Search

The present study aims to examine the protective effects and mechanism of a velvet antler polypeptide (VAP) against lithocholic acid (LCA)-induced cholestatic liver injury in mice. A 7.0 kDa VAP was orally administered at doses of 10 and 20 mg kg-1 day-1. Hematoxylin and eosin (H&E) staining of the liver showed that VAP7.0 reduced LCA-induced infiltration of inflammatory cells and areas of necrotic hepatocytes. In addition, VAP7.0 greatly reduced the levels of alanine aminotransferase (ALT), total bile acid (TBA) and total bilirubin (TBIL) in LCA mouse serum and prolonged the survival time of mice with LCA. VAP7.0 reduced the production of reactive oxygen species (ROS), decreased malondialdehyde (MDA) and increased the superoxide dismutase (SOD) levels in LCA mice. VAP7.0 also reduced OGG1 expression, which is a biochemical indicator of oxidative stress. Mechanistic analysis revealed that VAP7.0 significantly inhibited LCA-induced disruption of tight junction integrity, as determined by observing the morphology of the bile canaliculus, and this finding was confirmed by observation of the bile canalicular structure and tight junction proteins Occludin and ZO-1 expression. Moreover, we also found that VAP7.0 maintained the stability of hepatic paracellular permeability, as determined by Evans blue dye assays and horseradish peroxidase (HRP) tracer distribution through inhibiting the activation of the PI3K pathway in LCA mouse livers. In addition, VAP7.0 ameliorated H2O2-induced barrier dysfunction and tight junction disruption via inhibiting the PI3K activity in human HepG2 and SMMC7721 cells, which was confirmed by the PI3K activator 740Y-P. H2O2 disturbed the localization of the tight junction proteins ZO-1 and Occludin, resulting in the transfer of these proteins from the membrane to the cytoplasm of cells, whereas pretreatment of cells with VAP7.0 prevented the disruption of the localization of these proteins, as determined by immunofluorescence staining and western blot analysis. These results demonstrate that VAP7.0 reduces liver injury by inhibiting oxidative stress and maintains the stability of hepatic tight junctions via suppressing the activation of the intracellular signaling molecule PI3K in LCA mice and hepatocellular carcinoma cells.

Cell polarity and formation of bile canaliculi can be achieved in hepatocytes which are generated from patient-derived induced pluripotent stem cells. This allows for the study of endogenous mutant proteins, patient-specific pathogenesis, and drug responses for diseases where hepatocyte polarity and bile canaliculi play a key role. Here, we describe a step-by-step protocol for the generation of bile canaliculi-forming hepatocytes from induced pluripotent stem cells and their evaluation.

Hepatocytes display a unique biaxial polarity with shared apical luminal connections between adjacent hepatocytes that merge into a network of bile canaliculi. Belicova et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202103003) discovered that hepatocyte apical membranes generate Rab35-dependent extensions that traverse the lumen and are essential for bile canalicular formation and maintenance.

Clarification - We do not comment on water flow from hepatocytes "into the bile canaliculus" but instead, have studied bile flux within the canalicular network. Validation of Photoactivation - We validated the mentioned photoactivation technique in biological structures, provided raw data and have discussed the imaging technicalities in detail; for example, this intravital video shows how photoactivated material moves unidirectionally in the blood stream.

Peroxisome proliferator-activated receptor alpha (PPARα) controls lipid homeostasis through regulation of lipid transport and catabolism. PPARα activators are clinically used for hyperlipidemia treatment. The role of PPARα in bile acid (BA) homeostasis is beginning to emerge. Herein, Ppara-null and hepatocyte-specific Ppara-null (Ppara(ΔHep)) as well as the respective wild-type mice were treated with the potent PPARα agonist Wy-14,643 (Wy) and global metabolomics performed to clarify the role of hepatocyte PPARα in the regulation of BA homeostasis. Levels of all serum BAs were markedly elevated in Wy-treated wild-type mice but not in Ppara-null and Ppara(ΔHep) mice. Gene expression analysis showed that PPARα activation (1) down-regulated the expression of sodium-taurocholate acid transporting polypeptide and organic ion transporting polypeptide 1 and 4, responsible for the uptake of BAs into the liver; (2) decreased the expression of bile salt export pump transporting BA from hepatocytes into the bile canaliculus; (3) upregulated the expression of multidrug resistance-associated protein 3 and 4 transporting BA from hepatocytes into the portal vein. Moreover, there was a notable increase in the compositions of serum, hepatic and biliary cholic acid and taurocholic acid following Wy treatment, which correlated with the upregulated expression of the Cyp8b1 gene encoding sterol 12α-hydroxylase. The effects of Wy were identical between the Ppara(ΔHep) and Ppara-null mice. Hepatocyte PPARα controlled BA synthesis and transport not only via direct transcriptional regulation but also via crosstalk with hepatic farnesoid X receptor signaling. These findings underscore a key role for hepatocyte PPARα in the control of BA homeostasis.

Background: Neonates on long-term parenteral nutrition (PN) may develop parenteral nutrition-associated liver disease (PNALD). Aluminum (Al) is a known contaminant of infant PN, and we hypothesize that it substantially contributes to PNALD. In this study, we aim to assess the impact of Al on hepatocytes in a piglet model. Methods: We conducted a randomized control trial using a Yucatan piglet PN model. Piglets, aged 3-6 days, were placed into two groups. The high Al group (n=8) received PN with 63 µg/kg/day of Al, while the low Al group (n=7) received PN with 24 µg/kg/day of Al. Serum samples for total bile acids (TBA) were collected over two weeks, and liver tissue was obtained at the end of the experiment. Bile canaliculus morphometry was studied by transmission electron microscopy (TEM) and Image-J software analysis. In December 2019, the American Academy of Pediatrics issued a key technical report on Al effects in infants and children. This report was revised and commented on in this chapter. Results: The canalicular space was smaller, and the microvilli were shorter in the high Al group than in the low Al group. There was no difference in the TBA between the groups. Conclusions: Al causes structural changes in the hepatocytes despite unaltered serum bile acids. High Al in PN is associated with short microvilli, which could decrease the functional excretion area of the hepatocytes and impair bile flow. Long-term toxicity studies from the Al content in PN components supported the US Food and Drug Administration (FDA) in the decision to implement specific rules. It has been required that large-volume ingredients reduce the Al concentration and all small volume components be specifically labeled with the Al concentration. However, both US FDA and we consider that despite these rules, the total Al concentration from some components remains above the recommended final concentration. US FDA and our concerns about toxicity from the Al present in infant PN formulas are grounded, requiring more research on this topic.

Vartak et al's recent publicatioin reviews their previous provocative data that water flow into the bile canaliculus is not in response to an osmotic gradient, as initially proposed by Sperber. However their technique, photoactivation of a non-fluorescent precursor of fluorescein, has not been validated. In contrast, a similar technique, enzymatic activation of a non-fluorescent precursor of fluorescein supported the concept of osmotically generated canalicular water flow and confirmed that peristalsis also participates in the flow of fluid from the canaliculi into the duct system as originally described by Philips.

Many of the liver biopsy problems in paediatrics are unique to childhood, including establishing the cause of neonatal jaundice, determining the presence of extrahepatic biliary atresia and diagnosing a wide variety of metabolic disorders that present with hepatosplenomegaly. The neonatal liver biopsy to exclude biliary atresia as a cause of jaundice has important histopathological features, namely portal tract oedema, ductular reaction (proliferation of bile ductular structures), inspissated bile within periportal ductular structures, and usually some degree of fibrosis. Total parenteral nutrition and α1-antitrypsin deficiency can demonstrate the identical changes, so these conditions must remain in the differential diagnosis until clinically excluded. A wide variety of storage disorders, including Gaucher’s disease, glycogen storage disorders, and Niemann–Pick disease may present in the neonate or older child, and these disorders benefit from the availability of a liver specimen fixed in glutaraldehyde for transmission electron microscopy. The knowledge base of the variety of progressive familial intrahepatic cholestasis (PFIC) conditions has now expanded beyond three types to six (if mutation of the MYO5D gene is nominally included as type 6). The added types include PFIC-4 with mutation of the tight junction protein 2 gene, resulting in diminished expression of claudin-1 on bile canaliculi, and PFIC-5 with mutation of the NR1H4 gene encoding the farnesoid X receptor (FXR), which is diminished in expression (along with bile-salt export pump) on the bile canaliculus. PFIC-6 is often associated with intestinal microvillous inclusion disease, but not always.