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BACKGROUND: The pathophysiology of Graves' disease (GD) involves imbalances between follicular helper T (Tfh) and follicular regulatory T (Tfr) cells, as well as oxidative stress (OS). Prunella vulgaris L. (Xia Ku Cao, XKC) and its primary bioactive compound, luteolin, are recognized for their potential in treating GD. Yet, the mechanism accounting for the immune-modulatory and antioxidant effects of XKC remains elusive. PURPOSE: This study aims to evaluate the pharmacological effects and elucidate the underlying mechanism of XKC and luteolin in a GD mouse model induced by recombinant adenovirus of TSH receptor A subunit (Ad-hTSHR-289). METHODS: High-Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (HPLC-QTOF MS) was used to detect the constituents of XKC. The GD model was established through inducing female BALB/c mice with three intramuscular injections of Ad-TSHR-289. Thyroid function, autoantibody and OS parameters were measured by ELISA. Changes of Tfh cells and Tfr cells were detected by flow cytometry. RT-qPCR, Western Blotting, immunohistochemistry were used to explore the related molecular mechanisms. RESULTS: A total of 37 chemical components from XKC were identified by HPLC-QTOF MS, represented by flavonoids, steroids, terpenoids, and luteolin. XKC and luteolin reduced T4, TRAb levels and facilitated the recovery from thyroid damage in GD mice. Meanwhile, XKC and luteolin effectively alleviated OS by decreasing the levels of MDA, NOX2, 4-HNE, 8-OHdG, while increasing GSH level. Flow cytometry showed that XKC and luteolin restored the abnormal proportions of Tfh/Tfr and Tfh/Treg, and the mRNA levels of IL-21, Bcl-6 and Foxp3 in GD mice. In addition, XKC and luteolin inhibited PI3K, Akt, p-PI3K and p-Akt, but activated Nrf2 and HO-1. CONCLUSION: XKC and luteolin could inhibit the development of GD in vivo by rebalancing Tfh/Tfr cells and alleviating OS. This therapeutic mechanism may involve the Nrf2/HO-1 and PI3K/Akt signaling pathways. Luteolin is the main efficacy material basis of XKC in countering GD. For the first time, we revealed the mechanism of XKC and luteolin in the treatment of GD from the perspective of autoimmune and OS.
Assuntos
Modelos Animais de Doenças , Doença de Graves , Luteolina , Camundongos Endogâmicos BALB C , Estresse Oxidativo , Prunella , Linfócitos T Reguladores , Animais , Luteolina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Doença de Graves/tratamento farmacológico , Feminino , Linfócitos T Reguladores/efeitos dos fármacos , Prunella/química , Camundongos , Células T Auxiliares Foliculares/efeitos dos fármacos , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Glândula Tireoide/efeitos dos fármacos , Antioxidantes/farmacologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacosRESUMO
In contrast to the decreasing trends in developed countries, the incidence and mortality rates of cervical squamous cell carcinoma in China have increased significantly. The screening and identification of reliable biomarkers and candidate drug targets for cervical squamous cell carcinoma are urgently needed to improve the survival rate and quality of life of patients. In this study, we demonstrated that the expression of MUC1 was greater in neoplastic tissues than in non-neoplastic tissues of the cervix, and cervical squamous cell carcinoma patients with high MUC1 expression had significantly worse overall survival than did those with low MUC1 expression, indicating its potential for early diagnosis of cervical squamous cell carcinoma. Next, we explored the regulatory mechanism of MUC1 in cervical squamous cell carcinoma. MUC1 could upregulate ITGA2 and ITGA3 expression via ERK phosphorylation, promoting the proliferation and metastasis of cervical cancer cells. Further knockdown of ITGA2 and ITGA3 significantly inhibited the tumorigenesis of cervical cancer cells. Moreover, we designed a combination drug regimen comprising MUC1-siRNA and a novel ERK inhibitor in vivo and found that the combination of these drugs achieved better results in animals with xenografts than did MUC1 alone. Overall, we discovered a novel regulatory pathway, MUC1/ERK/ITGA2/3, in cervical squamous cell carcinoma that may serve as a potential biomarker and therapeutic target in the future.
MUC1 is overexpressed in cervical squamous cell carcinoma. MUC1 regulates ERK phosphorylation, and subsequently upregulates ITGA2 and ITGA3 expression to promote tumorigenesis in cervical squamous cell carcinoma. A combination drug regimen targeting MUC1 and ERK achieved better results compared than MUC1 alone.
Assuntos
Carcinoma de Células Escamosas , Proliferação de Células , Integrina alfa2 , Integrina alfa3 , Mucina-1 , Neoplasias do Colo do Útero , Humanos , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico , Feminino , Integrina alfa2/metabolismo , Integrina alfa2/genética , Animais , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/tratamento farmacológico , Mucina-1/metabolismo , Mucina-1/genética , Camundongos , Fosforilação , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Ensaios Antitumorais Modelo de Xenoenxerto , Sistema de Sinalização das MAP Quinases , Camundongos Nus , MAP Quinases Reguladas por Sinal Extracelular/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: For numerous years, the Xiehuo Xiaoying decoction (XHXY), a traditional Chinese medicine formula, has demonstrated substantial promise in treating Graves' disease (GD) in clinical settings, showcasing significant potential. However, the therapeutic mechanism and efficacy material basis of XHXY remains obscure. AIM OF THE STUDY: This work aims to investigate the underlying mechanisms and to study the efficacy material basis of XHXY in anti-GD effect using a combination of TMT quantitative proteomics and molecular docking method. MATERIALS AND METHODS: GD model was initiated by administering Ad-TSH289. Subsequently, the mice underwent a four-week regimen that included oral gavage of XHXY at doses of 17 g/kg·d and 34 g/kg·d, along with intraperitoneal injections of Gentiopicroside (GPS). Utilizing the principles of pharmacological chemistry in traditional Chinese medicine, we employed high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-QTOF/MS) to discern prescribed prototype composition of XHXY in serum samples from mouse. TMT proteomics research provided evidence of XHXY's putative targets and important pathways in vivo. The binding activity of probable action targets and prototype composition was detected by molecular docking. Finally, Immunohistochemistry (IHC) and TUNEL staining were used to verify the mechanism of XHXY and GPS in anti-GD. RESULTS: XHXY and GPS alleviated GD by ameliorating the pathological changes and reducing thyroxine and TRAb levels. In mouse serum, a total of 31 prototypical XHXY ingredients were detected, and the majority of these components were from monarch and minister medicine. Proteomics study results indicated that the XHXY may mainly regulate targets including FAS-associated death domain protein (FADD), Apolipoprotein C-III, etc. and main pathways are Apoptosis, Cholesterol metabolism, TNF signalling pathway, etc. Strong binding activity of the prototypical active ingredient and GPS towards FADD, Caspase 8, and Caspase 3 was demonstrated by molecular docking. XHXY and its primary component, GPS, elevated the expression of FADD, Caspase 8, and Caspase 3, and enhance apoptosis in thyroid cells, as lastly validated by TUNEL and IHC staining. CONCLUSIONS: XHXY exhibits a favorable therapeutic effect in treating GD by promoting apoptosis in thyroid cells through the upregulation of FADD, Caspase 8, and Caspase 3 expression. And GPS is the main efficacy material basis for its therapeutic effect in anti-GD.
Assuntos
Medicamentos de Ervas Chinesas , Doença de Graves , Animais , Camundongos , Caspase 3/metabolismo , Caspase 8/metabolismo , Simulação de Acoplamento Molecular , Proteômica , Doença de Graves/tratamento farmacológico , Doença de Graves/metabolismo , Apoptose , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêuticoRESUMO
Cholestatic liver disease is characterized by disturbances in the intestinal microbiota and excessive accumulation of toxic bile acids (BA) in the liver. Melatonin (MT) can improve liver diseases. However, the underlying mechanism remains unclear. This study aimed to explore the mechanism of MT on hepatic BA synthesis, liver injury, and fibrosis in 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed and Mdr2-/- mice. MT significantly improved hepatic injury and fibrosis with a significant decrease in hepatic BA accumulation in DDC-fed and Mdr2-/- mice. MT reprogramed gut microbiota and augmented fecal bile salt hydrolase activity, which was related to increasing intestinal BA deconjugation and fecal BA excretion in both DDC-fed and Mdr2-/- mice. MT significantly activated the intestinal farnesoid X receptor (FXR)/fibroblast growth factor 15 (FGF-15) axis and subsequently inhibited hepatic BA synthesis in DDC-fed and Mdr2-/- mice. MT failed to improve DDC-induced liver fibrosis and BA synthesis in antibiotic-treated mice. Furthermore, MT provided protection against DDC-induced liver injury and fibrosis in fecal microbiota transplantation mice. MT did not decrease liver injury and fibrosis in DDC-fed intestinal epithelial cell-specific FXR knockout mice, suggesting that the intestinal FXR mediated the anti-fibrosis effect of MT. In conclusion, MT ameliorates cholestatic liver diseases by remodeling gut microbiota and activating intestinal FXR/FGF-15 axis-mediated inhibition of hepatic BA synthesis and promotion of BA excretion in mice.
Assuntos
Colestase , Hepatopatias , Melatonina , Camundongos , Animais , Melatonina/farmacologia , Melatonina/metabolismo , Fígado/metabolismo , Colestase/tratamento farmacológico , Colestase/metabolismo , Colestase/patologia , Hepatopatias/metabolismo , Hepatopatias/patologia , Ácidos e Sais Biliares/metabolismo , Ácidos e Sais Biliares/farmacologia , Camundongos Knockout , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Camundongos Endogâmicos C57BLRESUMO
OBJECTIVE: To investigate the applicability of carboxymethyl starch (CMS) as a carrier to prepare solid dispersions (SDs) of piperine (PIP) and 18ß-glycyrrhetinic acid (ß-GA) (PIP-CMS and ß-GA-CMS SDs) and to explore the influence of drug properties on carrier selection. SIGNIFICANCE: The low oral bioavailability of natural therapeutic molecules, including PIP and ß-GA, severely restricts their pharmaceutical applications. Moreover, CMS, a natural polymer, is rarely reported as a carrier for SDs. METHODS: PIP-CMS and ß-GA-CMS SDs were prepared using the solvent evaporation method. Differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), Fourier transform infrared (FT-IR) spectroscopy, and scanning electron microscopy (SEM) were used for formulation characterization. Additionally, drug release characteristics were investigated. RESULTS: In vitro dissolution studies showed that the dissolutions of PIP-CMS and ß-GA-CMS SDs were 1.90-2.04 and 1.97-2.22 times higher than pure PIP and ß-GA, respectively, at a drug:polymer ratio of 1:6. DSC, XRPD, FT-IR, and SEM analyses confirmed the formation of SDs in their amorphous states. Significant improvements in Cmax and AUC0-24 h of PIP-CMS and ß-GA-CMS SDs (17.51 ± 8.15 µg/mL and 210.28 ± 117.13 µg·h/mL, respectively) and (32.17 ± 9.45 µg/mL and 165.36 ± 38.75 µg·h/mL, respectively) were observed in the pharmacokinetic study. Compared with weakly acidic ß-GA, loading weakly basic PIP seemed to have a profound effect on stability through intermolecular forces. CONCLUSIONS: Our findings showed CMS could be a promising carrier for SDs, and loading weakly basic drug may be more suitable, especially in binary SDs system.
Assuntos
Excipientes , Polímeros , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Disponibilidade Biológica , Solubilidade , Excipientes/química , Varredura Diferencial de CalorimetriaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Xiehuo Xiaoying decoction (XHXY) has shown great potential in the treatment of GD, but its mechanism remains obscure. Increase of follicular helper T (Tfh) cells and reduction of follicular regulatory T (Tfr) cells contribute to a high thyrotropin receptor antibodies (TRAb) level and possible Graves' disease (GD). Oxidative stress (OS) disrupts T helper cell differentiation and aggravates autoimmunity. AIM OF THE STUDY: This study aimed to investigate whether XHXY decoction can ameliorate autoimmunity in GD via inhibiting OS and regulating Tfh and Tfr cells. MATERIALS AND METHODS: The main XHXY bioactive compounds were identified using high-performance liquid chromatography quadrupole time-of-flight mass spectrometry. GD was induced in the mice through three intramuscular injections of adenovirus expressing the TSH receptor. Then, the mice received oral gavage of XHXY (17 g/kg·d) and 34 g/kg·d) for 4 weeks. OS indicators were assessed. Flow cytometry was used to confirm the proportion of Tfh and Tfr cells in the lymph nodes and spleens of the mice. Cytokine expression levels were determined using enzyme-linked immunosorbent assay. Factors including interleukin-21, B-cell lymphoma-6, and forkhead box P3 (Foxp3) were detected using quantitative polymerase chain reaction. The mRNA and protein expression levels of Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid-2-related factor 2 (Nrf2), and haem oxygenase 1 (HO-1) were detected using quantitative polymerase chain reaction and Western blotting, respectively. RESULTS: Twelve main ingredients of XHXY were identified. XHXY relieved GD by lowering thyroxine (p < 0.01) and TRAb levels (p < 0.01). XHXY ameliorated OS by decreasing the levels of NADPH oxidase 2 (p < 0.05), 4-hydroxynonenal (p < 0.01), and 8-oxo-2'-deoxyguanosine (p < 0.001). It inhibited Tfh cell expansion (p < 0.05), as well as the production of cytokine interleukin -21 (p < 0.01), interleukin -4 (p < 0.01) and transcription factor B-cell lymphoma 6 (p < 0.05). XHXY also induced Tfr cell amplification (p < 0.05), increased the production of interleukin -10 (p < 0.05) and transforming growth factor ß (p < 0.05) and the mRNA levels of Foxp3 (p < 0.05). Finally, the Tfh/Tfr ratio returned to normal. In addition, XHXY activated Nrf2 and HO-1 expression, but inhibited Keap1 activation. CONCLUSIONS: XHXY relieves autoimmunity in GD via inhibiting Tfh cell amplification and Tfr cell reduction, a mechanism which probably involves the Keap1/Nrf2 signaling pathway.
Assuntos
Doença de Graves , Linfoma de Células B , Animais , Camundongos , Citocinas/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Doença de Graves/tratamento farmacológico , Doença de Graves/metabolismo , Interleucinas/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Linfoma de Células B/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , RNA Mensageiro/metabolismo , Células T Auxiliares Foliculares , Linfócitos T Auxiliares-Indutores , Linfócitos T Reguladores , Medicina Tradicional ChinesaRESUMO
BACKGROUND: Acute cerebral infarction is a severe type of ischemic stroke that can be divided into anterior circulation cerebral infarction and posterior circulation cerebral infarction (PCCI). PCCI affects the structure of the posterior circulation brain, because posterior part of the brain, which has more complex anatomical structures and more prone to posterior circulation vascular variation. Therefore, improving the prognosis of PCCI patients is necessary. AIM: To explore the effect of medical care linkage-continuous management mode (MCLMM) on endovascular interventional therapy (EIT) for PCCI. METHODS: Sixty-nine patients with PCCI who received EIT and conventional nursing intervention were selected as the control group, and 78 patients with PCCI who received EIT and MCLMM intervention were selected as the observation group. The incidence of postoperative complications, compliance and disease self-management behavior after six months of intervention, modified Rankin scale (mRS) and Barthel index (BI) scores in the acute phase and after one year of intervention, and recurrence within one year were compared between the two groups. RESULTS: The total incidence rate of postoperative complications in the observation group (7.69%) was lower than that in the control group (18.84%) (P < 0.05). The scores for medical compliance behavior (regular medication, appropriate diet, and rehabilitation cooperation rates) and disease self-management behavior (self-will, disease knowledge, and self-care ability) in the observation group were higher than those in the control group (P < 0.05). After one year of intervention, in the observation group, the mRS score was significantly lower, and the BI score was significantly higher than those in the control group (P < 0.05). The recurrence rate within one year in the observation group (3.85%) was significantly lower than that in the control group (13.04%) (P < 0.05). CONCLUSION: MCLMM can reduce the incidence of complications after EIT for PCCI, improve patient compliance behavior and disease self-management ability, and promote the recovery of neurological function.
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Corydalis Bungeanae Herba is often used to treat a variety of inflammatory diseases in traditional Chinese medicine. In order to determine its chemical material basis, the components of Corydalis Bungeanae Herba were isolated by automated purification system. Flavonoids and alkaloids were prepared, and all such components were identified by mass spectrometry. The effects of the components on the production of inflammatory mediators and pharmacological mechanisms in the lipopolysaccharide(LPS)-induced RAW264.7 cell inflammation model were examined. Mouse macrophages(RAW264.7) were first treated with LPS. The relationship between cell viability and LPS concentration was observed. Then, the effects of flavonoids components and alkaloid components with different administration concentrations on cell viability were detected to determine the maximum administration concentration. Secondly, 2.5, 5, 10 and 20 µg·mL~(-1) flavonoids components and alkaloid components were added respectively to observe the effects and mechanism of different concentrations of flavonoids components and alkaloid components on LPS-induced inflammation of RAW264.7 macrophages. Griess reagent assay was used to detect NO content in cell supernatant. The inflammatory cytokines(TNF-α, IL-1ß and IL-6) in cell supernatant were determined by ELISA method. Western blot method was used to detect the intracellular nuclear factor(NF-κB) IκBα phosphorylation(p-IκBα), p65 phosphorylation(p-p65) and protein expression of TLR4, TLR2. The results showed that the alkaloid components inhibited the production of NO, TNF-α, IL-1ß and IL-6 in a dose-dependent mannerin the concentration range of 2.5-20 µg·mL~(-1). In inflammation upstream pathways, the inhibitory effect of the alkaloid components on the TLR2 expression level was weaker than that of TLR4. In inflammation downstream, alkaloid components significantly inhibited phosphorylation of IκBα and p65 in a dose-dependent manner. These data suggested that the alkaloid components were the material basis components of Corydalis Bungeanae Herba, and its anti-inflammatory mechanism might be related to inhibiting the transmission of inflammatory signals in TLRs/NF-κB signaling pathways dominated by TLR4, interfering with the activation of inflammatory genes and inhibiting their over expression, and down-regulating the secretion level of inflammatory factors.
Assuntos
Corydalis , Animais , Anti-Inflamatórios/uso terapêutico , Inflamação/tratamento farmacológico , Lipopolissacarídeos , Camundongos , NF-kappa B , Células RAW 264.7RESUMO
BACKGROUND AND AIMS: Cholestatic liver disease is characterized by gut dysbiosis and excessive toxic hepatic bile acids (BAs). Modification of gut microbiota and repression of BA synthesis are potential strategies for the treatment of cholestatic liver disease. The purpose of this study was to examine the effects and to understand the mechanisms of the probiotic Lactobacillus rhamnosus GG (LGG) on hepatic BA synthesis, liver injury, and fibrosis in bile duct ligation (BDL) and multidrug resistance protein 2 knockout (Mdr2-/- ) mice. APPROACH AND RESULTS: Global and intestine-specific farnesoid X receptor (FXR) inhibitors were used to dissect the role of FXR. LGG treatment significantly attenuated liver inflammation, injury, and fibrosis with a significant reduction of hepatic BAs in BDL mice. Hepatic concentration of taurine-ß-muricholic acid (T-ßMCA), an FXR antagonist, was markedly increased in BDL mice and reduced in LGG-treated mice, while chenodeoxycholic acid, an FXR agonist, was decreased in BDL mice and normalized in LGG-treated mice. LGG treatment significantly increased the expression of serum and ileum fibroblast growth factor 15 (FGF-15) and subsequently reduced hepatic cholesterol 7α-hydroxylase and BA synthesis in BDL and Mdr2-/- mice. At the molecular level, these changes were reversed by global and intestine-specific FXR inhibitors in BDL mice. In addition, LGG treatment altered gut microbiota, which was associated with increased BA deconjugation and increased fecal and urine BA excretion in both BDL and Mdr2-/- mice. In vitro studies showed that LGG suppressed the inhibitory effect of T-ßMCA on FXR and FGF-19 expression in Caco-2 cells. CONCLUSION: LGG supplementation decreases hepatic BA by increasing intestinal FXR-FGF-15 signaling pathway-mediated suppression of BA de novo synthesis and enhances BA excretion, which prevents excessive BA-induced liver injury and fibrosis in mice.
Assuntos
Ácidos e Sais Biliares , Colestase , Fatores de Crescimento de Fibroblastos/metabolismo , Lacticaseibacillus rhamnosus/metabolismo , Cirrose Hepática , Receptores Citoplasmáticos e Nucleares , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Ácidos e Sais Biliares/biossíntese , Ácidos e Sais Biliares/metabolismo , Ácido Quenodesoxicólico/farmacologia , Colestase/complicações , Colestase/metabolismo , Colestase/terapia , Ácidos Cólicos/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Humanos , Intestinos/microbiologia , Cirrose Hepática/etiologia , Cirrose Hepática/prevenção & controle , Camundongos , Camundongos Knockout , Probióticos/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
OBJECTIVES: High fructose feeding changes fibroblast growth factor 21 (FGF21) regulation. Lactobacillus rhamnosus GG (LGG) supplementation reduces fructose-induced non-alcoholic fatty liver disease (NAFLD). The aim of this study was to determine the role of FGF21 and underlying mechanisms in the protective effects of LGG. METHODS: FGF21 knockout (KO) mice and C57BL/6 wild type (WT) mice were fed 30% fructose for 12 weeks. LGG was administered to the mice in the last 4 weeks during fructose feeding. FGF21-adiponectin (ADPN)-mediated hepatic lipogenesis and inflammation were investigated. RESULTS: FGF21 expression was robustly increased after 5-weeks of feeding and significantly decreased after 12-weeks of feeding in fructose-induced NAFLD mice. LGG administration reversed the depressed FGF21 expression, increased adipose production of ADPN, and reduced hepatic fat accumulation and inflammation in the WT mice but not in the KO mice. Hepatic nuclear carbohydrate responsive-element binding protein (ChREBP) was increased by fructose and reduced by LGG, resulting in a reduction in the expression of lipogenic genes. The methylated form of protein phosphatase 2A (PP2A) C, which dephosphorylates and activates ChREBP, was upregulated by fructose and normalized by LGG. Leucine carboxyl methyltransferase-1, which methylates PP2AC, was also increased by fructose and decreased by LGG. However, those beneficial effects of LGG were blunted in the KO mice. Hepatic dihydrosphingosine-1-phosphate, which inhibits PP2A, was markedly increased by LGG in the WT mice but attenuated in the KO mice. LGG decreased adipose hypertrophy and increased serum levels of ADPN, which regulates sphingosine metabolism. This beneficial effect was decreased in the KO mice. CONCLUSION: LGG administration increases hepatic FGF21 expression and serum ADPN concentration, resulting in a reduced ChREBP activation through dihydrosphingosine-1-phosphate-mediated PP2A deactivation, and subsequently reversed fructose-induced NAFLD. Thus, our data suggest that FGF21 is required for the beneficial effects of LGG in reversal of fructose-induced NAFLD.
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Dieta da Carga de Carboidratos , Fatores de Crescimento de Fibroblastos/metabolismo , Lacticaseibacillus rhamnosus/fisiologia , Hepatopatia Gordurosa não Alcoólica/patologia , Proteínas Quinases Ativadas por AMP/metabolismo , Adiponectina/sangue , Adiponectina/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Modelos Animais de Doenças , Feminino , Fatores de Crescimento de Fibroblastos/deficiência , Fatores de Crescimento de Fibroblastos/genética , Peroxidação de Lipídeos , Lipogênese , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/metabolismo , Proteína Fosfatase 2/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Triglicerídeos/metabolismoRESUMO
In this paper,ultra performance liquid chromatography coupled with time-of-flight mass spectrometry( UPLC-Q-TOFMS) technique was used to study the effects of steamed notoginseng on endogenous markers in plasma of rats with hemolytic anemia induced by N-acetyl phenyl hydrazine( APH). The aim was to find out the potential biomarkers and possible blood enriching mechanism of steamed notoginseng on hemolytic anemia rats. In the experiment,steamed notoginseng medicine pair( steamed notoginseng-ginseng)and compound medicines( Sanqi Yangxue Capsules) were used respectively to intervene in APH-induced hemolytic anemia model rats.Then blood routine indexes such as red blood cells( RBC),hemoglobin( Hb) and related organ indexes were determined. As compared with the blank group,the RBC and Hb levels in the model group were substantially decreased( P< 0. 01),while the liver and spleen organ indexes were increased( P< 0. 05). The results of blood routine and organ index demonstrated that the blood deficiency model was successfully established. Steamed notoginseng can significantly increase the RBC level of rats( P<0. 01),and the related indicators of each drug group had a trend of returning to normal levels,verifying the blood enriching effect of steamed notoginseng. The UPLC-Q-TOF-MS technique,principal component analysis( PCA) and partial least squares-discrimination analysis( PLS-DA) were used to analyze the metabolic profiles between the normal group and the model group. Twenty-six potential biomarkers for hemolytic anemia were screened in plasma. Nine metabolites such as retinol,L-valine,and arachidonic acid were down-regulated in the blood deficiency rats,and 17 metabolites such as protoporphyrin â ¨ and niacinamide were up-regulated. The metabolic level of biomarkers could be changed to a normal state after rats were given with steamed notoginseng,drug pairs,and compound prescriptions. It can be speculated that steamed notoginseng may play a role of blood tonifying by improving biosynthesis of valine,leucine and isoleucine,as well as metabolic pathways such as retinol metabolism and arachidonic acid metabolism.
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Anemia Hemolítica/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Metabolômica , Panax notoginseng/química , Animais , Biomarcadores , Espectrometria de Massas , Metaboloma , Ratos , VaporRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Gynura segetum (GS) is an herbal medicine containing Pyrrolizidine Alkaloids (PAs) that causes hepatic sinusoidal obstruction syndrome (HSOS). AIM OF THE STUDY: To discover potential biomarkers and metabolic mechanisms involved in the hepatotoxicity induced by GS. METHODS: SD rats were randomly divided into 4 groups including Saline, the decoction of GS high, medium and low dosage at dosages of 3.75g ⢠kg-1, 7.5g ⢠kg-1 and 15g ⢠kg-1. A metabolomics approach using Ultraperformance Liquid Chromatography -Quadrupole-Time-of-Flight / Mass Spectrometry (UPLC-Q-TOF/MS) was developed to perform the plasma and urinary metabolic profiling analysis, and identified differential metabolites by comparing the saline control group and decoction of GS groups. RESULTS: The herbal was presented dosage-dependent led to ingravescence of hepatotoxicity after the rats were consecutively given with the decoction of GS at varied dosages. A total of 18 differential metabolites of decoction of GS-induced hepatotoxicity were identified, while 10 of them including arginine, proline, glutamate, creatine, valine, linoleic acid, arachidonic acid, sphinganine, phytosphingosine, and citric acid could be discovered in urine and plasma, and primarily involved in Amino acid metabolism, Lipids metabolism and Energy metabolism. CONCLUSIONS: The results suggested that the differential metabolites of arginine, creatine, valine, glutamine and citric acid were verified as potential markers of GS-induced hepatotoxicity via the regulation of multiple metabolic pathways primarily involving in Amino acids metabolism and Energy metabolism.
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Doença Hepática Induzida por Substâncias e Drogas/sangue , Doença Hepática Induzida por Substâncias e Drogas/urina , Medicamentos de Ervas Chinesas/toxicidade , Fígado/efeitos dos fármacos , Fígado/metabolismo , Metabolômica/métodos , Aminoácidos/sangue , Aminoácidos/urina , Animais , Biomarcadores/sangue , Biomarcadores/urina , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Metabolismo Energético/efeitos dos fármacos , Feminino , Masculino , Fitoterapia , Plantas Medicinais/toxicidade , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Ginsenosides F4 and Rg6 (GF4 and GRg6), two main active components of steamed notoginseng or red ginseng, are dehydrated disaccharide saponins. In this work, biotransformation of ginsenosides F4 and Rg6 in zebrafish was investigated by qualitatively identifying their metabolites and then proposing their possible metabolic pathways. The prediction of possible metabolism of ginsenosides F4 and Rg6 using zebrafish model which can effectively simulate existing mammals model was early and quickly performed. Metabolites of ginsenosides F4 and Rg6 after exposing to zebrafish for 24 h were identified by Ultraperformance Liquid Chromatography/Quadrupole-Time-of-Flight Mass Spectrometry. A total of 8 and 6 metabolites of ginsenosides F4 and Rg6 were identified in zebrafish, respectively. Of these, 7 and 5, including M1, M3-M5, M7-M9 and N1 (N5), N2, N4 (N9), N7-N8 were reported for the first time as far as we know. The mechanisms of their biotransformation involved were further deduced to be desugarization, glucuronidation, sulfation, dehydroxylation, loss of C-17 and/or C-23 residue pathways. It was concluded that loss of rhamnose at position C-6 and glucuronidation at position C-3 in zebrafish were considered as the main physiologic and metabolic processes of ginsenosides F4 and ginsenosides Rg6, respectively.
Assuntos
Ginsenosídeos/metabolismo , Peixe-Zebra/metabolismo , Animais , Biotransformação , Feminino , Masculino , Panax/química , Extratos Vegetais/químicaRESUMO
UPLC-QTOF-MS/MS was used to identify metabolites in rat blood, urine and feces after the administration of n-butanol extract derived from steamed notoginseng. The metabolic process of saponins came from steamed notoginseng was analyzed. The metabolites were processed by PeakView software, and identified according to the structural characteristics of prototype compounds and the accurate qualitative and quantitative changes of common metabolic pathways. Four saponins metabolites were identified based on MS/MS information of metabolites, namely ginsenoside Rh4, Rk3, Rk1, Rg5,and their 15 metabolites were verified. The metabolic pathways of the four ginsenosides in n-butanol extract included glucuronidation, desugar, sulfation, dehydromethylation, and branch loss. The metabolites of main active saponin components derived from steamed Panax notoginseng were analyzed from the perspective of qualitative analysis. And the material basis for the efficacy of steamed notoginseng was further clarified.
Assuntos
Ginsenosídeos/farmacocinética , Panax notoginseng/química , Saponinas/farmacocinética , Animais , Sangue , Cromatografia Líquida de Alta Pressão , Fezes , Ratos , Espectrometria de Massas em Tandem , UrinaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Nauclea officinalis Pierrc ex Pitard. is a Chinese medicinal herb that contains high level of alkaloids which is the most abundant and active constituent. Strictosamide isolated from Nauclea officinalis Pierrc ex Pitard. showed significant effects on inflammatory response, compared with pumiloside, 3-epi-pumiloside, vincosamide, 3α,5α-tetrahydrodeoxycordifoline lactam and naucleamide A-10-O-ß-D-glucopyranoside of this plant. AIM OF STUDY: we investigated the biological activities of the six compounds mentioned-above, and the underlying molecular mechanism exerted by the most potent one, strictosamide. MATERIALS AND METHODS: The effects of strictosamide and other five compounds on the inhibitory activity of nitric oxide (NO) were screened by Griess test. The contents of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in media were detected by using Enzyme-linked immunosorbent (ELISA) kits. The effects on the mRNA expression of nitric oxide synthase (iNOS), TNF-α and IL-1ß of strictosamide were further investigated by RT-qPCR. Western blot assay was conducted to illustrate the effects of strictosamide on iNOS and phosphorylation of p65, inhibitor of NF-κB (IκB)-α, IκB-kinase (IKK)-α as well as p-extracellular signal-regulated kinase (ERK), p-c-jun N-terminal kinase (JNK) and p-p38 in the protein levels. RESULTS: Strictosamide potently suppressed the productions of NO, TNF-α and IL-1ß in LPS-induced RAW 264.7 macrophages, and it dose-dependently alleviated the LPS-simulated protein level of iNOS as well as the mRNA expressions of iNOS, TNF-α and IL-1ß. In addition, molecular data revealed that strictosamide markedly decreased the expressions of p-p65, p-IκBα and p-IKKα. Furthermore, strictosamide significantly attenuated LPS-induced the phosphorylation of p38, ERK and JNK. CONCLUSIONS: At present study, the results indicated that the anti-inflammatory activity of strictosamide was associated with the restraint of NO, TNF-α and IL-1ß via negative regulation of both NF-κB and mitogen-activated protein kinases (MAPKs) in LPS-induced RAW 264.7 cells.
Assuntos
Alcaloides/farmacologia , Anti-Inflamatórios/farmacologia , Macrófagos/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Rubiaceae , Animais , Sobrevivência Celular/efeitos dos fármacos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopolissacarídeos , Macrófagos/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Fosforilação/efeitos dos fármacos , Células RAW 264.7 , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Alcaloides de VincaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Corydalis bungeana Turcz. (C. bungeana) is one of traditionally used medicines in China and possesses various biological effects, such as anti-inflammatory, antibacterial activity and inhibition of the immune function of the host. AIM OF THE STUDY: we studied the anti-inflammatory effect and molecular mechanism involved of C. bungeana both in vitro and in vivo model system in which the inflammatory response was induced by LPS treatment. MATERIALS AND METHODS: Anti-inflammatory activity of C. bungeana was investigated by LPS-induced RAW 264.7 macrophages and BALB/c mice. The production and expression of pro-inflammatory cytokines were evaluated by Griess reagent, ELISA kits and RT-qPCR, respectively. Phosphorylation status of IκBα and p65 was illustrated by western blot assay. RESULTS: C. bungeana reduced the secretion of NO, TNF-α, IL-6 and IL-1ß through inhibiting the protein expression of iNOS, TNF-α, IL-6 and IL-1ß in vitro and in vivo. Western blot analysis suggested that C. bungeana supressed NF-κB activation via regulating the phosphorylation of IκBα and p65. Immunohistochemical assay also demostrated the histological inflammatory change in liver tissue. CONCLUSIONS: The results indicate that C. bungeana supresses the activation of NF-κB signaling pathway through inhibiting phosphorylation of IκBα and p65, which results in good anti-inflammatory effect. In addition, C. bungeana attenuates inflammatory reaction by supressing the expression of various inflammatory cytokines both in vivo and in vitro.
Assuntos
Corydalis/química , Inflamação/prevenção & controle , Lipopolissacarídeos/toxicidade , NF-kappa B/metabolismo , Extratos Vegetais/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Citocinas/biossíntese , Citocinas/genética , Inflamação/induzido quimicamente , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , FosforilaçãoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Nauclea officinalis has been traditionally used in China for the treatment of fever, pneumonia and enteritidis etc. This study aims to investigate effects of N. officinalis on the inflammatory response as well as the possible molecular mechanism in LPS-stimulated RAW 264.7 murine macrophage cells. MATERIALS AND METHODS: Anti-inflammatory activity of N. officinalis (10, 20, 50, and 100µg/mL) was investigated by using LPS-induced RAW 264.7 macrophages. The NO production was determined by assaying nitrite in culture supernatants with the Griess reagent. The levels of TNF-α, IL-6 and IL-1ß in culture media were measured with ELISA kits. Real time fluorescence quantitative PCR was detected for mRNA expression of iNOS, TNF-α, IL-6 and IL-1ß. Western blot assay was performed to illustrate the inhibitory effects of N. officinalis on phosphorylation of IκB-α and NF-κB p65. RESULTS: Treatment with N. officinalis (10-100µg/mL) dose-dependently inhibited the production as well as mRNA expression of NO, TNF-α, IL-6 and IL-1ß in RAW 264.7 macrophages. Western blot assay suggested that the mechanism of the anti-inflammatory effect was associated with the inhibition of phosphorylation of IκB-α and NF-κB p65. CONCLUSIONS: The results indicated that N. officinalis potentially inhibited the activation of upstream mediator NF-κB signaling pathway via suppressing phosphorylation of IκB-α and NF-κB p65 to inhibit LPS-stimulated inflammation.
Assuntos
Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , NF-kappa B/metabolismo , Rubiaceae/química , Transdução de Sinais/efeitos dos fármacos , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Células Cultivadas , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Camundongos , Óxido Nítrico Sintase Tipo II/metabolismo , Óxidos de Nitrogênio/metabolismo , Fitoterapia/métodos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Nauclea officinalis has long been used in China for the treatment of cold, fever, swelling of throat, pink eyes, and so on; however, the in vivo integrated metabolism of its multiple bioactive components remains unknown. In this paper, an ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) method was established to identify chemical constituents in N. officinalis and metabolites in rat biological fluids after oral administration of N. officinalis. First, 40 chemical constituents in N. officinalis were detected within 19min by UPLC-QTOF/MS. Among them, 18 alkaloids and 7 phenolic acids and iridoids were identified or tentatively characterized. Secondly, 22 metabolites were identified after oral administration of N. officinalis extract, including 3, 9, 6 and 4 metabolites in the plasma, feces, urine and bile samples, respectively. Finally, the metabolic pathway was proposed, which were the hydroxylation, the hydroxylation of deglycosyation product of parent compound, the hydroxylation and dehydrogenation product of parent compound, and acetylation. Among these, hydroxylation was considered as the main metabolic processes. This work suggests that the integrative metabolism approach makes a useful template for drug metabolism research of traditional Chinese medicine (TCM).
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Extratos Vegetais/administração & dosagem , Rubiaceae/química , Administração Oral , Animais , Fezes , Masculino , Extratos Vegetais/metabolismo , Ratos , Ratos Sprague-Dawley , Rubiaceae/metabolismoRESUMO
Danmu injection and Danmu tablet are two widely used traditional Chinese medicine made of Nauclea officinalis (commonly known as Danmu), in which the alkaloids are the major active substances. In this paper, an ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method was developed for simultaneous determination and the pharmacokinetic characteristics study of six main active alkaloids (naucleamide A-10-O-ß-d-glucopyranosid, naucleamide G, pumiloside, 3-epi-pumiloside, strictosamide and vincosamide) of the two above-mentioned Danmu preparations in rat plasma. In the course of the experiment, following sample preparation by protein precipitation with methanol-ethyl acetate (2:1, v/v), the nitrogen-dried extraction was reconstituted in methanol and assayed on a C18 column using a gradient elution program with mobile phase consisting of acetonitrile and water containing 0.1% formic acid. The MS detection was performed in positive ionization mode with selected ion transitions. The established method was fully validated and proved to be sensitive and specific with lower limits of quantification (LLOQs) all less than 0.32ng/mL in rat plasma and matrix effects ranged from 88.87 to 108.27%. Good linearities of six alkaloids were obtained in respective concentration ranges (r(2)>0.995). The average extract recoveries for each compound at three quality control concentration levels were no less than 79.70%, and the precision and accuracy were within the acceptable limits. The validated method was successfully applied to the pharmacokinetic study of six alkaloid components of Danmu injection and tablet in rat plasma. The obtained results may be helpful to reveal the action mechanism and guide the clinical application of Danmu preparations.