Oxoglaucine Suppresses Hepatic Fibrosis by Inhibiting TGFß-Induced Smad2 Phosphorylation and ROS Generation.

Hepatic fibrosis is the first stage of liver disease, and can progress to a chronic status, such as cirrhosis or hepatocellular carcinoma. Excessive production of extracellular matrix (ECM) components plays an important role in the development of fibrosis. Mechanistically, transforming growth factor beta (TGFß)-induced phosphorylation of Smad is thought to be a key signaling pathway in the development of liver fibrosis. Although the natural isoquinoline alkaloid oxoglaucine (1,2,9,10-tetramethoxy-7H-dibenzo(de,g)quinolin-7-one) exerts numerous beneficial effects, including anti-cancer, anti-inflammatory, and anti-osteoarthritic effects in diverse cell types, the effects of oxoglaucine on liver fibrosis and fibrogenic gene expression have not been fully elucidated. The aim of this study is to evaluate the signaling pathway and antifibrotic activity of isoquinoline alkaloid oxoglaucine in TFGß-induced hepatic fibrosis in vitro. Using Hepa1c1c7 cells and primary hepatocytes, we demonstrated that oxoglaucine treatment resulted in inhibition of the expression of fibrosis markers such as collagen, fibronectin, and alpha-SMA. Subsequent experiments showed that oxoglaucine suppressed TGFß-induced phosphorylation of Smad2 and reactive oxygen species (ROS) generation, without altering cell proliferation. We further determined that the increase in Smad7 by oxoglaucine treatment is responsible for the inhibition of Smad2 phosphorylation and the anti-fibrogenic effects. These findings indicate that oxoglaucine plays a crucial role in suppression of fibrosis in hepatocytes, thereby making it a potential drug candidate for treatment of liver fibrosis.

Oxoglaucine mediates Ca2+ influx and activates autophagy to alleviate osteoarthritis through the TRPV5/calmodulin/CAMK-II pathway.

BACKGROUND AND PURPOSE: Stimulation of calcium influx and suppression of autophagy play important roles in the pathogenesis of osteoarthritis (OA). In this study, we used a novel inhibitor of TRPV5 cation channels - oxoglaucine to attenuate progression of deterioration and pathological changes in OA patient-derived chondrocytes and OA animal model, by activating autophagy. EXPERIMENTAL APPROACH: Inhibition by oxoglaucine of calcium influx was assessed in cells.. Analyses were also carried out to investigate the effect of oxoglaucine on OA by detection of anti-inflammatory response, TRPV5/CAMK-II/calmodulin pathway, autophagy, and cartilage protection both in vitro and in vivo. demonstrated by macroscopic evaluation and histological findings. KEY RESULTS: Oxoglaucine suppressed expression of proinflammatory and apoptosis-related proteins, including TNF-α, IL-6, IL-1ß, MMP-13, CASP-3, and BAX, and prevented matrix degradation in OA chondrocytes. It also successfully blocked Ca2+ influx, activating autophagy dose-dependently asshown by up-regulated expression of LC-3II/I, Beclin-1, ATG5, ATG7, higher autophagic influx and formation of autophagic vesicles. It also decreased expression of mRNA and protein of TRPV5, CAMK-II, and calmodulin. Conversely, 1,25-dihydroxyvitamin D3, anagonist of TRPV5 channels, reversed the oxoglaucine-induced calcium influx inhibition and autophagy activation, demonstrating the association of oxoglaucine with TRPV5. Further, oxoglaucine prevented the apoptosis and matrix degradation of articular cartilage in a rat model of OA. CONCLUSION AND IMPLICATIONS: Oxoglaucine protects against cartilage damage by blocking the TRPV5/CAMK-II/calmodulin pathway to inhibit Ca2+ influx and activate autophagy. Our results indicate that oxoglaucine has the potential to become a candidate drug for treatment of OA.

Effect of Consecutive Alternating Administration of a Triple Combination of Anti-Enteroviral Compounds in Mice Infected with Coxsackievirus B3.

A novel approach for treatment of enterovirus infections was characterized. Application of treatment course of consecutive alternating administration (CAA) of triple combination of enterovirus replication inhibitors in experimental infections (20 MLD50) with coxsackievirus B3 (CVB3) strains in newborn mice is presented. It was established that in infection with cardiotropic Woodruff strain the combination of pleconaril, МDL-860 and oxoglaucine (PMO) subjected to the CAA scheme, a significant protective effect was observed. Monotherapeutic courses as well as simultaneously daily applied PMO were without effect. Analogous data were observed at experimental infection with the neurotriopic Nancy strain of CVB3. Following IC50 values of virus samples taken every day from target organs of infected animals during the whole period of study, a drug-resistance was established in monotherapy with compounds-partners in the PMO combination. At courses by the treatment scheme CAA of PMO development of drug-resistance was not established, but an increased susceptibility to the effect of the inhibitor-components in the combination was proven. Toxicity of PMO applied via the CAA scheme and in the monotherapeutic courses in both healthy and CVB3 infected animals was recorded. All data obtained prove the potential of the CAA treatment scheme for development of effective chemotherapy of enterovirus infections.

Efecto de la estructura molecular sobre el espectro electrónico vertical de la oxoglaucina / Effects of the molecular structure on the electronic vertical spectrum of oxoglaucine / Efeito da estrutura molecular no espectro eletrônico vertical da oxoglaucina

Resumen Se investigó el espectro electrónico vertical de la oxoglaucina utilizando el método de interacción de configuraciones multi-referencial (DFT/MRCI). Se analizó el efecto de los grupos metilo y metoxilo sobre la geometría y la distribución energética de los estados excitados de baja energía utilizando la base TZVP. Se encontró que, en los mínimos del estado fundamental de oxoglaucina, oxoglaucina sin grupos metilo y sin grupos metoxilo, los estados excitados singulete de más baja energía son de tipo nП* (S1) y ПП* (S2) y que los estados triplete ПП* (T1) y nП* (T2) son energéticamente accesibles desde S1 Desde el punto de vista energético, se encontró que el canal más eficiente para el entrecruzamiento de sistemas singulete-triplete es 1(nП*)3(ПП*). Aunque la presencia de los grupos metilo y metoxilo distorsionan la planaridad del sistema de anillos, su efecto en la distribución (vertical) de los estados singulete y triplete de más baja energía es mínimo.

Abstract The vertical electronic spectrum of oxoglaucine by means of a multireference configuration interaction method (DFT/ MRCI) was studied. The effect of both methyl and methoxy groups on the geometries and energetic distribution of the low-lying excited states was analyzed. The results show that, by means of the TZVP basis set, at the ground state minima of oxoglaucine, oxoglaucine without methyl and methoxy groups, the lowest excited singlet states are of nП* (S1) and ПП* (S2) type. Triplet states of ПП* (T1) and nП* (T2) type are energetically accessible from S1. From the energetic point of view, it can be proposed that the channel for an efficient intersystem crossing 1(nП*)3(ПП*) is plausible. Although the presence of the methyl and methoxyl groups distort the planarity of the rings system, the effect in the vertical distribution of the lowest lying singlet and triplet states can be considered as negligible.

Resumo O espectro eletrônico vertical da oxoglaucina foi investigado por meio do método de interação de configurações multi referência (DFT/MRCI). Foi estudado o efeito dos grupos metilo e metoxi nas geometrias e na distribuição energética dos estados excitados de baixa energia utilizando a base TZVP. Os resultados mostram que nos mínimos do estado fundamental da oxoglaucina, a oxoglaucina sem grupos metilo e metoxi os estados excitados singletos de mais baixa energia são de tipo nП* (S1) y ПП* (S2) e que os estados tripletos do tipo ПП* (T1) e nП* (T2) são energeticamente acessíveis a partir de S1 Do ponto de vista energético, pode-se propor que o canal para um cruzamento intersistema eficiente 1(nП*)3(ПП*) seja plausível. Embora a presença dos grupos metilo e metoxilo distorçam a planaridade do sistema de anéis, o efeito na distribuição vertical dos estados de singleto e de tripleto de mais baixa energía pode ser considerado negligenciável.

Development and Validation of a HPLC-ESI-MS/MS Method for Simultaneous Quantification of Fourteen Alkaloids in Mouse Plasma after Oral Administration of the Extract of Corydalis yanhusuo Tuber: Application to Pharmacokinetic Study.

The tuber of Corydalis yanhusuo is a famous traditional Chinese medicine and found to have potent pharmacological effects, such as antinociceptive, antitumor, antibacterial, anti-inflammatory, and anti-depressive activities. Although there are several methods to be developed for the analysis and detection of the bioactive ingredients' alkaloids, so far, only few prominent alkaloids could be quantified, and in vitro and in vivo changes of comprehensive alkaloids after oral administration are still little known. In this study, we first developed a simple and sensitive high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method to quantify the comprehensive alkaloids of extracts of C. yanhusuo in mouse plasma, using nitidine chloride as an internal standard. As results, at least fourteen alkaloids, including an aporphine (oxoglaucine), a protopine (protopine), five tertiary alkaloids (corydaline, tetrahydroberberine, tetrahydropalmatine, tetrahydrocolumbamine, and tetrahydrocoptisine) and seven quaternary alkaloids (columbamine, palmatine, berberine, epiberberine, coptisine, jatrorrhizine, and dehydrocorydaline) could be well quantified simultaneously in mouse plasma. The lower limits of quantification were greater than, or equal to, 0.67 ng/mL, and the average matrix effects ranged from 96.4% to 114.3%. The mean extraction recoveries of quality control samples were over 71.40%, and the precision and accuracy were within the acceptable limits. All the analytes were shown to be stable under different storage conditions. Then the established method was successfully applied to investigate the pharmacokinetics of these alkaloids after oral administration of the extract of Corydalis yanhusuo in mice. To the best of our knowledge, this is the first document to report the comprehensive and simultaneous analyses of alkaloids of C. yanhusuo in mouse plasma. It was efficient and useful for comprehensive pharmacokinetic and metabolomic analyses of these complex alkaloids after drug administration.

Effect of consecutive alternating administration (CAA) of a triple anti-enteroviral combination on Coxsackievirus B1 neuroinfection in mice.

Currently, clinically effective antivirals for use in the treatment of enteroviral (EV) infections do not exist. The main reason is the development of drug resistance, the principle obstacle in the development of EV infection chemotherapy, based til now on monotherapy. The most important achievement of our previous studies was the development of a novel scheme for in vivo application of a triple combination of EV inhibitors with different modes of action against Coxsackievirus B (CVB) infections in mice. It consists of consecutive alternating administration (CAA) of the substances in the combination. Here, we tested the effect of the triple combination pleconaril, guanidine-HCl, and oxoglaucine (PGO) via CAA in newborn mice infected with a neurotropic strain of CVB1 (20 LD50 per mouse). This combination manifested a considerable protective effect with pleconaril doses of 25-200mg/kg: it decreased mortality rate (protection index, PI, between 31.3% and 67.7%) and increased mean survival time (MST) by 4-6days. Pleconaril monotherapy demonstrated activity similar to that of PGO via CAA, as measured by PI values, but MST values were slightly lower. However, it also greatly suppressed growth of infected suckling mice, especially at 200mg/kg. This toxic effect was avoided with CAA of PGO at pleconaril doses of 25-100mg/kg. Pleconaril monotherapy administered every 3days was ineffective. The PGO with CAA treatment course decreased infectious virus content, whereas pleconaril monotherapy did not. Analysis of drug-sensitivity in brain samples from CVB1 infected mice, based on IC50 (50% inhibitory concentration) values from cell culture experiments, showed that the CAA course counteracted the development of drug resistance to pleconaril and oxoglaucine in the triple PGO combination and increased drug sensitivity. In contrast, pleconaril and oxoglaucine monotherapies resulted in drug resistance. This data clearly proves the effectiveness of the proposed novel approach-the CAA treatment course-for combined application of EV replication inhibitors.

Phosphatidylinositol 4-kinase III beta is the target of oxoglaucine and pachypodol (Ro 09-0179) for their anti-poliovirus activities, and is located at upstream of the target step of brefeldin A.

In recent years, phosphatidylinositol 4-kinase III beta (PI4KB) has emerged as a conserved target of anti-picornavirus compounds. In the present study, PI4KB was identified as the direct target of the plant-derived anti-picornavirus compounds, oxoglaucine and pachypodol (also known as Ro 09-0179). PI4KB was also identified as the target via which pachypodol interferes with brefeldin A (BFA)-induced Golgi disassembly in non-infected cells. Oxysterol-binding protein (OSBP) inhibitor also has interfering activity against BFA. It seems that this interference is not essential for the anti-poliovirus (PV) activities of BFA and PI4KB/OSBP inhibitors. BFA inhibited early to late phase PV replication (0 to 6 hr postinfection) as well as PI4KB inhibitor, but with some delay compared to guanidine hydrochloride treatment. In contrast with PI4KB/OSBP inhibitors, BFA inhibited viral nascent RNA synthesis, suggesting that BFA targets some step of viral RNA synthesis located downstream of the PI4KB/OSBP pathway in PV replication. Our results suggest that PI4KB is a major target of anti-picornavirus compounds identified in vitro for their anti-picornavirus activities and for some uncharacterized biological phenomena caused by these compounds, and that BFA and PI4KB/OSBP inhibitors synergistically repress PV replication by targeting distinct steps in viral RNA replication.

Oxoglaucine-lanthanide complexes: synthesis, crystal structure and cytotoxicity.

AIM: To evaluate the in vitro cytotoxicity of oxoglaucine (OG) complexes: [Sm(OG)2(NO3)3]•H2O (1), [Eu(OG)2(NO3)3]•1.5CH3OH (2) and [Er(OG)2(NO3)3]•H2O (3) through comparison to oxoglaucine and lanthanide salts. MATERIALS AND METHODS: The reactions of OG with corresponding lanthanide salts gave rise to complexes 1-3. The crystal structures of complexes 1-3 were determined by single-crystal X-ray diffraction analysis. The in vitro cytotoxicity of oxoglaucine and complexes 1-3 against five human cancer cell lines were evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium Bromide (MTT) method. RESULTS: Complexes 1-3 have similar mononuclear structures. The 50% inhibitory concentration (IC50) of complex 1 against SGC7901 cells was 32.1 µM; that of complex 2 against MCF-7 cells was 3.2 µM; those of complex 3 on HeLa and MCF-7 cells were 8.3 and 1.4 µM, respectively. CONCLUSION: The three OG-lanthanide complexes exhibited significantly enhanced cytotoxicity vs. OG and corresponding lanthanide salts.