RESUMO
BACKGROUND: Mume Fructus (MF) is the fruit of Prunus mume Sieb. et Zucc, a plant of Rosaceae family. Previous studies demonstrated that MF was capable of ameliorating ulcerative colitis (UC) in mice, its action mechanism needs to be clarified. PURPOSE: This study deciphered whether and how MF extract accelerates colonic mucosal healing, the therapeutic endpoint of UC. METHODS: Biochemical, histopathological and qRT-PCR analyses were utilized to define the therapeutic efficacy of MF on dextran sulfate sodium (DSS)-induced colitis in mice. UHPLC-QTOF-MS/MS-based metabolomics technique was adopted to explore the changes of endogenous metabolites associated with UC and responses to MF intervention. qRT-PCR analysis was performed to confirm the molecular pathway in vivo. The effects of MF and lysophosphatidylcholine (LPC) on cell viability, wound healing, proliferation, and migration were examined through a series of in vitro experiments. Moreover, the effects of different subtypes of phospholipase A2 (PLA2) inhibitors on MF-treated colonic epithelial cells were detected by wound healing test and transwell assay. RESULTS: Orally administered MF could alleviate colitis in mice mainly by accelerating the healing of colonic mucosa. Guided by an unbiased metabolomics screen, we identified LPC synthesis as a major modifying pathway in colitis mice after MF treatment. Notably, MF facilitated the synthesis of LPC by enhancing the expression of PLA2 in colitis mice. Mechanistically, MF and LPC accelerated wound closure by promoting cell migration. Moreover, the promotion of MF on wound healing and migration of colonic epithelial cells was blunted by a cytosolic phospholipase A2 (cPLA2) inhibitor. CONCLUSION: MF can facilitate colonic mucosal healing of mice with colitis through cPLA2-mediated intestinal LPC synthesis, which may become a novel therapeutic agent of UC.
Assuntos
Colite Ulcerativa , Colite , Prunus , Camundongos , Animais , Sulfato de Dextrana/efeitos adversos , Lisofosfatidilcolinas/metabolismo , Prunus/química , Frutas/química , Espectrometria de Massas em Tandem , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/metabolismo , Colo/patologia , Colite Ulcerativa/tratamento farmacológico , Cicatrização , Mucosa Intestinal/metabolismo , Fosfolipases A2 Citosólicas/análise , Fosfolipases A2 Citosólicas/metabolismo , Fosfolipases A2 Citosólicas/farmacologia , Modelos Animais de Doenças , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: 3, 3'-diindolylmethane (DIM), a classical aryl hydrocarbon receptor (AhR) agonist, has been shown to relieve neuropathic pain, but few studies have reported the efficacy of DIM in visceral pain under colitis condition. PURPOSE: This study aimed to investigate the effect and mechanism of DIM on visceral pain under colitis condition. METHODS: Cytotoxicity was performed using the MTT assay. RT-qPCR and ELISA assays were applied to determine the expression and release of algogenic substance P (SP), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Flow cytometry was used to examine the apoptosis and efferocytosis. The expression of Arg-1-arginine metabolism-related enzymes was detected using western blotting assays. ChIP assays were used to examine the binding of Nrf2 to Arg-1. Mouse models of dextran sulfate sodium (DSS) were established to illustrate the effect of DIM and validate the mechanism in vivo. RESULTS: DIM did not directly affect expressions and release of algogenic SP, NGF and BDNF in enteric glial cells (EGCs). However, when co-cultured with DIM-pre-treated RAW264.7 cells, the release of SP and NGF was decreased in lipopolysaccharides-stimulated EGCs. Furthermore, DIM increased the number of PKH67+ F4/80+ cells in the co-culture system of EGCs and RAW264.7 cells in vitro and alleviated visceral pain under colitis condition by regulating levels of SP and NGF as well as values of electromyogram (EMG), abdominal withdrawal reflex (AWR) and tail-flick latency (TFL) in vivo, which was significantly inhibited by efferocytosis inhibitor. Subsequently, DIM was found to down-regulate levels of intracellular arginine, up-regulate levels of ornithine, putrescine and Arg-1 but not extracellular arginine or other metabolic enzymes, and polyamine scavengers reversed the effect of DIM on efferocytosis and release of SP and NGF. Moving forward, Nrf2 transcription and the binding of Nrf2 to Arg-1-0.7 kb was enhanced by DIM, AhR antagonist CH223191 abolished the promotion of DIM on Arg-1 and efferocytosis. Finally, nor-NOHA validated the importance of Arg-1-dependent arginine metabolism in DIM-alleviated visceral pain. CONCLUSION: DIM enhances macrophage efferocytosis in an arginine metabolism-dependent manner via "AhR-Nrf2/Arg-1" signals and inhibits the release of SP and NGF to relieve visceral pain under colitis condition. These findings provide a potential therapeutic strategy for the treatment of visceral pain in patients with colitis.
Assuntos
Colite , Dor Visceral , Camundongos , Animais , Receptores de Hidrocarboneto Arílico/metabolismo , Fator 2 Relacionado a NF-E2 , Fator Neurotrófico Derivado do Encéfalo , Dor Visceral/tratamento farmacológico , Fator de Crescimento Neural , Macrófagos/metabolismo , Colite/induzido quimicamente , Colite/tratamento farmacológicoRESUMO
Intestinal mucus barrier dysfunction is closely involved in the pathogenesis of inflammatory bowel diseases (IBD). To investigate the protective effect and underlying mechanism of arctigenin, a phytoestrogen isolated from the fruits of Arctium lappa L., on the intestinal mucus barrier under colitis condition. The role of arctigenin on the intestinal mucus barrier and the apoptosis of goblet cells were examined by using both in vitro and in vivo assays. Arctigenin was demonstrated to promote the mucus secretion and maintain the integrity of mucus barrier, which might be achieved by an increase in the number of goblet cells via inhibiting apoptosis. Arctigenin selectively inhibited the mitochondrial pathway-mediated apoptosis. Moreover, arctigenin elevated the protein level of prohibitin 1 (PHB1) through blocking the ubiquitination via activation of estrogen receptor ß (ERß) to competitively interact with PHB1 and disrupt the binding of tripartite motif 21 (TRIM21) with PHB1. ERß knock down in the colons of mice with DSS-induced colitis resulted in significant reduction of the protection of arctigenin and DPN against the mucosal barrier. Arctigenin can maintain the integrity of the mucus barrier by inhibiting the apoptosis of goblet cells through the ERß/TRIM21/PHB1 pathway.
Assuntos
Colite , Doenças Inflamatórias Intestinais , Animais , Apoptose , Colite/induzido quimicamente , Receptor beta de Estrogênio/metabolismo , Furanos , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/patologia , Lignanas , Camundongos , Camundongos Endogâmicos C57BL , Muco/metabolismo , Fitoestrógenos , ProibitinasRESUMO
Oral administration of curcumin has been shown to inhibit pulmonary fibrosis (PF) despite its extremely low bioavailability. In this study, we investigated the mechanisms underlying the anti-PF effect of curcumin in focus on intestinal endocrine. In bleomycin- and SiO2-treated mice, curcumin (75, 150 mg· kg-1 per day) exerted dose-dependent anti-PF effect when administered orally or rectally but not intravenously, implying an intestinal route was involved in the action of curcumin. We speculated that curcumin might promote the generation of gut-derived factors and the latter acted as a mediator subsequently entering the lungs to ameliorate fibrosis. We showed that oral administration of curcumin indeed significantly increased the expression of gut-derived hepatocyte growth factor (HGF) in colon tissues. Furthermore, in bleomycin-treated mice, the upregulated protein level of HGF in lungs by oral curcumin was highly correlated with its anti-PF effect, which was further confirmed by coadministration of c-Met inhibitor SU11274. Curcumin (5-40 µM) dose-dependently increased HGF expression in primary mouse fibroblasts, macrophages, CCD-18Co cells (fibroblast cell line), and RAW264.7 cells (monocyte-macrophage cell line), but not in primary colonic epithelial cells. In CCD-18Co cells and RAW264.7 cells, curcumin dose-dependently activated PPARγ and CREB, whereas PPARγ antagonist GW9662 (1 µM) or cAMP response element (CREB) inhibitor KG-501 (10 µM) significantly decreased the boosting effect of curcumin on HGF expression. Finally, we revealed that curcumin dose-dependently increased the production of 15-deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2) in CCD-18Co cells and RAW264.7 cells, which was a common upstream of the two transcription factors. Moreover, both the in vitro and in vivo effects of curcumin were diminished by coadministration of HPGDS-inhibitor-1, an inhibitor of 15d-PGJ2 generation. Together, curcumin promotes the expression of HGF in colonic fibroblasts and macrophages by activating PPARγ and CREB via an induction of 15d-PGJ2, and the HGF enters the lungs giving rise to an anti-PF effect.
Assuntos
Colo/efeitos dos fármacos , Curcumina/uso terapêutico , Fator de Crescimento de Hepatócito/metabolismo , Prostaglandina D2/análogos & derivados , Fibrose Pulmonar/tratamento farmacológico , Administração Oral , Animais , Colo/citologia , Colo/metabolismo , Curcumina/administração & dosagem , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos ICR , PPAR gama/metabolismo , Prostaglandina D2/metabolismo , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Células RAW 264.7 , Regulação para Cima/efeitos dos fármacosRESUMO
Lindera aggregata (Sims) Kosterm root has been used in traditional Chinese medicine for the treatment of rheumatism palsy, dyspepsia and frequent urination for a long time. Norisoboldine, the main active constituent of this herb drug, possesses outstanding anti-arthritis activity. However, the in vivo disposition of norisoboldine is known to a limited extent, especially under the pathological condition of rheumatoid arthritis (RA). The aim of this study is to investigate whether and how the absorption of norisoboldine is altered in adjuvant-induced arthritis (AIA) rats. Comparative studies of the intestinal absorption of norisoboldine in normal and AIA rats at different pathological stages of the arthritis were performed using in situ single-pass intestinal perfusion, and the effects of an inhibitor of efflux proteins were also investigated. Norisoboldine was shown to be a substrate of P-glycoprotein (P-gp), as P-gp inhibitor verapamil markedly increased the permeability coefficient (Peff ) of norisoboldine by 88% in the intestine of normal rats. Compared with normal rats, AIA rats displayed increased Peff values of norisoboldine by 84% and 86% on day 5 and day 10 after the appearance of the secondary response of arthritis, respectively. Verapamil could eliminate the difference of intestinal absorption of norisoboldine between normal and AIA rats. Further studies showed that impaired expression and activity of P-gp in AIA rats play a decisive role in the absorption enhancement of norisoboldine. Notably, the impairment of P-gp function positively correlated with the severity of arthritis. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Alcaloides/farmacocinética , Anti-Inflamatórios/farmacocinética , Artrite Experimental/metabolismo , Duodeno/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Regulação para Baixo , Absorção Intestinal , Masculino , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Índice de Gravidade de Doença , Verapamil/farmacologiaRESUMO
AIM: Paeoniflorin has shown to attenuate bleomycin-induced pulmonary fibrosis (PF) in mice. Because the epithelial-mesenchymal transition (EMT) in type 2 lung endothelial cells contributes to excessive fibroblasts and myofibroblasts during multiple fibrosis of tissues, we investigated the effects of paeoniflorin on TGF-ß mediated pulmonary EMT in bleomycin-induced PF mice. METHODS: PF was induced in mice by intratracheal instillation of bleomycin (5 mg/kg). The mice were orally treated with paeoniflorin or prednisone for 21 d. After the mice were sacrificed, lung tissues were collected for analysis. An in vitro EMT model was established in alveolar epithelial cells (A549 cells) incubated with TGF-ß1 (2 ng/mL). EMT identification and the expression of related proteins were performed using immunohistochemistry, transwell assay, ELISA, Western blot and RT-qPCR. RESULTS: In PF mice, paeoniflorin (50, 100 mg·kg(-1)·d(-1)) or prednisone (6 mg·kg(-1)·d(-1)) significantly decreased the expression of FSP-1 and α-SMA, and increased the expression of E-cadherin in lung tissues. In A549 cells, TGF-ß1 stimulation induced EMT, as shown by the changes in cell morphology, the increased cell migration, and the increased vimentin and α-SMA expression as well as type I and type III collagen levels, and by the decreased E-cadherin expression. In contrast, effects of paeoniflorin on EMT disappeared when the A549 cells were pretreated with TGF-ß1 for 24 h. TGF-ß1 stimulation markedly increased the expression of Snail and activated Smad2/3, Akt, ERK, JNK and p38 MAPK in A549 cells. Co-incubation with paeoniflorin (1-30 µmol/L) dose-dependently attenuated TGF-ß1-induced expression of Snail and activation of Smad2/3, but slightly affected TGF-ß1-induced activation of Akt, ERK, JNK and p38 MAPK. Moreover, paeoniflorin markedly increased Smad7 level, and decreased ALK5 level in A549 cells. CONCLUSION: Paeoniflorin suppresses the early stages of TGF-ß mediated EMT in alveolar epithelial cells, likely by decreasing the expression of the transcription factors Snail via a Smad-dependent pathway involving the up-regulation of Smad7.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glucosídeos/uso terapêutico , Pulmão/efeitos dos fármacos , Monoterpenos/uso terapêutico , Fibrose Pulmonar/tratamento farmacológico , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Células A549 , Animais , Anti-Inflamatórios não Esteroides/química , Bleomicina , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Glucosídeos/química , Humanos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Monoterpenos/química , Paeonia/química , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Bergenin, isolated from the herb of Saxifrage stolonifera Curt. (Hu-Er-Cao) has hepatoprotective, anti-inflammatory, antitussive, and neuroprotective activities. The aim of the present study was to establish a simple, rapid, and sensitive RP-HPLC method for determination of bergenin in rat plasma and compare its oral pharmacokinetic behaviors in normal and CCl4-induced hepatic injury rats. With norisoboldine as an internal standard, chromatographic separation was performed on a C18 analytical column with acetonitrile and water (11 : 89, V/V) containing 0.1% formic acid as the mobile phase. A good linearity was obtained over the range of 100-10 000 ng·mL-1. The lower limit of quantification was 50 ng·mL-1. The developed method was successfully applied to a study of the pharmacokinetic difference of bergenin (100 mg·kg-1) between normal and hepatic injury rats after oral administration. Marked alterations of pharmacokinetic parameters in hepatic injury rats were observed. Compared to normal rats, the AUC(0-∞) of bergenin in hepatic injury rats was elevated to 2.11-fold and Cmax was increased by 130%, whereas CL value was only 55% of the normal rats, suggesting that the systemic exposure of bergenin was significantly increased under hepatic injury status.
Assuntos
Benzopiranos/farmacocinética , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacocinética , Saxifragaceae/química , Animais , Benzopiranos/administração & dosagem , Tetracloreto de Carbono , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Medicamentos de Ervas Chinesas/administração & dosagem , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem/métodosRESUMO
Tetrandrine (Tet), the main active constituent of Stephania tetrandra root, has been demonstrated to alleviate adjuvant-induced arthritis in rats. The present study was designed to investigate the effects of Tet on the migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and explore the underlying mechanisms. By using cultures of primary FLS isolated from synoviums of RA patients and cell line MH7A, Tet (0.3, 1 µmol·L(-1)) was proven to significantly impede migration and invasion of RA-FLS, but not cell proliferation. Tet also greatly reduced the activation and expressions of matrix degrading enzymes MMP-2/9, the expression of F-actin and the activation of FAK, which controlled the morphologic changes in migration process of FLS. To identify the key signaling pathways by which Tet exerts anti-migration effect, the specific inhibitors of multiple signaling pathways LY294002, Triciribine, SP600125, U0126, SB203580, and PDTC (against PI3K, Akt, JNK, ERK, p38 MAPK and NF-κB-p65, respectively) were used. Among them, LY294002, Triciribine, and SP600125 were shown to obviously inhibit the migration of MH7A cells. Consistently, Tet was able to down-regulate the activation of Akt and JNK as demonstrated by Western blotting assay. Moreover, Tet could reduce the expressions of migration-related proteins Rho GTPases Rac1, Cdc42, and RhoA in MH7A cells. In conclusion, Tet can impede the migration and invasion of RA-FLS, which provides a plausible explanation for its protective effect on RA. The underlying mechanisms involve the reduction of the expressions of Rac1, Cdc42, and RhoA, inhibition of the activation of Akt and JNK, and subsequent down-regulation of activation and/or expressions of MMP-2/9, F-actin, and FAK.
Assuntos
Artrite Reumatoide/metabolismo , Benzilisoquinolinas/farmacologia , Movimento Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Stephania/química , Membrana Sinovial/efeitos dos fármacos , Animais , Artrite , Artrite Reumatoide/prevenção & controle , Benzilisoquinolinas/uso terapêutico , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo , Fibroblastos/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Fosfatidilinositol 3-Quinases/metabolismo , Fitoterapia , Extratos Vegetais/uso terapêutico , Raízes de Plantas , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Membrana Sinovial/citologia , Membrana Sinovial/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Norisoboldine (NOR), the primary isoquinoline alkaloid constituent of the root of Lindera aggregata, has previously been demonstrated to attenuate osteoclast (OC) differentiation. Accumulative evidence has shown that aryl hydrocarbon receptor (AhR) plays an important role in regulating the differentiation of various cells, and multiple isoquinoline alkaloids can modulate AhR. In the present study, we explored the role of NOR in the AhR signaling pathway. These data showed that the combination of AhR antagonist resveratrol (Res) or α-naphthoflavone (α-NF) nearly reversed the inhibition of OC differentiation through NOR. NOR could stably bind to AhR, up-regulate the nuclear translocation of AhR, and enhance the accumulation of the AhR-ARNT complex, AhR-mediated reporter gene activity and CYP1A1 expression in RAW 264.7 cells, suggesting that NOR might be an agonist of AhR. Moreover, NOR inhibited the nuclear translocation of NF-κB-p65, resulting in the evident accumulation of the AhR-NF-κB-p65 complex, which could be markedly inhibited through either Res or α-NF. Although NOR only slightly affected the expression of HIF-1α, NOR markedly reduced VEGF mRNA expression and ARNT-HIF-1α complex accumulation. In vivo studies indicated that NOR decreased the number of OCs and ameliorated the bone erosion in the joints of rats with collagen-induced arthritis, accompanied by the up-regulation of CYP1A1 and the down-regulation of VEGF mRNA expression in the synovium of rats. A combination of α-NF nearly completely reversed the effects of NOR. In conclusion, NOR attenuated OC differentiation and bone erosion through the activation of AhR and the subsequent inhibition of both NF-κB and HIF pathways.
Assuntos
Alcaloides/farmacologia , Alcaloides/uso terapêutico , Artrite/metabolismo , Lindera/química , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Artrite/tratamento farmacológico , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Masculino , Camundongos , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Ligante RANK/farmacologia , Ratos , Ratos Wistar , Receptores de Hidrocarboneto Arílico/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Arctigenin is the main active ingredient of Fructus Arctii for the treatment of type 2 diabetes. In this study, the pharmacokinetics of arctigenin in normal and type 2 diabetic rats following oral and intravenous administration was investigated. As compared to normal rats, Cmax and AUC(0-10h) values of oral arctigenin in diabetic rats increased by 356.8% and 223.4%, respectively. In contrast, after intravenous injection, the Cmax and AUC(0-10h) values of arctigenin showed no significant difference between diabetic and normal rats. In order to explore how the bioavailability of oral arctigenin increased under diabetic condition, the absorption behavior of arctigenin was evaluated by in situ single-pass intestinal perfusion (SPIP). The results indicated that arctigenin was a substrate of P-glycoprotein (P-gp). The absorption difference of arctigenin in the normal and diabetic rats could be eliminated by the pretreatment of classic P-gp inhibitor verapamil, suggesting that P-gp might be the key factor causing the absorption enhancement of arctigenin in diabetic rats. Further studies revealed that the uptake of rhodamine 123 (Rho123) in diabetic rats was significantly higher, indicating that diabetes mellitus might impair P-gp function. Consistently, a lower mRNA level of P-gp in the intestine of diabetic rats was found. In conclusion, the absorption of arctigenin after oral administration was promoted in diabetic rats, which might be partially attribute to the decreased expression and impaired function of P-gp in intestines.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Furanos/farmacocinética , Absorção Intestinal , Lignanas/farmacocinética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Administração Intravenosa , Administração Oral , Animais , Disponibilidade Biológica , Masculino , Estrutura Molecular , Ratos , Ratos Sprague-DawleyRESUMO
This study aimed to explore the protective effects of madecassoside (Mad), a triterpenoid saponin isolated from Centella asiatica herbs, on experimental pulmonary fibrosis (PF) and underlying mechanisms. PF model was established in mice by endotracheal instillation with bleomycin (5 mg/kg). Mice were orally administered with Mad (10, 20, 40 mg/kg) and prednisone (5 mg/kg) for 7 or 21 days. Mad (20, 40 mg/kg) significantly improved lung pathological changes and reduced collagen deposition. In the aspect of collagen synthesis, Mad (20, 40 mg/kg) reduced the expressions of α-smooth muscle actin and transforming growth factor-ß1 (TGF-ß1), and inhibited the phosphorylations of Smad2 and Smad3 in the lung tissues. However, in vitro, Mad showed little effect on TGF-ß1-induced phosphorylation of either Smad2 or Smad3 in primary mouse lung fibroblasts. Moreover, Mad (20, 40 mg/kg) attenuated oxidative damage and inflammation presented at the early stage of PF, evidenced by reduced total leukocytes in the bronchoalveolar lavage fluid, decreased myeloperoxidase activity and malondialdehyde level, and increased super-oxide dismutase activity and glutathione level in lung tissues. On the other hand, Mad (40 mg/kg) elevated the matrix metalloproteinase 1/tissue inhibitor of metalloproteinase 1 ratio in lung tissues of PF mice mainly by downregulating tissue inhibitor of metalloproteinase 1 expression. The present study demonstrated that Mad can ameliorate PF by preventing the deposition of extracellular matrix, which might be achieved mainly through attenuating inflammation and oxidative stress and consequent TGF-ß1 overexpression.
Assuntos
Colágeno/metabolismo , Pulmão/efeitos dos fármacos , Fibrose Pulmonar/tratamento farmacológico , Triterpenos/farmacologia , Actinas/metabolismo , Animais , Bleomicina/efeitos adversos , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Pulmão/patologia , Metaloproteinase 13 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
To develop a simple and highly sensitive high performance liquid chromatography with electrospray ionization mass spectrometric (LC-ESI-MS) method for the simultaneous determination of madecassoside and its major metabolite madecassic acid in rat plasma, and compare the pharmacokinetics of the two compounds in normal and collagen-induced arthritis (CIA) rats. Glycyrrhetinic acid was used as the internal standard (IS). Chromatographic separation was accomplished on an Inertsil ODS-3 column, using a gradient elution with the mobile phase composed of acetonitrile and water acidified with 0.1% (V/V) formic acid. Detection was achieved by ESI-MS under the negative selected ion monitoring (SIM) mode. In normal and CIA rats, madecassoside (30 mg·kg(-1)) was orally administered for 21 consecutive days from the day of arthritis onset. For madecassoside, the linear range was 10-1 000 ng·mL(-1) with the square regression coefficient (r) of 0.998 9, while for madecassic acid, the linear range was 10-500 ng·mL(-1) with the square regression coefficient (r) of 0.996 1. The lower limit of quantification was 10 ng·mL(-1) for both analytes. The intra- and inter-day precision ranged from 1.78% to 13.42% for madecassoside and 2.30% to 14.90% for madecassic acid, and the accuracy was between -0.95% and 6.30% for madecassoside and between -1.48% and 5.34% for madecassic acid. The average recoveries of madecassoside, madecassic acid and IS from spiked plasma samples were > 81%. The developed method was successfully applied to the pharmacokinetic study of madecassoside and madecassic acid in rats after an oral administration of madecassoside. During initial 7 days of dosing, the cmax and AUC of madecassoside were greatly decreased and Vd/F was markedly increased in CIA rats, and no significant difference was observed on the first day of dosing. In contrast, the T1/2, cmax and AUC of madecassic acid were significantly increased, and Ke of madecassic acid was greatly decreased in CIA rats compared with normal rats. Along with repeated administration of madecassoside, the differences of pharmacokinetic parameters of both madecassoside and madecassic acid between CIA and normal rats gradually subsided. The pharmacokinetic characteristics of both madecassoside and madecassic acid in rats were significantly altered by arthritis status, and the differences of pharmacokinetic parameters between arthritis and normal rats coincide with the severity of arthritis.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Centella/química , Fitoterapia , Extratos Vegetais/farmacocinética , Triterpenos/sangue , Triterpenos/farmacocinética , Animais , Antirreumáticos/sangue , Antirreumáticos/farmacocinética , Antirreumáticos/uso terapêutico , Área Sob a Curva , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Cromatografia Líquida/métodos , Colágeno , Feminino , Extratos Vegetais/sangue , Extratos Vegetais/uso terapêutico , Ratos Wistar , Valores de Referência , Índice de Gravidade de Doença , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Triterpenos/uso terapêuticoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Centella asiatica (L.) Urb. herb is frequently used in traditional Chinese medicine for many indications, such as traumatic injuries, keloid and scar. Madecassoside is the main active ingredient of this herb drug with higher content than other triterpenoid constituents. Understandings of pharmacokinetic profiles of madecassoside should be beneficial for its development as a therapeutic agent. MATERIALS AND METHODS: Sprague-Dawley rats were intravenously and orally administered madecassoside (100 mg/kg), respectively. Plasma, heart, liver, spleen, lung, kidney, brain, bile, urine and feces were collected at the designed time points. Madecassoside concentrations in biological samples were determined by a sensitive and well-validated liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method. A liquid chromatography coupled with time-of-flight mass spectrometry (LC-TOF-MS) method was established to identify its major metabolites in feces. To further pursue the disposition characteristics of madecassoside in rats, the involvement of the hepatobiliary efflux transporters in biliary elimination were studied by combination with digoxin (P-glycoprotein inhibitor) or probenecid (multidrug resistance-associated protein inhibitor). A linked-rat model was also used to assess the role of enterohepatic circulation in the pharmacokinetics of madecassoside. RESULTS: After a single oral dosing, madecassoside was widely distributed in heart, liver, spleen, lung and kidney of rats, and the levels of madecassoside in liver and kidney were relatively higher than other organs. The excretions of madecassoside in bile, urine and feces were 7.16% (0-12 h), 0.25% (0-72 h) and 24.68% (0-72 h), respectively. The findings suggested that madecassoside might excrete mainly by metabolites. The combination with either digoxin or probenecid significantly attenuated the excretion of madecassoside as parent from bile, indicating that P-glycoprotein and multidrug resistance-associated protein might contribute to the hepatobiliary elimination of madecassoside. The presence of enterohepatic circulation, as implied by double-humped profiles in plasma and tissue concentration-time curves, was confirmed by a linked-rat model. Furthermore, three metabolities of madecassoside were indentified in rat feces and the possible metabolic pathways were proposed. CONCLUSIONS: These findings provide valuable information regarding in vivo process of madecassoside, and help us to recognize the efficacy and safety of madecassoside itself, the relevant herbs or herbal preparations.
Assuntos
Centella , Triterpenos/farmacocinética , Animais , Bile/química , Encéfalo/metabolismo , Fezes/química , Rim/metabolismo , Fígado/metabolismo , Pulmão/metabolismo , Miocárdio/metabolismo , Ratos , Ratos Sprague-Dawley , Baço/metabolismo , Distribuição Tecidual , Triterpenos/sangue , Triterpenos/urinaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: In the theory of traditional Chinese medicine, pulmonary fibrosis (PF) belongs to pulmonary arthralgia, which means blood stasis in lung tissue. The roots of Paeonia lactiflora Pall are usually used to relieve the symptoms of this disease by promoting blood circulation and removing blood stasis. Paeoniflorin, the main active ingredient of P. lactiflora, may have anti-PF potential. AIM OF STUDY: This study aimed to investigate the effects and underlying mechanisms of paeoniflorin on bleomycin (BLM)-induced PF in mice. MATERIALS AND METHODS: The PF model was established in mice by an intratracheal instillation of BLM. Paeoniflorin (25, 50, 100mg/kg) and prednisone (6mg/kg), as a positive control, were orally administered for consecutive 21 days. Histopathological changes were evaluated by hematoxylin and eosin stain and Masson's trichrome stain. The content of hydroxyproline was detected by using kits. The contents of type I collagen, TGF-ß1 and IFN-γ were detected by ELISA. The levels of α-SMA, Smad4, Smad7 and the phosphorylations of Smad2/3 were detected by western blot. The mRNA expressions of MMP-1 and TIMP-1 were detected by RT-PCR. RESULTS: In mice treated with BLM, paeoniflorin (50mg/kg) significantly prolonged the survival periods, attenuated infiltration of inflammatory cells, interstitial fibrosis, and deposition of extracellular matrix in lung tissues. It also decreased the contents of hydroxyproline (a marker of collagens), type I collagen and α-SMA (an indicator of myofibroblasts) in lung tissues of mice. Paeoniflorin down-regulated the expressions of TGF-ß1, Smad4 and the phosphorylations of Smad2/3, while up-regulated the expression of Smad7 in lung tissues. Moreover, paeoniflorin increased the content of IFN-γ. But, it only slightly affected mRNA expressions of MMP-1 and TIMP-1 in lung tissues of mice. CONCLUSIONS: Paeoniflorin attenuates PF by suppressing type I collagen synthesis via inhibiting the activation of TGF-ß/Smad pathway and increasing the expression of IFN-γ.
Assuntos
Benzoatos/uso terapêutico , Hidrocarbonetos Aromáticos com Pontes/uso terapêutico , Colágeno Tipo I/antagonistas & inibidores , Medicamentos de Ervas Chinesas/uso terapêutico , Glucosídeos/uso terapêutico , Pulmão/efeitos dos fármacos , Paeonia/química , Fibrose Pulmonar/tratamento farmacológico , Animais , Benzoatos/isolamento & purificação , Bleomicina/farmacologia , Hidrocarbonetos Aromáticos com Pontes/isolamento & purificação , Colágeno Tipo I/biossíntese , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/isolamento & purificação , Glucosídeos/isolamento & purificação , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Estrutura Molecular , Monoterpenos , Raízes de Plantas/química , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologiaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Puerarin is the most abundant isoflavonoid in Radix Puerariae (Gegen), which has been prescribed as a medicinal herb for treating fever in China for a long history. AIM OF THE STUDY: The present study aimed at evaluating the antipyretic effect of puerarin and revealing the related mechanisms. MATERIALS AND METHODS: Lipopolysaccharide (LPS)-induced fever in rats was used to assess the antipyretic effect of puerarin. After an intraperitoneal injection of LPS (100µg/kg), body temperature was tested every 30min up to 8h. Different doses of puerarin (25, 50, 100mg/kg) were intraperitoneally administered 30min before LPS injection. In vitro, LPS-stimulated RAW 264.7 cells were treated with various concentrations of puerarin (25-200µM). The pyrogenic mediators, including interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), prostaglandin E(2) (PGE(2)) and nitric oxide (NO), were examined on both transcription and expression levels. Furthermore, the influences of the activation of nuclear factor-kappa B (NF-κB) and the phosphorylation of mitogen-activated protein kinases (MAPKs) by puerarin were assayed by western blot. RESULTS: The intraperitoneal administration of puerarin at test doses clearly demonstrated apparent antipyretic effect through the declines in body temperature elevated by LPS in rats. The in vitro data showed that puerarin inhibited the production of IL-1ß, TNF-α, IL-6, PGE(2) and NO; moreover, the RT-PCR analysis and the western blot analysis indicated that puerarin regulated the transcriptional level via suppression of NF-κB activation and blockade of MAPK signal pathway. CONCLUSIONS: In summary, the antipyretic property of puerarin might result, at least in part, from an inhibition of endogenous pyrogen production and expression. Taken in this sense, our findings provide an explanation for puerarin acting as an important constituent in Gegen, thus, provide scientific basis for the wide use of Radix Puerariae in China as a traditional antipyretic.
Assuntos
Antipiréticos/farmacologia , Regulação da Temperatura Corporal/efeitos dos fármacos , Febre/tratamento farmacológico , Isoflavonas/farmacologia , Lipopolissacarídeos , Macrófagos/efeitos dos fármacos , Animais , Antipiréticos/administração & dosagem , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Febre/induzido quimicamente , Febre/genética , Febre/imunologia , Febre/fisiopatologia , Regulação da Expressão Gênica , Mediadores da Inflamação/metabolismo , Injeções Intraperitoneais , Isoflavonas/administração & dosagem , Macrófagos/imunologia , Masculino , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fosforilação , Fitoterapia , Plantas Medicinais , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transcrição GênicaRESUMO
The present study was performed to investigate the anti-septic effects of Qi-Shao-Shuang-Gan (QSSG), a combination of Astragalus membranaceus saponins (SAM) and Paeonia lactiflora glycosides (GPL), in septic mice induced by cecal ligation and puncture. QSSG was shown to elevate the survival rate of mice, decrease infiltration of polymorphonuclear leukocytes into livers and lungs, lower serum levels of myeloperoxidase, nitric oxide, and lactate dehydrogenase, and decrease mRNA expressions of inducible nitric oxide synthase and interleukin-1ß in livers. It also restored the impaired expressions of protein C (PC) mRNA in mouse livers and expressions of thrombomodulin and endothelial PC receptor mRNA in endothelial cells. Neither SAM nor GPL alone could significantly increase the survival rate of septic mice. The findings indicate that QSSG exerts protective action against polymicrobial sepsis by inhibiting systemic inflammatory response and upregulating PC pathway, and there are synergistic effects between SAM and GPL.
Assuntos
Medicamentos de Ervas Chinesas/uso terapêutico , Glicosídeos/uso terapêutico , Saponinas/uso terapêutico , Sepse/tratamento farmacológico , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Astragalus propinquus/química , Fatores de Coagulação Sanguínea/genética , Fatores de Coagulação Sanguínea/metabolismo , Ceco/cirurgia , Combinação de Medicamentos , Interleucina-1beta/genética , L-Lactato Desidrogenase/sangue , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Pulmão/imunologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Infiltração de Neutrófilos/efeitos dos fármacos , Óxido Nítrico/sangue , Óxido Nítrico Sintase/genética , Paeonia/química , Peroxidase/sangue , Proteína C/genética , Proteína C/metabolismo , Edema Pulmonar/prevenção & controle , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Aleatória , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Sepse/metabolismo , Sepse/patologia , Trombomodulina/genética , Trombomodulina/metabolismoRESUMO
Scopoletin is the main constituent of coumarin found in the stems of Erycibe obtusifolia Benth, a traditional Chinese medicine used in the treatment of rheumatoid arthritis. We have previously demonstrated that scopoletin is able to decrease the serum level of uric acid in hyperuricemic mice induced by potassium oxonate, and attenuate croton oil-induced inflammation. In the present study, we evaluated the anti-arthritic effects of scopoletin in rat adjuvant-induced arthritis by assessing paw swelling, pathology, and synovial angiogenesis. It was found that scopoletin, injected intraperitoneally at doses of 50, 100 mg/kg, reduced both inoculated and non-inoculated paw swelling as well as articular index scores, and elevated the mean body weight of adjuvant-induced arthritic rats. Rats treated with higher dose of scopoletin showed a near-normal histological architecture of the joints and a reduced new blood vessel formation in the synovial tissues. Furthermore, scopoletin downregulated the overexpression of vascular endothelial growth factor, basic fibroblast growth factor and interleukin 6 in the synovial tissues of adjuvant-induced arthritic rats. In conclusion, scopoletin is capable of ameliorating clinical symptoms of rat adjuvant-induced arthritis, by reducing numbers of new blood vessels in the synovium and the production of important endogenous angiogenic inducers. Therefore, this compound may be a potential agent for angiogenesis-related diseases and could serve as a structural base for screening more potent synthetic analogs.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Artrite Experimental/tratamento farmacológico , Convolvulaceae/química , Neovascularização Patológica/prevenção & controle , Escopoletina/uso terapêutico , Membrana Sinovial/efeitos dos fármacos , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/isolamento & purificação , Animais , Artrite Experimental/patologia , Imuno-Histoquímica , Masculino , Estrutura Molecular , Neovascularização Patológica/metabolismo , Caules de Planta/química , Ratos , Ratos Sprague-Dawley , Escopoletina/administração & dosagem , Escopoletina/isolamento & purificação , Membrana Sinovial/irrigação sanguínea , Membrana Sinovial/metabolismo , Membrana Sinovial/patologiaRESUMO
Sepsis remains the leading cause of death in intensive care units. Uncontrolled systemic inflammation and an impaired protein C pathway are two important contributors to sepsis pathophysiology. Based on the beneficial effects of the saponin fraction from Astragalus membranaceus roots (SAM) against inflammation, liver dysfunction, and endothelium injury, we investigated the potential protective roles and underlying mechanisms of SAM on polymicrobial sepsis induced by cecal ligation and puncture (CLP) in mice. SAM, orally administered 1 h before and after CLP, significantly elevated the survival rate of mice. At 96 h after CLP operation, all mice in the model group died, whereas 33.3% of mice in the SAM (400 mg/kg)-treated group survived. SAM attenuated both inflammatory factors and their abilities to induce tissue dysfunction, which was mainly evidenced by decreased infiltration of polymorphonuclear leukocytes, tissue edema, and lung wet-to-dry weight ratio, lowered levels of myeloperoxidase (MPO), nitric oxide (NO), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in serum, as well as downregulated expressions of iNOS and IL-1beta mRNA in livers. Furthermore, we addressed the effects of SAM on the protein C (PC) pathway, closely linked with sepsis. In CLP-induced septic mice, SAM elevated the impaired expression of PC mRNA in livers. In vitro, SAM reversed the decreased expressions of thrombomodulin (TM) and endothelial PC receptor (EPCR) mRNA induced by lipopolysaccharide (LPS) in endothelial cells. These findings suggest that SAM is able to restore the impaired protein C pathway. Taken together, the current study demonstrates that SAM has protective effects on polymicrobial sepsis in mice. The mechanisms of action involve anti-inflammation and upregulation of the PC pathway.
Assuntos
Astragalus propinquus/química , Extratos Vegetais/farmacologia , Raízes de Plantas/química , Proteína C/metabolismo , Saponinas/farmacologia , Sepse/prevenção & controle , Animais , Antígenos CD/genética , Ceco/lesões , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Receptor de Proteína C Endotelial , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Extratos Vegetais/química , Proteína C/genética , Punções , Receptores de Superfície Celular/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saponinas/química , Sepse/microbiologia , Sepse/mortalidade , Transdução de Sinais/efeitos dos fármacos , Trombomodulina/genéticaRESUMO
Sepsis continues to be a challenge in clinic. Therapeutic strategies focus on local host defenses and the inhibition of overwhelming inflammation response. The present study aimed to investigate the protective effects and the underlying mechanisms of the ethanol extract of Alpinia katsumadai Hayata seeds (EAKH) on polymicrobial sepsis induced by cecal ligation and puncture (CLP) in mice. It was shown that oral administration of EAKH at 1 h before and 2 h after CLP significantly elevated the survival rate and the mean arterial pressure of mice. Histological examination and serum ALT/AST assessment demonstrated that EAKH protected the animals from lung and liver tissue injury and dysfunction. Although EAKH was devoid of direct bacteriostatic or bacteriocidal activities, it facilitated peritoneal bacteria clearance and increased leukocyte migration into peritoneal cavity of septic mice. Furthermore, EAKH remarkably decreased serum pro-inflammatory cytokine (TNF-alpha, IL-1beta and NO) levels in septic mice. These findings demonstrated that EAKH has preventive effects on mouse sepsis induced by CLP, which may be attributed to elevating local defense via promoting leukocyte migration to infection focus and attenuating systemic inflammation.
Assuntos
Alpinia/química , Antibacterianos/farmacologia , Anti-Inflamatórios/farmacologia , Extratos Vegetais/farmacologia , Sepse/prevenção & controle , Animais , Antibacterianos/imunologia , Anti-Inflamatórios/imunologia , Ceco/lesões , Movimento Celular/imunologia , Modelos Animais de Doenças , Interleucina-1beta/imunologia , Masculino , Medicina Tradicional Chinesa , Camundongos , Camundongos Endogâmicos ICR , Infiltração de Neutrófilos/efeitos dos fármacos , Óxido Nítrico/imunologia , Extratos Vegetais/imunologia , Sepse/tratamento farmacológico , Sepse/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
OBJECTIVE: To study absorption kinetics of scopoletin in rat stomachs and intestines. METHOD: Rats was cannulated for in situ recirculation. UV and HPLC methods were used to determine the concentrations of phenolsulfonphthalein and scopoletin, respectively. RESULT: The absorption rates in rat stomachs at 2 h after administration was 76.31%; The absorption rates at colon, duodenum, ileum and jejunum were 46.25%, 40.54%, 38.21%, 32.77%, respectively. The absorption rate constant (Ka) at concentrations of 10.0144, 20.0288-40.0576 mg x L(-1) in intestine were 0.6434, 0.6137, 0.5970 h(-1), respectively. The Ka of scopoletin at pH of 6.0, 6.8 and 7.4 in intestine were 0.6217, 0.6033, 0.6137 h(-1), respectively. CONCLUSION: The concentrations and pH values of scopoletin solution had no distinctive effect on the absorption kinetics. The absorption of scopoletin was a first-order process with passive diffusion mechanism. Scopoletin was well absorbed at stomachs and intestines in rats. Colon was the best absorption site of scopoletin, which suggest that a sustained-release preparation should be suitable for this compound.