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Cells ; 9(1)2020 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-31940841


Activation of hepatic macrophages represents the critical driving force to promote cholestatic liver injury. Exosomes, as important small extracellular vesicles released by almost all types of cells, contribute to intercellular communication. We previously reported that cholangiocyte-derived exosomal long noncoding RNA (lncRNA) H19 plays a vital role in disrupting bile acid homeostasis in hepatocytes and promoting the activation of hepatic stellate cells (HSCs). Exosomal H19 derived from cholangiocytes was rapidly taken up by Kupffer cells. However, the mechanistic links between exosomal lncRNA H19 and macrophage-driven inflammation in cholestasis remain unclear. Here, we reported that the hepatic H19 level was closely correlated with macrophage activation and hepatic fibrosis in both Mdr2-/- and bile duct ligation (BDL) cholestatic mouse models, as well as in human primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) patients. Exosomal H19 significantly induced the expression and secretion of chemokine (C-C motif) ligand 2 (CCL-2) and interleukin 6 (IL-6) in Kupffer cells. H19-enriched exosomes enhanced the activation M1 polarization of Kupffer cells and promoted the recruitment and differentiation of bone marrow-derived macrophages, which were inhibited by a CCL-2 pharmacological inhibitor. In conclusion, Cholangiocyte-derived exosomal H19 played a critical role in macrophage activation, differentiation, and chemotaxis through CCL-2/CCR-2 signaling pathways, which represent a therapeutic target for cholestatic liver diseases.

Hepatology ; 70(4): 1317-1335, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30985008


Activation of hepatic stellate cells (HSCs) represents the primary driving force to promote the progression of chronic cholestatic liver diseases. We previously reported that cholangiocyte-derived exosomal long noncoding RNA-H19 (lncRNA-H19) plays a critical role in promoting cholestatic liver injury. However, it remains unclear whether cholangiocyte-derived lncRNA-H19 regulates HSC activation, which is the major focus of this study. Both bile duct ligation (BDL) and Mdr2 knockout (Mdr2-/- ) mouse models were used. Wild-type and H19maternalΔExon1/+ (H19KO) mice were subjected to BDL. Mdr2-/- H19maternalΔExon1/+ (DKO) mice were generated. Exosomes isolated from cultured mouse and human cholangiocytes or mouse serum were used for in vivo transplantation and in vitro studies. Fluorescence-labeled exosomes and flow cytometry were used to monitor exosome uptake by hepatic cells. Collagen gel contraction and bromodeoxyuridine assays were used to determine the effect of exosomal-H19 on HSC activation and proliferation. Mouse and human primary sclerosing cholangitis (PSC)/primary biliary cholangitis (PBC) liver samples were analyzed by real-time PCR, western blot analysis, histology, and immunohistochemistry. The results demonstrated that hepatic H19 level was closely correlated with the severity of liver fibrosis in both mouse models and human patients with PSC and PBC. H19 deficiency significantly protected mice from liver fibrosis in BDL and Mdr2-/- mice. Transplanted cholangiocyte-derived H19-enriched exosomes were rapidly and preferentially taken up by HSCs and HSC-derived fibroblasts, and promoted liver fibrosis in BDL-H19KO mice and DKO mice. H19-enriched exosomes enhanced transdifferentiation of cultured mouse primary HSCs and promoted proliferation and matrix formation in HSC-derived fibroblasts. Conclusion: Cholangiocyte-derived exosomal H19 plays a critical role in the progression of cholestatic liver fibrosis by promoting HSC differentiation and activation and represents a potential diagnostic biomarker and therapeutic target for cholangiopathies.

Dig Liver Dis ; 51(8): 1154-1163, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31003959


Alcoholic liver disease (ALD) is one of the most common liver diseases worldwide. However, the exact mechanisms underlying ALD remain unclear. Previous studies reported that sphingosine kinase 2 (SphK2) plays an essential role in regulating hepatic lipid metabolism. In the current study, we demonstrate that compared to wild-type (WT) mice, SphK2 deficient (SphK2-/-) mice exhibited a greater degree of liver injury and hepatic lipid accumulation after feeding with an alcohol diet for 60 days. This is accompanied by a down-regulation of steroid 7-alpha-hydroxylase (Cyp7b1) and an up-regulation of pro-inflammatory mediators (Tnfα, F4/80, Il-1ß). In vitro experiments showed that alcohol induced SphK2 expression in mouse primary hepatocytes and cultured mouse macrophages. Furthermore, alcohol feeding induced a more severe intestinal barrier dysfunction in SphK2-/- mice than WT mice. Deficiency of SphK2 impaired the growth of intestinal organoids. Finally, SphK2 expression levels were down-regulated in the livers of human patients with alcoholic cirrhosis and hepatocellular carcinoma compared to healthy controls. In summary, these findings suggest that SphK2 is a crucial regulator of hepatic lipid metabolism and that modulating the SphK2-mediated signaling pathway may represent a novel therapeutic strategy for the treatment of ALD and other metabolic liver diseases.

Hepatol Commun ; 3(1): 63-73, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30619995


Patients with cirrhosis are growing older, which could have an impact on brain dysfunction beyond hepatic encephalopathy. Our aim was to study the effect of concomitant aging and cirrhosis on brain inflammation and degeneration using human and animal experiments. For the human study, age-matched patients with cirrhosis and controls between 65 and 85 years underwent cognitive testing, quality of life (QOL) assessment, and brain magnetic resonance (MR) spectroscopy and resting state functional MR imaging (rs-fMRI) analysis. Data were compared between groups. For the animal study, young (10-12 weeks) and old (1.5 years) C57BL/6 mice were given either CCl4 gavage to develop cirrhosis or a vehicle control and were followed for 12 weeks. Cortical messenger RNA (mRNA) expression of inflammatory mediators (interleukin [IL]-6, IL-1ß, transforming growth factor ß [TGF-ß], and monocyte chemoattractant protein 1), sirtuin-1, and gamma-aminobutyric acid (GABA)-ergic synaptic plasticity (neuroligin-2 [NLG2], discs large homolog 4 [DLG4], GABA receptor, subunit gamma 1/subunit B1 [GABRG1/B1]) were analyzed and compared between younger/older control and cirrhotic mice. The human study included 46 subjects (23/group). Patients with cirrhosis had worse QOL and cognition. On MR spectroscopy, patients with cirrhosis had worse changes related to ammonia and lower N-acetyl aspartate, whereas rs-fMRI analysis revealed that these patients demonstrated functional connectivity changes in the frontoparietal cortical region compared to controls. Results of the animal study showed that older mice required lower CCl4 to reach cirrhosis. Older mice, especially with cirrhosis, demonstrated higher cortical inflammatory mRNA expression of IL-6, IL-1ß, and TGF-ß; higher glial and microglial activation; and lower sirtuin-1 expression compared to younger mice. Older mice also had lower expression of DLG4, an excitatory synaptic organizer, and higher NLG2 and GABRG1/B1 receptor expression, indicating a predominantly inhibitory synaptic organization. Conclusion: Aging modulates brain changes in cirrhosis; this can affect QOL, cognition, and brain connectivity. Cortical inflammation, microglial activation, and altered GABA-ergic synaptic plasticity could be contributory.

Hepatology ; 67(4): 1441-1457, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28926118


Impaired intestinal barrier function promotes the progression of various liver diseases, including cholestatic liver diseases. The close association of primary sclerosing cholangitis (PSC) with inflammatory bowel disease highlights the importance of the gut-liver axis. It has been reported that bile duct ligation (BDL)-induced liver fibrosis is significantly reduced in C/EBP homologous protein knockout (CHOP-/- ) mice. However, the underlying mechanisms remain unclear. In the current study, we demonstrate that BDL induces striking and acute hepatic endoplasmic reticulum (ER) stress responses after 1 day, which return to normal after 3 days. No significant hepatocyte apoptosis is detected 7-14 days following BDL. However, the inflammatory response is significantly increased after 7 days, which is similar to what we found in human PSC liver samples. BDL-induced loss of stemness in intestinal stem cells (ISCs), disruption of intestinal barrier function, bacterial translocation, activation of hepatic inflammation, M2 macrophage polarization and liver fibrosis are significantly reduced in CHOP-/- mice. In addition, intestinal organoids derived from CHOP-/- mice contain more and longer crypt structures than those from wild-type (WT) mice, which is consistent with the upregulation of stem cell markers (leucine-rich repeat-containing G-protein-coupled receptor 5, olfactomedin 4, and SRY [sex determining region Y]-box 9) and in vivo findings that CHOP-/- mice have longer villi and crypts as compared to WT mice. Similarly, mRNA levels of CD14, interleukin-1ß, tumor necrosis factor-alpha, and monocyte chemotactic protein-1 are increased and stem cell proliferation is suppressed in the duodenum of patients with cirrhosis. CONCLUSION: Activation of ER stress and subsequent loss of stemness of ISCs plays a critical role in BDL-induced systemic inflammation and cholestatic liver injury. Modulation of the ER stress response represents a potential therapeutic strategy for cholestatic liver diseases as well as other inflammatory diseases. (Hepatology 2018;67:1441-1457).

Ductos Biliares/patologia , Colestase/patologia , Mucosa Intestinal/patologia , Células-Tronco/metabolismo , Fator de Transcrição CHOP/metabolismo , Animais , Apoptose/genética , Técnicas de Cultura de Células , Estresse do Retículo Endoplasmático/genética , Feminino , Hepatócitos/patologia , Humanos , Ligadura/efeitos adversos , Fígado/patologia , Hepatopatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco/fisiologia , Fator de Transcrição CHOP/genética
Alcohol Clin Exp Res ; 41(11): 1857-1865, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28925102


BACKGROUND: Cirrhosis and alcohol can independently affect the gut-liver axis with systemic inflammation. However, their concurrent impact in humans is unclear. METHODS: Our aim was to determine the effect of continued alcohol misuse on the gut-liver axis in cirrhotic patients. Age- and MELD-balanced cirrhotic patients who were currently drinking (Alc) or abstinent (NAlc) and healthy controls underwent serum and stool collection. A subset underwent upper endoscopy and colonoscopy for biopsies and duodenal fluid collection. The groups were compared regarding (i) inflammation/intestinal barrier: systemic tumor necrosis factor levels, intestinal inflammatory cytokine (duodenum, ileum, sigmoid), and ileal antimicrobial peptide expression; (ii) microbiota composition: 16SrRNA sequencing of duodenal, ileal, and colonic mucosal and fecal microbiota; and (iii) microbial functionality: duodenal fluid and fecal bile acid (BA) profile (conjugation and dehydroxylation status), intestinal BA transporter (ASBT, FXR, FGF-19, SHP) expression, and stool metabolomics using gas chromatography/mass spectrometry. RESULTS: Alc patients demonstrated a significant duodenal, ileal, and colonic mucosal and fecal dysbiosis, compared to NAlc and controls with lower autochthonous bacterial taxa. BA profile skewed toward a potentially toxic profile (higher secondary and glycine-conjugated BAs) in duodenal fluid and stool in Alc patients. Duodenal fluid demonstrated conjugated secondary BAs only in the Alc group. There was a greater expression of all ileal BA transporters in Alc patients. This group also showed higher endotoxemia, systemic and ileal inflammatory expression, and lower amino acid and bioenergetic-associated metabolites, without change in antimicrobial peptide expression. CONCLUSIONS: Despite cirrhosis, continued alcohol misuse predisposes patients to widespread dysbiosis with alterations in microbial functionality such as a toxic BA profile, which can lead to intestinal and systemic inflammation.

Alcoolismo/fisiopatologia , Disbiose/fisiopatologia , Trato Gastrointestinal/fisiopatologia , Cirrose Hepática/fisiopatologia , Alcoolismo/diagnóstico , Alcoolismo/epidemiologia , Disbiose/diagnóstico , Disbiose/epidemiologia , Endoscopia do Sistema Digestório/métodos , Feminino , Humanos , Cirrose Hepática/diagnóstico , Cirrose Hepática/epidemiologia , Masculino , Microbiota/fisiologia , Pessoa de Meia-Idade