RESUMO
Cholestasis is a common condition in which the flow of bile from the liver to the intestines is inhibited. It has been shown that organic anion-transporting polypeptide 3A1 (OATP3A1) is upregulated in cholestasis to promote bile acid efflux transport. We have previously shown that the growth factor fibroblast growth factor 19 and inflammatory mediator tumor necrosis factor α (TNFα) increased OATP3A1 mRNA levels in hepatoma peritoneal lavage cell/PRF/5 cell lines. However, the mechanism underlying TNFα-stimulated OATP3A1 expression in cholestasis is unknown. To address this, we collected plasma samples from control and obstructive cholestasis patients and used ELISA to detect TNFα levels. We found that the TNFα levels of plasma and hepatic mRNA transcripts were significantly increased in obstructive cholestatic patients relative to control patients. A significant positive correlation was also observed between plasma TNFα and liver OATP3A1 mRNA transcripts in patients with obstructive cholestasis. Further mechanism analysis revealed that recombinant TNFα induced OATP3A1 expression and activated NF-κB and extracellular signal-regulated kinase (ERK) signaling pathways as well as expression of related transcription factors p65 and specificity protein 1 (SP1). Dual-luciferase reporter and chromatin immunoprecipitation assays showed that recombinant TNFα upregulated the binding activities of NF-κB p65 and SP1 to the OATP3A1 promoter in peritoneal lavage cell/PRF/5 cells. These effects were diminished following the application of NF-κB and ERK inhibitors BAY11-7082 and PD98059. We conclude that TNFα stimulates hepatic OATP3A1 expression in human obstructive cholestasis by activating NF-κB p65 and ERK-SP1 signaling. These results suggest that TNFα-activated NF-κB p65 and ERK-SP1 signaling may be a potential target to ameliorate cholestasis-associated liver injury.
Assuntos
Colestase , Transportadores de Ânions Orgânicos , Fator de Necrose Tumoral alfa , Ácidos e Sais Biliares/metabolismo , Colestase/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , NF-kappa B/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
BACKGROUND: Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders affecting women of reproductive age. Cangfu Daotan Decoction (CFDTT) is one of the prescriptions in the stagnation of obesity-type PCOS. Our previous study showed that CFDTT treatment of obese PCOS was correlated with organic anion transporting polypeptides (OATPs). METHODS: Here, we studied the effects of CFDTT on obese PCOS and its underlying mechanism. We built an obese PCOS rat model and treated the rats with CFDTT. Then, we detected the serum levels of TCHO, TG, LDL-c, HDL-c, luteinizing hormone (LH), follicle stimulating growth hormone (FSH), testosterone (T), estradiol (E2), IL-1ß, IL-6, and TNF-α in each group and adopted RT-PCR, western blotting and immunohistochemical staining to investigate the effects of CFDTT treatment on the expression of OATP2B1 and OATP3A1 in ovarian and uterine tissues. In addition, we compared the pregnancy outcomes of each group. RESULTS: We found that CFDTT decreased the serum levels of TCHO, TG, LDL-c, LH, T, IL-1ß, IL-6, and TNF-α and increased the levels of HDL-c, FSH and E2 in a dose-dependent manner. CFDTT could induce the expression of OATP2B1 and OATP3A1 in ovarian and uterine tissues. Moreover, CFDTT could improve pregnancy outcomes. CONCLUSION: These data suggested that the therapeutic mechanism of Cangfu Daotan Decoction on PCOS may be correlated with regulating lipid metabolism, sex hormone secretion and the inflammatory response and increasing OATP2B1 and OATP3A1 expression. Cangfu Daotan Decoction can be developed as a PCOS treatment drug.
Assuntos
Síndrome do Ovário Policístico , Animais , Medicamentos de Ervas Chinesas , Feminino , Hormônio Luteinizante , Obesidade , Gravidez , RatosRESUMO
Polycystic ovary syndrome (PCOS) is a common endocrinal metabolic disease, and its pathogenesis has not yet been thoroughly studied. The purpose of this experiment was to investigate the effect of RNA interference on Oatp3a1 gene expression on the biological viability and immune factors of ovarian granulosa cells in rats with PCOS. First, rats were intragastrically administered 1 mg/kg letrozole to successfully construct PCOS model. Western blot, qRT-PCR, CCK8 and flow cytometry were used to detect the gene expression, immune factor protein expression, cell proliferation and apoptosis in ovarian granulosa cells transfected with siRNA Oatp3a1 in rats with PCOS, respectively. The results showed that follicle stimulating hormone receptor (FSHR) was located on the cell membrane of rat ovarian granulosa cells, and letrozole successfully induced PCOS rat model. In PCOS rat ovarian granulosa cells, the mRNA expression level of Oapta1 was higher than that in normal rat ovarian granulosa cells. At the same time, compared with the sham group, the protein expression of NF-κB, TGF-ß1 and VEGF in si-Oatp3a1 group was significantly down-regulated (P < 0.05), and the cell proliferation rate was significantly decreased in si-Oatp3a1 group (P < 0.05) in comparison with the sham group. The apoptotic rate was increased obviously (P < 0.05), which was about 2.5 times that of the sham group. This indicates that in the ovarian granulosa cells of rats with PCOS, the interference of Oatp3a1 gene expression can significantly inhibit cell proliferation and promote apoptosis, while inhibiting the expression of immune factors TGF-ß1 and VEGF can reduce the expression of NF-κB protein, thereby suppressing the activation of the NF-κB signaling pathway.
RESUMO
Human organic anion transporting polypeptide 3A1 (OATP3A1) is predominately expressed in the heart. The ability of OATP3A1 to transport statins into cardiomyocytes is unknown, although other OATPs are known to mediate the uptake of statin drugs in liver. The pleiotropic effects and uptake of simvastatin acid were analyzed in primary human cardiomyocytes and HEK293 cells transfected with the OATP3A1 gene. Treatment with simvastatin acid reduced indoxyl sulfate-mediated reactive oxygen species and modulated OATP3A1 expression in cardiomyocytes and HEK293 cells transfected with the OATP3A1 gene. We observed a pH-dependent effect on OATP3A1 uptake, with more efficient simvastatin acid uptake at pH5.5 in HEK293 cells transfected with the OATP3A1 gene. The Michaelis-Menten constant (Km) for simvastatin acid uptake by OATP3A1 was 0.017±0.002µM and the Vmax was 0.995±0.027fmol/min/105 cells. Uptake of simvastatin acid was significantly increased by known (benzylpenicillin and estrone-3-sulfate) and potential (indoxyl sulfate and cyclosporine) substrates of OATP3A1. In conclusion, the presence of OATP3A1 in cardiomyocytes suggests that this transporter may modulate the exposure of cardiac tissue to simvastatin acid due to its enrichment in cardiomyocytes. Increases in uptake of simvastatin acid by OATP3A1 when combined with OATP substrates suggest the potential for drug-drug interactions that could influence clinical outcomes.
Assuntos
Transportadores de Ânions Orgânicos/metabolismo , Sinvastatina/metabolismo , Sinvastatina/farmacocinética , Interações Medicamentosas , Células HEK293 , Humanos , Indicã/metabolismo , Indicã/farmacocinética , Miócitos Cardíacos/metabolismo , Espécies Reativas de OxigênioRESUMO
Epidemiological studies indicate that 17ß-estradiol (E2) prevents colorectal cancer (CRC). Organic anion transporting polypeptides (OATPs) are involved in the cellular uptake of various endogenous and exogenous substrates, including hormone conjugates. Because transfer of estrone sulfate (E1-S) can contribute to intra-tissue conversion of estrone to the biologically active form -E2, it is evident that the expression patterns of OATPs may be relevant to the analysis of CRC incidence and therapy. We therefore evaluated DNA methylation and transcript levels of two members of the OATP family, OATP3A1 and OATP4A1, that may be involved in E1-S transport in colorectal cancer patients. We detected a significant reduction in OATP3A1 and a significant increase in OATP4A1 mRNA levels in cancerous tissue, compared with histopathologically unchanged tissue (n=103). Moreover, we observed DNA hypermethylation in the OATP3A1 promoter region in a small subset of CRC patients and in HCT116 and Caco-2 colorectal cancer cell lines. We also observed increased OATP3A1 transcript following treatment with 5-aza-2-deoxycytidine and sodium butyrate. The OATP4A1 promoter region was hypomethylated in analyzed tissues and CRC cell lines and was not affected by these treatments. Our results suggest a potential mechanism for OATP3A1 downregulation that involves DNA methylation during colorectal carcinogenesis.
Assuntos
Neoplasias Colorretais/patologia , Metilação de DNA , Transportadores de Ânions Orgânicos/genética , Idoso , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Ácido Butírico/farmacologia , Células CACO-2 , Neoplasias Colorretais/genética , Decitabina , Regulação para Baixo , Estrona/análogos & derivados , Estrona/metabolismo , Feminino , Células HCT116 , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismoRESUMO
Objective To investigate the expression variation of kidney OATP-3 in rats relating to rocuronium metabolism in anhepatic phase and to primarily explain the reason of extrahepatic metabolism characters of rocuronium. Methods Twelve rats were distributed to 2 groups randomly with 6 in each: Group A (control group) and Group B in which the hepatic portal devascularization was performed for 60 min. Kidney tissues of the rats were taken. oatp-3 mRNA was detected by RT-PCR and OATP-3 protein by Western blotting. Results The expression levels of kidney OATP-3 mRNA and protein in Group B were significantly higher than those in Group A(P