RESUMEN

BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is a chronic liver disease characterized by progressive biliary inflammation and bile duct injury. Berberine (BBR) is a bioactive isoquinoline alkaloid found in various herbs and has multiple beneficial effects on metabolic and inflammatory diseases, including liver diseases. This study aimed to examine the therapeutic effect of BBR on cholestatic liver injury in a PSC mouse model (Mdr2-/- mice) and elucidate the underlying mechanisms. METHODS: Mdr2-/-mice (12-14 weeks old, both sexes) received either BBR (50 mg/kg) or control solution daily for eight weeks via oral gavage. Histological and serum biochemical analyses were used to assess fibrotic liver injury severity. Total RNAseq and pathway analyses were used to identify the potential signaling pathways modulated by BBR in the liver. The expression levels of key genes involved in regulating hepatic fibrosis, bile duct proliferation, inflammation, and bile acid metabolism were validated by qRT-PCR or Western blot analysis. The bile acid composition and levels in the serum, liver, small intestine, and feces and tissue distribution of BBR were measured by LC-MS/MS. Intestinal inflammation and injury were assessed by gene expression profiling and histological analysis. The impact on the gut microbiome was assessed using 16S rRNA gene sequencing. RESULTS: BBR treatment significantly ameliorated cholestatic liver injury, evidenced by decreased serum levels of AST, ALT, and ALP, and reduced bile duct proliferation and hepatic fibrosis, as shown by H&E, Picro-Sirius Red, and CK19 IHC staining. RNAseq and qRT-PCR analyses indicated a substantial inhibition of fibrotic and inflammatory gene expression. BBR also mitigated ER stress by downregulating Chop, Atf4 and Xbp-1 expression. In addition, BBR modulated bile acid metabolism by altering key gene expressions in the liver and small intestine, resulting in restored bile acid homeostasis characterized by reduced total bile acids in serum, liver, and small intestine and increased fecal excretion. Furthermore, BBR significantly improved intestinal barrier function and reduced bacterial translocation by modulating the gut microbiota. CONCLUSION: BBR effectively attenuates cholestatic liver injury, suggesting its potential as a therapeutic agent for PSC and other cholestatic liver diseases.

RESUMEN

BACKGROUND: NAFLD has become the most common chronic liver disease worldwide. Human antigen R (HuR), an RNA-binding protein, is an important post-transcriptional regulator. HuR has been reported as a key player in regulating lipid homeostasis in the liver and adipose tissues by using tissue-specific HuR knockout mice. However, the underlying mechanism by which hepatocyte-specific HuR regulates hepatic lipid metabolism under metabolic stress remains unclear and is the focus of this study. METHODS: Hepatocyte-specific HuR deficient mice (HuRhKO) and age-/gender-matched control mice, as well as long-noncoding RNA H19 knockout mice (H19-/-), were fed a Western Diet plus sugar water (WDSW). Hepatic lipid accumulation, inflammation and fibrosis were examined by histology, RNA transcriptome analysis, qRT-PCR, and Western blot analysis. Bile acid composition was measured using LC-MS/MS. RESULTS: Hepatocyte-specific deletion of HuR not only significantly increased hepatic lipid accumulation by modulating fatty acid synthesis and metabolism but also markedly induced inflammation by increasing immune cell infiltration and neutrophil activation under metabolic stress. In addition, hepatic deficiency of HuR disrupted bile acid homeostasis and enhanced liver fibrosis. Mechanistically, HuR is a repressor of H19 expression. Analysis of a recently published dataset (GSE143358) identified H19 as the top-upregulated gene in liver-specific HuR knockout mice. Similarly, hepatocyte-specific deficiency of HuR dramatically induced the expression of H19 and sphingosine-1 phosphate receptor 2 (S1PR2), but reduced the expression of sphingosine kinase 2 (SphK2). WDSW-induced hepatic lipid accumulation was alleviated in H19-/- mice. Furthermore, the downregulation of H19 alleviated WDSW-induced NAFLD in HuRhKO mice. CONCLUSIONS: HuR not only functions as an RNA binding protein to modulate post-transcriptional gene expression but also regulates H19 promoter activity. Hepatic HuR is an important regulator of hepatic lipid metabolism via modulating H19 expression.

RESUMEN

The disease progression of nonalcoholic fatty liver disease (NAFLD) from simple steatosis (NAFL) to nonalcoholic steatohepatitis (NASH) is driven by multiple factors. Berberine (BBR) is an ancient Chinese medicine and has various beneficial effects on metabolic diseases, including NAFLD/NASH. However, the underlying mechanisms remain incompletely understood due to the limitation of the NASH animal models used. Methods: A high-fat and high-fructose diet-induced mouse model of NAFLD, the best available preclinical NASH mouse model, was used. RNAseq, histological, and metabolic pathway analyses were used to identify the potential signaling pathways modulated by BBR. LC-MS was used to measure bile acid levels in the serum and liver. The real-time RT-PCR and Western blot analysis were used to validate the RNAseq data. Results: BBR not only significantly reduced hepatic lipid accumulation by modulating fatty acid synthesis and metabolism but also restored the bile acid homeostasis by targeting multiple pathways. In addition, BBR markedly inhibited inflammation by reducing immune cell infiltration and inhibition of neutrophil activation and inflammatory gene expression. Furthermore, BBR was able to inhibit hepatic fibrosis by modulating the expression of multiple genes involved in hepatic stellate cell activation and cholangiocyte proliferation. Consistent with our previous findings, BBR's beneficial effects are linked with the downregulation of microRNA34a and long noncoding RNA H19, which are two important players in promoting NASH progression and liver fibrosis. Conclusion: BBR is a promising therapeutic agent for NASH by targeting multiple pathways. These results provide a strong foundation for a future clinical investigation.

Asunto(s)

Berberina/uso terapéutico , Progresión de la Enfermedad , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/patología , Transducción de Señal , Animales , Berberina/farmacología , Ácidos y Sales Biliares/metabolismo , Dieta Occidental , Ácidos Grasos/metabolismo , Perfilación de la Expresión Génica , Ontología de Genes , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Ratones Endogámicos C57BL , Modelos Biológicos , Enfermedad del Hígado Graso no Alcohólico/sangre , Enfermedad del Hígado Graso no Alcohólico/genética , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Transducción de Señal/efectos de los fármacos , Transcriptoma/genética

RESUMEN

Inflammation plays an essential role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Berberine (BBR), an isoquinoline alkaloid isolated from Chinese medicinal herbs, has been widely used to treat various diseases, including liver diseases for hundreds of years. The previous studies have shown that BBR inhibits high fat-diet-induced steatosis and inflammation in rodent models of NAFLD. However, the underlying molecular mechanisms remain unclear. This study is aimed to identify the potential mechanisms by which BBR inhibits free fatty acid (FFA) and LPS-induced inflammatory response in mouse macrophages and hepatocytes. Mouse RAW264.7 macrophages and primary mouse hepatocytes were treated with palmitic acid (PA) or LPS or both with or without BBR (0-10 µM) for different periods (0-24 h). The mRNA and protein levels of proinflammatory cytokines (TNF-α, IL-6, IL-1ß, MCP-1) and ER stress genes (CHOP, ATF4, XBP-1) were detected by real-time RT-PCR, Western blot and ELISA, respectively. The results indicated that BBR significantly inhibited PA and LPS-induced activation of ER stress and expression of proinflammatory cytokines in macrophages and hepatocytes. PA/LPS-mediated activation of ERK1/2 was inhibited by BBR in a dose-dependent manner. In summary, BBR inhibits PA/LPS-induced inflammatory responses through modulating ER stress-mediated ERK1/2 activation in macrophages and hepatocytes.

Asunto(s)

Berberina/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Inflamación/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Animales , Berberina/uso terapéutico , Citocinas/metabolismo , Inflamación/inducido químicamente , Lipopolisacáridos/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/inducido químicamente , Ácido Palmítico/toxicidad , Células RAW 264.7

RESUMEN

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.

Asunto(s)

Conductos Biliares/metabolismo , Colangitis Esclerosante/metabolismo , Inflamación/metabolismo , Activación de Macrófagos , Macrófagos/metabolismo , ARN Largo no Codificante/metabolismo , Animales , Conductos Biliares/citología , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Exosomas/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Largo no Codificante/genética

RESUMEN

Biliary atresia (BA) is a neonatal liver disease featuring cholestasis and severe liver fibrosis (LF). Despite advances in the development of surgical treatment, lacking an early diagnostic marker and intervention of LF invariably leads to death from end-stage liver disease in the early years of life. We previously reported that knockout of sphingosine 1-phosphate receptor 2 (S1PR2) protected mice from bile duct ligation (BDL)-induced cholangiocyte proliferation and LF. Our recent studies further showed that both hepatic and serum exosomal long noncoding RNA H19 (lncRNAH19) levels are correlated with cholestatic injury in multidrug resistance 2 knockout (Mdr2-/- ) mice. However, the role of lncRNAH19 in BA progression remains unclear. Here, we show that both hepatic and serum exosomal H19 levels are positively correlated with severity of fibrotic liver injuries in BA patients. H19 deficiency protects mice from BDL-induced cholangiocyte proliferation and LF by inhibiting bile-acid-induced expression and activation of S1PR2 and sphingosine kinase 2 (SphK2). Furthermore, H19 acts as a molecular sponge for members of the microRNA let-7 family, which results in up-regulation of high-mobility group AT-hook 2 (HMGA2), a known target of let-7 and enhancement of biliary proliferation. Conclusion: These results indicate that H19 plays a critical role in cholangiocyte proliferation and cholestatic liver injury in BA by regulating the S1PR2/SphK2 and let-7/HMGA2 axis. Serum exosomal H19 may represent a noninvasive diagnostic biomarker and potential therapeutic target for BA.

Asunto(s)

Conductos Biliares/citología , Atresia Biliar/complicaciones , Proliferación Celular , Colestasis/complicaciones , Células Epiteliales/fisiología , Cirrosis Hepática/etiología , ARN Largo no Codificante/fisiología , Animales , Atresia Biliar/metabolismo , Células Cultivadas , Colestasis/metabolismo , Exosomas/metabolismo , Femenino , Humanos , Lactante , Cirrosis Hepática/metabolismo , Masculino , Ratones , ARN Largo no Codificante/análisis , ARN Largo no Codificante/biosíntesis

RESUMEN

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.

Asunto(s)

Hepatopatías Alcohólicas/enzimología , Hígado/enzimología , Hígado/patología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Células Cultivadas , Femenino , Hepatocitos/enzimología , Humanos , Intestinos/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Esteroide Hidroxilasas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba

RESUMEN

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.

Asunto(s)

Colangitis Esclerosante/genética , Colestasis/patología , Exosomas/genética , Regulación de la Expresión Génica , Cirrosis Hepática/genética , ARN Largo no Codificante/genética , Animales , Proliferación Celular/genética , Células Cultivadas , Colangitis Esclerosante/patología , Colestasis/genética , Modelos Animales de Enfermedad , Citometría de Flujo , Células Estrelladas Hepáticas/patología , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Cirrosis Hepática/patología , Masculino , Ratones , Ratones Noqueados , Distribución Aleatoria , Especificidad de la Especie

RESUMEN

Cholestatic liver injury is an important clinical problem with limited understanding of disease pathologies. Exosomes are small extracellular vesicles released by a variety of cells, including cholangiocytes. Exosome-mediated cell-cell communication can modulate various cellular functions by transferring a variety of intracellular components to target cells. Our recent studies indicate that the long noncoding RNA (lncRNA), H19, is mainly expressed in cholangiocytes, and its aberrant expression is associated with significant down-regulation of small heterodimer partner (SHP) in hepatocytes and cholestatic liver injury in multidrug resistance 2 knockout (Mdr2-/- ) mice. However, how cholangiocyte-derived H19 suppresses SHP in hepatocytes remains unknown. Here, we report that cholangiocyte-derived exosomes mediate transfer of H19 into hepatocytes and promote cholestatic injury. Hepatic H19 level is correlated with severity of cholestatic injury in both fibrotic mouse models, including Mdr2-/- mice, a well-characterized model of primary sclerosing cholangitis (PSC), or CCl4 -induced cholestatic liver injury mouse models, and human PSC patients. Moreover, serum exosomal-H19 level is gradually up-regulated during disease progression in Mdr2-/- mice and patients with cirrhosis. H19-carrying exosomes from the primary cholangiocytes of wild-type (WT) mice suppress SHP expression in hepatocytes, but not the exosomes from the cholangiocytes of H19-/- mice. Furthermore, overexpression of H19 significantly suppressed SHP expression at both transcriptional and posttranscriptional levels. Importantly, transplant of H19-carrying serum exosomes of old fibrotic Mdr2-/- mice significantly promoted liver fibrosis (LF) in young Mdr2-/- mice. CONCLUSION: Cholangiocyte-derived exosomal-H19 plays a critical role in cholestatic liver injury. Serum exosomal H19 represents a noninvasive biomarker and potential therapeutic target for cholestatic diseases. (Hepatology 2018).

Asunto(s)

Conductos Biliares/patología , Colestasis/genética , Hígado/patología , ARN Largo no Codificante/metabolismo , Animales , Conductos Biliares/metabolismo , Colangitis Esclerosante/patología , Colestasis/patología , Exosomas/genética , Femenino , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Receptores Citoplasmáticos y Nucleares/metabolismo

RESUMEN

The multidrug resistance 2 knockout (Mdr2-/- ) mouse is a well-established model of cholestatic cholangiopathies. Female Mdr2-/- mice develop more severe hepatobiliary damage than male Mdr2-/- mice, which is correlated with a higher proportion of taurocholate in the bile. Although estrogen has been identified as an important player in intrahepatic cholestasis, the underlying molecular mechanisms of gender-based disparity of cholestatic injury remain unclear. The long noncoding RNA H19 is an imprinted, maternally expressed, and estrogen-targeted gene, which is significantly induced in human fibrotic/cirrhotic liver and bile duct-ligated mouse liver. However, whether aberrant expression of H19 accounts for gender-based disparity of cholestatic injury in Mdr2-/- mice remains unknown. The current study demonstrated that H19 was markedly induced (∼200-fold) in the livers of female Mdr2-/- mice at advanced stages of cholestasis (100 days old) but not in age-matched male Mdr2-/- mice. During the early stages of cholestasis, H19 expression was minimal. We further determined that hepatic H19 was mainly expressed in cholangiocytes, not hepatocytes. Both taurocholate and estrogen significantly activated the extracellular signal-regulated kinase 1/2 signaling pathway and induced H19 expression in cholangiocytes. Knocking down H19 not only significantly reduced taurocholate/estrogen-induced expression of fibrotic genes and sphingosine 1-phosphate receptor 2 in cholangiocytes but also markedly reduced cholestatic injury in female Mdr2-/- mice. Furthermore, expression of small heterodimer partner was substantially inhibited at advanced stages of liver fibrosis, which was reversed by H19 short hairpin RNA in female Mdr2-/- mice. Similar findings were obtained in human primary sclerosing cholangitis liver samples. CONCLUSION: H19 plays a critical role in the disease progression of cholestasis and represents a key factor that causes the gender disparity of cholestatic liver injury in Mdr2-/- mice. (Hepatology 2017;66:869-884).

Asunto(s)

Colangitis Esclerosante/patología , Colestasis/patología , Regulación de la Expresión Génica , ARN Largo no Codificante/genética , Receptores de Lisoesfingolípidos/genética , Animales , Colangitis Esclerosante/genética , Colestasis/genética , Modelos Animales de Enfermedad , Regulación hacia Abajo , Estrógenos/farmacología , Femenino , Técnicas de Inactivación de Genes , Genes MDR , Humanos , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Masculino , Ratones , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Rol , Factores Sexuales , Receptores de Esfingosina-1-Fosfato , Ácido Taurocólico/farmacología

RESUMEN

Bile duct obstruction is a potent stimulus for cholangiocyte proliferation, especially for large cholangiocytes. Our previous studies reported that conjugated bile acids (CBAs) activate the protein kinase B (AKT) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathways through sphingosine 1-phosphate receptor (S1PR) 2 in hepatocytes and cholangiocarcinoma cells. It also has been reported that taurocholate (TCA) promotes large cholangiocyte proliferation and protects cholangiocytes from bile duct ligation (BDL)-induced apoptosis. However, the role of S1PR2 in bile-acid-mediated cholangiocyte proliferation and cholestatic liver injury has not been elucidated. Here, we report that S1PR2 is the predominant S1PR expressed in cholangiocytes. Both TCA- and sphingosine-1-phosphate (S1P)-induced activation of ERK1/2 and AKT were inhibited by JTE-013, a specific antagonist of S1PR2, in cholangiocytes. In addition, TCA- and S1P-induced cell proliferation and migration were inhibited by JTE-013 and a specific short hairpin RNA of S1PR2, as well as chemical inhibitors of ERK1/2 and AKT in mouse cholangiocytes. In BDL mice, expression of S1PR2 was up-regulated in whole liver and cholangiocytes. S1PR2 deficiency significantly reduced BDL-induced cholangiocyte proliferation and cholestatic injury, as indicated by significant reductions in inflammation and liver fibrosis in S1PR2 knockout mice. Treatment of BDL mice with JTE-013 significantly reduced total bile acid levels in serum and cholestatic liver injury. CONCLUSION: This study suggests that CBA-induced activation of S1PR2-mediated signaling pathways plays a critical role in obstructive cholestasis and may represent a novel therapeutic target for cholestatic liver diseases. (Hepatology 2017;65:2005-2018).

Asunto(s)

Colangiocarcinoma/patología , Colangitis Esclerosante/patología , Colestasis/patología , Cirrosis Hepática/patología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Análisis de Varianza , Animales , Ácidos y Sales Biliares/farmacología , Neoplasias de los Conductos Biliares/metabolismo , Neoplasias de los Conductos Biliares/patología , Conductos Biliares/cirugía , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Colangiocarcinoma/metabolismo , Colangitis Esclerosante/metabolismo , Colestasis/complicaciones , Modelos Animales de Enfermedad , Ligadura , Hígado/lesiones , Hígado/patología , Cirrosis Hepática/metabolismo , Lisofosfolípidos/metabolismo , Masculino , Ratones , Ratones Endogámicos CBA , Ratones Noqueados , Distribución Aleatoria , Rol , Transducción de Señal , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato , Regulación hacia Arriba

RESUMEN

BACKGROUND: Apigenin is a non-toxic natural flavonoid that is abundantly present in common fruits and vegetables. It has been reported that apigenin has various beneficial health effects such as anti-inflammation and chemoprevention. Multiple studies have shown that inflammation is an important risk factor for atherosclerosis, diabetes, sepsis, various liver diseases, and other metabolic diseases. Although it has been long realized that apigenin has anti-inflammatory activities, the underlying functional mechanisms are still not fully understood. METHODOLOGY AND PRINCIPAL FINDINGS: In the present study, we examined the effect of apigenin on LPS-induced inflammatory response and further elucidated the potential underlying mechanisms in human THP-1-induced macrophages and mouse J774A.1 macrophages. By using the PrimePCR array, we were able to identify the major target genes regulated by apigenin in LPS-mediated immune response. The results indicated that apigenin significantly inhibited LPS-induced production of pro-inflammatory cytokines, such as IL-6, IL-1ß, and TNF-α through modulating multiple intracellular signaling pathways in macrophages. Apigenin inhibited LPS-induced IL-1ß production by inhibiting caspase-1 activation through the disruption of the NLRP3 inflammasome assembly. Apigenin also prevented LPS-induced IL-6 and IL-1ß production by reducing the mRNA stability via inhibiting ERK1/2 activation. In addition, apigenin significantly inhibited TNF-α and IL-1ß-induced activation of NF-κB. CONCLUSION AND SIGNIFICANCE: Apigenin Inhibits LPS-induced Inflammatory Response through multiple mechanisms in macrophages. These results provided important scientific evidences for the potential application of apigenin as a therapeutic agent for inflammatory diseases.

Asunto(s)

Antiinflamatorios/farmacología , Apigenina/farmacología , Inflamación/prevención & control , Lipopolisacáridos , Macrófagos/efectos de los fármacos , Animales , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Flavonoides/farmacología , Células HEK293 , Humanos , Inflamasomas/efectos de los fármacos , Inflamasomas/genética , Inflamasomas/metabolismo , Inflamación/inducido químicamente , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , FN-kappa B/genética , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética

RESUMEN

Alcohol abuse with/without cirrhosis is associated with an impaired gut barrier and inflammation. Gut microbiota can transform primary bile acids (BA) to secondary BAs, which can adversely impact the gut barrier. The purpose of this study was to define the effect of active alcohol intake on fecal BA levels and ileal and colonic inflammation in cirrhosis. Five age-matched groups {two noncirrhotic (control and drinkers) and three cirrhotic [nondrinkers/nonalcoholics (NAlc), abstinent alcoholic for >3 mo (AbsAlc), currently drinking (CurrAlc)]} were included. Fecal and serum BA analysis, serum endotoxin, and stool microbiota using pyrosequencing were performed. A subgroup of controls, NAlc, and CurrAlc underwent ileal and sigmoid colonic biopsies on which mRNA expression of TNF-α, IL-1ß, IL-6, and cyclooxygenase-2 (Cox-2) were performed. One hundred three patients (19 healthy, 6 noncirrhotic drinkers, 10 CurrAlc, 38 AbsAlc, and 30 NAlc, age 56 yr, median MELD: 10.5) were included. Five each of healthy, CurrAlc, and NAlc underwent ileal/colonic biopsies. Endotoxin, serum-conjugated DCA and stool total BAs, and secondary-to-primary BA ratios were highest in current drinkers. On biopsies, a significantly higher mRNA expression of TNF-α, IL-1ß, IL-6, and Cox-2 in colon but not ileum was seen in CurrAlc compared with NAlc and controls. Active alcohol use in cirrhosis is associated with a significant increase in the secondary BA formation compared with abstinent alcoholic cirrhotics and nonalcoholic cirrhotics. This increase in secondary BAs is associated with a significant increase in expression of inflammatory cytokines in colonic mucosa but not ileal mucosa, which may contribute to alcohol-induced gut barrier injury.

Asunto(s)

Alcoholismo/complicaciones , Ácidos y Sales Biliares/metabolismo , Enfermedades del Colon/inducido químicamente , Inflamación/etiología , Cirrosis Hepática/complicaciones , Ácidos y Sales Biliares/química , Enfermedades del Colon/patología , Heces/química , Humanos , Inflamación/patología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Persona de Mediana Edad

RESUMEN

BACKGROUND: HIV protease inhibitor (PI), the core component of highly active antiretroviral treatment (HAART) for HIV infection, has been implicated in HAART-associated cardiovascular complications. Our previous studies have demonstrated that activation of endoplasmic reticulum (ER) stress is linked to HIV PI-induced inflammation and foam cell formation in macrophages. Raltegravir is a first-in-its-class HIV integrase inhibitor, the newest class of anti-HIV agents. We have recently reported that raltegravir has less hepatic toxicity and could prevent HIV PI-induced dysregulation of hepatic lipid metabolism by inhibiting ER stress. However, little information is available as to whether raltegravir would also prevent HIV PI-induced inflammatory response and foam cell formation in macrophages. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, we examined the effect of raltegravir on ER stress activation and lipid accumulation in cultured mouse macrophages (J774A.1), primary mouse macrophages, and human THP-1-derived macrophages, and further determined whether the combination of raltegravir with existing HIV PIs would potentially exacerbate or prevent the previously observed activation of inflammatory response and foam cell formation. The results indicated that raltegravir did not induce ER stress and inflammatory response in macrophages. Even more interestingly, HIV PI-induced ER stress, oxidative stress, inflammatory response and foam cell formation were significantly reduced by raltegravir. High performance liquid chromatography (HPLC) analysis further demonstrated that raltegravir did not affect the uptake of HIV PIs in macrophages. CONCLUSION AND SIGNIFICANCE: Raltegravir could prevent HIV PI-induced inflammatory response and foam cell formation by inhibiting ER stress. These results suggest that incorporation of this HIV integrase inhibitor may reduce the cardiovascular complications associated with current HAART.

Asunto(s)

Estrés del Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , Inhibidores de la Proteasa del VIH/efectos adversos , Macrófagos/efectos de los fármacos , Raltegravir Potásico/uso terapéutico , Animales , Terapia Antirretroviral Altamente Activa/efectos adversos , Enfermedades Cardiovasculares/inducido químicamente , Enfermedades Cardiovasculares/prevención & control , Línea Celular , Cromatografía Líquida de Alta Presión , Citocinas/metabolismo , Retículo Endoplásmico/metabolismo , Ensayo de Inmunoadsorción Enzimática , Inhibidores de Integrasa VIH/efectos adversos , Inhibidores de Integrasa VIH/uso terapéutico , Inhibidores de la Proteasa del VIH/uso terapéutico , Homeostasis , Humanos , Inflamación/metabolismo , Interleucina-6/metabolismo , Macrófagos del Hígado/metabolismo , Metabolismo de los Lípidos , Lípidos/química , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Especies Reactivas de Oxígeno/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

UNLABELLED: Human immunodeficiency virus (HIV) protease inhibitors (HIV PIs) are the core components of highly active antiretroviral therapy, which has been successfully used in the treatment of HIV-1 infection in the past two decades. However, benefits of HIV PIs are compromised by clinically important adverse effects, such as dyslipidemia, insulin resistance, and cardiovascular complications. We have previously shown that activation of endoplasmic reticulum (ER) stress plays a critical role in HIV PI-induced dys-regulation of hepatic lipid metabolism. HIV PI-induced hepatic lipotoxicity is closely linked to the up-regulation of CCAAT enhancer binding protein homologous protein (CHOP) in hepatocytes. To further investigate whether CHOP is responsible for HIV PI-induced hepatic lipotoxicity, C57BL/6J wild-type (WT) or CHOP knockout (CHOP(-/-) ) mice or the corresponding primary mouse hepatocytes were used in this study. Both in vitro and in vivo studies indicated that HIV PIs (ritonavir and lopinavir) significantly increased hepatic lipid accumulation in WT mice. In contrast, CHOP(-/-) mice showed a significant reduction in hepatic triglyceride accumulation and liver injury, as evidenced by hematoxylin and eosin and Oil Red O staining. Real-time reverse-transcriptase polymerase chain reaction and immunoblotting data showed that in the absence of CHOP, HIV PI-induced expression of stress-related proteins and lipogenic genes were dramatically reduced. Furthermore, tumor necrosis factor alpha and interleukin-6 levels in serum and liver were significantly lower in HIV PI-treated CHOP(-/-) mice, compared to HIV PI-treated WT mice. CONCLUSION: Taken together, these data suggest that CHOP is an important molecular link of ER stress, inflammation, and hepatic lipotoxicity, and that increased expression of CHOP represents a critical factor underlying events leading to hepatic injury. (HEPATOLOGY 2013).

Asunto(s)

Terapia Antirretroviral Altamente Activa/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , Inhibidores de la Proteasa del VIH/efectos adversos , Factor de Transcripción CHOP/genética , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/fisiología , Femenino , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Hepatocitos/efectos de los fármacos , Hepatocitos/fisiología , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Transcripción CHOP/metabolismo

RESUMEN

Hyperlipidemia associated with the HIV protease inhibitor (PI), the major component of highly active antiretroviral treatment (HAART) for HIV infection, has stimulated interest in developing new agents that minimize these side effects in the clinic. HIV integrase inhibitor is a new class of anti-HIV agents. Raltegravir is a first-in-its-class oral integrase inhibitor and has potent inhibitory activity against HIV-1 strains that are resistant to other antiretroviral regimens. Our previous studies have demonstrated that HIV PI-induced endoplasmic reticulum (ER) stress links to dysregulation of lipid metabolism. However, little information is available as to whether raltegravir would have similar effects as the HIV PIs. In this study, we examined the effect of raltegravir on lipid metabolism both in primary rat hepatocytes and in in vivo mouse models, and we further determined whether the combination of raltegravir with existing HIV PIs would potentially exacerbate or prevent the previously observed development of dyslipidemia. The results indicated that raltegravir did not induce ER stress or disrupt lipid metabolism either in vitro or in vivo. However, HIV PI-induced ER stress and lipid accumulation were significantly inhibited by raltegravir both in in vitro primary rat hepatocytes and in in vivo mouse liver. High-performance liquid chromatography analysis further demonstrated that raltegravir did not affect the uptake and metabolism of HIV PIs in hepatocytes. Thus, raltegravir has less hepatic toxicity and could prevent HIV PI-induced dysregulation of lipid metabolism by inhibiting ER stress. These results suggest that incorporation of this HIV integrase inhibitor may reduce the side effects associated with current HAART.

Asunto(s)

Retículo Endoplásmico/fisiología , Inhibidores de Integrasa VIH/farmacología , Inhibidores de la Proteasa del VIH/efectos adversos , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/efectos de los fármacos , Pirrolidinonas/farmacología , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Antagonismo de Drogas , Inhibidores de Integrasa VIH/efectos adversos , Inhibidores de Integrasa VIH/farmacocinética , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Hiperlipidemias/metabolismo , Hiperlipidemias/patología , Hiperlipidemias/prevención & control , Metabolismo de los Lípidos/genética , Hígado/metabolismo , Lopinavir , Masculino , Ratones , Ratones Endogámicos C57BL , Pirimidinonas/efectos adversos , Pirrolidinonas/efectos adversos , Pirrolidinonas/farmacocinética , Raltegravir Potásico , Ratas , Ritonavir/efectos adversos , Transducción de Señal

RESUMEN

HIV protease inhibitor (PI)-associated cardiovascular risk, especially atherosclerosis, has become a major concern in the clinic. Macrophages are key players in the inflammatory response and atherosclerosis formation. We have previously shown that HIV PIs induce endoplasmic reticulum (ER) stress, activate the unfolded protein response (UPR), and increase the synthesis of the inflammatory cytokines, TNF-alpha and IL-6, by regulating the intracellular translocation of RNA binding protein HuR in macrophages. However, the underlying signaling mechanisms remain unclear. We show here that the HIV PI lopinavir significantly activated the extracellular-signal regulated protein kinase (ERK), but not c-Jun N-terminal kinase (JNK) and p38 MAPK. Lopinavir-induced cytosolic translocation of HuR and TNF-alpha and IL-6 synthesis was attenuated by specific chemical inhibitor of MEK (PD98058) or over-expression of dominant negative mutant of MEK1. In addition, we demonstrated that lopinavir-induced ERK activation and TNF-alpha and IL-6 expression were completely inhibited in macrophages from CHOP null mice. Taken together, these results indicate activation of the UPR plays an essential role in HIV PI-induced inflammatory cytokine synthesis and release by activating ERK, which increases the cytosolic translocation of HuR and subsequent binding to the 3'UTR of TNF-alpha and IL-6 mRNAs in macrophages.

Asunto(s)

Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Inhibidores de la Proteasa del VIH/farmacología , Interleucina-6/genética , Macrófagos/fisiología , Pirimidinonas/farmacología , Factor de Necrosis Tumoral alfa/genética , Animales , Línea Celular Tumoral , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Leucemia Monocítica Aguda , Lopinavir , Sistema de Señalización de MAP Quinasas , Macrófagos/efectos de los fármacos , Ratones , Ratones Noqueados , FN-kappa B/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Desnaturalización Proteica , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología

RESUMEN

Curcumin (diferuloylmethane) is an orange-yellow compound from turmeric (Curcuma longa), a spice found in curry powder. Traditionally known for its anti-inflammatory effects, curcumin has established itself in the last two decades to be a potent immunomodulatory agent that can regulate the activation of a variety of immunocytes and the expression of inflammatory factors. Considering that the beta-diketone moiety of curcumin may result in its instability and poor metabolic property, we previously designed a series of mono-carbonyl analogues of curcumin with enhanced stability by deleting this moiety. These compounds demonstrate improved pharmacokinetic profiles both in vitro and in vivo. In this study, we reported a total of 44 mono-carbonyl analogues, which have been evaluated for the inhibitory activities against LPS-induced TNF-alpha and IL-6 release in the macrophages. Based on the screening results of these analogues, five active compounds A01, A03, A13, B18 and C22 were investigated to inhibit TNF-alpha and IL-6 release in a dose-dependent manner, three of which further demonstrated inhibitory effects on LPS-induced TNF-alpha, IL-1beta, IL-6, MCP-1, COX-2, PGES, iNOS and p65 NF-kappaB mRNA production. The results indicated that these mono-carbonyl analogues may possess anti-inflammatory activities similar to curcumin despite the absence of the beta-diketone. These mono-carbonyl analogues may be a favourable alternative for the development of curcumin-based anti-inflammatory drugs both pharmacokinetically and pharmacologically. We further examined the biological properties of A13, the only hydrosoluble analogue when combined with hydrochloric acid. The results showed a dose-dependent inhibition of LPS-induced cytokine production. These data further indicated that compound A13 may be explored as a promising anti-inflammatory molecule.

Asunto(s)

Carbono/química , Curcumina/química , Lipopolisacáridos/metabolismo , Animales , Curcuma , Inflamación , Interleucina-6/metabolismo , Cetonas/metabolismo , Macrófagos/metabolismo , Ratones , Modelos Químicos , Extractos Vegetales/farmacología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

HIV protease inhibitors (PIs) have been associated with the serious Metabolic Syndrome, which is the major risk factor of atherosclerotic cardiovascular disease. Atherosclerosis is widely considered to be a chronic inflammatory disease. Macrophages are the most prominent cell type present in atherosclerotic lesions and play essential roles in both early lesion development and late lesion complications. We previously reported that HIV PIs induced accumulation of intracellular free cholesterol and lipids, decreased endoplasmic reticulum (ER) calcium stores, activated the unfolded protein response (UPR), significantly increased apoptosis, and promoted foam cell formation in macrophages. HIV PI-induced ER stress and subsequent activation of the UPR, represents an important cell signaling mechanism of HIV PI-induced metabolic syndromes. Here we show that all HIV PIs, except amprenavir, increased expression of the major mediators of inflammatory response, TNF-alpha and IL-6, to varying degrees. Furthermore, we show that the RNA-binding protein, HuR, plays an important role in HIV PI-induced expression of TNF-alpha and IL-6. Atazanavir increased the cytoplasmic levels of HuR and enhanced the binding of HuR to 3'-UTR of TNF-alpha and IL-6. Down regulation of HuR expression by siRNA prevented atazanavir-induced increase of TNF-alpha and IL-6. These results suggest that HuR might have an impact on pathophysiological processes of HIV PI-induced atherosclerosis.

Asunto(s)

Antígenos de Superficie/metabolismo , Regulación de la Expresión Génica , Inhibidores de la Proteasa del VIH/farmacología , Interleucina-6/biosíntesis , Macrófagos/metabolismo , Proteínas de Unión al ARN/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Sulfato de Atazanavir , Aterosclerosis/patología , Proteínas ELAV , Proteína 1 Similar a ELAV , Retículo Endoplásmico/metabolismo , Humanos , Interleucina-6/metabolismo , Lentivirus/metabolismo , Ratones , Oligopéptidos/farmacología , Piridinas/farmacología , Factores de Riesgo , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Treatment of human immunodeficiency virus (HIV)-infected patients with HIV protease inhibitors (PIs) has been associated with serious lipid disturbances. However, the incidence and degree of impaired lipid metabolism observed in the clinic vary considerably between individual HIV PIs. Our previous studies demonstrated that HIV PIs differ in their ability to increase the levels of transcriptionally active sterol regulatory element-binding proteins (SREBPs), activate the unfolded protein response (UPR), induce apoptosis, and promote foam cell formation in macrophages. In the present study, we examined the effects of three HIV PIs, including amprenavir, atazanavir, and ritonavir, on the UPR activation and the expression of key genes involved in lipid metabolism in primary rodent hepatocytes. Both atazanavir and ritonavir activated the UPR, induced apoptosis, and increased nuclear SREBP levels, but amprenavir had no significant effect at the same concentrations. In rat primary hepatocytes, cholesterol 7alpha-hydroxylase (CYP7A1) mRNA levels were significantly decreased by atazanavir (38%) and ritonavir (56%) but increased by amprenavir (90%); 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase mRNA levels were increased by amprenavir (23%) but not by ritonavir and atazanavir; low-density lipoprotein receptor mRNA was increased by atazanavir (20%) but not by amprenavir and ritonavir. Similar results were obtained in mouse primary hepatocytes. Atazanavir and ritonavir also decreased CYP7A1 protein levels and bile acid biosynthesis, while amprenavir had no significant effect. The current results may help provide a better understanding of the cellular mechanisms of HIV PI-induced dyslipidemia and also provide useful information to help predict clinical adverse effects in the development of new HIV PIs.

Asunto(s)

Acilcoenzima A/metabolismo , Colesterol 7-alfa-Hidroxilasa/metabolismo , Inhibidores de la Proteasa del VIH/administración & dosificación , Hepatocitos/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Receptores de LDL/metabolismo , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Metabolismo de los Lípidos/fisiología , Masculino , Desnaturalización Proteica/efectos de los fármacos , Pliegue de Proteína , Ratas , Ratas Sprague-Dawley