RESUMEN
BACKGROUND AND AIMS: The study aimed to describe the clinical course and outcomes, and analyze the genotype-phenotype correlation in patients with tight junction protein 2 (TJP2) deficiency. APPROACH AND RESULTS: Data from all children with chronic cholestasis and either homozygous or compound heterozygous mutations in TJP2 were extracted and analyzed. The patients were categorized into 3 genotypes: TJP2-A (missense mutations on both alleles), TJP2-B (missense mutation on one allele and a predicted protein-truncating mutation [PPTM] on the other), and TJP2-C (PPTMs on both alleles). A total of 278 cases of genetic intrahepatic cholestasis were studied, with TJP2 deficiency accounting for 44 cases (15.8%). Of these, 29 were homozygous and 15 were compound heterozygous variants of TJP2 . TJP2-A genotype was identified in 21 (47.7%), TJP2-B in 7 cases (15.9%), and TJP2-C in 16 cases (36.4%), respectively. Patients with the TJP2-C genotype were more likely to experience early infantile cholestasis (87.5% vs. 53.5%, p =0.033), less likely to clear jaundice (12.5% vs. 52.2%, p =0.037), more likely to develop ascites, and had higher serum bile acids. Patients with the TJP2-C genotype were more likely to die or require liver transplantation (native liver survival: 12.5% vs. 78.6%, p <0.001), with a median age at death/liver transplantation of 2.5 years. Cox regression analysis revealed that TJP2-C mutations ( p =0.003) and failure to resolve jaundice ( p =0.049) were independent predictors of poor outcomes. CONCLUSIONS: Patients with the TJP2-C genotype carrying PPTMs in both alleles had a rapidly progressive course, leading to early decompensation and death if they did not receive timely liver transplantation.
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Colestasis Intrahepática , Genotipo , Proteína de la Zonula Occludens-2 , Humanos , Proteína de la Zonula Occludens-2/genética , Masculino , Femenino , Lactante , Colestasis Intrahepática/genética , Preescolar , Niño , Trasplante de Hígado , Mutación , Estudios de Asociación GenéticaRESUMEN
There are no published data on the use of odevixibat, a selective ileal bile acid transporter (IBAT) inhibitor, in children with tight junction protein 2 (TJP2) deficiency (also named as PFIC-4). We describe a case series of five children treated with odevixibat. After treatment, serum bile acids (sBA) decreased compared to baseline [mean value: 244 (±125), vs 38 (±34) µmol/L; p = 0.007]; reduction in sBA was >70% from baseline (or <70 µmol/L) in all. Improvements in pruritus were reported in all patients. The drug was well tolerated. IBAT inhibitors should be considered a valuable treatment option in patients with TJP2 deficiency.
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Proteínas Portadoras , Colestasis Intrahepática , Niño , Humanos , Glicoproteínas de Membrana , Benzodiazepinas , Ácidos y Sales Biliares , Proteína de la Zonula Occludens-2/metabolismoRESUMEN
Zonula occludens-1 (ZO-1) is a scaffolding protein of tight junctions, which seal adjacent epithelial cells, that is also expressed in adherens junctions. The distribution pattern of ZO-1 differs among stratified squamous epithelia, including that between skin and oral buccal mucosa. However, the causes for this difference, and the mechanisms underlying ZO-1 spatial regulation, have yet to be elucidated. In this study, we showed that epithelial turnover and proliferation are associated with ZO-1 distribution in squamous epithelia. We tried to verify the regulation of ZO-1 by comparing normal skin and psoriasis, known as inflammatory skin disease with rapid turnover. We as well compared buccal mucosa and oral lichen planus, known as an inflammatory oral disease with a longer turnover interval. The imiquimod (IMQ) mouse model, often used as a psoriasis model, can promote cell proliferation. On the contrary, we peritoneally injected mice mitomycin C, which reduces cell proliferation. We examined whether IMQ and mitomycin C cause changes in the distribution and appearance of ZO-1. Human samples and mouse pharmacological models revealed that slower epithelial turnover/proliferation led to the confinement of ZO-1 to the uppermost part of squamous epithelia. In contrast, ZO-1 was widely distributed under conditions of faster cell turnover/proliferation. Cell culture experiments and mathematical modelling corroborated these ZO-1 distribution patterns. These findings demonstrate that ZO-1 distribution is affected by epithelial cell dynamics.
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Carcinoma de Células Escamosas , Psoriasis , Ratones , Animales , Humanos , Uniones Estrechas/metabolismo , Mitomicina/metabolismo , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-2/metabolismo , Proliferación Celular , Carcinoma de Células Escamosas/metabolismoRESUMEN
Recent evidences have suggested that Zinc finger protein 582 (ZNF582) plays different important roles in various tumors, but its clinical role, biological function and regulatory mechanism in clear cell renal cell carcinoma (ccRCC) are still vague. Through analyzing GEO and TCGA-KIRC data and validation with local samples, we identified the low expression pattern of ZNF582 in ccRCC. Decreased ZNF582 expression is correlated with higher tumor stage and grade, distant metastasis and poor prognosis. By analyzing the DNA methylation data of ccRCC in TCGA-KIRC and using Massarray DNA methylation and demethylation analysis, we confirmed the hypermethylation status of ZNF582 in ccRCC and its negative regulation on ZNF582 expression. Using cell phenotype experiments and orthotopic kidney tumor growth models, we determined the inhibitory effect of ZNF582 overexpression on ccRCC growth and metastasis in vivo and in vitro. Mechanistically, using TMT (Tandem mass tags) quantitative proteomics test, Co-IP (Co-immunoprecipitation) and Western Blot experiments, we clarified that ZNF582 binds to TJP2 and up-regulates TJP2 protein expression. Increased TJP2 protein combines with ERK2 to promote ERK2 protein expression and suppresses the phosphorylation of ERK2, thereby inhibiting the growth and metastasis of ccRCC. In general, our findings provide the first solid theoretical rationale for targeting ZNF582/TJP2/ERK2 axis to improve ccRCC treatment.
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Carcinoma de Células Renales , Neoplasias Renales , Humanos , Carcinoma de Células Renales/metabolismo , Fosforilación , Neoplasias Renales/metabolismo , Riñón/patología , Proteínas/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Proteína de la Zonula Occludens-2/genética , Proteína de la Zonula Occludens-2/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismoRESUMEN
Tight junctions (TJs) regulate the transit of ions and molecules through the paracellular pathway in epithelial cells. Zonula occludens 2 (ZO-2) is a cytoplasmic TJ protein. Here, we studied the ubiquitination of hZO-2 employing mutants of SUMOylation site K730 present in the GuK domain and the putative ubiquitination residues K759 and K992 located at the GuK domain and proline-rich region, respectively. In immunoprecipitation experiments done with MDCK cells transfected with wild-type (WT) hZO-2 or the ubiquitination-site mutants hZO-2-K759R or -K992R, we observed diminished ubiquitination of the mutants, indicating that residues K759 and K992 in hZO-2 are acceptors for ubiquitination. Moreover, using TUBES, we found that residues K759 and K992 of hZO-2 are targets of K48 polyubiquitination, a signal for proteasomal degradation. Accordingly, compared to WT hZO-2, the half-life of hZO-2 mutants K759R and K992R augmented from 19.9 to 37.3 and 23.3 h, respectively. Instead, the ubiquitination of hZO-2 mutant K730R increased, and its half-life diminished to 6.7 h. The lack of these lysine residues in hZO-2 affects TJ sealing as the peak of TER decreased in monolayers of MDCK cells transfected with any of these mutants. These results highlight the importance of ZO-2 ubiquitination and SUMOylation to maintain a healthy and stable pool of ZO-2 molecules at the TJ.
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Sumoilación , Uniones Estrechas , Proteína de la Zonula Occludens-2/metabolismo , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-1/metabolismo , Lisina/metabolismo , Fosfoproteínas/metabolismo , Línea Celular , Prolina/metabolismoRESUMEN
ZO-2 is a peripheral tight junction (TJ) protein whose silencing in renal epithelia induces cell hypertrophy. Here, we found that in ZO-2 KD MDCK cells, in compensatory renal hypertrophy triggered in rats by a unilateral nephrectomy and in liver steatosis of obese Zucker (OZ) rats, ZO-2 silencing is accompanied by the diminished activity of LATS, a kinase of the Hippo pathway, and the nuclear concentration of YAP, the final effector of this signaling route. ZO-2 appears to function as a scaffold for the Hippo pathway as it associates to LATS1. ZO-2 silencing in hypertrophic tissue is due to a diminished abundance of ZO-2 mRNA, and the Sp1 transcription factor is critical for ZO-2 transcription in renal cells. Treatment of OZ rats with metformin, an activator of AMPK that blocks JNK activity, augments ZO-2 and claudin-1 expression in the liver, reduces the paracellular permeability of hepatocytes, and serum bile acid content. Our results suggest that ZO-2 silencing is a common feature of hypertrophy, and that ZO-2 is a positive regulator of the Hippo pathway that regulates cell size. Moreover, our observations highlight the importance of AMPK, JNK, and ZO-2 as therapeutic targets for blood-bile barrier dysfunction.
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Proteínas Quinasas Activadas por AMP , Hígado Graso , Proteína de la Zonula Occludens-2/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Vía de Señalización Hippo , Hipertrofia , Ratas , Ratas Zucker , Proteínas de Uniones EstrechasRESUMEN
MAGUK protein ZO-2 is present at tight junctions (TJs) and nuclei. In MDCK ZO-2 knockdown (KD) cells, nuclei exhibit an irregular shape with lobules and indentations. This condition correlates with an increase in DNA double strand breaks, however cells are not senescent and instead become resistant to UV-induced senescence. The irregular nuclear shape is also observed in isolated cells and in those without TJs, due to the lack of extracellular calcium. The aberrant nuclear shape of ZO-2 KD cells is not accompanied by a reduced expression of lamins A/C and B and lamin B receptors. Instead, it involves a decrease in constitutive and facultative heterochromatin, and microtubule instability that is restored with docetaxel. ZO-2 KD cells over-express SUN-1 that crosses the inner nuclear membrane and connects the nucleoskeleton of lamin A to nesprins, which traverse the outer nuclear membrane. Nesprins-3 and -4 that indirectly bind on their cytoplasmic face to vimentin and microtubules, respectively, are also over-expressed in ZO-2 KD cells, whereas vimentin is depleted. SUN-1 and lamin B1 co-immunoprecipitate with ZO-2, and SUN-1 associates to ZO-2 in a pull-down assay. Our results suggest that ZO-2 forms a complex with SUN-1 and lamin B1 at the inner nuclear membrane, and that ZO-2 and cell-cell contacts are required for a normal nuclear shape.
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Comunicación Celular/inmunología , Epitelio/metabolismo , Proteína de la Zonula Occludens-2/metabolismo , Humanos , TransfecciónRESUMEN
Claudin-2 is a major component of tight junctions (TJs), which play an important role in reovirus entry into host cells. The Bombyx mori cytoplasmic polyhedosis virus (BmCPV) relates to the cypovirus strain of the reovirus family. So far, the role of claudin-2 in the process of BmCPV infection is not known. In the present study, it was observed that increasing expression of the claudin-2 gene (CLDN2) may concomitantly elevate BmCPV infection. Contrarily, knockdown of CLDN2 expression by siRNAs can reduce BmCPV infection. Similarly, antibody-based blockage of claudin-2 could also decrease BmCPV cell entry. These results suggest that claudin-2 can promote BmCPV infection in vitro. Moreover, immunofluorescence (IF) assays showed that claudin-2 can interact with BmCPV during viral infection. Specifically, co-immunoprecipitation experiments indicated that claudin-2 binds the BmCPV VP7 (instead of VP3 proteins). The interaction between VP7 and claudin-2 was further confirmed by bimolecular fluorescence complementation (BIFC). Altogether, our results suggest that BmCPV cell entry can be promoted upon interaction of VP7 with claudin-2. These findings provide new mechanistic insights related to BmCPV infection. KEY POINTS: â¢Claudin-2 could promote BmCPV infection of cells. â¢Claudin-2 interacted with BmCPV during BmCPV infection. â¢Claudin-2 could interact with BmCPV VP7 protein, but not with VP3 proteins.
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Bombyx , Reoviridae , Animales , Claudina-2 , Claudinas/genética , Interacciones Huésped-Patógeno , Proteínas de Insectos , Internalización del Virus , Proteína de la Zonula Occludens-2RESUMEN
The epithelial cell tight junction structure is the site of the transepithelial movement of solutes and water between epithelial cells (paracellular permeability). Paracellular permeability can be divided into two distinct pathways, the Pore Pathway mediating the movement of small ions and solutes and the Leak Pathway mediating the movement of large solutes. Claudin proteins form the basic paracellular permeability barrier and mediate the movement of small ions and solutes via the Pore Pathway. The Leak Pathway remains less understood. Several proteins have been implicated in mediating the Leak Pathway, including occludin, ZO proteins, tricellulin, and actin filaments, but the proteins comprising the Leak Pathway remain unresolved. Many aspects of the Leak Pathway, such as its molecular mechanism, its properties, and its regulation, remain controversial. In this review, we provide a historical background to the evolution of the Leak Pathway concept from the initial examinations of paracellular permeability. We then discuss current information about the properties of the Leak Pathway and present current theories for the Leak Pathway. Finally, we discuss some recent research suggesting a possible molecular basis for the Leak Pathway.
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Células Epiteliales/metabolismo , Uniones Estrechas/metabolismo , Animales , Claudinas/metabolismo , Células Epiteliales/fisiología , Humanos , Ocludina/metabolismo , Permeabilidad , Uniones Estrechas/fisiología , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-2/metabolismoRESUMEN
PROBLEM: This study aims to investigate the effects of alpha-linolenic acid (ALA) on the gut microbiota (GM) and the abdominal environment in mice with endometriosis (EMS). METHODS: The effects of faecal microbiota transplantation (FMT) from EMS mice on mice treated with antibiotic cocktail were conducted. The 16S rRNA sequencing and PICRUSt software were used to detect the structure and function of GM respectively. The protein levels of Claudin 4 and ZO-2 in the intestinal wall were detected using the western blotting. The level of LPS in the abdominal cavity was detected using enzyme-linked immunosorbent assay (ELISA). The content of macrophages in the abdominal cavity was detected using flow cytometry. RESULTS: The exogenous supplementation of ALA could restore the abundance of Firmicutes and Bacteroidota in EMS mice. After the ALA treatment, the abundance of 125 functional pathways and 50 abnormal enzymes related to GM in EMS mice was significantly improved (p < .05). The expression of the ZO-2 protein in the intestinal wall was decreased, and the level of LPS in the abdominal cavity was significantly increased after FMT from EMS mice (p < .05). ALA could increase the expression of the ZO-2 protein in the intestinal wall of EMS mice, reduce the level of LPS in the abdominal cavity (p < .05) and reduce the aggregation of peritoneal macrophages (p < .05). CONCLUSION: Alpha-linolenic acid can improve the GM, intestinal wall barrier and abdominal inflammatory environment and reduce the level of LPS in mice with EMS.
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Endometriosis/metabolismo , Microbioma Gastrointestinal/efectos de los fármacos , Ácido alfa-Linolénico/farmacología , Animales , Modelos Animales de Enfermedad , Femenino , Ratones , Proteína de la Zonula Occludens-2/metabolismoRESUMEN
The presence of tight junction protein zonula occludens 2 (ZO-2) at the nucleus inhibits the transcription of genes regulated by TEAD transcription factor. Here, we analyzed whether the movement of ZO-2 into the nucleus modulates the nuclear concentration of TEAD. In sparse cultures of ZO-2 knockdown Madin-Darby canine kidney cells, nuclear TEAD was diminished, as in parental cells transfected with a ZO-2 construct without nuclear localization signals, indicating that ZO-2 facilitates the entry of TEAD into the nucleus. Inhibition of nPKCδ in parental cells triggers the interaction between ZO-2 and TEAD at the cytoplasm and facilitates TEAD/ZO-2 complex nuclear importation. Using proximity ligation, immunoprecipitation, and pull-down assays, TEAD/ZO-2 interaction was confirmed. Nuclear TEAD is phosphorylated, and its exit in parental cells is enhanced by activation of a ZO-2 nuclear exportation signal by nPKCε, while the nuclear accumulation of ZO-2 triggered by the mutation of ZO-2 nuclear export signals induces no change in TEAD nuclear concentration. In summary, our results indicate that the movements of ZO-2 in and out of the nucleus modulate the intracellular traffic of TEAD through a process regulated by nPKCδ and ε and provide a novel role of ZO-2 as a nuclear translocator of TEAD.
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Núcleo Celular/metabolismo , Células Epiteliales/metabolismo , Factores de Transcripción de Dominio TEA/metabolismo , Proteína de la Zonula Occludens-2/metabolismo , Animales , Línea Celular , Perros , Células HEK293 , Humanos , Señales de Localización Nuclear , Fosforilación , Unión Proteica , Proteína Quinasa C-epsilon/metabolismo , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Ratas , Transducción de SeñalRESUMEN
Constriction of the apical plasma membrane is a hallmark of epithelial cells that underlies cell shape changes in tissue morphogenesis and maintenance of tissue integrity in homeostasis. Contractile force is exerted by a cortical actomyosin network that is anchored to the plasma membrane by the apical junctional complexes (AJC). In this study, we present evidence that MAGI proteins, structural components of AJC whose function remained unclear, regulate apical constriction of epithelial cells through the Par polarity proteins. We reveal that MAGIs are required to uniformly distribute Partitioning defective-3 (Par-3) at AJC of cells throughout the epithelial monolayer. MAGIs recruit ankyrin-repeat-, SH3-domain- and proline-rich-region-containing protein 2 (ASPP2) to AJC, which modulates Par-3-aPKC to antagonize ROCK-driven contractility. By coupling the adhesion machinery to the polarity proteins to regulate cellular contractility, we propose that MAGIs play essential and central roles in maintaining steady state intercellular tension throughout the epithelial cell sheet.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Moléculas de Adhesión Celular/metabolismo , Polaridad Celular , Forma de la Célula , Células Epiteliales/enzimología , Guanilato-Quinasas/metabolismo , Uniones Intercelulares/enzimología , Proteína Quinasa C/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Moléculas de Adhesión Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Guanilato-Quinasas/genética , Células HEK293 , Homeostasis , Humanos , Uniones Intercelulares/genética , Transducción de Señal , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-2/genética , Proteína de la Zonula Occludens-2/metabolismo , Quinasas Asociadas a rho/metabolismoRESUMEN
BACKGROUND & AIMS: Liver tight junctions (TJs) establish tissue barriers that isolate bile from the blood circulation. TJP2/ZO-2-inactivating mutations cause progressive cholestatic liver disease in humans. Because the underlying mechanisms remain elusive, we characterized mice with liver-specific inactivation of Tjp2. METHODS: Tjp2 was deleted in hepatocytes, cholangiocytes, or both. Effects on the liver were assessed by biochemical analyses of plasma, liver, and bile and by electron microscopy, histology, and immunostaining. TJ barrier permeability was evaluated using fluorescein isothiocyanate-dextran (4 kDa). Cholic acid (CA) diet was used to assess susceptibility to liver injury. RESULTS: Liver-specific deletion of Tjp2 resulted in lower Cldn1 protein levels, minor changes to the TJ, dilated canaliculi, lower microvilli density, and aberrant radixin and bile salt export pump (BSEP) distribution, without an overt increase in TJ permeability. Hepatic Tjp2-defcient mice presented with mild progressive cholestasis with lower expression levels of bile acid transporter Abcb11/Bsep and detoxification enzyme Cyp2b10. A CA diet tolerated by control mice caused severe cholestasis and liver necrosis in Tjp2-deficient animals. 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene ameliorated CA-induced injury by enhancing Cyp2b10 expression, and ursodeoxycholic acid provided partial improvement. Inactivating Tjp2 separately in hepatocytes or cholangiocytes showed only mild CA-induced liver injury. CONCLUSION: Tjp2 is required for normal cortical distribution of radixin, canalicular volume regulation, and microvilli density. Its inactivation deregulated expression of Cldn1 and key bile acid transporters and detoxification enzymes. The mice provide a novel animal model for cholestatic liver disease caused by TJP2-inactivating mutations in humans.
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Canalículos Biliares/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Colestasis/genética , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-2/genética , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/genética , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/metabolismo , Animales , Hidrocarburo de Aril Hidroxilasas/metabolismo , Ácidos y Sales Biliares/metabolismo , Canalículos Biliares/patología , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Colagogos y Coleréticos/uso terapéutico , Ácido Cólico , Claudina-1/metabolismo , Familia 2 del Citocromo P450/metabolismo , Proteínas del Citoesqueleto/metabolismo , Células Epiteliales , Femenino , Fibrosis , Predisposición Genética a la Enfermedad , Hepatocitos , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Mutación , Oxazoles/uso terapéutico , Permeabilidad , Factores Protectores , ARN Mensajero/metabolismo , Esteroide Hidroxilasas/metabolismo , Uniones Estrechas/ultraestructura , Ácido Ursodesoxicólico/uso terapéutico , Proteína de la Zonula Occludens-2/deficienciaRESUMEN
The hepatic barrier is indispensable for the physiological functions of the liver and is impaired under various pathological conditions. Tight junctions reportedly play a central role in hepatic barrier regulation; however, there is limited direct evidence supporting this observation, with few in vivo models or confirmations of the implicated molecular mechanisms presented to date. We inactivated the tight junction component gene, Tjp2/ZO-2, and the related molecule, Tjp1/ZO-1, in mouse livers. In humans, TJP2/ZO-2 mutations have been implicated in the development of human progressive familial intrahepatic cholestasis 4 (PFIC4). The mice deficient in either ZO-1 or ZO-2 in the liver did not exhibit major abnormalities. However, the ablation of both molecules impaired the molecular architecture as well as the structure and function of hepatocyte tight junctions, which disrupted the hepatic barrier and was lethal to the mice by 6â¯weeks of age. In mutant mice, bile canaliculus formation and cellular polarity were compromised; also, transporter expression and localization were deregulated. Moreover, typical hepatic zonation and bile duct formation were inhibited, and sinusoidal vessels were disorganized. These findings clarify the role of tight junctions and polarity in the hepatic barrier as well as the effect that their disruption has on liver tissue. The observations also suggest that liver-specific ZO-1-/- and ZO-2-/- mice could be used as models for PFIC4, and this will provide new insights into liver pathophysiology and clinical applications.
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Conductos Biliares/metabolismo , Colestasis Intrahepática/metabolismo , Hígado/metabolismo , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-2/metabolismo , Animales , Conductos Biliares/patología , Colestasis Intrahepática/genética , Colestasis Intrahepática/patología , Humanos , Hígado/patología , Ratones , Ratones Noqueados , Uniones Estrechas/genética , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-2/genéticaRESUMEN
A 15-year-old boy who had a history of on and off pruritus and jaundice since many years found to have a novel mutation in TJP2 gene. On examination, he had clubbing, splenomegaly, grade 3 oesophageal varices and short stature. Investigation revealed direct hyperbirubinemia with elevated liver enzymes with normal gamma-glutamyl transferase (GGT). Antinuclear antibody (ANA), smooth muscle antibody (SMA) anti-liver kidney microsomal (anti-LKM) and viral markers for hepatitis were negative. However, IgG was elevated and anti-smooth muscle antibody (ASMA) was weekly positive (1:20). He was also given a trial of steroid and azathioprine for 1 year on the basis of liver biopsy findings, raised IgG and positive ASMA but finding no improvement stopped. Genetic testing by next-generation sequencing found a novel compound heterozygous missense variation in exon 17 of the TJP2 gene confirming progressive familial intrahepatic cholestasis type 4 as the aetiology of cholestatic liver disease.
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Colestasis Intrahepática/diagnóstico , Colestasis Intrahepática/genética , Mutación/genética , Proteína de la Zonula Occludens-2/genética , Adolescente , Colestasis Intrahepática/terapia , Heterocigoto , Humanos , India , MasculinoRESUMEN
PURPOSE: The nodlike receptor family pyrin domain containing 3 (NLRP3) inflammasome is a critical player in vascular pathology as it regulates caspase-1-mediated interleukin (IL)-1ß processing. Physical activity ameliorates obesity-induced inflammation and vascular dysfunction, but the mechanisms responsible for these positive changes are incompletely understood. Here, the protective effect of physical activity on the inflammasome-associated vascular dysfunction in obesity and its putative mechanisms were investigated. METHODS: Mice were fed a control low-fat diet (LFD) or a high-fat diet (HFD; 45% of calories from fat) and provided with running wheel access (LF-RUN or HF-RUN) or denied wheel access for our sedentary condition (LF-SED or HF-SED). The NLRP3 inflammasome-associated pathway, including NLRP3, caspase-1, and IL-1ß, in mice aorta was examined by RT-qPCR and FLICA and DAB staining. The protein expression of zonula occluden-1 (ZO-1), ZO-2, adiponectin (APN), and adiponectin receptor 1 (AdipoR1) in aortic endothelial cells was determined by immunofluorescence double staining. Intracellular reactive oxidative stress and nitric oxide (NO) production were monitored with fluorescence probes, dihydroethidium, and diaminofluorecein. RESULTS: HFD increased caspase-1 and IL-1ß at mRNA and protein levels in endothelial cells of the aorta, and this was attenuated by voluntary running. HFD decreased ZO-1 and ZO-2 expression and reduced APN and AdipoR1 signaling; these were restored by running. The elevated intracellular superoxide (O2) production observed in HF-SED was ameliorated in HF-RUN. Finally, HF-RUN improved NO production in the aorta compared with HF-SED. CONCLUSIONS: Our findings suggest that voluntary running ameliorates mechanisms associated with vascular dysfunction by suppressing NLRP3 inflammasome, improving NO production, and reducing oxidative stress. Such benefits of physical activity may be, at least in part, associated with APN-AdipoR1 signaling and tight junction protein expression.
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Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Óxido Nítrico/metabolismo , Obesidad/metabolismo , Estrés Oxidativo , Condicionamiento Físico Animal/fisiología , Adiponectina/metabolismo , Animales , Aorta/metabolismo , Caspasa 1/metabolismo , Dieta con Restricción de Grasas , Dieta Alta en Grasa , Endotelio Vascular/metabolismo , Interleucina-1beta/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Actividad Motora , ARN Mensajero/metabolismo , Receptores de Adiponectina/metabolismo , Superóxidos/metabolismo , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-2/metabolismoRESUMEN
Recent studies have revealed that aloe emodin (AE), a natural compound from the root and rhizome of Rheum palmatum L., exhibits significant pharmacologic activities. However, the pharmacologic relevance of the compound, particularly for cardiovascular disease, remains largely unknown. Here, we hypothesized that AE could improve endothelial junction dysfunction through inhibiting the activation of NOD-like receptor family pyrin domain containing-3 (NLRP3) inflammasome regulated by NLRP3 ubiquitination, and ultimately prevent cardiovascular disease. In vivo, we used confocal microscopy to study the expression of tight junction proteins zonula occludens-1/2 (ZO-1/2) and the formation of NLRP3 inflammasome in coronary arteries of hypertension. And the experimental serum was used to detect the activation of NLRP3 inflammasome by ELISA assay. We found that AE could restore the expression of the endothelial connective proteins ZO-1/2 and decrease the release of high mobility group box1 (HMGB1), and also inhibited the formation and activation of NLRP3 inflammasome. Similarly, in vitro, our findings demonstrated that AE could restore the expression of the tight junction proteins ZO-1/2 and decrease monolayer cell permeability that related to endothelial function after stimulation by angiotensin II (Ang II) in microvascular endothelial cells (MECs). We also demonstrated that AE could inhibit Ang II-induced NLRP3 inflammasome formation and activation, which were regulated by NLRP3 ubiquitination in MECs, as shown by fluorescence confocal microscopy and Western blot. Together with these changes, we revealed a new protection mechanism of AE that inhibited NLRP3 inflammasome activation and decreased the release of HMGB1 by promoting NLRP3 ubiquitination. Our findings implicated that AE exhibited immense potential and specific therapeutic value in hypertension-related cardiovascular disease in the early stage and the development of innovative drugs.