untreated or inadequately treated maternal infections such as syphilis and malaria, and maternal conditions including hypertensive disorders, are known risk factors for stillbirth. METHODS: We undertook a systematic review of the evidence for 16 antenatal interventions with the potential to prevent stillbirths. We searched a range of sources including PubMed and the Cochrane Library. For interventions with prior Cochrane reviews, we conducted additional meta-analyses including eligible newer randomised controlled trials following the Cochrane protocol. We focused on interventions deliverable at the community level in low-/middle-income countries, where the burden of stillbirths is greatest. RESULTS: Few of the studies we included reported stillbirth as an outcome; most that did were underpowered to assess this outcome. While Cochrane reviews or meta-analyses were available for many interventions, few focused on stillbirth or perinatal mortality as outcomes, and evidence was frequently conflicting. Several interventions showed clear evidence of impact on stillbirths, including heparin therapy for certain maternal indications; syphilis screening and treatment; and insecticide-treated bed nets for prevention of malaria. Other interventions, such as management of obstetric intrahepatic cholestasis, maternal anti-helminthic treatment, and intermittent preventive treatment of malaria, showed promising impact on stillbirth rates but require confirmatory studies. Several interventions reduced known risk factors for stillbirth (e.g., anti-hypertensive drugs for chronic hypertension), yet failed to show statistically significant impact on stillbirth or perinatal mortality rates. Periodontal disease emerged as a clear risk factor for stillbirth but no interventions have reduced stillbirth rates. CONCLUSION: Evidence for some newly recognised risk factors for stillbirth, including periodontal disease, suggests the need for large, appropriately designed randomised trials to test whether intervention can minimise these risks and prevent stillbirths. Existing evidence strongly supports infection control measures, including syphilis screening and treatment and malaria prophylaxis in endemic areas, for preventing antepartum stillbirths. These interventions should be incorporated into antenatal care programs based on attributable risks and burden of disease.

Vitamin A-induced cholestatic hepatitis: a case report.

We report a case of intrahepatic cholestasis due to chronic vitamin A supplementation. A 70-year-old woman was admitted to the hospital for jaundice and reduced nutritional and general status with a 2-month history of increasing cholestasis. Some years previously she had suffered from breast and ovarian cancer with subsequent surgery and chemotherapy. Chemotherapy was terminated one month before elevated serum transaminase activities and cholestatic serum markers were noted. Following the chemotherapy, supportive care included weekly vitamin A injections (100,000 IU per injection). Liver biopsy showed an acute toxic liver injury with focal parenchymal necrosis, sinusoidal lesions, inflammatory infiltrate (round cells, macrophages), and activation and proliferation of stellate cells. The hepatic vitamin A concentration was found to be significantly elevated. There were no signs of intrahepatic metastasis or liver cirrhosis. Treatment with ursodeoxycholic acid rapidly improved the cholestasis and led to a total recovery after three weeks.

Bilirubin rinse: A simple protectant against the rat liver graft injury mimicking heme oxygenase-1 preconditioning.

Heme oxygenase (HO)-1 preconditioning through genetic or pharmacologic interventions was shown experimentally to improve posttransplant outcome of liver grafts. However, its clinical application requires careful consideration because of the complexity and economic costs of the procedures. This study aimed to examine if graft preconditioning with HO-1 could be substituted by a simple treatment with heme-degrading products such as bilirubin. Rats were pretreated with or without hemin, an HO-1 inducer for preconditioning. Their livers were harvested as grafts in University of Wisconsin (UW) solution for 16 hours at 4 degrees C and followed by reperfusion ex vivo or by transplantation in vivo. The control grafts were also treated with a rinse buffer containing varied concentrations of unconjugated bilirubin with different time intervals. The HO-1-preconditioned grafts ex vivo exhibited a marked improvement of bile output and cell injury that was cancelled by blocking HO with zinc protoporphyrin-IX. The aggravation of the graft viability by the inhibitor was repressed by supplementation of bilirubin but not by that of carbon monoxide. Furthermore, a short-term rinse treatment with micromolar levels of bilirubin attenuated biliary dysfunction and cell injury of the grafts both ex vivo and in vivo even without HO-1 preconditioning. The protective effects of HO-1 preconditioning or bilirubin rinse appeared to involve its inhibitory effects on lipid peroxidation in hepatocytes. In conclusion, these results suggest that bilirubin rinse serves as a simple strategy to ameliorate hyperacute oxidative stress and hepatobiliary dysfunction of the transplanted grafts, mimicking effects of HO-1-mediated preconditioning.

The inhibitory effect of SA3443, a novel cyclic disulfide compound, on alpha-naphthyl isothiocyanate-induced intrahepatic cholestasis in rats.

1. We investigated the effect of SA3443, a novel cyclic disulfide compound, on alpha-naphthyl isothiocyanate (ANIT)-induced intrahepatic cholestasis in rats. 2. SA3443 given orally at doses of 100 or 300 mg/kg, three times at 4h intervals, dose-dependently suppressed the elevation of serum total bilirubin, alkaline phosphatase activity and transaminase activities induced by administration of ANIT (80 mg/kg) 3h before the second dosages of SA3443. 3. SA3443 also significantly inhibited the reduction of bile flow in the ANIT-treated rats. 4. These findings indicated that SA3443 had a markedly inhibitory effect on ANIT-induced intrahepatic cholestasis in rats.

Metronidazole in prevention of cholestasis associated with total parenteral nutrition.

The effect of metronidazole on liver function tests was studied in patients given total parenteral nutrition (TPN) for an acute attack of Crohn's disease. In those receiving TPN and metronidazole, serum activities of alkaline phosphatase and gamma-glutamyl-transferase decreased or remained normal after 30 days of TPN. In contrast, serum activities of these enzymes increased significantly after 30 days of TPN alone. These results suggest that intrahepatic cholestasis associated with TPN may be related to intestinal overgrowth of anaerobic bacteria.