RESUMO
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and devastating lung disease with a median survival of only 3-5 years. Due to the lack of effective therapy, IPF threatens human health. Recently, increasing reports have indicated that Rho-associated coiled-coil protein kinases (ROCKs) play important roles in the development of IPF and might represent a novel target for the treatment of IPF. Herein, a new series of selective ROCK2 inhibitors based on indoline were designed and synthesized. Structural modification resulted in optimized compound 9b with an IC50 value of 6 nM against ROCK2 and the inhibition of collagen gel contraction. Cellular assays demonstrated that 9b could significantly suppress the expression of collagen I and α-SMA, and inhibited ROCK signaling pathway. Oral administration of compound 9b (10 mg/kg) exerted more significant anti-pulmonary fibrosis effects than nintedanib (100 mg/kg) and KD025 (100 mg/kg) in a bleomycin-induced IPF rat model, suggesting that 9b could serve as a potential lead compound for the treatment of IPF.
Assuntos
Fibrose Pulmonar Idiopática , Humanos , Ratos , Animais , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose , Colágeno/efeitos adversos , Colágeno Tipo I , Quinases Associadas a rhoRESUMO
Eight new flavonoids, including two ß-hydroxy/methoxychalcones, velutones A and B (1 and 2), two 1,3-diarylpropan-1-ols, velutols C and D (3 and 4), a dihydroxychalcone, velutone E (5), a chalcone, velutone F (6), a furanoflavanone, velutone G (7), and a furanoflavonol, velutone H (8), and 14 known compounds were isolated from Millettia velutina. Their structures were determined by high-resolution electrospray ionisation mass spectrometry (HR-ESIMS) and spectroscopic data analyses and time-dependent density functional theory electronic circular dichroism (TD-DFT-ECD) calculations. Among the isolated constituents, compound 6 exhibited the most potent inhibitory effect (IC50: 1.3 µM) against nigericin-induced IL-1ß release in THP-1 cells. The initial mechanism of action study revealed that compound 6 suppressed NLRP3 inflammasome activation via blocking ASC oligomerization without affecting the priming step, which subsequently inhibited caspase-1 activation and IL-1ß secretion. Most importantly, compound 6 exerted potent protective effects in the LPS-induced septic shock mice model by improving the survival rate of mice and suppressing serum IL-1ß release. These results demonstrated that compound 6 had the potential to be developed as a broad-spectrum NLRP3 inflammasome inhibitor for the treatment of NLRP3-related disease.
Assuntos
Flavonoides/farmacologia , Millettia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Caspase 1 , Humanos , Inflamassomos , Inflamação , Lipopolissacarídeos , Macrófagos , Camundongos , Estrutura Molecular , Células THP-1RESUMO
Millettia pulchra is a renowned anti-inflammatory herbal medicine in southeast provinces of China. However, the underlying anti-inflammation mechanism remained incompletely understood. Herein, four new isoflavones, pulvones A-D and eleven reported constituents were isolated from the stems of Millettia pulchra with their structures being elucidated by HRMS and NMR analysis. The anti-inflammatory activities of pulvones A and C were further evaluated due to the better inhibitory activity on nitric oxide production in LPS-stimulated RAW264.7 cells and no obvious cytotoxicity to RAW264.7 cells. Western blot showed that pulvones A significantly decreased the levels of iNOS and COX-2 proteins and pulvones C only decreased the level of iNOS protein. ELISA analysis demonstrated that pulvones A inhibited the production of both interleukin-6 (IL-6) and IL-1ß while pulvones C showed better suppression effect on IL-1ß production in LPS-stimulated RAW264.7 cells. Then, their potential inhibitory effects on NF-κB pathway were tested in LPS-stimulated RAW264.7 cells. Immunofluorescence and western blot assay showed that pulvones A and C reduced the nuclear translocation of NF-κB(p65) and interrupted IκB phosphorylation. The ADP-Glo™ kinase assay showed pulvones A and C could directedly inhibit the IKKß kinase activity with the inhibitory rate of 40%, which were also verified by docking study. Collectively, these results suggested that pulvones A and C's anti-inflammatory effects were relevant to the interruption of NF-κB activation by inhibiting IKKß kinase.
Assuntos
Anti-Inflamatórios/farmacologia , Inflamação/tratamento farmacológico , Isoflavonas/farmacologia , Macrófagos/efeitos dos fármacos , Millettia/química , Animais , Anti-Inflamatórios/química , Inflamação/imunologia , Inflamação/patologia , Isoflavonas/química , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Simulação de Acoplamento Molecular , NF-kappa B , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacosRESUMO
Inhibitors that bind to the paclitaxel- or vinblastine-binding sites of tubulin have been part of the pharmacopoeia of anticancer therapy for decades. However, tubulin inhibitors that bind to the colchicine-binding site are not used in clinical cancer therapy, because of their low therapeutic index. To address multidrug resistance to many conventional tubulin-binding agents, numerous efforts have attempted to clinically develop inhibitors that bind the colchicine-binding site. Previously, we have found that millepachine (MIL), a natural chalcone-type small molecule extracted from the plant Millettia pachycarpa, and its two derivatives (MDs) SKLB028 and SKLB050 have potential antitumor activities both in vitro and in vivo However, their cellular targets and mechanisms are unclear. Here, biochemical and cellular experiments revealed that the MDs directly and irreversibly bind ß-tubulin. X-ray crystallography of the tubulin-MD structures disclosed that the MDs bind at the tubulin intradimer interface and to the same site as colchicine and that their binding mode is similar to that of colchicine. Of note, MDs inhibited tubulin polymerization and caused G2/M cell-cycle arrest. Comprehensive analysis further revealed that free MIL exhibits an s-cis conformation, whereas MIL in the colchicine-binding site in tubulin adopts an s-trans conformation. Moreover, introducing an α-methyl to MDs to increase the proportion of s-trans conformations augmented MDs' tubulin inhibition activity. Our study uncovers a new class of chalcone-type tubulin inhibitors that bind the colchicine-binding site in ß-tubulin and suggests that the s-trans conformation of these compounds may make them more active anticancer agents.
Assuntos
Chalconas/química , Chalconas/farmacologia , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Colchicina/farmacologia , Cristalografia por Raios X , Células Hep G2 , Humanos , Simulação de Acoplamento Molecular , Polimerização/efeitos dos fármacos , Tubulina (Proteína)/químicaRESUMO
Development of agents to overcome multidrug resistance (MDR) is one of the important strategies in cancer chemotherapy, and P-glycoprotein (P-gp) correlates with the degree of resistance. As a naturally occurring isoflavone, whether barbigerone (BA) could reverse MDR, is unknown. In this paper, we evaluated effects of BA on reversing P-gp mediated MDR of adriamycin (ADR)-resistant human breast carcinoma (MCF-7/ADR) cells. BA (0.5 µM) treatment showed strong potency to increase ADR cytotoxicity toward MCF-7/ADR cells. It was also demonstrated that BA time- and dose-dependently increased accumulations of ADR and reduced the efflux in MCF-7/ADR cells, pretreatment of these cells with BA might relocalized ADR to the nuclei. Furthermore, the results also revealed that BA did not affect P-gp, but alter P-gp ATPase activity. Intravenous administration of BA significantly increased anticancer efficacy of ADR to MCF-7/ADR xenograft model in nude mice. These results revealed that BA might reverse P-gp mediated MDR through inhibition of ATPase activity, which indicated a novel use of BA as a potent candidate for cancer chemotherapy.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Isoflavonas/uso terapêutico , Animais , Neoplasias da Mama/patologia , Humanos , Isoflavonas/farmacologia , Células MCF-7 , Camundongos , Camundongos NusRESUMO
Eleven compounds were successfully separated from Asteris souliei by using a two-step high-performance counter-current chromatography method. The first step involved a reversed phase isocratic counter-current chromatography separation using hexane/ethyl acetate/methanol/water (1:0.8:1:1 v/v/v/v), which produced three fractions, the first two of which were mixtures. The second step used step-gradient reversed-phase counter-current chromatography with hexane/butanol/ethyl acetate/methanol/water (1:0.5:3.5:1:4 v/v/v/v/v) initially followed by hexane/ethyl acetate/methanol/water (1:2:1:2 v/v/v/v) to separate Fraction 1 into seven compounds; and hexane/ethyl acetate/methanol/water (1:1:1:1.2 v/v/v/v) to separate Fraction 2 into three further compounds. The chemical structures of the separated compounds were identified by ESI-MS and NMR spectroscopy (1 H and 13 C). Baicalin (5), eriodictyol (7), apigenin-7-glycoside (8), quercetin (9), luteolin (10), and apigenin (11) showed obvious inhibitory effects on lipopolysaccharide-induced nitric oxide production in RAW264.7 cells at a concentration of 10 µg/mL.
Assuntos
Anti-Inflamatórios/isolamento & purificação , Asteraceae/química , Flavonoides/isolamento & purificação , Ácido Quínico/análogos & derivados , Animais , Anti-Inflamatórios/farmacologia , Cromatografia Líquida de Alta Pressão , Distribuição Contracorrente , Flavonoides/farmacologia , Camundongos , Ácido Quínico/isolamento & purificação , Ácido Quínico/farmacologia , Células RAW 264.7RESUMO
Selective inhibition of Janus kinase 3 (JAK3) has been identified as an important strategy for the treatment of autoimmune disorders. Based on the unique cysteine 909 residue (Cys909) of JAK3 at the gatekeeper position, we have developed a new irreversible covalent inhibitor, III-4, which is highly potent and selective in targeting JAK3. Importantly, III-4 selectively inhibited JAK3 (IC50 = 57 ± 1.21 nM) over other JAKs (IC50 > 10 µM) and Cys909 kinome members (IC50 > 1 µM). A cellular selectivity study also confirmed that III-4 preferentially inhibited JAK3 over JAK1 in JAK/STAT signaling. Moreover, the fact that III-4 covalently modified the Cys909 residue in JAK3 was clearly validated by mass spectrometry and covalent docking analysis. Based on the favorable target profiles, the pharmacokinetic properties and its low toxicity, III-4 exhibited better efficacy than tofacitinib in impeding disease progression in CIA mice, without any significant adverse effects. Taken together, III-4 is a potent, selective, and durable inhibitor of JAK3 and has the potential for the treatment of inflammatory disorders and autoimmune diseases, such as rheumatoid arthritis.
Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Janus Quinase 3/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Animais , Antirreumáticos/administração & dosagem , Antirreumáticos/farmacologia , Antirreumáticos/toxicidade , Artrite Experimental/enzimologia , Artrite Reumatoide/enzimologia , Progressão da Doença , Feminino , Humanos , Concentração Inibidora 50 , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/toxicidade , Pirimidinas/farmacologia , Pirróis/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Five new flavonoids, griffinones A-E (1-5), a new biphenylneolignan, griffilignan A (6) and ten known compounds were isolated from the n-hexane and EtOAc extracts of Millettia griffithii. The structures of the new compounds were determined by 1D and 2D NMR, and by HRMS. The anti-inflammatory activity of the isolated compounds was evaluated on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 cells. Among the isolated compounds, compounds 1, 2 and 14 showed significant anti-inflammatory activity with IC50 values of 20.4, 2.1 and 35.7µM, respectively and no obvious toxicities were observed at 100µM. Western blot and PCR assay further showed that inhibition of nitric oxide production by compound 2 was associated with suppression of iNOS expression. Modeling studies suggested that the amino group, phenyl ring as well as the isopentenyl tails of compound 2 could help bind to iNOs.
Assuntos
Anti-Inflamatórios/farmacologia , Flavonoides/farmacologia , Lignanas/farmacologia , Millettia/química , Caules de Planta/química , Animais , Linhagem Celular , Lipopolissacarídeos/toxicidade , Espectroscopia de Ressonância Magnética , Camundongos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
TNF-α is a dominant inflammatory mediator in the pathogenesis of inflammatory diseases including rheumatoid arthritis. In our research, we discovered 2-chloro-N-(4-(2-morpholinoethoxy)phenyl)quinazolin-4-amine (9c) exhibited an outstanding anti-inflammatory activity on inhibiting TNF-α production with an IC50 of 8.86 µM in RAW264.7 cells. Interestingly, 9c had no effect on mRNA level of TNF-α but up-regulated the precursor of TNF-α (pro-TNF-α). Then, we studied TNF-α converting enzyme (TACE), which is the most important proteases responsible for the release of TNF-α from pro-TNF-α to soluble TNF-α. The results showed 9c reduced TACE both on the levels of mRNA and protein in a dose-dependent manner. In vivo study, collagen-induced arthritis (CIA) mice were treated by 9c orally. 9c exhibited significant anti-arthritis effect by ameliorating arthritic score, reducing inflammatory cell infiltration, protecting joints from destruction and decreasing the production of systemic TNF-α, IL-6, IL-1ß. The underlying mechanism of 9c on CIA was coincided with the in vitro, which was mediated by TACE. In conclusion, we discovered a novel quinazoline derivative which ameliorates arthritis through inhibiting production of TNF-α mediated by TACE for the first time.
Assuntos
Proteínas ADAM/metabolismo , Artrite Experimental/tratamento farmacológico , Colágeno/administração & dosagem , Quinazolinas/uso terapêutico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Proteína ADAM17 , Animais , Linhagem Celular , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Masculino , Camundongos , Camundongos Endogâmicos DBA , Quinazolinas/farmacologia , RNA Mensageiro/genética , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genéticaRESUMO
Honokiol (HK), a biphenolic neolignan isolated from Magnolia officinalis, has been reported to possess anti-inflammatory and anti-angiogenic activaties. In this study, our aim was to investigate anti-psoriatic activities of HK and the involved mechanisms. In vitro, the effects of HK on the regulation of Th1/Th2 and TNF-α-induced NF-κB (p65) activation were analyzed by respective FCS and immunofluorescence. Additionally, the K14-VEGF transgenic model was used for the in vivo study. ELISA and Q-PCR were performed to evaluate serum levels of Th1/Th2 cytokines and their corresponding mRNA expressions. Effects on VEGFR-2 and p65 activation, as well as other angiogenic and inflammatory parameters were studied by immunostainings. Importantly, we found that HK significantly decreased the ratio of Th1/Th2-expression CD4(+) T cells and inhibited TNF-α-induced activation of NF-κB. The morphology and histological features of psoriasis were effectively improved by HK treatment. The expression of TNF-α and IFN-γ, and their corresponding mRNA levels were down-regulated and the expression of nuclear p65, VEGFR-2, as well as related phosphorylated proteins (p-VEGFR-2, p-ERK1/2, p-AKT and p-p38) were also suppressed. Overall, these results in our study suggested that HK exhibits anti-psoriatic effects through the inhibition of NF-κB and VEGFR-2.
Assuntos
Compostos de Bifenilo/farmacologia , Compostos de Bifenilo/uso terapêutico , Lignanas/farmacologia , Lignanas/uso terapêutico , Terapia de Alvo Molecular , NF-kappa B/antagonistas & inibidores , Fitoterapia , Psoríase/tratamento farmacológico , Psoríase/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Inibidores da Angiogênese , Animais , Anti-Inflamatórios , Compostos de Bifenilo/isolamento & purificação , Linfócitos T CD4-Positivos , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Lignanas/isolamento & purificação , Magnolia , Camundongos Transgênicos , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Psoríase/patologia , Equilíbrio Th1-Th2/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Diabetes mellitus, commonly characterized by hyperglycemia, is a group of metabolic diseases. Some oral anti-diabetic drugs show poor tolerability during chronic treatment, and associate with undesired side effects. Recent advances in the understanding of physiological functions of incretins and their degrading enzyme dipeptidyl peptidase DPP-IV have led to the discovery of DPP-IV inhibitors as a new class of oral anti-diabetic drugs. Several DPP-IV inhibitors have different chemical structures of which the xanthine scaffold has specific advantages. Combining previous work with the research strategy of pharmacophore hybridization, we retained this scaffold and synthesized a new series of amino-alcohol or diamino-modified xanthine compounds. Some xanthines exhibited submicromolar inhibitory activities against DPP-IV. The most potent compound 40 [Formula: see text] exhibits a good in vivo efficacy in reducing glucose excursion at a single dose and a better chronic effect in reducing body weight than metformin in DIO mice. In other words, the combined effect improved the pathological state of DIO mice.
Assuntos
Dipeptidil Peptidase 4/química , Inibidores da Dipeptidil Peptidase IV/química , Desenho de Fármacos , Glucose/metabolismo , Homeostase , Xantina/química , Animais , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Homeostase/efeitos dos fármacos , Camundongos , Fatores de Tempo , Xantina/farmacologiaRESUMO
Millettia griffithii is a unique Chinese plant located in the southern part of Yunnan Province. Up to now, there is no report about its phytochemical or related bioactivity research. In our previous study, the n-hexane crude extract of Millettia griffithii revealed significant anti-inflammatory activity at 100 µg/mL, inspiring us to explore the anti-inflammatory constituents. Four fractions (I, II, III, and A) were fractionated from n-hexane crude extract by high-performance counter-current chromatography with solvent system composed of n-hexane/ethyl acetate/methanol/water (8:9:8:9, v/v) and then were investigated for the potent anti-inflammatory activity. Fraction A, with the most potent inhibitory activity was further separated to give another four fractions (IV, V, VI, and B) with solvent system composed of n-hexane/ethyl acetate/methanol/water (8:4:8:4, v/v). Compound V and fraction B exhibited remarkable anti-inflammatory activity with nitric oxide inhibitory rate of 80 and 65%, which was worth further fractionation. Then, three fractions (VII, VIII, and IX) were separated from fraction B with a solvent system composed of n-hexane/ethyl acetate/methanol/water (8:1:8:1, v/v), with compound VIII demonstrating the most potent inhibitory activity (80%). Finally, the IC50 values of compound V and VIII were tested as 38.2 and 14.9 µM. The structures were identified by electrospray ionization mass spectrometry and(1)H and (13)C NMR spectroscopy.
Assuntos
Anti-Inflamatórios não Esteroides/isolamento & purificação , Anti-Inflamatórios não Esteroides/farmacologia , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Millettia/química , Extratos Vegetais/química , Animais , Anti-Inflamatórios não Esteroides/química , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Distribuição Contracorrente , Relação Dose-Resposta a Droga , Flavonoides/química , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Espectroscopia de Ressonância Magnética , Camundongos , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Extratos Vegetais/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-AtividadeRESUMO
A series of tert-butyl-5-methylpyrimidin-piperazine derivatives were synthesized and their anti-obesity activities were evaluated. Compounds 4g and 5j were found to have significant effects in down-regulating the triglyceride level of 3T3-L1 adipocytes. 5j exhibited remarkable therapeutic effects on the diet-induced obesity (DIO) mouse model at 20 mg kg(-1) day(-1) for 4 weeks by decreasing the weights of body, liver, and fat. 5j also regulated serum biomarkers to appropriate ranges, exerted therapeutic activity of steatosis in liver tissue and ameliorated the obese-related symptoms. In addition, 5j significantly decreased the blood glucose levels in oral glucose tolerance tests and improved the insulin sensitivity in insulin tolerance tests. These results suggest that 5j could be a candidate for obesity treatment.
Assuntos
Fármacos Antiobesidade/síntese química , Fármacos Antiobesidade/farmacologia , Hipolipemiantes/síntese química , Hipolipemiantes/farmacologia , Obesidade/prevenção & controle , Piperazinas/síntese química , Piperazinas/farmacologia , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adiposidade/efeitos dos fármacos , Animais , Biomarcadores/sangue , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Desenho de Fármacos , Fígado Gorduroso/etiologia , Fígado Gorduroso/prevenção & controle , Insulina/sangue , Resistência à Insulina , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Obesidade/sangue , Obesidade/etiologia , Obesidade/fisiopatologia , Relação Estrutura-Atividade , Fatores de Tempo , Triglicerídeos/metabolismo , Redução de Peso/efeitos dos fármacosRESUMO
Leptin, the product of the obese gene, is an adipocyte-secreted protein hormone playing a key role in the progression of obesity and hepatic steatosis. In this study, 28 novel (thio)urea and guanidine-based analogues have been synthesized and N-(1-(4-(3-(2-chloroethyl)ureido)benzyl)piperidin-4-yl)-3-(trifluoromethyl) benzamide (7i) was found to be a potent regulator of leptin expression in 3T3-L1 adipocytes. Treatment with 7i at a dose of 50 mg/kg/day for 35 days reduced the body weight and liver weight of diet-induced obesity mice by 13.5% and 18.4%, respectively, while also improving the serum levels of triglyceride, total cholesterol, leptin, adiponectin, LDL-c, HDL-c. Hematoxylin-eosin (H&E) and Oil Red O staining also confirmed that 7i ameliorated fat deposition in liver tissue and restricted the size of adipocytes in obesity-related fatty liver disease.
Assuntos
Fígado Gorduroso/tratamento farmacológico , Guanidina/administração & dosagem , Obesidade/tratamento farmacológico , Ureia/administração & dosagem , Células 3T3-L1 , Animais , Dieta Hiperlipídica , Fígado Gorduroso/etiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Guanidina/síntese química , Guanidina/química , Humanos , Leptina/antagonistas & inibidores , Leptina/biossíntese , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Obesidade/complicações , Ureia/síntese química , Ureia/químicaRESUMO
Mai Tong Fang (MTF), a Chinese herbal combination, has been used for the treatment of diabetic nephropathy in traditional medical clinics in China. However, the anti-adipogenic and anti-hyperglycemic effects of MTF have not been fully elucidated, so this study explored these pharmacological activities in 3T3-L1 adipocytes and ob/ob mice, respectively, of the water fraction of milkvetch root, salviae miltiorrhizae and mulberry as key components of MTF. MTF was found to inhibit adipogenesis and triglyceride accumulation in 3T3-L1 adipocytes. Oral administration of MTF in ob/ob mice for 8 weeks, exhibited positive controls on blood glucose and body weight, and further improved glucose tolerance according to an oral glucose tolerance test. Importantly, MTF extract alleviated fat deposition and ballooning degeneration in liver tissue and blocked the increase of adipocyte size in adipose tissue from treated ob/ob mice. These results indicated that the extract of key components in the traditional Chinese prescription MTF continue a potent anti-adipogenic and glucose-lowering agent.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , Medicamentos de Ervas Chinesas/administração & dosagem , Obesidade/tratamento farmacológico , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Animais , Glicemia , China , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Medicamentos de Ervas Chinesas/química , Teste de Tolerância a Glucose , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/química , Medicina Tradicional Chinesa , Camundongos , Obesidade/metabolismoRESUMO
Inflammatory bowel disease (IBD) is a chronic and incurable disease with an increasing incidence rate and low mortality rate. Selectively inhibiting JAK1 and TYK2 has been proposed as a strategy to enhance the efficacy of such inhibitors while minimizing the potential side effects on other JAK isoforms. Our previous studies identified small molecule 18 as a JAK1/TYK2 inhibitor with high selectivity and a new structure. Specifically, the IC50 of 18 at the kinase level reached 39 nM and 21 nM for JAK1 and TYK2, respectively, with 10-fold selectivity over both JAK2 and JAK3. In in vitro studies, 18 dose-dependently inhibited cytokine-induced STAT phosphorylation downstream of the JAK1 and TYK2 signaling pathway. In pharmacokinetic experiments, 18 demonstrated an oral bioavailability of 59.82%, making it a promising candidate for further in vivo studies. Using two mouse models of acute ulcerative colitis (UC) induced by the administration of dextran sulfate sodium (DSS) or oxazolone (OXA), 18 dose-dependently showed a better therapeutic effect than the positive control drug tofacitinib. Additionally, after long-term administration for 32 days, 18 displayed low toxicity to mice and a high safety profile. Taken together, these findings suggest that 18 is a JAK1/TYK2 dual inhibitor with therapeutic effects superior to those of tofacitinib in the treatment of IBD. Moreover, 18 is also a suitable clinical candidate for further investigation in diseases with strong involvement from interferon and/or IL-12/IL-23 in their pathogenesis. This study confirmed the therapeutic effect and long-term safety of inhibiting JAK1 and TYK2 to treat IBD.
Assuntos
Doenças Inflamatórias Intestinais , Inibidores de Janus Quinases , Camundongos , Animais , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/química , Janus Quinase 1 , Inibidores de Janus Quinases/farmacologia , Inibidores de Janus Quinases/uso terapêutico , Doenças Inflamatórias Intestinais/tratamento farmacológico , Citocinas , Interleucina-12RESUMO
The NLRP3 inflammasome is a multiprotein complex that is a component of the innate immune system, involved in the production of pro-inflammatory cytokines. Its abnormal activation is associated with many inflammatory diseases. In this study, we designed and synthesized a series of NLRP3 inflammasome inhibitors based on pyridazine scaffolds. Among them, P33 exhibited significant inhibitory effects against nigericin-induced IL-1ß release in THP-1 cells, BMDMs, and PBMCs, with IC50 values of 2.7, 15.3, and 2.9 nM, respectively. Mechanism studies indicated that P33 directly binds to NLRP3 protein (KD = 17.5 nM), inhibiting NLRP3 inflammasome activation and pyroptosis by suppressing ASC oligomerization during NLRP3 assembly. Additionally, P33 displayed excellent pharmacokinetic properties, with an oral bioavailability of 62%. In vivo efficacy studies revealed that P33 significantly ameliorated LPS-induced septic shock and MSU crystal-induced peritonitis in mice. These results indicate that P33 has great potential for further development as a candidate for treating NLRP3 inflammasome-mediated diseases.
RESUMO
In this study, we reported millepachine (MIL), a novel chalcone compound for the first time isolated from Millettia pachycarpa Benth (Leguminosae), induced cell cycle arrest and apoptosis in human hepatocarcinoma cells in vitro and in vivo. In in vitro screening experiments, MIL showed strong antiproliferation activity in several human cancer cell lines, especially in HepG2 cells with an IC50 of 1.51 µM. Therefore, we chose HepG2 and SK-HEP-1 cells to study MIL's antitumor mechanism. Flow cytometry showed that MIL induced a G2/M arrest and apoptosis in a dose-dependent manner. Western blot demonstrated that MIL-induced G2/M arrest was correlated with the inhibition of cyclin-dependent kinase 1 activity, including a remarkable decrease in cell division cycle (cdc) 2 synthesis, the accumulation of phosphorylated-Thr14 and decrease of phosphorylation at Thr161 of cdc2. This effect was associated with the downregulation of cdc25C and upmodulation of checkpoint kinase 2 in response to DNA damage. MIL also activated caspase 9 and caspase 3, and significantly increased the ratio of Bax/Bcl-2 and stimulated the release of cytochrome c into cytosol, suggesting MIL induced apoptosis via mitochondrial apoptotic pathway. Associated with those effects, MIL also induced the generation of reactive oxygen species. In HepG2 tumor-bearing mice models, MIL remarkably and dose dependently inhibited tumor growth. Treatment of mice with MIL (20mg/kg intravenous [i.v.]) caused more than 65% tumor inhibition without cardiac damage compared with 47.57% tumor reduction by 5mg/kg i.v. doxorubicin with significant cardiac damage. These effects suggested that MIL and its easily modified structural derivative might be a potential lead compound for antitumor drug.
Assuntos
Apoptose , Proteína Quinase CDC2/metabolismo , Carcinoma Hepatocelular/patologia , Chalconas/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular , Mitocôndrias/patologia , Animais , Antineoplásicos Fitogênicos/farmacologia , Proteína Quinase CDC2/genética , Carcinoma Hepatocelular/metabolismo , Chalcona/farmacologia , Chalconas/química , Ciclina B/genética , Ciclina B/metabolismo , Quinases Ciclina-Dependentes , Citosol/metabolismo , Dano ao DNA , Relação Dose-Resposta a Droga , Ativação Enzimática , Feminino , Citometria de Fluxo , Células Hep G2 , Humanos , Concentração Inibidora 50 , Neoplasias Hepáticas/patologia , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Millettia/química , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Isoflavones possess many biological activities, including anti-inflammatory and anticancer effects. Microtubules (composed of αß-tubulin heterodimers) are described as one possible cellular target of some of these isoflavones. However, the binding of tubulin to isoflavones has not been extensively studied, and until now, no crystal structure of the tubulin-isoflavone complex has been solved, and details of the isoflavone-tubulin interaction remain elusive. PURPOSE: Barbigerone is an isoflavone mainly found in the genus Milletti, such as the edible leguminous plant Millettia ferruginea, with anticancer activity. This study aims to confirm the cellular target of barbigerone and to study its anticancer mechanism. METHOD: Surface plasmon resonance assays and X-ray crystallography were used to study the interaction of barbigerone with tubulin protein. Immunofluorescence, in-cell and in vitro tubulin polymerization assays were employed to investigate the mechanism. MTT assays, cell clonal formation assays, wound healing assays, tube formation assays and H460 xenograft models were conducted to evaluate the in vitro and in vivo anticancer activities of barbigerone and one of its derivatives, 0412. RESULTS: Here, we found that barbigerone binds to tubulin to inhibit tubulin polymerization. Moreover, we solved the X-ray crystal structure of the tubulin-barbigerone complex at 2.33 Å resolution, which unambiguously determined the orientation and position of barbigerone in the colchicine-binding site. Illuminated by the X-ray data, we synthetized and obtained a more active isoflavone, 0412. Both barbigerone and 0412 inhibit cancer cell proliferation, tubulin polymerization, migration of HeLa cells and capillary-like tube formation of HUVECs, induce G2/M phase cell cycle arrest and apoptosis, and exhibit anticancer activity in an H460 xenograft model. CONCLUSION: In all, through biochemical and X-ray crystal structure results, we identified tubulin as the cellular target of one isoflavone, barbigerone, and proved that the tubulin-barbigerone complex plays a guiding role in obtaining a more active compound, 0412. These studies provide a crucial research basis for the development of isoflavones as anticancer candidate compounds.
Assuntos
Antineoplásicos , Isoflavonas , Humanos , Tubulina (Proteína)/metabolismo , Células HeLa , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/química , Isoflavonas/farmacologia , Isoflavonas/química , Proliferação de Células , Esqueleto/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Relação Estrutura-Atividade , Estrutura MolecularRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Piper wallichii (family: Piperaceae), a folk herbal medicine with anti-inflammatory and anti-thrombotic properties, has been traditionally used to treat rheumatic arthralgia, lumbocrural pain, gastrointestinal flatulence, and other intestinal diseases in China, Thailand, and India. However, there is no scientific report on the efficacy and potential mechanisms of Piper wallichii for ulcerative colitis (UC). AIM OF THE STUDY: The study aims to investigate the therapeutic effect and possible molecular mechanisms of the ethanol extract of Piper wallichii (EEPW) on DSS-induced UC in BALB/c mice. MATERIALS AND METHODS: The main components in EEPW were characterized by UPLC-QE-Orbitrap-MS. Subsequently, the anti-inflammatory effect of EEPW in vitro was preliminarily evaluated in RAW264.7 cells stimulated with LPS. UC model mice were triggered by free access to 4% DSS aqueous solution for 12 consecutive days, and simultaneously, EEPW (25, 50, and 100 mg/kg) and tofacitinib (positive control, 30 mg/kg) were orally administrated, respectively. The therapeutic efficacy of EEPW on UC was assessed by body weight, DAI, colon length, and pathological morphology. Besides, we investigated the effects of EEPW on intestinal barrier function, inflammatory factors, and immune systems of UC mice through immunohistochemistry (IHC), flow cytometry, and other techniques. Moreover, the expression of related proteins in the TLR4/NF-κB/COX-2 pathway was analyzed by Western blot. RESULTS: A total of 14 components were identified in the positive and negative modes, including isofutoquinol A (11), hancinone C (12), and futoquinol (14) which characterized by references. In the RAW264.7 cells experiments, the extract significantly suppressed the levels of TNF-α and IL-6. More importantly, EEPW distinctly improved the symptoms of DSS-induced UC mice as reflected by a significant recovery from body weight, colon length, pathological injuries of the colon, and so on. Further research found that EEPW remarkably restored the levels of occludin, promoted proliferation, and inhibited apoptosis in colon to maintain the integrity of intestinal barrier. In addition, the down-regulation of TNF-α and IL-1ß in colon, Th1 and Th17 cells in spleen, as well as the up-regulation of IL-10 in colon and Th2 cells in spleen were distinctly observed in EEPW-treated groups. Furthermore, the protein expression of TLR4, p-IκB-α, p-p65, and COX-2 were significantly inhibited by EEPW. CONCLUSIONS: This study confirmed for the first time that EEPW effectively ameliorated DSS-induced UC in mice, which might be related to improving intestinal barrier function, maintaining the levels of inflammatory factors, and regulating the immune system. In addition, we found that the anti-inflammatory effect of EEPW on UC mice was involved in the TLR4/NF-κB/COX-2 signaling pathway. In conclusion, Piper wallichii can be used as a candidate for the treatment of UC.