RESUMEN
In the brain, neurons, glial cells, vascular endothelial cells (ECs), and mural cells form a functional structure referred to as the neurovascular unit (NVU). The functions of the NVU become impaired with aging. To gain insight into the mechanism underlying the aging-related changes in the NVU, we characterized in the present study the gliovascular interface in transgenic mice expressing a dominant-negative form of the telomeric repeat-binding factor 2 (TERF2) specifically in ECs using the Tie2 promoter. In these transgenic mice, senescence occurred in the cerebral ECs and was accompanied by upregulation of the mRNAs of proinflammatory cell adhesion molecules and cytokines. It is noteworthy that in the deep layers of the cerebral cortex, astrocytes exhibited an increase in the signals for S100ß as well as a decrease in the polarization of the water channel aquaporin-4 (AQP4) to the perivascular endfeet of the astrocytes. Mechanistically, the perivascular localization of dystroglycan and its ligand, laminin α2, was decreased, and their localization correlated well with the perivascular localization of AQP4, which supports the notion that their interaction regulates the perivascular localization of AQP4. The diminished perivascular localization of laminin α2 may be attributed to its proteolytic degradation by the matrix metalloproteinase-2 released by senescent ECs. Pericyte coverage was increased and negatively correlated with the decrease in the perivascular localization of AQP4. We propose that aging-related changes in ECs induce a mild morphological alteration of astrocytes and affect the localization of AQP4 at the gliovascular interface.
Asunto(s)
Senescencia Celular , Células Endoteliales , Laminina , Metaloproteinasa 2 de la Matriz , Neuroglía , Animales , Ratones , Acuaporina 4/genética , Acuaporina 4/metabolismo , Astrocitos/metabolismo , Células Endoteliales/metabolismo , Laminina/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones Transgénicos , Neuroglía/metabolismoRESUMEN
The efficacy of cisplatin (CDDP) has been demonstrated in the treatment of various cancers as monotherapy and combination therapy with immunotherapy. However, acquired CDDP resistance is a major obstacle to successful treatment. In the present study, the mechanisms underlying acquired CDDP resistance were examined using ACR20 cells, which are CDDPresistant cells derived from A549 lung cancer cells. CDDP induces cytotoxicity by binding nuclear DNA and generating reactive oxygen species (ROS). Contrary to our expectation, ROS levels were elevated in ACR20 cells not treated with CDDP. Pretreatment with an ROS inhibitor enhanced the sensitivity of ACR20 cells to CDDP and prevented the activation of nuclear factor (NF)кB signaling and upregulation of inhibitor of apoptosis proteins (IAPs). Notably, evaluation of the mitochondrial oxygen consumption rate and mitochondrial superoxide levels revealed a deterioration of mitochondrial function in ACR20 cells. Mitochondrial DNA PCRRFLP analysis revealed four mutations with varying percentage levels in ACR20 cells. In addition, in cytoplasmic hybrids with mitochondria from ACR20 cells, intrinsic ROS levels were elevated, expression of IAPs was increased, and complex I activity and sensitivity to CDDP were decreased. Analysis of threedimensional structure data indicated that a mutation (ND2 F40L) may impact the proton translocation pathway, thereby affecting mitochondrial complex I activity. Together, these findings suggest that intrinsic ROS levels were elevated by mitochondrial DNA mutations, which decreased the sensitivity to CDDP via activation of NFκB signaling and induction of IAP expression in ACR20 cells. These findings indicate that newly identified mutations in mitochondrial DNA may lead to acquired cisplatin resistance in cancer.
Asunto(s)
Cisplatino/farmacología , ADN Mitocondrial/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Células A549 , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Humanos , Mutación , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Regulación hacia ArribaRESUMEN
Vascular inflammation via T-cell-mediated immune responses has been shown to be critically involved in the pathogenesis of abdominal aortic aneurysm (AAA). T-cell coinhibitory molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) is known to act as a potent negative regulator of immune responses. However, the role of this molecule in the development of AAA remains completely unknown. We determined the effects of CTLA-4 overexpression on experimental AAA. We continuously infused CTLA-4 transgenic (CTLA-4-Tg)/apolipoprotein E-deficient (Apoe-/-) mice or control Apoe-/- mice fed a high-cholesterol diet with angiotensin II by implanting osmotic mini-pumps and evaluated the development of AAA. Ninety percent of angiotensin II-infused mice developed AAA, with 50% mortality because of aneurysm rupture. Overexpression of CTLA-4 significantly reduced the incidence (66%), mortality (26%), and diameter of AAA. These protective effects were associated with a decreased number of effector CD4+ T cells and the downregulated expression of costimulatory molecules CD80 and CD86, ligands for CTLA-4, on CD11c+ dendritic cells in lymphoid tissues. CTLA-4-Tg/Apoe-/- mice had reduced accumulation of macrophages and CD4+ T cells, leading to attenuated aortic inflammation, preserved vessel integrity, and decreased susceptibility to AAA and aortic rupture. Our findings suggest T-cell coinhibitory molecule CTLA-4 as a novel therapeutic target for AAA.
Asunto(s)
Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/inmunología , Rotura de la Aorta/inmunología , Aterosclerosis/complicaciones , Antígeno CTLA-4/metabolismo , Angiotensina II/toxicidad , Animales , Aorta Abdominal/efectos de los fármacos , Aorta Abdominal/inmunología , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/diagnóstico , Aneurisma de la Aorta Abdominal/patología , Rotura de la Aorta/inducido químicamente , Rotura de la Aorta/patología , Aterosclerosis/inmunología , Antígeno CTLA-4/genética , Antígeno CTLA-4/inmunología , Dieta Aterogénica/efectos adversos , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Noqueados para ApoE , Índice de Severidad de la Enfermedad , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Nonalcoholic steatohepatitis (NASH) progresses from nonalcoholic fatty liver disease (NAFLD); however, efficacious drugs for NASH treatment are lacking. Sodium alginate (SA), a soluble dietary fiber extracted from brown algae, could protect the small intestine from enterobacterial invasion. NASH pathogenesis has been suggested to be associated with enterobacterial invasion, so we examined the effect of SA on methionine- and choline-deficient (MCD) diet-induced steatohepatitis in mice (the most widely-used model of NASH). The mice (n = 31) were divided into three groups (mice fed with regular chow, MCD diet, and MCD diet premixed with 5% SA) for 4 and 8 weeks. The MCD diet increased lipid accumulation and inflammation in the liver, the NAFLD Activity Score and hepatic mRNA expression of tumor necrosis factor-ï¡ and collagen 1ï¡1, and induced macrophage infiltration. Villus shortening, disruption of zonula occludens-1 localization and depletion of mucus production were observed in the small intestine of the MCD-group mice. SA administration improved lipid accumulation and inflammation in the liver, and impaired barrier function in the small intestine. Collectively, these results suggest that SA is useful for NASH treatment because it can prevent hepatic inflammation and fatty degeneration by maintaining intestinal barrier function.
Asunto(s)
Alginatos/farmacología , Hígado Graso/tratamiento farmacológico , Metionina/deficiencia , Animales , Deficiencia de Colina/tratamiento farmacológico , Deficiencia de Colina/metabolismo , Deficiencia de Colina/patología , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Modelos Animales de Enfermedad , Hígado Graso/etiología , Hígado Graso/metabolismo , Hígado Graso/patología , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Intestino Delgado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Here, we developed a novel in vitro co-culture model, in which process-bearing astrocytes and isolated cerebral microvessels from mice were co-cultured. Astrocytes formed contacts with microvessels from both adult and neonatal mice. However, concentrated localization of the immunofluorescence signal for aquaporin-4 (AQP4) at contact sites between perivascular endfoot processes and blood vessels was only detected with neonatal mouse microvessels. Contact between astrocytic processes and microvessels was retained, whereas concentrated localization of AQP4 signal at contact sites was lost, by knockdown of dystroglycan or α-syntrophin, reflecting polarized localization of AQP4 at perivascular regions in the brain. Further, using our in vitro co-culture model, we found that astrocytes predominantly extend processes to pericytes located at the abluminal surface of microvessels, providing additional evidence that this model is representative of the in vivo situation. Altogether, we have developed a novel in vitro co-culture model that can reproduce aspects of the in vivo situation and is useful for assessing contact formation between astrocytes and blood vessels.
Asunto(s)
Astrocitos/fisiología , Encéfalo/fisiología , Microvasos/fisiología , Animales , Acuaporina 4/metabolismo , Astrocitos/metabolismo , Encéfalo/metabolismo , Proteínas de Unión al Calcio/metabolismo , Estructuras de la Membrana Celular/metabolismo , Estructuras de la Membrana Celular/fisiología , Técnicas de Cocultivo/métodos , Distroglicanos/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Microvasos/metabolismo , Proteínas Musculares/metabolismo , Pericitos/metabolismo , Pericitos/fisiologíaRESUMEN
OBJECTIVE: Prior to heart transplant, sensitization to human leukocyte antigen can occur after blood transfusions used during implantation of ventricular assist devices. The result is an increased risk of antibody-mediated rejection (AMR) after heart transplant. While plasmapheresis (PPH) treats serious AMR cases, what subsequent changes occur in the blood concentrations of immunosuppressive agents is still unknown. We investigated pre- and post-PPH changes in blood concentrations of tacrolimus (FK506) and mycophenolic acid (MPA) in a heart-transplant patient experiencing AMR. CASE: A 40-year-old woman with a history of dilated cardiomyopathy had heart transplantation for advanced heart failure. Since the patient was donor-specific antibody-positive and at risk for AMR, intravenous immunoglobulin therapy and PPH were performed just before transplantation. Triple combination immunosuppressive therapy was initiated, but 4 days after transplantation, panel-reactive antibody increased drastically, and AMR was diagnosed by biopsy. Multidisciplinary therapy, including PPH, was performed. Blood samples were collected to measure blood concentrations of FK506 and MPA before and after passage through the plasma separator. RESULTS: The elimination efficiency of FK506 from PPH was -6.25 - 2.25%, while the elimination efficiency of MPA was much greater at 32.35 - 51.43%. CONCLUSION: These results show the necessity of carefully considering changes in blood concentrations that occur in immunosuppressive agents due to PPH, including the pharmacokinetics of the particular drug. However, proper timing of the PPH relative to drug administration can also minimize immunosuppressant loss.â©.
Asunto(s)
Trasplante de Corazón , Inmunosupresores/sangre , Ácido Micofenólico/sangre , Plasmaféresis , Tacrolimus/sangre , Adulto , Femenino , Rechazo de Injerto/prevención & control , HumanosRESUMEN
Cisplatin (CDDP) is widely used as an anti-cancer platinum agent but its therapeutic efficacy is limited by acquired drug resistance. To develop a new therapeutic strategy that could overcome this resistance, it is important to characterize CDDP-resistant cancer cells. Here we established human lung cancer A549 cell-derived low- and high-grade CDDP-resistant sublines, termed ACR4 and ACR20â¯cells, by stepwise increasing CDDP concentrations up to 4 and 20⯵M, respectively. ACR4 and ACR20â¯cells showed 6- and 16-fold higher resistance to CDDP than A549â¯cells, respectively. Cell migration, invasion, and sphere formation were significantly decreased, whereas expression of the stem cell marker CD44v was increased in order of A549, ACR4, and ACR20â¯cells. The expression of the cystine-glutamate transporter xCT, which is encoded by SLC7A11, was upregulated because of the increased cell surface expression of CD44v in ACR20â¯cells. Treatment with the xCT inhibitor salazosulfapyridine and knockdown of SLC7A11 mRNA by a specific siRNA significantly improved sensitivity to CDDP in A549, ACR4, and ACR20â¯cells. Thus, our results suggest that CD44v overexpression is not involved in cancer stem cell properties but increases xCT expression, which leads to the acquisition of CDDP-resistance. This mechanism may contribute to the development of a new therapeutic strategy that can overcome resistance.
Asunto(s)
Sistema de Transporte de Aminoácidos y+/genética , Cisplatino/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Receptores de Hialuranos/metabolismo , Neoplasias Pulmonares/genética , Regulación hacia Arriba , Células A549 , Sistema de Transporte de Aminoácidos y+/metabolismo , Biomarcadores de Tumor/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Humanos , Concentración 50 Inhibidora , Neoplasias Pulmonares/patología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
In the central nervous system, astrocytes extend endfoot processes to ensheath synapses and microvessels. However, the mechanisms underlying this astrocytic process extension remain unclear. A limitation of the use of 2D cultured astrocytes for such studies is that they display a flat, epithelioid morphology, with no or very few processes, which is markedly different from the stellate morphology observed in vivo. In this study, we obtained 2D cultured astrocytes with a rich complexity of processes using differentiation of neurospheres in vitro. Using these process-bearing astrocytes, we showed that laminin, an extracellular matrix molecule abundant in perivascular sites, efficiently induced process formation and branching. Specifically, the numbers of the first- and second-order branch processes and the maximal process length of astrocytes were increased when cultured on laminin, compared with when they were cultured on poly-L-ornithine or type IV collagen. Knockdown of dystroglycan or α-syntrophin, constituent proteins of the dystrophin-glycoprotein complex that provides a link between laminin and the cytoskeleton, using small interference RNAs inhibited astrocyte process formation and branching, and down-regulated expression of the water channel aquaporin-4 (AQP4). Direct knockdown and a specific inhibitor of AQP4 also inhibited, whereas over-expression of AQP4 enhanced astrocyte process formation and branching. Knockdown of AQP4 decreased phosphorylation of focal adhesion kinase (FAK) that is critically implicated in actin remodeling. Collectively, these results indicate that the laminin-dystroglycan-α-syntrophin-AQP4 axis is important for process formation and branching of 2D cultured astrocytes. OPEN PRACTICES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Read the Editorial Highlight for this article on page 436.
Asunto(s)
Acuaporina 4/metabolismo , Astrocitos/efectos de los fármacos , Proteínas de Unión al Calcio/metabolismo , Distroglicanos/metabolismo , Laminina/farmacología , Proteínas de la Membrana/metabolismo , Proteínas Musculares/metabolismo , Animales , Astrocitos/metabolismo , Células Cultivadas , Quinasa 1 de Adhesión Focal/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/efectos de los fármacos , ARN Interferente Pequeño/farmacología , TransfecciónRESUMEN
BACKGROUND: Exosomes, small-membrane vesicles, are secreted by cells and include several types of proteins and nucleic acids. Exosomes transfer cellular information derived from donor cells and are involved in various physiological and pathological events, such as organ-specific metastasis. Elucidating the exosome uptake mechanisms is important for understanding the progression processes of organ-specific metastasis. However, whether the exosomes secreted by the donor cells are selectively or non-selectively incorporated into the recipient cells is unknown. METHODS: In this study, three human carcinoma cell lines, A549 (lung), HCT116 and COLO205 (colon), were used. The exosome isolation efficiency was compared between three methods: ultracentrifugation, ExoQuick-TC and Total Exosome Isolation kits. Recipient cells were treated with Pitstop 2, an inhibitor of clathrin-dependent endocytosis, or genistein, an inhibitor of caveolae-dependent endocytosis, and then incubated with DiO-labeled exosomes. RESULTS: Among the three methods examined, ultracentrifugation was the most efficient and reproducible. Exosomes derived from a donor cell line are incorporated into the three cell lines, but the exosome uptake capability was different depending on the recipient cell type and did not depend on the donor cell type. Exosome uptake in COLO205 was inhibited by Pitstop 2 and genistein. Exosome uptake in HCT116 was inhibited by Pitstop 2, but not genistein, while that in A549 cells was not inhibited by these inhibitors. Taken together, these results suggest that the exosomes secreted by donor cells are non-selectively incorporated into recipient cells and that the exosome uptake mechanism is different depending on the recipient cells. CONCLUSIONS: Different recipient cells' exosome uptake capabilities may be involved in organ-specific metastasis.
Asunto(s)
Exosomas/metabolismo , Genisteína/farmacología , Neoplasias/metabolismo , Sulfonamidas/farmacología , Tiazolidinas/farmacología , Células A549 , Transporte Biológico/genética , Endocitosis/genética , Células HCT116 , Humanos , Metástasis de la Neoplasia , Neoplasias/tratamiento farmacológico , Neoplasias/patologíaRESUMEN
Recent advances in diagnostic technologies have revealed that nonsteroidal anti-inflammatory drugs (NSAIDs) can cause serious mucosal injury in the upper and lower gastrointestinal tract (including the small intestine). A drug to treat NSAID-induced small-intestinal injury (SII) is lacking. Sodium alginate is a soluble dietary fiber extracted from brown seaweed and its solution has been used as a hemostatic agent to treat gastrointestinal bleeding due to gastric ulcers. Whether sodium alginate has therapeutic effects on NSAID-induced SII and its mechanism of action are not known. Here, we investigated if administration of two forms (high-molecular-weight (HMW) and low-molecular-weight (LMW)) of sodium alginate could ameliorate indomethacin-induced SII. Pretreatment with HMW sodium alginate or LMW sodium alginate before indomethacin administration improved ulceration and the resultant intestinal shortening was associated with reduced histological severity of mucosal injury and ameliorated mRNA expression of inflammation-related molecules in the small intestine. We found that mRNAs of secretory Muc2 and membrane-associated Muc1, Muc3 and Muc4 were expressed in the small intestine. mRNA expression of Muc1-4 was increased in indomethacin-induced SII, and these increases were prevented by sodium alginate. Thus, administration of sodium alginate could be a therapeutic approach to prevent indomethacin-induced SII.
Asunto(s)
Alginatos/administración & dosificación , Mucosa Intestinal/efectos de los fármacos , Intestino Delgado/efectos de los fármacos , Úlcera Gástrica/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Motilidad Gastrointestinal/efectos de los fármacos , Ácido Glucurónico/administración & dosificación , Ácidos Hexurónicos/administración & dosificación , Humanos , Indometacina/efectos adversos , Mucosa Intestinal/lesiones , Mucosa Intestinal/patología , Intestino Delgado/lesiones , Ratones , Úlcera Gástrica/inducido químicamente , Úlcera Gástrica/patologíaRESUMEN
The liver and the small intestine are closely related in the processes of drug absorption, metabolism and excretion via the enterohepatic circulation. Small intestinal ulcers are a serious adverse effect commonly occurring in patients taking nonsteroidal anti-inflammatory drugs. However, the influence of small intestinal ulcers on drug metabolism has not been established. This study examined the expressional changes of cytochrome P450 (CYP) in the liver using an indomethacin-induced small intestinal ulcer rat model and in cultured cells. After the administration of indomethacin to rats, ulcers were observed in the small intestine and expression of CYP3A1, the major isoform of hepatic CYP, was significantly down-regulated in the liver, accompanied by increased expression of inducible nitric oxide synthase, tumor necrosis factor α, interleukin (IL)-1ß and IL-6, in the small intestine and the liver. The indomethacin-induced small intestinal ulceration, the increase in inflammatory mediators in the small intestine and the liver, and the down-regulation of CYP3A1 expression in the liver were inhibited by co-administration of ampicillin, an antibacterial agent. In the human hepatic HepG2 cell line, IL-1ß, IL-6 and NOC-18, an NO donor, caused down-regulation of CYP3A4, the major isoform of human CYP3A. Thus, this study suggests that after indomethacin treatment small intestinal ulcers cause the down-regulation of CYP3A1 in the rat liver through an increase in ulcer-derived inflammatory mediators. Copyright © 2016 John Wiley & Sons, Ltd.
Asunto(s)
Citocromo P-450 CYP3A/biosíntesis , Modelos Animales de Enfermedad , Regulación Enzimológica de la Expresión Génica , Indometacina/toxicidad , Intestino Delgado/metabolismo , Úlcera/metabolismo , Animales , Antiinflamatorios no Esteroideos/toxicidad , Citocromo P-450 CYP3A/genética , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Células Hep G2 , Humanos , Intestino Delgado/efectos de los fármacos , Intestino Delgado/patología , Masculino , Ratas , Ratas Sprague-Dawley , Úlcera/inducido químicamente , Úlcera/patologíaRESUMEN
AIMS: Cisplatin (CDDP) is a platinum-based drug that is widely used in cancer chemotherapy, but the development of resistance in tumor cells is a major weakness of these treatments. Several mechanisms have been proposed to explain cisplatin resistance, and disruption of certain cellular pathways could modulate drug sensitivity to cisplatin. A lower level of cross-resistance to cisplatin leads to better outcomes in clinical use. MAIN METHODS: Cross-resistance was assessed using cisplatin resistant lung cancer cell line A549/CDDP. Cell cycle analysis was used to examine the effect of cisplatin on cell signaling pathways regulating G2/M transition in cisplatin resistant cells. KEY FINDINGS: A549/CDDP cells exhibited cross-resistance to carboplatin, but not oxaliplatin, which is often found in platinum analogues. Flow cytometry showed that nocodazole treatment caused a G2/M block in both A549/CDDP cells and cisplatin susceptible cells. However, A549/CDDP cells escaped the G2/M block following exposure to cisplatin. Activation of the Cdc2/CyclinB complex is required for transition from G2 to M phase, and the inactive form of phosphorylated Cdc2 is activated by Cdc25C dephosphorylation of Tyr15. In the cisplatin-treated susceptible cells, the levels of phosphorylated Cdc2 and Cdc25C were markedly decreased, leading to a loss of Cdc2 activity and G2/M arrest. In A549/CDDP cells, however, Cdc2 activity was supported by the expression of Cdc2 and Cdc25C after the addition of cisplatin, which resulted in G2/M progression. SIGNIFICANCE: The resistance phenotype of G2/M progression has been correlated with dysregulation of Cdc2 in a human lung cancer cell line selected for cisplatin.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Quinasas Ciclina-Dependientes/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Compuestos Organoplatinos/farmacología , Proteína Quinasa CDC2 , Proteínas de Ciclo Celular/metabolismo , División Celular/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos , Fase G2/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Neoplasias Pulmonares/patología , Puntos de Control de la Fase M del Ciclo Celular , Nocodazol/farmacología , Oxaliplatino , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Non-steroidal anti-inflammatory drugs induce the serious side effect of small intestinal ulcerations (SIUs), but little information is available regarding the consequences to drug metabolism and absorption. AIM: We examined the existence of secondary hepatic inflammation in rats with indomethacin (INM)-induced SIUs and assessed its relationship to the cytochrome P450 (CYP) and P-glycoprotein (mdr1a), the major drug-metabolizing factors in the small intestine and the liver. METHODS: Gene expression of the CYP family of enzymes and mdr1a was measured with quantitative real-time polymerase chain reaction (qPCR). Vancomycin (VCM), a poorly absorbed drug, was administered intraduodenally to rats with SIUs. RESULTS: INM induced SIUs predominantly in the lower region of the small intestine with high expression of inflammatory markers. Liver dysfunction was also observed, which suggested a secondary inflammatory response in rats with SIUs. In the liver of rats with SIUs, the expression of CYP2C11, CYP2E1, and CYP3A1 was significantly decreased, and loss of CYP3A protein was observed. Although previous studies have shown a direct effect of INM on CYP3A activity, we could not confirm any change in hepatic CY3A4 expression (major isoform of human CYP3A) in vitro. The plasma VCM concentration was increased in rats with SIUs due to partial absorption from the mucosal injury, but not in normal mucosa. CONCLUSIONS: INM-induced SIUs had a subtle effect on intestinal CYP expression, but had an apparent action on hepatic CYP, which was influenced, at least in part, by the secondary inflammation. Furthermore, drug absorption was increased in rats with SIUs.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Antiinflamatorios no Esteroideos/toxicidad , Sistema Enzimático del Citocromo P-450/genética , Indometacina/toxicidad , Enfermedades Intestinales/inducido químicamente , Enfermedades Intestinales/genética , Úlcera/inducido químicamente , Úlcera/genética , Animales , Hidrocarburo de Aril Hidroxilasas/genética , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP3A/genética , Familia 2 del Citocromo P450 , Modelos Animales de Enfermedad , Expresión Génica/efectos de los fármacos , Células Hep G2 , Humanos , Enfermedades Intestinales/metabolismo , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Esteroide 16-alfa-Hidroxilasa/genética , Úlcera/metabolismo , Vancomicina/administración & dosificación , Vancomicina/sangre , Vancomicina/farmacocinéticaRESUMEN
CYP3A and P-glycoprotein (P-gp) play important roles in drug metabolism and excretion; however, their functions in pathological conditions remain unclear. Hepatobiliary abnormalities have been described in patients with ulcerative colitis, which may affect drug metabolism and excretion in the liver and small intestine. We examined the functions of CYP3A and P-gp in the liver and small intestine of mice with dextran sodium sulfate (DSS)-induced colitis. Up to day 7, inflammatory markers were significantly increased in the livers of DSS-treated mice, accompanied by decreased CYP3A. Additionally hepatobiliary transporters and Pregnane X receptor, which regulates the transcriptional activation of CYP3A, were reduced. Both CYP3A and P-gp were significantly decreased in the upper small intestine of DSS-treated mice on day 7. This was associated with the increased expression of inducible nitric oxide synthase, but not changes in nuclear receptor expression. On day 7 of DSS treatment, the concentrations of cyclosporine A (CsA), a substrate of both CYP3A and P-gp, were significantly higher than controls. These results indicated the existence of a second inflammatory response in the liver and upper small intestine of mice with DSS-induced colitis, and bioavailability of CsA was increased by the dysfunction of CYP3A and P-gp in these organs.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Colitis Ulcerosa/metabolismo , Ciclosporina/sangre , Citocromo P-450 CYP3A/fisiología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Disponibilidad Biológica , Colitis Ulcerosa/inducido químicamente , Citocromo P-450 CYP3A/metabolismo , Sulfato de Dextran , Regulación hacia Abajo , Humanos , Intestino Delgado/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo II/metabolismo , Receptor X de Pregnano , Receptores de Esteroides/metabolismoRESUMEN
Non-steroidal anti-inflammatory drugs (NSAIDs)-induced small intestinal injury is a serious clinical event with recent advances of diagnostic technologies, but a successful therapeutic method to treat such injuries is still lacking. Licorice, a traditional herbal medicine, and its derivatives have been widely used for the treatment of a variety of diseases due to their extensive biological actions. However, it is unknown whether these derivatives have an effect on NSAIDs-induced small intestinal damage. Previously, the anti-inflammatory effects of three compounds extracted from the licorice root, glycyrrhizin, 18ß-glycyrrhetinic acid, and dipotassium glycyrrhizinate, were compared in vitro cell culture. The most prominent inhibitory effect on the tumor necrosis factor-α (TNF-α) production was observed with the administration of 18ß-glycyrrhetinic acid as an active metabolite of glycyrrhizin. In this study, a complex compound of 18ß-glycyrrhetinic acid and hydroxypropyl γcyclodextrin was examined to improve the oral bioavailability. After administration of this complex to indomethacin treated mice, a significantly high plasma concentration of 18ß-glycyrrhetinic acid was detected using the tandem mass spectrometry coupled with the HPLC. Furthermore, the complex form of 18ß-glycyrrhetinic acid and hydroxypropyl γcyclodextrin reduced mRNA expressions of TNF-α, interleukin (IL)-1ß, and IL-6, which was histologically confirmed in the improvement of indomethacin-induced small intestinal damage. These results suggest that the complex of 18ß-glycyrrhetinic acid and hydroxypropyl γcyclodextrin has the potential therapeutic value for preventing the adverse effects of indomethacin-induced small intestinal injury.
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Ácido Glicirretínico/análogos & derivados , Indometacina/efectos adversos , Intestino Delgado/efectos de los fármacos , Intestino Delgado/lesiones , gamma-Ciclodextrinas/farmacología , Animales , Disponibilidad Biológica , Regulación de la Expresión Génica/efectos de los fármacos , Ácido Glicirretínico/química , Ácido Glicirretínico/farmacocinética , Ácido Glicirretínico/farmacología , Interleucina-1beta/genética , Interleucina-6/genética , Intestino Delgado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Solubilidad , Factor de Necrosis Tumoral alfa/genética , gamma-Ciclodextrinas/química , gamma-Ciclodextrinas/farmacocinéticaRESUMEN
In our previous study, it was found that linoleoyl ethanolamide (LE) is present in sake lees, which are produced as a byproduct during the making of Japanese sake. LE is a fatty acid ethanolamide, which have been demonstrated to exert a variety of biological functions, and in this study, the anti-inflammatory effects of LE were examined using in vitro cell culture and in vivo animal experiments. In mouse RAW264.7 macrophages, LE suppressed the lipopolysaccharide (LPS)-induced expression of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6. In addition, LE inhibited LPS-induced increases in the levels of cyclooxygenase enzyme-2 and prostaglandin E(2), which are indicators of inflammation. The inhibitory effect of LE on the release of TNF-α was stronger than that of dipotassium glycyrrhizinate, which is widely used in external human skin care treatments. LE also suppressed the LPS-induced activation of Toll-like receptor 4 signaling and nuclear translocation of nuclear factor-κB (NF-κB) p65. In a contact dermatitis animal model, applying LE to affected ear skin ameliorated 2,4-dinitrofluorobenzene-induced contact dermatitis and pro-inflammatory cytokine expression at inflamed sites. These results indicate that LE exerts anti-inflammatory effects by inhibiting NF-κB signaling, and LE is proposed to be a useful therapeutic agent against contact dermatitis.
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Antiinflamatorios/farmacología , Dermatitis por Contacto/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Ácidos Linoleicos/farmacología , Alcamidas Poliinsaturadas/farmacología , Animales , Línea Celular , Citocinas/metabolismo , Dermatitis por Contacto/patología , Dinitrofluorobenceno/toxicidad , Modelos Animales de Enfermedad , Femenino , Ácido Glicirrínico/farmacología , Inflamación/patología , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/toxicidad , Macrófagos/efectos de los fármacos , Macrófagos/patología , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Duodenal HCO3- secretion increases in response to luminal acid, mediated by endogenous nitric oxide (NO) as well as prostaglandins (PGs). In this study, we examined the effects of various inhibitors of cyclooxygenase (COX) or NO synthase (NOS) on the acid-induced HCO3- secretion in rats and determined the enzyme isoforms responsible for this response. A proximal duodenal loop was perfused with saline under urethane anesthesia, and the HCO3- secretion was measured at pH 7.0 using a pH-stat method and by adding 10 mM HCl. Mucosal acidification was performed by exposing the loop to 10 mM HCl for 10 min. Indomethacin, SC-560 (a selective COX-1 inhibitor) and rofecoxib (a selective COX-2 inhibitor) were given intraduodenally 1 hr before exposure to 10 mM HCl, while N(G)-nitro-L-arginine methyl ester (L-NAME: a nonselective NOS inhibitor) and aminoguanidine (a relatively selective inhibitor of iNOS) were given subcutaneously 3 hr before the acidification. The mucosal acidification increased the HCO3- secretion, with a rise in mucosal PGE2 content and luminal release of NO. The HCO3- secretory and PGE2 biosynthetic responses were significantly inhibited by indomethacin and SC-560, while rofecoxib had no effect on these responses. On the other hand, L-NAME, but not aminoguanidine, attenuated NO release following the acidification, resulting in inhibition of the acid-induced HCO3- secretion in a L-arginine-sensitive manner. Neither COX-2 nor iNOS mRNAs were observed in the mucosa before and 1 hr after acidification, while the gene expression of COX-1 and nNOS was constitutively detected in the mucosa and appeared to be slightly up-regulated after the acid stimulation. These results suggest that COX-1 and cNOS play as the respective key enzyme responsible for producing PG and NO following the duodenal acidification, both of which are involved in the mechanism for the acid-induced HCO3- secretion in the duodenum.
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Bicarbonatos/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Duodeno/metabolismo , Isoenzimas/fisiología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/fisiología , Prostaglandina-Endoperóxido Sintasas/fisiología , Animales , Ciclooxigenasa 1 , Guanidinas/farmacología , Indometacina/farmacología , Lactonas/farmacología , Masculino , Proteínas de la Membrana , NG-Nitroarginina Metil Éster/farmacología , Pirazoles/farmacología , Ratas , Ratas Sprague-Dawley , SulfonasRESUMEN
AIM:To examine whether nizatidine stimulates duodenal HCO(3)(-) secretion in rats by inhibiting AChE activity.METHODS:Under pentobarbital anesthesia, a proximal duodenal loop was perfused with saline, and the HCO(3)(-) secretion was measured at pH 7.0 using a pH-stat method and by adding 10mM HCl. Nizatidine, neostigmine, carbachol or famotidine was administered i.v. as a single injection.RESULTS:Intravenous administration of nizatidine (3-30mg/kg) dose-dependently increased duodenal HCO(3)(-) secretion, and the effect at 10mg/kg was equivalent to that obtained by carbachol at 0.01mg/kg. This nizatidine action was observed at the same dose range that inhibited acid secretion and enhanced gastric motility, mimicked by i.v. injection of neostigmine(0.03mg/kg), and significantly attenuated by bilateral vagotomy and prior s.c. administration of atropine but not by indomethacin, a cyclooxygenase inhibitor, or NG-nitro-L-arginine methyl ester, a NO synthase inhibitor. The HCO(3)(-) secretory response to acetylcholine (0.001mg/kg) was significantly potentiated by the concurrent administration of nizatidine (3mg/kg,i.v.). The IC(50) of nizatidine for AChE of rat erythrocytes was 1.4·10(-6) M, about 12 times higher than that of neostigmine. Neither famotidine (> 10(-3) M, 30mg/kg, i.v.) nor cisapride (> 10(-3) M,3mg/kg, i.v.) had any influence on AChE activity or duodenal HCO(3)(-) secretion. Duodenal damage induced by acid perfusion (100mM HCl for 4h) in the presence of indomethacin was significantly prevented by nizatidine and neostigmine, at the doses that increased the HCO(3)(-)secretion.CONCLUSION:Nizatidine stimulates duodenal HCO(3)(-) secretion, in both vagal dependent and atropine sensitive manners, and the action is associated with the anti-AChE activity of this agent.