Bile Duct Ligation Mice Research Articles

Kinin B1 receptor blockade attenuates hepatic fibrosis and portal hypertension in chronic liver diseases in mice

Background and aimsKinin B1 receptors (B1Rs) are implicated in the pathogenesis of fibrosis. This study examined the anti-fibrotic effects of B1R blockade with BI 113823 in two established mouse models of hepatic fibrosis induced by intraperitoneal carbon tetrachloride (CCl4) injection or bile duct ligation (BDL). The mechanisms underlying the protection afforded by B1R inhibition were examined using human peripheral blood cells and LX2 human hepatic stellate cells (HSCs).MethodsFibrotic liver diseases were induced in mice by intraperitoneal carbon tetrachloride (CCl4) injection for 6 weeks, and by bile duct ligation (BDL) for 3 weeks, respectively. Mice received daily treatment of vehicle or BI 113823 (B1R antagonist) from onset of the experiment until the end of the study.ResultsB1Rs were strongly induced in fibrotic mouse liver. BI 113823 significantly attenuated liver fibrosis and portal hypertension (PH), and improved survival in both CCl4 and BDL mice. BI 113823 significantly reduced the expression of fibrotic proteins α-SMA, collagens 1, 3, 4, and profibrotic growth factors PDGF, TGFβ, CTGF, VEGF, proliferating cell nuclear antigen; and reduced hepatic Akt phosphorylation in CCl4- and BDL-induced liver fibrosis. BI 113823 also reduced expression of Cytokines IL-1, IL-6; chemokines MCP-1, MCP-3 and infiltration of inflammatory cells; and inhibited human monocyte and neutrophil activation, transmigration, TNF-α & MPO production in vitro. BI 113823 inhibited TGF-β and B1R agonist-stimulated human-HSC activation, contraction, proliferation, migration and fibrosis protein expression, and inhibited activation of PI3K/Akt signalling pathway.ConclusionsB1Rs merits consideration as a novel therapeutic target for chronic liver fibrosis and PH.

This Month in AJP

The cellular mechanisms leading to cognitive symptoms in rodent bile duct ligation (BDL) models of cholestasis are unclear. Using BDL mice and human neuronal cell cultures, Gee et al (Am J Pathol, AJPA-D-22-00224) studied these mechanisms. BDL reduced spatial learning capabilities as well as exhibited cellular changes, which were partly restored by treatment with obeticholic acid (OCA)—farnesoid X receptor (FXR) agonist and a clinically approved treatment agent. OCA therapy or FXR agonism may limit cholestasis-induced neuronal senescence.

Rare earth cerium oxide nanoparticles attenuated liver fibrosis in bile duct ligation mice model

Liver fibrosis is one of the major liver complications which eventually progresses to liver cirrhosis and liver failure. Cerium oxide nanoparticles, also known as nanoceria (NC) are nanoparticles with potential antioxidant and anti-inflammatory activities. Herein, we evaluated the hepatoprotective and anti-fibrotic effects of nanoceria (NC) against bile duct ligation (BDL) induced liver injury. NC were administered i.p. for 12 days (0.5 and 2 mg/kg) to C57BL/6J mice. The biochemical markers of liver injury, oxidative and nitrosative stress markers, inflammatory cytokines were evaluated. Fibrosis assessment and mechanistic studies were conducted to assess the hepatoprotective effects of NC. Administration of NC proved to significantly ameliorate liver injury as evident by reduction in SGOT, SGPT, ALP and bilirubin levels in the treated animals. NC treatment significantly reduced the hydroxyproline levels and expression of fibrotic markers. In summary, our findings establish the hepatoprotective and anti-fibrotic effects of NC against BDL induced liver injury and liver fibrosis. These protective effects were majorly ascribed to their potential ROS inhibition and antioxidant activities through catalase, superoxide dismutase (SOD)-mimetic properties and auto-regenerating capabilities.

Vitamin D Receptor Activation Targets ROS-Mediated Crosstalk Between Autophagy and Apoptosis in Hepatocytes in Cholestasic Mice

Observational epidemiologic studies have associated vitamin D deficiency with cholestasis. We reported previously that activation of the vitamin D/vitamin D receptor (VDR) axis in cholangiocytes mitigates cholestatic liver injury by remodeling the damaged bile duct. However, the function of VDR in hepatocytes during cholestasis remains unclear. Paricalcitol (VDR agonist, 200 ng/kg) was injected intraperitoneally into bile duct-ligated mice every other day for 5 days. Primary hepatocytes and HepG2 hepatoma cells were transfected with Vdr short hairpin RNA, control short hairpin RNA, Vdr plasmid, control vector, Atg5 small interfering RNA (siRNA), and control siRNA. Liver histology, cell proliferation, and autophagy were evaluated. Treatment with the VDR agonist paricalcitol improved liver injury in bile duct-ligated mice by up-regulating VDR expression in hepatocytes, which in turn reduced hepatocyte apoptosis by inhibiting reactive oxygen species (ROS) generation via suppressing the Ras-related C3 botulinum toxin substrate 1/reduced nicotinamide adenine dinucleotide phosphate oxidase 1 pathway. Mechanistically, upon exposure to an ROS-inducing compound, Vdr siRNA contributed to apoptosis, whereas the Vdr overexpression caused resistance to apoptosis. Interestingly, up-regulated VDR expression also increased the generation of autophagosomes and macroautophagic/autophagic flux, which was the underlying mechanism for reduced apoptosis following VDR activation. Autophagy depletion impaired the positive effects of VDR overexpression, whereas autophagy induction was synergystic with VDR overexpression. Importantly, up-regulation of VDR promoted autophagy activation by suppressing the activation of the extracellular signal-regulated kinase (ERK)/p38 mitogen-activated protein kinase (p38MAPK) pathway. Thus, a p38MAPK inhibitor abrogated the Vdr siRNA-induced decrease in autophagy and the Vdr siRNA-induced increase in apoptosis. In contrast, a Mitogen-activated protein kinase kinase (MEK)/ERK activator prevented the enhancement of autophagy and decreased apoptosis following Vdr overexpression. Moreover, the ROS inhibitor N-acetylcystein (NAC) blocked Vdr siRNA-enhanced activation of the ERK/p38MAPK pathway. VDR activation mitigated liver cholestatic injury by reducing autophagy-dependent hepatocyte apoptosis and suppressing the activation of the ROS-dependent ERK/p38MAPK pathway. Thus, VDR activation may be a potential target for the treatment of cholestatic liver disease.

A mouse model of hepatic encephalopathy: bile duct ligation induces brain ammonia overload, glial cell activation and neuroinflammation

Hepatic encephalopathy (HE) is a common complication of chronic liver disease, characterized by an altered mental state and hyperammonemia. Insight into the brain pathophysiology of HE is limited due to a paucity of well-characterized HE models beyond the rat bile duct ligation (BDL) model. Here, we assess the presence of HE characteristics in the mouse BDL model. We show that BDL in C57Bl/6j mice induces motor dysfunction, progressive liver fibrosis, liver function failure and hyperammonemia, all hallmarks of HE. Swiss mice however fail to replicate the same phenotype, underscoring the importance of careful strain selection. Next, in-depth characterisation of metabolic disturbances in the cerebrospinal fluid of BDL mice shows glutamine accumulation and transient decreases in taurine and choline, indicative of brain ammonia overload. Moreover, mouse BDL induces glial cell dysfunction, namely microglial morphological changes with neuroinflammation and astrocyte reactivity with blood–brain barrier (BBB) disruption. Finally, we identify putative novel mechanisms involved in central HE pathophysiology, like bile acid accumulation and tryptophan–kynurenine pathway alterations. Our study provides the first comprehensive evaluation of a mouse model of HE in chronic liver disease. Additionally, this study further underscores the importance of neuroinflammation in the central effects of chronic liver disease.

Anti–Cholestatic Therapy with Obeticholic Acid Improves Short-Term Memory in Bile Duct–Ligated Mice

Patients with cholestatic liver disease, including those with primary biliary cholangitis, can experience symptoms of impaired cognition or brain fog. This phenomenon remains unexplained and is currently untreatable. Bile duct ligation (BDL) is an established rodent model of cholestasis. In addition to liver changes, BDL animals develop cognitive symptoms early in the disease process (before development of cirrhosis and/or liver failure). The cellular mechanisms underpinning these cognitive symptoms are poorly understood. Herein, the study explored the neurocognitive symptom manifestations, and tested potential therapies, in BDL mice, and used human neuronal cell cultures to explore translatability to humans. BDL animals exhibited short-term memory loss and showed reduced astrocyte coverage of the blood-brain barrier, destabilized hippocampal network activity, and neuronal senescence. Ursodeoxycholic acid (first-line therapy for most human cholestatic diseases) did not reverse symptomatic or mechanistic aspects. In contrast, obeticholic acid (OCA), a farnesoid X receptor agonist and second-line anti-cholestatic agent, normalized memory function, suppressed blood-brain barrier changes, prevented hippocampal network deficits, and reversed neuronal senescence. Co-culture of human neuronal cells with either BDL or human cholestatic patient serum induced cellular senescence and increased mitochondrial respiration, changes that were limited again by OCA. These findings provide new insights into the mechanism of cognitive symptoms in BDL animals, suggesting that OCA therapy or farnesoid X receptor agonism could be used to limit cholestasis-induced neuronal senescence.

Ganfule capsule alleviates bile duct ligation-induced liver fibrosis in mice by inhibiting glutamine metabolism

Background: Liver fibrosis is a pathological outcome of a variety of liver diseases, and it can also progress into liver cirrhosis and liver cancer. Specific liver antifibrotic drugs have not been clinically approved yet. Studies have demonstrated the protective effects of Ganfule capsule (GFL) on the liver and its therapeutic potential in hepatic cancer. However, the mechanism of GFL is not clear in the treatment of liver fibrosis. Objective: This article aims to study the protective effect of GFL on liver fibrosis and its possible mechanism. Methods: The cholestatic liver fibrosis model was prepared by subjecting C57BL/6 mice to bile duct ligation (BDL). The GFL groups were treated with different concentrations of GFL for 14 days. Pathological analysis, serum biochemical index detection, metabonomic analysis, immunohistochemistry, Western blot, and real-time PCR were carried out. Results: GFL could alleviate liver injury and liver fibrosis caused by BDL in mice. Metabonomic analysis of mice serum showed postoperative metabolic disorder, which could be alleviated by GFL through glutamine metabolism; valine, leucine, and isoleucine biosynthesis; aminoacyl-tRNA biosynthesis; and other metabolic pathways. GFL affected glutamine metabolism by inhibiting the activity of glutaminase 1 (GLS1). The activation of GLS1 is regulated by the NF-κB pathway, and experiments showed that GFL could inhibit IκB-α and NF-κB p65 phosphorylation. Conclusion: This study confirms the protective effect of GFL on liver injury and shows that GFL inhibits glutamine metabolism, which was correlated with the NF-κB pathway, and eventually alleviates liver fibrosis. These results are conducive to the development of new therapeutic drugs for liver fibrosis.

Enhanced anxiolytic and analgesic effectiveness or a better safety profile of morphine and tramadol combination in cholestatic and addicted mice.

The involvement of the opioidergic system on anxiolytic and antinociceptive responses induced by cholestasis was investigated in cholestatic and addicted mice. Elevated plus-maze and tail-flick devices were used to assess anxiety and pain levels, respectively. The data indicated that induction of cholestasis and injection of opioid drugs including morphine and tramadol enhanced %OAT and %OAE but naloxone reduced %OAT and %OAE in the sham-operated and bile duct ligation (BDL) mice. Induction of cholestasis and addiction to morphine and tramadol prolonged tail-flick latency, which was reversed by naloxone. Coadministration of morphine and tramadol enhanced anxiolytic and analgesic effects in the sham-operated and BDL mice. It seems (a) cholestasis and addiction affect anxiety and pain behaviors, (b) μ-opioid receptors play a key role in anxiolytic and analgesic effects induced by cholestasis, and (c) cotreatment with morphine and tramadol augmented the effectiveness of them for induction of anxiolytic and analgesic effects both in cholestatic and addicted mice.

Slc25a39 and Slc25a40 Expression in Mice with Bile Duct Ligation or Lipopolysaccharide Treatment.

SLC25A39/40, involved in mitochondrial GSH (mGSH) import from the cytoplasm, is essential for protection against oxidative stress and mitochondrial dysfunction. We examined the effects of cholestasis, through bile duct ligation (BDL) and lipopolysaccharide (LPS)-induced inflammation in mice, on Slc25a39/40 expression. Additionally, we used human clear cell renal carcinoma (KMRC-1) cells to elucidate the mechanism of regulation of SLC25A39/40 expression in the kidneys after LPS treatment. BDL resulted in a decrease in Slc25a39 mRNA in the liver and a decrease in Slc25a39/40 mRNA and protein in the kidneys. Consequently, there was a significant decrease in mGSH levels in the kidneys of BDL mice compared with those in sham mice. LPS treatment resulted in increased Slc25a40 expression in the kidneys. In KMRC-1 cells, the combination treatment of LPS-RS or FPS-ZM1 with LPS suppressed the LPS-induced increase in SLC25A40, suggesting that SLC25A40 expression could be regulated by the signaling pathway via toll-like receptor 4 and the receptor for advanced glycation end products, respectively. Our findings contribute to understanding the role of mGSH in the maintenance of the mitochondrial redox state. To the best of our knowledge, this is the first study that demonstrates the changes in Slc25a39/40 expression in mice with cholestasis-associated renal injury and LPS-induced inflammation.

Transcriptome Profile of Thyroid Glands in Bile Duct Ligation Mouse Model.

Thyroid hormone (TH) contributes to multiple cellular mechanisms in the liver, muscle cells, adipose tissue, and brain, etc. In particular, the liver is an important organ in TH metabolism for the conversion of thyronine (T4) into triiodothyronine (T3) by the deiodinase enzyme. TH levels were significantly decreased and thyroid-stimulating hormone (TSH) levels were significantly increased in patients with liver failure compared with normal subjects. Among liver failure diseases, hepatic encephalopathy (HE) deserves more attention because liver damage and neuropathologies occur simultaneously. Although there is numerous evidence of TH dysregulation in the HE model, specific mechanisms and genetic features of the thyroid glands in the HE model are not fully understood. Here, we investigated the significantly different genes in the thyroid glands of a bile duct ligation (BDL) mouse model as the HE model, compared to the thyroid glands of the control mouse using RNA sequencing. We also confirmed the alteration in mRNA levels of thyroid gland function-related genes in the BDL mouse model. Furthermore, we evaluated the increased level of free T4 and TSH in the BDL mouse blood. Thus, we emphasize the potential roles of TH in liver metabolism and suggest that thyroid dysfunction-related genes in the HE model should be highlighted for finding the appropriate solution for an impaired thyroid system in HE.

Therapeutic Effect of Acer tegmentosum Maxim Twig Extract in Bile Duct Ligation-Induced Acute Cholestasis in Mice.

Cholestatic liver disease, or cholestasis, is a condition characterized by liver inflammation and fibrosis following a bile duct obstruction and an intrahepatic accumulation of bile acids. Inhibiting inflammation is a promising therapeutic strategy for cholestatic liver diseases. Acer tegmentosum Maxim extract (ATE) is best known for its anti-inflammatory and antioxidative properties. In this study, we investigated the effects of ATE on liver injury and fibrosis in mice with bile duct ligation (BDL)-induced cholestasis through analysis of gene expression, cytokines, and histological examination. Oral administration of ATE (20 or 50 mg/kg) for 14 days significantly attenuated hepatocellular necrosis compared to vehicle-treated BDL mice, which was accompanied by the reduced level of serum bile acids and bilirubin. We determined that ATE treatment reduced liver inflammation, oxidative stress, and fibrosis. These beneficial effects of ATE were concurrent with the decreased expression of genes involved in the NF-κB pathway, suggesting that the anti-inflammatory effect of ATE could be a possible mechanism against cholestasis-associated liver injury. Our findings substantiate ATE's role as an alternative therapeutic agent for cholestasis-induced liver injury and fibrosis.

The Role of Gut Microbiota in Mice With Bile Duct Ligation-Evoked Cholestatic Liver Disease-Related Cognitive Dysfunction.

The pathogenesis of Hepatic Encephalopathy (HE) is complex and multifactorial. The development of metagenomics sequencing technology led to show the significant role of gut microbiota in the pathogenesis of cognitive dysfunction, which paved the way for further research in this field. However, it is unknown whether gut microbiota plays a role in bile duct ligation (BDL)-evoked cholestatic liver disease-related cognitive dysfunction. The aim of this investigation is to assess BDL mice induced cognitive dysfunction and meanwhile to delineate the alteration of gut microbiota in cognitive dysfunction mice, which may underline the role of gut microbiota in BDL mice induced cognitive dysfunction. Our study was carried out in male C57BL/6 J mice with bile duct ligation. The liver functions were assessed via different biochemical markers [alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), and total bile acid (TBA)] and a histopathological examination of the liver tissue. We used the novel object recognition test (NORT) to assess cognitive dysfunction. And BDL mice were divided into BDL with cognitive dysfunction (BDL-CD) or BDL without cognitive dysfunction (BDL-NCD groups) by the result of hierarchical cluster analysis of NORT. Then, 16S ribosomal RNA (rRNA) gene sequencing was used to compare the gut bacterial composition between BDL-CD and BDL-NCD groups. According to our results, we concluded that bile duct ligation can significantly change the gut microbiota composition, and Bacteroides fragilis, Bacteroides ovatus V975, and Bacteroides thetaiotaomicron play a vital role in BDL-evoked cholestatic liver disease-related cognitive dysfunction.

MiR-200c-3p targets SESN1 and represses the IL-6/AKT loop to prevent cholangiocyte activation and cholestatic liver fibrosis

AbstractCholestasis causes ductular reaction in the liver where the reactive cholangiocytes not only proliferate but also gain a neuroendocrine-like phenotype, leading to inflammatory cell infiltration and extracellular matrix deposition and contributing to the development and progression of cholestatic liver fibrosis. This study aims to elucidate the role of miR-200c in cholestasis-induced biliary liver fibrosis and cholangiocyte activation. We found that miR-200c was extremely abundant in cholangiocytes but was reduced by cholestasis in a bile duct ligation (BDL) mouse model; miR-200c was also decreased by bile acids in vitro. Phenotypically, loss of miR-200c exacerbated cholestatic liver injury, including periductular fibrosis, intrahepatic inflammation, and biliary hyperplasia in both the BDL model and the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) model. We identified sestrin 1 (SESN1) as a target of miR-200c. Sesn1−/−-BDL mice showed mitigation of cholestatic liver injury. On a molecular level, the pro-proliferative IL-6/AKT feedback loop was activated in Mir200c−/− livers but was inhibited in Sesn1−/− livers upon cholestasis in mice. Furthermore, rescuing expression of miR-200c by the adeno-associated virus serotype 8 ameliorated BDL-induced liver injury in Mir200c−/− mice. Taken together, this study demonstrates that miR-200c restrains the proliferative and neuroendocrine-like activation of cholangiocytes by targeting SESN1 and inhibiting the IL-6/AKT feedback loop to protect against cholestatic liver fibrosis. Our findings provide mechanistic insights regarding biliary liver fibrosis, which may help to reveal novel therapeutic targets for the treatment of cholestatic liver injury and liver fibrosis.Cholangiocytes are the primary targets of cholangiopathies. This study elucidates the role of miR-200c in maintaining cholangiocyte homeostasis making use of cell culture and mouse models of cholestasis. MiR-200c restrains the proliferative and neuroendocrine-like activation of cholangiocytes by targeting sestrin 1(SESN1) and inhibiting the IL-6/AKT feedback loop to prevent cholestatic liver injury. The findings provide critical mechanistic insights into biliary liver fibrosis and suggest miR-200c may be a novel therapeutic target of cholangiopathies.

Activation of α7nAChR preserves intestinal barrier integrity by enhancing the HO-1 / STAT3 signaling to inhibit NF-κB activation in mice

BackgroundActivation of alpha-7 nicotinic acetylcholine receptor (α7nAChR) can inhibit a systemic inflammatory response and preserve intestinal barrier integrity. This study aimed at elucidating the molecular mechanisms by which α7nAChR activation could inhibit intestinal barrier injury and cholestatic liver fibrosis in mice following bile duct ligation (BDL). Materials and methodsThe intestine-specific heme oxygenase-1 (HO-1) knockout VillinCreHmox1-/- and control Hmox1flox/flox C57BL/6 mice were subjected to the BDL procedure. The therapeutic effects of GST-21, a specific ligand for α7nAChR, on systemic and intestinal inflammation, intestinal barrier integrity, liver fibrosis and injury, HO-1 expression, STAT3, AKT and NF-κBp65 activation were examined in these mice and intestinal epithelial cells after being co-cultured with macrophages. ResultsCompared with the vehicle-injected BDL group, treatment with GST-21 to activate α7nAChR decreased intestinal and liver injury and fibrosis in BDL mice, accompanied by reducing serum cytokine levels. In addition, activation of α7nAChR preserved the levels of tight junction protein expression and intestinal epithelial barrier integrity in BDL mice and epithelial cells following co-cultured with macrophages. The therapeutic effects of α7nAChR activation were mediated by enhancing HO-1 expression and STAT3 phosphorylation, and reducing the NF-κBp65 activation in intestinal tissues and epithelial cells co-cultured with macrophages. Finally, activation of α7nAChR induced HO-1 expression and STAT3 phosphorylation in an interdependent manner, independent of the PI3K/AKT signaling. ConclusionActivation of α7nAChR enhanced HO-1 expression and STAT3 signaling to inhibit NF-κB activation, preserving the intestinal barrier integrity, and reducing inflammation and liver fibrosis in cholestatic mice. Therefore, targeting α7nAChR may be a promising interventional strategy for primary biliary cholangitis.

Angelica keiskei Root Extract Attenuates Bile Duct Ligation-Induced Liver Injury in Mice.

Although multiple studies have shown that Angelica keiskei of the Umbelliferae family has potent anti-inflammatory and antioxidative activities and that it reduces the serum bile acids in humans, whether A. keiskei has protective effects against cholestasis-induced liver injury remains unexplored until now. This study tests the hypothesis that Angelica keiskei root extract (AKE) alleviates liver injury, inflammation, and fibrosis in mouse models of acute cholestasis induced by bile duct ligation (BDL). Oral administration of AKE (200 or 500 mg/kg) attenuated hepatocellular necrosis and significantly reduced serum levels of bile acids and bilirubin in BDL mice. The critical enzyme of bile acid synthesis, CYP7A1, was repressed by AKE, suggesting that reduced bile acid production may contribute to liver protection. Moreover, we determined through gene expression and cytokine analysis and histological examination that AKE treatment decreased liver inflammation, oxidative stress, and fibrosis. AKE also suppressed the NF-κB pathway, suggesting this as a possible mediator of its anti-inflammatory effect. Our findings substantiate that AKE may be promising for treating cholestatic liver diseases in the future.

Upregulation of cadherin-11 contributes to cholestatic liver fibrosis.

ABSTRACTImportanceCadherin‐11 (CDH11), a cell‐to‐cell adhesion molecule, is implicated in the fibrotic process of several organs. Biliary atresia (BA) is a common cholestatic liver disease featuring cholestasis and progressive liver fibrosis in children. Cholestatic liver fibrosis may progress to liver cirrhosis and lacks effective therapeutic strategies. Currently, the role of CDH11 in cholestatic liver fibrosis remains unclear.ObjectiveThis study aimed to explore the functions of CDH11 in cholestatic liver fibrosis.MethodsThe expression of CDH11 in BA livers was evaluated by database analysis and immunostaining. Seven BA liver samples were used for immunostaining. The wild type (Wt) and CDH11 knockout (CDH11–/– ) mice were subjected to bile duct ligation (BDL) to induce cholestatic liver fibrosis. The serum biochemical analysis, liver histology, and western blotting were used to assess the extent of liver injury and fibrosis as well as activation of transforming growth factor‐β (TGF‐β)/Smad pathway. The effect of CDH11 on the activation of hepatic stellate cell line LX‐2 cells was investigated.ResultsAnalysis of public RNA‐seq datasets showed that CDH11 expression levels were significantly increased in livers of BA, and CDH11 was correlated with liver fibrosis in BA. BDL‐induced liver injury and liver fibrosis were attenuated in CDH11–/– mice compared to Wt mice. The protein expression levels of phosphorylated Smad2/3 were decreased in livers of CDH11–/– BDL mice compared to Wt BDL mice. CDH11 knockdown inhibited the activation of LX‐2 cells.InterpretationCDH11 plays an important role in cholestatic liver fibrosis and may represent a potential therapeutic target for cholestatic liver disease, such as BA.

Fibroblast activation protein activated antifibrotic peptide delivery attenuates fibrosis in mouse models of liver fibrosis

In liver fibrosis, activated hepatic stellate cells are known to overexpress fibroblast activation protein. Here we report a targeted antifibrotic peptide-delivery system in which fibroblast activation protein, which is overexpressed in fibrotic regions of the liver, liberates the antifibrotic peptide melittin by cleaving a fibroblast activation protein-specific site in the peptide. The promelittin peptide is linked to pegylated and maleimide-functionalized liposomes, resulting in promelittin-modified liposomes. The promelittin-modified liposomes were effective in reducing the viability of activated hepatic stellate cells but not that of control cells. In three types of liver fibrosis mouse models, intravenously administered promelittin-modified liposomes significantly reduces fibrotic regions. In addition, in the bile duct ligation mouse model promelittin-modified liposome-treatment increases overall survival. Although this peptide-delivery concept was tested for liver fibrosis, it can potentially be adapted to other fibrotic diseases.

Downregulation of Ca2+-Activated Cl- Channel TMEM16A Mediated by Angiotensin II in Cirrhotic Portal Hypertensive Mice.

Portal hypertension is defined as an increased pressure in the portal venous system and occurs as a major complication in chronic liver diseases. The pathological mechanism underlying the pathogenesis and development of portal hypertension has been extensively investigated. Vascular tone of portal vein smooth muscles (PVSMs) is regulated by the activities of several ion channels, including Ca2+-activated Cl− (ClCa) channels. TMEM16A is mainly responsible for ClCa channel conductance in vascular smooth muscle cells, including portal vein smooth muscle cells (PVSMCs). In the present study, the functional roles of TMEM16A channels were examined using two experimental portal hypertensive models, bile duct ligation (BDL) mice with cirrhotic portal hypertension and partial portal vein ligation (PPVL) mice with non-cirrhotic portal hypertension. Expression analyses revealed that the expression of TMEM16A was downregulated in BDL-PVSMs, but not in PPVL-PVSMs. Whole-cell ClCa currents were smaller in BDL-PVSMCs than in sham- and PPVL-PVSMCs. The amplitude of spontaneous contractions was smaller and the frequency was higher in BDL-PVSMs than in sham- and PPVL-PVSMs. Spontaneous contractions sensitive to a specific inhibitor of TMEM16A channels, T16Ainh-A01, were reduced in BDL-PVSMs. Furthermore, in normal PVSMs, the downregulation of TMEM16A expression was mimicked by the exposure to angiotensin II, but not to bilirubin. This study suggests that the activity of ClCa channels is attenuated by the downregulation of TMEM16A expression in PVSMCs associated with cirrhotic portal hypertension, which is partly mediated by increased angiotensin II in cirrhosis.

Electroacupuncture Ameliorates Intestinal Barrier Destruction in Mice With Bile Duct Ligation–Induced Liver Injury by Activating the Cholinergic Anti-Inflammatory Pathway

ObjectivesElectroacupuncture (EA) at Zusanli (ST36) can attenuate inflammation in different rodent models. However, the therapeutic mechanisms underlying its action in inhibiting intestinal barrier destruction and liver injury in cholestasis mice have not been clarified. This study aimed at investigating whether EA at ST36 could activate the cholinergic anti-inflammatory pathway to inhibit intestinal barrier destruction and liver injury in cholestasis mice. Materials and MethodsMale Hmox1floxp/floxp C57BL/6 mice were randomized and subjected to a sham or bile duct ligation (BDL) surgery. The BDL mice were randomized and treated with, or without (BDL group), sham EA at ST36 (BDL+sham-ST36) or EA at ST36 (BDL+ST36), or received α-bungarotoxin (α-BGT), a specific inhibitor of nicotinic acetylcholine receptor α7 subunit (α7nAChR), before stimulation (BDL+ST36+α-BGT). These mice, together with a group of intestine-specific heme oxygenase-1 (HO-1) knockout (KO) Villin-Cre-HO-1−/− mice, were monitored for their body weights before and 14 days after BDL. The levels of plasma cytokines and liver injury–related alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by enzyme-linked immunoassay, and pathological changes in the intestinal mucosa and liver fibrosis as well as intestinal barrier permeability in individual mice were examined by histology and immunohistochemistry. The levels of α7nAChR, HO-1, ZO-1, Occludin, Claudin-1, and NF-κBp65 expression and NF-κBp65 phosphorylation in intestinal tissues were quantified. ResultsCompared with the sham group, BDL significantly increased the levels of plasma interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor α, ALT, and AST and caused intestinal mucosal damages, high permeability, and liver fibrosis in mice, which were remarkably mitigated, except for further increased levels of plasma IL-10 in the BDL+ST36 group of mice. Similarly, EA at ST36 significantly up-regulated α7nAChR and HO-1 expression; mitigated the BDL-decreased ZO-1, Occludin, and Claudin-1 expression; and attenuated the BDL-increased NF-κBp65 phosphorylation in intestinal tissues of mice. The therapeutic effects of EA at ST36 were significantly abrogated by pretreatment with α-BGT or HO-1 KO. ConclusionEA at ST36 inhibits the BDL-induced intestinal mucosal damage and liver fibrosis by activating the HO-1 cholinergic anti-inflammatory pathway in intestinal tissues of mice.

Long Noncoding RNAs Regulate Hyperammonemia-Induced Neuronal Damage in Hepatic Encephalopathy

Background Hyperammonemia can result in various neuropathologies, including sleep disturbance, memory loss, and motor dysfunction in hepatic encephalopathy. Long noncoding RNA (lncRNA) as a group of noncoding RNA longer than 200 nucleotides is emerging as a promising therapeutic target to treat diverse diseases. Although lncRNAs have been linked to the pathogenesis of various diseases, their function in hepatic encephalopathy has not yet been elucidated. Research Design and Methods. To identify the roles of lncRNAs in hepatic encephalopathy brain, we used a bile duct ligation (BDL) mouse model and examined the alteration of neuronal cell death markers and neuronal structure-related proteins in BDL mouse cortex tissue. Furthermore, analysis of the transcriptome of BDL mouse brain cortex tissues revealed several lncRNAs critical to the apoptosis and neuronal structural changes associated with hepatic encephalopathy. Results We confirmed the roles of the lncRNAs, ZFAS1, and GAS5 as strong candidate lncRNAs to regulate neuropathologies in hepatic encephalopathy. Our data revealed the roles of lncRNAs, ZFAS1, and GAS5, on neuronal cell death and neural structure in hyperammonemia in in vivo and in vitro conditions. Conclusion Thus, we suggest that the modulation of these lncRNAs may be beneficial for the treatment of hepatic encephalopathy.