Aberrant elevation of BACE1 levels in brains of Alzheimer’s disease (AD) patients may involve Aβ. In the present study, we used a neuron culture model system to investigate the effects of Aβ on BACE1 expression as well as the underlying mechanisms.Rat primary cortical neurons were treated with relatively low concentrations (2.5 μM) of Aβ42 oligomers (Aβ-O) or fibrils (Aβ-F) for 2–3 days. Aβ-O induced a significant increase in protein levels of BACE1, while Aβ-F only had a marginal effect. Levels of amyloid precursor protein (APP) and the major α-secretase, ADAM10, remained unaltered upon treatment with both types of Aβ. Aβ-O treatment resulted in activation of eIF2α and caspase 3 in a time-dependent manner, with no changes in the endoplasmic reticulum (ER) stress marker, GRP78, indicating that a typical ER stress response is not induced under our experimental conditions. Furthermore, Aβ-O did not affect BACE1 mRNA expression but augmented the levels of exogenous BACE1 expressed via recombinant adenoviruses, indicating regulation of BACE1 protein expression, not at the transcriptional or translational but the post-translational level. Immunocytochemical analysis revealed that Aβ-O causes a significant increase in BACE1 immunoreactivity in neurites (both axons and dendrites), but not soma of neurons; this change appears relevant to the mechanism of Aβ-O-induced BACE1 elevation, which may involve impairment of BACE1 trafficking and degradation. In contrast, Aβ-O had no effect on APP immunoreactivity.Our results collectively suggest that Aβ oligomers induce BACE1 elevation via a post-translational mechanism involving its altered subcellular distribution in neurons, which possibly triggers a vicious cycle of Aβ generation, thus contributing to the pathogenetic mechanism of AD.The online version of this article (doi:10.1186/s13041-015-0163-5) contains supplementary material, which is available to authorized users. Alzheimer’s disease (AD) is the most common neurodegenerative dementia characterized by two major pathological hallmarks, extracellular amyloid plaques and intracellular neurofibrillary tangles composed of amyloid β-protein (Aβ) and phosphorylated tau protein, respectively . The fintrans-Golgi network (TGN), and mainly degraded in lysosomes [Aβ is a cleavage product of APP by two proteases, specifically, β-site APP cleaving enzyme 1 (BACE1) and the γ-secretase complex. APP is initially cleaved by BACE1, producing secreted APP-β and C-terminal fragment (CTF) known as β-CTF. β-CTF is subsequently cleaved by γ-secretase to release Aβ. Alternatively, APP is cleaved by α-secretase, mainly constituting ADAM10, at the α-site within the Aβ sequence, precluding Aβ formation . BACE1 iysosomes , 8.BACE1 levels and activity are increased in the brains of AD patients –15 and mAβ42 oligomers (Aβ-O) and fibrils (Aβ-F) were prepared using previously established protocols . WesternIn designing an experimental protocol, we attempted to achieve a condition whereby Aβ treatment induces relatively modest neuronal damage, mimicking physiological conditions. In our pilot experiments, neurons displayed relatively modest responses to 2.5 μM Aβ-O, which was therefore employed for further experiments.To estimate the effects of Aβ on cell survival, the cell survival assay was performed using Cell Counting Kit-8. Cell survival remained almost unchanged upon treatment with Aβ-F, and was only very slightly (10–15 %) decreased upon Aβ-O treatment on days 2 and 3 Fig.  comparedNext, we examined the effects of Aβ on BACE1 protein levels. Primary cortical neurons were treated with 2.5 μM Aβ-O, Aβ-F or vehicle for 2 or 3 days, followed by Western blot analysis. Endogenous BACE1 levels in neurons treated with Aβ-O for 2 or 3 days were significantly increased by 20 and 31 %, respectively, compared with those in control Fig. , d. Aβ-FSince Aβ-O induces various cellular responses, including caspase activation, we analyzed its effects on proteins related to apoptotic and other stress responses. First, we examined cleaved caspase 3 (the activated form), an executor molecule in the apoptotic cascade. Levels of cleaved caspase 3 were significantly increased in neurons treated with Aβ-O compared with controls, in a time-dependent manner to 167 and 225 % on days 2 and 3, respectively Fig. , b. NextTo ascertain the molecular mechanism underlying the Aβ-O-induced increase in BACE1 protein, BACE1 mRNA levels were assessed in neurons treated with Aβ using semi-quantitative RT-PCR. BACE1 mRNA levels normalized by vimentin were not significantly altered in neurons treated with Aβ-O or Aβ-F at days 1 and 2 Fig. , b, compNext, we clarified whether the increase in BACE1 protein levels induced by Aβ-O is translationally regulated. To this end, we investigated the effects of Aβ-O on exogenously expressed BACE1. Primary cortical neurons were infected with recombinant adenoviruses expressing human BACE1 with a C-terminal rhodopsin tag , and aftTo gain further insights into the mechanism underlying Aβ-O-induced elevation of BACE1, we analyzed the influence of Aβ-O on intraneuronal localization of BACE1 using immunocytochemistry. In control untreated neurons, endogenous BACE1 immunoreactivity was observed in neuronal soma and neurites, consistent with recent findings , 24. IntIt is possible that Aβ-O may induce BACE1 accumulation in neurites by altering BACE1 degradation via lysosomal pathway, since BACE1 is suggested to be primarily degraded via this pathway , 8, 23. Finally, we examined whether APP processing is affected by treatment with Aβ-O. To this end, we analyzed APP CTFs in neurons treated with Aβ-O and/or LY2886721 (LY) , a potenIn the current study, we investigated whether Aβ augments BACE1 expression in neurons and the associated mechanisms. For this purpose, we established an experimental model in which primary cortical neurons were treated with relatively low concentrations of Aβ-O or Aβ-F for relatively long periods that facilitated analysis of cellular responses without obvious cell death, mimicking the pathological conditions of AD. Using this model, we initially showed that Aβ-O treatment induces a significant increase in BACE1 protein levels while Aβ-F produces only a marginal effect. In contrast, APP and ADAM10 levels remained unaltered, clearly indicating a specific effect of Aβ-O on BACE1. Our findings are consistent with previous studies describing Aβ42-induced BACE1 upregulation , 26, 27.Next we focused on the cellular mechanisms by which Aβ-O elevates BACE1 protein expression. BACE1 mRNA levels were unaltered by Aβ-O, indicating that elevation is not regulated at the transcriptional level. Several studies have demonstrated stimulation of BACE1 transcription upon Aβ-O treatment in cultured cells other than primary neurons , 26, 28.Taken together, the data strongly suggest that Aβ-O elevates BACE1 levels via a post-translational mechanism in our neuron model. To gain further insights into the mechanism of Aβ-O-mediated BACE1 upregulation, immunocytochemical analysis was performed to investigate the subcellular localization of BACE1. Intriguingly, Aβ-O treatment induced a significant increase in BACE1 immunoreactivity in neurites, but not soma, while the immunoreactivity of APP remained unaltered. Consistent with our findings, BACE1 is reported to accumulate in dystrophic neurites surrounding amyloid plaques in brains of AD patients and AD model mice , 23. TheIn our experiments, treatment with Aβ-O, but not Aβ-F, led to activation of the eIF2α pathway and the apoptosis cascade. We observed a significant time-dependent increase in cleaved caspase 3 in Aβ-O-treated neurons, consistent with the findings of numerous previous studies , 39. HowWe have presented evidence that oligomeric Aβ upregulates BACE1 via a post-translational mechanism involving its altered subcellular localization. Our data also suggest that Aβ oligomers modulate amyloidogenic APP processing. The present findings imply the direct involvement of Aβ oligomers in BACE1 elevation in brains of AD patients and AD model mice. A vicious cycle is proposed to exist whereby soluble Aβ oligomers augment Aβ production by promoting amyloidogenic processing of APP through BACE1 modulation , 19. ThuWe used a neuron model system to investigate the effects of Aβ-O and Aβ-F on BACE1 expression as well as the underlying mechanisms. The present findings collectively demonstrated that Aβ-O induces a significant and specific increase in protein levels of BACE1 via a post-translational mechanism. Immunocytochemical analysis further revealed that Aβ-O affects the subcellular distribution of BACE1 to augment its levels in neuritic compartments, which appears relevant for Aβ-O-mediated BACE1 elevation. We additionally found that Aβ-O induces activation of eIF2α and caspase 3, with no changes in GRP78, indicating that typical ER stress does not participate in cellular responses to Aβ-O in our experimental model. Thus, a vicious cycle appears to exist whereby soluble Aβ oligomers promote Aβ production through post-translational modulation of BACE1, contributing to the pathogenetic mechanism of AD.2. Cells were maintained in a humidified atmosphere of 5 % CO2/95 % air in Macs Neuro Medium  containing 0.5 mM L-glutamine, NeuroBrew-21 (Miltenyi Biotec), and penicillin-streptomycin. Half of the medium was replaced with fresh medium every 3–4 days.Primary cerebral cortical neurons were obtained from 17 day-old embryos of a Wistar rat as described previously , 49. NeuThe following primary antibodies were used in this study: anti-Aβ , anti-BACE1 , anti-APP ), Aβ preparations were diluted in regular medium and used to replace the entire medium. Control cultures were treated with the same concentration of DMSO.Aβ42 oligomers and fibrils were prepared as described previously , 52. BriRecombinant adenoviruses expressing BACE1 were prepared using an Adenovirus Dual Expression Vector Kit  as described previously . In reco2 incubator for 2 h. Absorbance was measured at 450 nm using a microplate reader. Absorbance of the medium was subtracted as a blank from that of each sample.Primary cortical neurons cultured on 12-well plates were treated with Aβ-O, Aβ-F or vehicle for 2 or 3 days. Cell Counting Kit-8 solution  was added to each well, and the plates left in a CO3VO4). After rocking for 1 h at 4 °C, samples were centrifuged at 100,000 x g for 30 min, and the supernatants used as cell lysates. The protein content in cell lysates was estimated with the bicinchoninic acid assay . Samples containing equal amounts of protein were mixed with 2x Laemmli sample buffer and incubated at 95 °C for 3 min, followed by separation on 9 or 12 % polyacrylamide gels and transfer to polyvinylidene difluoride (PVDF) membranes. Blots were blocked in 5 % non-fat dried milk in phosphate-buffer saline (PBS) containing 0.05 % Tween-20, and probed with primary antibodies, followed by secondary peroxidase-labeled anti-rabbit or mouse IgG. The Can Get Signal Immunoreaction Enhancer Solution  was occasionally incubated with primary antibodies to enhance immunoreaction. Protein expression was detected with a chemiluminescence reagent , and the resulting images examined with a LAS-1000  image analyzer. β-Actin was used as the internal control to normalize the levels of proteins of interest.Cells were lysed on ice in RIPA buffer  containing protease inhibitors  and phosphatase inhibitors , followed by Bonferroni’s multiple comparison test or Student’s


Argemone gracilenta’s methanol extract and its fractions. We identified those compounds of the most active fractions that displayed anti-proliferative activity.Cancer is one of the leading causes of death worldwide. Natural products have been regarded as important sources of potential chemotherapeutic agents. In this study, we evaluated the anti-proliferative activity of evaluated in vitro using the MTT colorimetric method. Identification of the active compounds present in the fractions with the highest activity was achieved by nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS) analyses.The anti-proliferative activity on different cancerous cell lines  was 50 values of 2.8, 2.5, 12.1, and 2.7, 2.4, 79.5 μg/mL on M12.C3F6, RAW 264.7, and HeLa cancerous cell lines, respectively. No activity was shown on the normal L-929 cell line. From the hexane fraction, a mixture of fatty acids and fatty acid esters of 16 or more carbon atoms with anti-proliferative activity was identified, showing a range of IC50 values of 16.8-24.9, 34.1-35.4, and 67.6-91.8 μg/mL on M12.C3F6, RAW 264.7, and HeLa cancerous cell lines, respectively. On the normal L-929 cell line, this mixture showed a range of IC50 values of 85.1 to 100 μg/mL.Both argemonine and berberine alkaloids, isolated from the ethyl acetate fraction, displayed high anti-proliferative activity with ICArgemone gracilenta.This is the first study that relates argemonine, berberine, and a mixture of fatty acids and fatty acid esters with the anti-proliferative activity displayed by  Argemone genus, locally known as “cardo or chicalote” ). Preparative TLC was performed on precoated silica gel 60 F254 plates (Merck).Melting points were determined on a Fisher Johns melting point apparatus. The infrared spectra were measured on a Bruker Vector 22 spectrometer. GC-MS spectra were acquired using an Agilent 6890 series GC system and Agilent 5973 mass selective detector, employing a fused-silica column, 30 m × 0.32 mm HP-5MS . The temperature of the column was varied from 40 to 250°C with a slope of 10°C/min and a stay of 5 min at this temperature. All NMR spectra were recorded on a Varian Unity 400 spectrometer at 400 MHz for Argemone gracilenta was collected in Guaymas, Sonora , Northwest of Mexico, in May 2011. The plant was taxonomically identified  by Jesús Sánchez Escalante, taxonomist at the Herbarium of the Universidad de Sonora.n-hexane, ethyl acetate, and ethanol (3 × 400 mL for each solvent) with constant agitation for 12 h. The volumes obtained (1200 mL) were concentrated to dryness under reduced pressure at 40°C to yield 26 g of n-hexane, 12.5 g of EtOAc, and 29.0 g of EtOH fractions. The methanol extract and fractions were stored to -4°C in amber glass vials until use [The plant was dried at room temperature and homogenized (1400 g) with a Whiley mill (200 mesh). An extract of the homogenized plant was obtained with methanol  during 10 days under periodic agitation. The extract was filtered and concentrated to dryness on a rotatory evaporator under reduced pressure at 40°C. The methanol extract (130.9 g) was suspended consecutively in ntil use .n-hexane-CH2Cl2 (100:0 to 0:100), then CH2Cl2-MeOH (100:0 to 0:100) mixtures of increasing polarity to yield 11 fractions.The EtOAc fraction was further chromatographed on a silica gel column (120 g) eluting with 2Cl2-MeOH (98:2), was obtained as a yellow-orange oil.The FAg-4A fraction , eluted with CH2Cl2-MeOH (98:2), was obtained as a colorless oil.The FAg-5B fraction , eluted with CH2Cl2-MeOH (85:15), was subjected to preparative TLC  to yield 6.3 mg of argemonine (Rf = 0.8) and 7.9 mg of berberine (Rf = 0.7).The FAg 7 fraction , eluted with CH1H NMR , δ ppm; 2.52 , 2.45 , 4.04  3.58 , 3.66 (2-OCH3 and 8-OCH3), 3.33 , 6.31 , 6.47 (H-4 and H-10). 13C NMR , δ ppm; 33.3 (C-5 and C-11), 39.7 (N-CH3), 56.3 (C-6 and C-12), 55.6 (3-OCH3 and 9-OCH3), 55.8 (2-OCH3 and 8-OCH3), 109.7 (C-4 and C-10), 111.3 (C-1 and C-7), 122.5 (C-4a and C-10a), 127.4 (C-1a and C-7a), 147.9 (C-3 and C-9), 148.4 (C21 and C-8).Argemonine. Mp 122.3-123.4°C (melting point of reference 147-148°C ), 1H NMR1H NMR , δ ppm; 3.26 , 4.12 , 4.35 , 4.95 , 6.13 , 6.89 , 7.45 , 7.95 , 8.00 , 8.61 , 9.78 . 13C NMR , δ ppm; 28.2 (C-5), 57.1 (C-6), 57.6 (9-OCH3), 62.5 (10-OCH3), 103.6 , 106.5 (C-1), 109.3 (C-4), 121.5 (C-13), 121.9 (C-4a), 123.3 (C-12a), 124.5 (C-12), 128 (C-11), 131.9 (C-14a), 135.1 (C-8a), 139.6 (C-14), 145.7 (C-9), 146.4 (C-8), 149.9 (C-3), 152 (C-2 and C-10).Berberine. Mp 144.3-146.1°C (melting point of reference 146°C ) 1H NMR The content of fatty acids and fatty acid esters in fractions FAg-4A and FAg-5B was analyzed by gas chromatography (Agilent 6890) coupled to a quadrupole mass detector in electron impact mode at 70 eV (Agilent 5973 N). A solution of 5 mg of each subfraction in 1 mL of solvent (methanol or dichloromethane) was prepared. Volatile compounds were separated on an HP 5MS capillary column . The oven temperature was set at 40°C for 2 min and then programmed from 40 to 260°C at 10°C/min, and kept for 20 min at 260°C. Mass detector conditions were as follows: interphase temperature was 200°C and mass acquisition ranged from 20 to 550. Temperatures of the injector and detector were set to 250°C and 280°C, respectively. The splitless injection mode was performed with 1 μL of the oily extract. The carrier gas was helium at a flow rate of 1 mL/min. Volatiles were identified by comparing their mass spectra with those of the National Institute of Standards and Technology NIST 1.7 library. Semi-quantitative data were calculated from the GC peak areas without using correction factors and were expressed as relative percentage (peak area %) of the total volatile constituents identified.2.The M12.C3F6 (murine B-cell lymphoma) and RAW 264.7  cells lines were kindly provided by Dr. Emil R. Unanue . Cell lines NCTC clone L-929  and HeLa (human cervix carcinoma) were purchased from the American Type Culture Collection . All cell cultures were carried out in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% heat- inactivated fetal calf serum and grown at 37°C in an atmosphere of 5% CO50 values  [The MTT assay was used to evaluate the anti-proliferative activity. It is a colorimetric assay based in the fact that mitochondrial oxidoreductase enzymes are capable of reducing the tetrazolium dye MTT 3--2,5-diphenyltetrazolium bromide to its insoluble formazan, which has a purple color. The cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present. Briefly, cells were seeded in a 96-well plate with DMEM medium  at a density of 10,000 cells/well. Different concentrations of methanol extract and fractions were added followed by 48 h incubation. All experiments were conducted in parallel with controls (0.06%-0.5% DMSO). Ten microliters of 3--2,5-diphenyltetrazolium bromide  were added to each well at the end of the treatment period and incubated at 37°C for 4 h. Formazan crystals were dissolved with acidic isopropanol, and the plates were read in an ELISA plate reader, using a test wavelength of 570 nm and a reference wavelength of 630 nm. Plates were normally read within 10 min after adding isopropanol. The anti-proliferative activity of extracts was reported as IC by 50%) .All data were expressed as mean ± SD. Data were subjected to statistical analysis of variance (ANOVA) by comparing means with Tukey test (p <0.05). IBM® SPSS® 20 statistical program was used for all statistical analyses.Anti-proliferative activity assays were performed using the MTT colorimetric assay on three cancerous cell lines  and a normal cell line (L-929) as control. The concentrations used of the methanol extract and fractions were from 25 to 200 μg/mL, each assay was performed in triplicate.in vitro anti-proliferative activity assays of the Argemone gracilenta’s methanol extract and its fractions on different cancerous cell lines. The results are expressed in IC50 values (mg/mL) ± SD. The United States National Cancer Institute (US-NCI) establishes that a crude extract that shows an IC50 value of less than 100 μg/mL is considered active. When the IC50 value is lower than 30 μg/mL, the US-NCI considers a crude extract promising for purification and a biological activity study [50 values are lower than 4 μg/mL [Table ty study . In the  4 μg/mL ,23.Table50 value of 46.20 ± 8.41 μg/mL; for RAW 264.7 and HeLa cell lines, the extract showed activity with IC50 values of 64.45 ± 8.97 and 78.87 ± 8.52 μg/mL, respectively. The methanol extract was not active on the normal cell line L-929 used as control, since it showed an IC50 value > 100 μg/mL.The methanol extract showed activity on the M12.C3F6 cell line with an IC50 values of 32.60 ± 1.10 and 41.27 ± 4.27 μg/mL respectively, and showing no activity on the normal cell line L-929, with an IC50 value > 100 μg/mL.After fractionation of the methanol extract, there was an increment in the activity of some fractions. The ethyl acetate fraction had greater activity, with an increase in the anti-proliferative activity mainly on cell lines RAW 264.7 and M12.C3F6 with IC50 values of 2.8 and 2.5 μg/mL on M12.C3F6 and RAW 264.7 cell lines, respectively  and berberine (7B) showed anti-proliferative activity on M12.C3F6, RAW 264.7, and HeLa cancer cell lines, and on the L929 control cell line . In oncosis, the cell demonstrates swelling, rapid membrane breakdown, swollen nuclei without DNA fragmentation, and organelle swelling. In autophagy, vacuolization (autophagic vacuoles), degradation of cytoplasm contents, and a slight chromatin condensation are observed .The cell lines were observed 48 h after exposure to argemonine and evidence of two different cell death pathways was found. In the M12.C3F6 cell line  growth, demonstrating that unsaturated 18 carbon fatty acids exert anti-proliferative activity on that cell line, whereas saturated fatty acids  show no cell inhibition activity . HoweverArgemone genus are scarce. For this reason, the results regarding the anti-proliferative activity of fatty acids, FAG-4A and FAG-5B fractions, could be the starting point for their further study as potential inhibitors of a wide range of human cancer cells, hence, pointing out their relevance in the battle against cancer.Studies of biological activities and the importance of fatty acids extracted from the Argemone gracilenta on cancerous cell lines and provides support for the traditional use of this plant against multiple diseases, as well as of other species of the Argemone genus. In addition, two alkaloids, berberine and argemonine, with important anti-poliferative activity were isolated. We present the first analysis of the argemonine alkaloid as an anti-proliferative compound, showing promising results for future studies as a potential anticancer drug. Fatty acids and fatty acids esters of 16 or more carbon atoms with anti-proliferative activity were also identified. This is also the first time that the anti-proliferative activity displayed by plants of the Argemone genus is associated to this type of compounds.This study presents the first analysis of the anti-proliferative activity of


The complex has a highly ruffled porphyrin core with mean absolute core-atom displacements Ca, Cb, Cm and Cav of 0.25 (5), 0.17 (12), 0.432 (16) and 0.25 (13) Å, respectively. One of the four phenyl groups of the porphyrin is disordered over two sets of sites with refined occupancy ratio of 0.718 (7):0.282 (7). The mean Fe—Np (Np is a porphyrin N atom) bond length [1.975 (9) Å] indicates the low-spin state of the iron atom. The two 2-methylimidazole ligands are nearly perpendicular and form a dihedral angle of 86.93 (10)°. The dihedral angles between the 2-methylimidazole ligands and the closest Fe—Np vector are 38.04 (9) and 35.00 (7)°. In the crystal, the complex cations interact with the perchlorate anions through N—H⋯O hydrogen bonds, forming chains running parallel to [110].In the title compound, [Fe(C L)2]+  and with the central FeIII atom in an octahedral coordination are known. The first ferric porphyrin crystal structure with two sterically hindered axial ligands is [Fe(OEP)(2-MeHIm)2]ClO4, which was reported by Geiger and co-workers 2]ClO4  (2-MeHIm)2]Cl . Herein, we report the structural properties of the iron(III) porphyrin complex [Fe(TTP)(2-MeHIm)2](ClO4)·1.5THF where the metal is likewise octahedrally coordinated.The structural characterization of metalloporphyrin complexes with steric nitrogen-donor ligands has been undertaken intensively in order to understand the control of structures, spin states, and other physical properties. Many structures of ferric porphyrins with general formula [Fe(Porph)(meso-tetrakis(p-tolyl)porphyrinato]iron(III) is a negatively charged perchlorate ion. One of the four phenyl groups of the porphyrin is disordered over two sets of sites [0.718 (7):0.282 (7)] and the dihedral angles between the disordered phenyl planes and the 24-atom mean plane are 72.4 (4) and 63.36 (12)°. Additional quantitative information on the structure is given in Fig. 2p vector. The relative orientation of the two 2-methylimidazole planes is nearly perpendicular, the dihedral angle being 86.93 (10)°. Fig. 2Ca, Cb, Cm, and Cav are 0.25 (5), 0.17 (12), 0.432 (16) and 0.25 (13) Å, respectively. The mean Fe—Np (Np is a porphyrin N atom) bond length is 1.975 (9) Å, similar to 1.974 (4) Å in (perp-[Fe(OEP)(2-MeHIm)2]Cl) 2]ClO4 2]ClO4, which hinders the rotation of the benzene groups.The dihedral angles between the mean planes of the phenyl rings and the 24-atom mean plane are 59.55 (6), 82.53 (7), 72.4 (4) [and/or 63.36 (12)] and 75.17 (5)°, smaller than the same angles of 89.7, 83.3, 87.2 and 87.9° in  were prepared according to the reported methods  were dissolved in 50 mL THF. After 12 h reaction, the solution was filtered and then evaporated to dryness under vacuum. The resulting purple solid, [Fe(TTP)ClO4], was harvested that was dried in vacuo . UV–vis (CH2Cl2): 411.89, 516.5 nm.[Fe(TTP)Cl]  and AgClO4]  and excess 2-methylimidazole  were dissolved in 7 mL THF. After 10 min, the solution was transferred into glass tubes which were layered with hexanes as nonsolvent. Dark-purple block-shaped crystals suitable for a single-crystal X-ray diffraction study were collected after 15 d. UV–vis (CH2Cl2): 415.44, 509.68, 571.87, 612.00 nm.[Fe(TTP)ClOUiso(H) = 1.5Ueq(C) for methyl H atoms or Uiso(H) = 1.2Ueq otherwise. One THF molecule is disordered over two sets of sites about an inversion center with an occupancy factor of 0.5. During the refinement, the O—C, C—C and C⋯C distances within the disordered THF molecule were constrained to be 1.42 (1), 1.50 (1) and 2.40 (1) Å, respectively. One of the four phenyl groups was found to be disordered over two orientations and the site occupancy factors (SOFs) of disordered moieties are refined by means of a ‘free variable’. The refined final SOFs were 0.718 (7) and 0.282 (7). Two carbon atoms (C39 and C43) of the tetrahydrofuran molecules and one carbon atom (C12) of a methyl group exhibited unusually large displacement parameters and thus were refined using SIMU and ISOR restraints. Seven outliers were omitted in the last cycles of refinement.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016010562/rz5191sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989016010562/rz5191Isup2.hklStructure factors: contains datablock(s) I. DOI: 1471779CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Today feature tracking (FT) is considered to be a robust assessment tool in cardiovascular magnetic resonance (CMR) for strain assessment. The FT algorithm is dependent on a high contrast between blood pool and myocardium. Extracellular contrast agents decrease blood-myocardial contrast in SSFP images and thus might affect FT results. However, in a routine CMR scan, SSFP-cine images including short axis views are partly acquired after contrast agent injection. The aim of this study was to investigate the effect of extracellular contrast agent (Gadobutrol) (CA) on the precision and reproducibility of the feature tracking algorithm.A total of 40 patient volunteers  were scanned in supine position on a clinical 1.5 T MR scanner (Philips Ingenia). SSFP-cine images in midventricular short axis view (SA) as well as horizontal long axis view (HLA) were acquired before and 10–15 min after injection of a double dose Gadobutrol. FT derived systolic circumferential and longitudinal strain parameters were then calculated for pre- and post-contrast images.FT derived midventricular peak systolic circumferential strain (PSCS) (-24.8 ± 6.4 % vs. -20.4 ± 6.3 %), apical PSCS (-28.67 ± 6.5 % vs. -24.06 ± 8.5 %), basal PSCS (-24.42 % ± 6.5 vs. -20.68 ± 7.1 %), peak systolic longitudinal strain (-19.57 ± 3.3 % vs. -17.24 ± 4.1 %), midventricular epicardial PSCS (-9.84 ± 3.4 % vs. -8.13 ± 3.4 %) , midventricular PSCS-rate (-1.52 ± 0.4 vs. -1.28 ± 0.5) and peak diastolic circumferential strain rate (1.4 ± 0.5 vs. 1.05 ± 0.5) were significantly reduced after CA application. Post CA strain assessment showed higher intra- and interobserver variability. Pre-CA: intraobserver: mean 0.21, Limits of agreement (LoA) -2.8 and 3.2; interobserver: mean 0.64, LoA -2.8 and 4.1. Post-CA: intraobserver: mean -0.11, LoA -5.1 to 4.9; interobserver: mean 4.93 LoA 2.4 to 12.2.The FT algorithm is dependent on a high contrast between blood and myocardium. Post CA strain results are significantly lower and less reproducible than pre-CA strain results. Myocardial strain imaging allows for objective quantification of cardiac contractility and is increasingly being recognized as a sensitive tool for analysis and diagnosis of various myocardial disorders , 2. Furt40 patient volunteers  were prospectively enrolled into the study. Blood pressure (BP) and heart rate (HR) were monitored during imaging. The study population included patients with suspected dilated cardiomyopathy, myocarditis and hypertrophic cardiomyopathy. None of the included patients had myocardial scar. Glomerular filtration rate (GFR) was controlled in all patients prior to contrast agent application, all patients had sufficient renal function (GFR: >40 ml/min). Written informed consent was obtained from all controls and patients. This study was approved by the institutional review board .CMR was performed on a clinical 1.5 T MR scanner  with a dedicated cardiac phased-array receiver coil. Scout images were acquired in axial, coronal and sagittal orientation. Cardiac functional imaging was performed using retrospectively gated SSFP sequences in the standard cardiac axes. For the assessment of ejection fraction, a minimum of 12 short axis slices (SA) were acquired per subject, with 30 phases reconstructed per slice. For assessment of strain additional retrospectively gated balanced-SSFP cine images in apical, midventricular and basal SA as well as horizontal long axis view (HLA) (each with 40 phases reconstructed per slice) were acquired before and 10–15 min after injection of a double dose contrast agent (CA) ) as used for late enhancement imaging. Typical scan parameters were: field of view 350 mm, slice thickness: 8 mm; NSA:1; TE 1.4 ms; TR 2.8 ms; Flip angle 50°; 40 phases per cardiac cycle.(Ɛcc) values were derived from the apical-, mid- and basal-left-ventricular short axis slice. Longitudinal strain values were derived from a horizontal long axis slice. For strain analysis an initial endocardial contour is drawn in an end-diastolic phase with optimal contrast between blood and myocardium. The FT software then propagates the contour throughout the cardiac cycle. In case of faulty contour propagation the software allows editing throughout the cardiac cycle. Additionally, endocardial shortening was assessed in 20 subjects by calculating the percentage of end-diastolic to end-systolic shortening of the endocardial contour lengths in a midventricular SSFP image pre- and post-CA application.CMR-FT strain analysis was performed using dedicated software  which has been previously validated –6. CircuTo investigate the effect of CA on FT strain assessment, established strain derived CMR indices , peak systolic circumferential strain rate (PSCSR), peak diastolic circumferential strain rate (PDCSR)) were calculated for the midmyocardial slice in 40 subjects. Additionally, apical and basal PSCS systolic longitudinal strain (PSLS) as well as midventricular epicardial PSCS (EPSCS) were calculated for 20 patients. The peak strain rates were defined as the minimum respectively maximum values of the strain rate curve .Intra- and interobserver reproducibility of midmyocardial strain was investigated by two independent blinded observers in 20 randomly selected subjects. For assessment of intraobsever reproducibility an interval of two weeks was chosen between the first and second analysis.2) were placed in the septum and the left ventricular lumen in an end-diastolic image. The signal average of the left ventricular lumen was divided by the signal average of the septum.To investigate the reduction of the blood-myocardium contrast, a blood-myocardium contrast quotient was calculated in 20 subjects in a midventricular SSFP image pre and post CA application. Regions of interest (ROI) . Results are expressed as mean ± SD. Normal distribution was tested with the D’Agostino-Pearson test. The Student t-test was employed for pre- and post-CA SSFP derived strain comparison if data were normally distributed, otherwise the Wilcoxon signed rank test was used. n method  to assesƐcc curves derived by FT in a subject pre- and post-CA application. Mean HR (67.5 ± 13.6 BPM vs. 69.2 ± 13.9 BPM), mean BP ( 136.1 ± 18.2/72 ± 9 mm Hg vs. 138 ± 20.1/72.9 ± 8.2 mm Hg) as well as mean LVEF( 61 ± 9 % vs. 60 ± 9 %) did not differ significantly pre- and post-CA application. The blood-myocardium contrast quotient significantly decreased post CA application (4.03 ± 0.6 vs. 2.16 ± 0.3 p < 0.0001). No significant correlation was found between the degree of contrast reduction and the degree of strain reduction following CA application (r = 0.32). Assessment of endocardial shortening revealed no significant difference between pre-CA and post-CA images (27.97 ± 8.02 % vs. 27.63 ± 8.2 %).The study protocol was completed by all participants. Table r = 0.81 , apical PSCS (-28.67 ± 6.5 % vs. -24.06 ± 8.5 %), basal PSCS (-24.42 % ± 6.5 vs. -20.68 ± 7.1 %), PSLS (-19.57 ± 3.3 % vs. -17.24 ± 4.1 %) and midventricular EPSCS (-9.84 ± 3.4 % vs. -8.13 ± 3.4 %) were significantly reduced in comparison to baseline strain analysis. Furthermore, midventricular PSCSR(-1.52 ± 0.4 vs. -1.28 ± 0.5) and PDCSR(1.4 ± 0.5 vs. 1.05 ± 0.5) were also significantly reduced after CA application (Tables r = 0.97) (Interobserver: r = 0.95) and superior in comparison to post-CA derived strain (Intraobserver: r = 0.91) (Interobserver: r = 0.91). The intra- and interobserver coefficients of variation were 4.5 and 5.4 %, for pre-CA strain assessment, 22.8 % and 20.9 % for post-CA strain assessment respectively.Bland Altman analysis of pre-CA intra-observer reproducibility yielded a better agreement (0.21 ± 1.5 with limits of agreement between -2.8 and 3.2) than post-CA intra-observer reproducibility (-0.11 ± 2.5 with limits of agreement between- -5.1 to 4.9). Correspondingly interobserver variability (0.64 ± 1.75 with limits of agreement between -2.8 and 4.1 vs. 4.93 ± 3.7 s with limits of agreement between- -2.4 to 12.2) was superior pre-CA  may increase blood myocardium contrast post CA injection. However, in this study pulse sequences were chosen to reflect the clinical routine. As discussed above endocardial shortening and ejection fraction were used as surrogate markers to rule out an effect of Gadobutrol on contractility, however, the gold standard for strain imaging -namely myocardial tissue tagging- was not employed.FT has become an established tool for rapid SSFP based strain analysis, however it is dependent on a high contrast to allow for precise voxel tracking from frame to frame. Assessing strain from post CA SSFP cine images, which leads to a reduction in the myocardium-blood contrast, leads to an unsystematic underestimation of strain with increased intra- and interobserver variability. The strain based evaluation of many ischemic- and non-ischemic- cardiomyopathies demands the highest possible precision and robustness, therefore FT based strain analysis should only be performed on pre CA SSFP cine images.


Their structures were elucidated on the basis of extensive spectroscopic analysis including NMR, UV and MS. The prenyl moieties in compounds 1–3 were further modified by cyclization and hydroxylation. The new compounds were tested for their cytotoxicity against four cancer cell lines  and showed IC50 values in the range of 13.76–22.27 μM.Three new minor prenylated flavonoids, named macadenanthins A–C (The online version of this article (doi:10.1007/s13659-015-0059-1) contains supplementary material, which is available to authorized users. Macaranga −), requiring 13° of unsaturation. The IR spectrum showed absorption bands for OH (3425 cm−1), carbonyl (1648 cm−1) and aromatic ring (1604 and 1488 cm−1) moieties. The UV spectrum showed absorption maxima at 270 and 380 nm, which indicated the presence of a flavonol skeleton     ; a prenymentclasspt{minimaentclass1pt{minima2) possessed a molecular formula of C25H24O6 as deduced from its HREIMS (m/z 420.1576 [M]+), suggesting 14° of unsaturation. The IR spectrum of 2 indicated the characteristic bands of hydroxyl (3423 cm−1) and conjugated carbonyl (1648 cm−1) groups. Comparing the NMR spectral data , 132.3 (d), 77.9 (s), 28.3 × 2 (q); δH 6.45 , 5.78 , 1.41 ] +), suggesting 13° of unsaturation. The UV spectrum showed the characteristic absorbances for a flavonol [λmax (log ε) 270 (4.47) and 376 (4.27) nm]    in 3. Th1–3 were evaluated against human breast adenocarcinoma (MCF-7), human hepatocellular (Hep G2), human cervical carcinoma (Hela), mouse leukemia (P388) cell lines by MTT method, with 5-FU used as a positive control −, 875 [2M–H]−; HRESIMS m/z 437.1609 .Yellow powder; mentclass2pt{minimc 0.34, MeOH); UV (MeOH) λmax (log ε) 211 (4.50), 236 (4.41), 361 (3.98), 430 (4.21) nm; IR (KBr) νmax 3423, 2974, 2924, 1648, 1624, 1602, 1564, 1483, 1362, 1320, 1278, 1195, 1157, 1135, 1091, 1042, 960, 807 cm−1; 1H and 13C NMR data see Table m/z 419 [M–H]−, 839 [2M–H]−; HREIMS m/z 420.1576 .Yellow powder; mentclass2pt{minimc 0.12, MeOH); UV (MeOH) λmax (log ε) 204 (4.83), 270 (4.47), 376 (4.27), 426 (4.21) nm; IR (KBr) νmax 3428, 2925, 2855, 1648, 1625, 1612, 1485, 1367, 1321, 1268, 1190, 1155, 1090, 1044, 822, 604, 543 cm−1; 1H and 13C NMR data see Table m/z 437 [M–H]−; HREIMS m/z 438.1675 .Yellow powder; mentclass2pt{minim1–3 were tested for their cytotoxicity against human breast adenocarcinoma (MCF-7), human hepatocellular (Hep G2), human cervical carcinoma (Hela), mouse leukemia (P388) by the MTT method, and 5-FU was used as a positive control. Briefly, 100 μL cell suspension (1 × 105 cells/mL) was seeded into 96-well microteter plates and cultured for 24 h before the compound was added. Then, different concentrations of the compounds were added to the plates, the cells were cultivated for 48 h, and 10 μL of MTT (5 mg/mL) was added to each well. After 4 h, the culture medium was removed and the formazan crystal was completely dissolved with 150 μL DMSO to each well by vigorously shaking the plate. Finally, formazan absorbance was assessed by a BioRad microplate reader at 570 nm.Compounds Supplementary material 1 (PDF 2534 kb)


Here we analyze if VA-esters can serve as prodrugs and if different ester structures have different in vitro/in vivo effects. Modulation of GABAA receptors expressed in Xenopus oocytes was studied with 2-microelectrode-voltage-clamp. Anxiolytic effects of the VA-esters were studied on male C57BL/6N mice by means of the elevated plus maze-test; anticonvulsant properties were deduced from changes in seizure threshold upon pentylenetetrazole infusion. VA was detected in plasma confirming hydrolysis of the esters and release of VA in vivo. Esterification significantly reduced the positive allosteric modulation of GABAA (α1β3γ2S) receptors in vitro. in vivo, the studied VA-ester derivatives induced similar or even stronger anxiolytic and anticonvulsant action than VA. While methylation and propylation of VA resulted in faster onset of anxiolysis, the action of VA-ethylester was longer lasting, but occurred with a significant delay. The later finding is in line with the longer lasting anticonvulsant effects of this compound. The estimated VA plasma concentrations provided first insight into the release kinetics from different VA-esters. This might be an important step for its future clinical application as a potential non-sedative anxiolytic and anticonvulsant.Valerenic acid (VA) is a β GABAA receptors belong to the superfamily of Cys-loop-type ligand-gated ion channels  modulating neuronal excitability and transmitter release  type A receptors  diseases such as generalized anxiety and panic disorders, epilepsy, and sleep disturbances  of recombinant receptors prevents IGABA enhancement while the “reversed mutation” in β1 (S266N) enhances current stimulation to extents observed on β2/3-subunit containing receptors (3(N265M) point-mutated mice supporting the hypothesis that the anxiolytic effects of VA are caused by interaction with β3-containing GABAA receptors , a constituent of affinity  and modusubunits . A pointeceptors . Valereneceptors . Anxiolyeceptors .A recently published pharmacokinetic study on rats revealed that approximately 34% of VA are absorbed after oral administration with a half-life between 2.7 and 5 h . This goIGABA through GABAA receptors? (ii) Do VA-esters represent prodrugs (i.e. are esters hydrolyzed in vivo and is VA detectable in the plasma? (iii) Are VA-esters active in vivo and – if so – does esterification affect the anxiolytic and anticonvulsant properties of VA?Together these findings make VA or one of its derivatives  interest2All experiments on animals were carried out in accordance to the Austrian Animal Experimental Law, which is in line with the EU directive 2010/63/EU.2.1t-butylmethylether were of p.a. quality and purchased from ROTH . For HPLC analysis double distilled water and acetonitrile, HPLC quality  were used.Valerenic acid (VA) was purchased from HWI Pharma Solutions  and converted into the aforementioned derivatives as described below . Reaction mixtures were magnetically stirred and monitored by thin layer chromatography using Merck Silica 60F254 plates . Flash chromatography was performed on a Sepacore Flash System  using Merck silica gel . Yields refer to chromatographically and spectroscopically pure compounds. 1H-NMR (200 MHz) and 13C-NMR (50 MHz) were recorded on Bruker AC 200 . The chemical shifts δ are reported relative to the residual solvent peaks. All 1H and 13C shifts are given in ppm . Specific rotation was measured on an Anton Paar MCP500 polarimeter  at 20 °C in DCM.All reactions were carried out in oven dried 4 ml-reaction vials under an argon atmosphere. DCM was predistilled and then desiccated on AlLC–MS/MS analyses were carried out on an Ultimate 3000 RSLC-series system  coupled to a triple quadrupol mass spectrometer API 4000 .2.22.2.14Cl solution (three times), saturated NaHCO3 solution (three times) and once with brine; it was then dried and concentrated under reduced pressure. Purification of the crude material via column chromatography (LP:EtOAc=30:1) provided 30.2 mg (94%) of Valerenic acid methylester as a colorless oil.•1H-NMR :δ=0.77 , 1.37–1.98 , 2.19 , 2.90–2.99 , 3.49–3.56 , 3.72 , 7.01 •13C-NMR δ=12.0 (s), 12.4 (s), 13.5 (s), 24.5 (d), 25.4 (d), 28.7(d), 33.0 (t), 34.3 (t), 37.4 (d), 47.4 (t), 51.7 (s), 125.7 (q), 130.9 (q), 133.4 (t), 169.0 (q)Valerenic acid  and 4-dimethylaminopyridine  were dissolved in 1.3 ml dry DCM under an Argon atmosphere and cooled to 0 °C, then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide  was added in one portion. After stirring the mixture for five min methanol  was added dropwise and the mixture was left warming to room temperature overnight. The solution was taken up in 50 ml ethylacetate (EtOAc) and was subsequently washed with saturated NHAnalytical data is consistent with the reported data for Valerenic acid methylester .2.2.2•1H-NMR :δ=0.78 , 1.29 , 1.37–2.02 , 2.19 , 2.92–2.98 , 3.46–3.56 , 4.17 , 7.01 •13C-NMR :δ=12.0 (s), 12.4 (s), 13.5 (s), 14.3 (s), 24.5 (d), 25.5 (d), 28.7(d), 33.1 (t), 34.3 (t), 37.4 (d), 47.4 (t), 60.4 (d), 125.7 (q), 130.9 (q), 133.4 (t), 169.0 (q)Using the analogous procedure as for the preparation of VA-ME, treatment of valerenic acid  with 4-dimethylaminopyridine , EDCI  and ethanol  yielded 20.2 mg (95%) of VA-EE as colorless oil.Analytical data is consistent with the reported data for Valerenic acid ethylester .2.2.3•1H-NMR :δ=0.77 , 0.95 , 1.37–2.01 , 2.19 , 2.91–2.97 , 3.48–3.55 , 4.07 , 7.02 •13C-NMR δ=10.5 (s), 12.0 (s), 12.4 (s), 13.5 (s), 24.5 (d), 25.5 (d), 28.7(d), 33.1 (t), 34.3 (t), 37.4 (d), 47.4 (t), 66.0 (d), 126.0 (q), 130.7 (q), 133.5 (q), 143.3 (d) 168.7 (q)Using the analogous procedure as for the preparation of VA-ME, treatment of valerenic acid  with 4-DMAP , EDCI  and propanol  yielded 35.1 mg (99%) of VA-PE as colorless oil.20D=−77.0 ;[α]2.2.44Cl and NaHCO3 solution and one time with brine. The organic phase was dried and concentrated in vacuo. Purification by column chromatography (LP:EtOAc=4:1) furnished 19.1 mg (85%) of VA-POM as a slightly yellow oil.•1H-NMR :δ=0.78 , 1.21 , 1.38–1.98 , 2.20 , 2.91–2.95 , 3.50–3.55 , 5.81 , 7.09 •13C-NMR δ=12.0 (s), 12.2 (s), 13.5 (s), 24.5 (d), 25.3 (d), 26.8 (s), 28.7(d), 33.0 (t), 34.3 (t), 37.4 (d), 38.8 (q), 47.4 (t), 79.9 (d), 125.0 (q), 131.2 (q), 133.1 (q), 145.6 (d) 167.0 (q), 177.2(q)Valerenic acid  and 1,8-diazabicycloundec-7-ene  were dissolved in dry DCM (1.3 ml) under an argon atmosphere; pivaloyloxymethyl chloride  was added dropwise . The mix20D=−70.4 ;[α]2.3Xenopus laevis , synthesis of capped off run-off poly(A+) cRNA transcripts from linearized cDNA templates (pCMV vector) was performed as described , before surgically removing parts of the ovaries. Follicle membranes from isolated oocytes were enzymatically digested with 2 mg/ml collagenase (Type 1 A). One day after isolation, the oocytes were injected with about 10–50 nl of diethylpyrocarbonate—treated water containing the different cRNAs at a concentration of approximately 150–3000 ng/μl/subunit. The amount of cRNA was determined by means of a NanoDrop ND-1000 . To ensure expression of the γ-subunit in the case of α1β3γ2S receptors, cRNAs were mixed in a ratio of 1:1:10 -1-piperazinyl)-ethanesulfonic acid . Microelectrodes were filled with 2 M KCl and had resistances between 1 and 3 M Ω  to 3 min (co-application of GABA EC3–7 and 1 µM compound) to 5–10 min (co-application of GABA EC3–7 and 10 µM compound) to 15–20 min (co-application of GABA EC3–7 and 30 µM compound) to 30 min (GABA EC3–7 and 100 µM compound). Oocytes with maximal current amplitudes >3 µA were discarded to exclude voltage-clamp errors . The EC3–7 was determined at the beginning of each experiment.Stimulation of chloride currents by modulators of the GABAI(GABA+Compound)/IGABA)−1, where I(GABA+Compound) is the current response in the presence of the compound and IGABA is the control GABA current. Each data point represents the mean±S.E.M from at least 5 oocytes and ≥2 oocyte batches.Enhancement of the chloride current was defined as  were obtained from Charles River Laboratories . For maintenance, mice were group-housed (maximum 5 mice per type IIL cage) with free access to food and water. At least 24 h before the commencement of experiments, mice were transferred to the testing facility, where they were given free access to food and water. The temperature in the maintenance and testing facilities was 22±2 °C; the humidity was 40–60%; a 12 h light-dark cycle was in operation (lights on from 07.00 to 19.00). Only male mice – aged 3–6 months – were tested. Compounds at a dose of 3 mg/kg bodyweight or solvent alone were applied by intraperitoneal (i.p.) injection. The dose was chosen according to a previously published dose–response curve . Testing solutions were prepared in a solvent composed of saline (0.9% NaCl solution with 10% DMSO and 3% Polysorbat 80). The final DMSO concentration was fixed to 10% . Applica2.6.2The animals’ behavior was tested over 5 min on an elevated plus maze 1 m above ground consisting of two closed and two open arms, each 50×5 cm in size. The test instrument was built from grey PVC; the height of closed arm walls was 20 cm. Illumination intensity was set to 180 lx. Animals were placed in the center, facing an open arm. Analysis of open and closed arm entries, distance and time on open arm was automatically done with Video-Mot 2 equipment and software . Drugs or solvent were applied 15, 30 or 60 min before testing.2.6.3Seizure threshold was determined by pentylenetetrazole (PTZ) tail-vein infusion on freely moving animals at a rate of 100 µl/min . Infusion was stopped when animals displayed generalized clonic seizures. Animals were killed by cervical displacement immediately after the first generalized seizure. The seizure threshold dose was calculated from the infused volume in relation to body weight. The compounds were injected 15, 30, 60, 90, 120 or 150 min before PTZ infusion. At the infusion rate of 100 µl/min, generalized seizures are induced within 90 s.2.72.7.1Blood samples were taken 15, 30, 60 and 120 min after i.p. injection of the compounds. 10 min after i.p injection of thiopental (150 mg/kg bodyweight in 0.9% sodium chloride solution) blood samples (500–800 µl) were collected and compiled into ethylenediamine tetra-acetic acid (EDTA)-coated micro tubes (1.6 mg EDTA/sample) and centrifuged at 12,000 rpm for 5 min at 4 °C. Plasma samples were transferred into 1.5 ml tubes and stored at −80 °C until analysis.2.7.2t-butylmethylether  and vortexed for 5 min. From the clear lower organic layer the solvent was removed through a constant nitrogen stream at room temperature (25 °C). The residue was dissolved with 100 µl of methanol, sonicated, centrifuged for 5 min  and the supernatant was finally transferred to autosampler vials .A liquid–liquid extraction method together with an internal standard (IS) acetoxyvalerenic acid  was applied for the quantification of VA in plasma. To 100 µl of plasma sample 10 µl of IS solution (1 µg/ml 10% aqueous DMSO) was added. These solutions were extracted by liquid–liquid partition with 400 µl of dichloromethane/2.7.3The samples (10 µl) were analyzed by liquid chromatography/mass spectrometry (LC–MS/MS) on an Ultimate 3000 RSLC-series system  coupled to a triple quadrupol mass spectrometer (AB Sciex Instruments API 4000) equipped with an orthogonal APCI source operated in negative mode and displayed with Analyst 1.5 software.m/z 233 [M–H]− (VA) and m/z 291 [M−H]− (ACVA). MRM m/z 233/84 (VA) as well as m/z 291/249 (ACVA) were used for calibration curves to give a linear concentration range from 0.1 ng/ml  to 500 ng/ml (correlation coefficient 0.9996). Extraction efficiencies (average 84%) were determined by comparison of peak areas between quality control (QC) and analysis samples. For validation, quality control (QC) samples were prepared in the same way as the calibration standards.LC separation was performed on an Acclaim RSLC 120C18 column , preceded by an Acclaim 120C18 guard cartridge , at a flow rate of 0.500 ml/min and a column temperature of 30 °C. The mobile phase consisted of a continuous linear gradient, mixed from aqueous formic acid, pH 3.5 (mobile phase A), and acetonitril (mobile phase B), to elute VA. The gradient ranged from 50% B (0 min) to 80% at 8 min, kept constant at 80% until 10 min, and finally decreased linearly to 50% again at 11 min. Between sampling, the column was purged with 98% B (acetonitrile) for 4 min before equilibrating for 6 min resulting in a total analysis time of 18 min. Within this setup valerenic acid eluted at 4.06 min., acetoxyvalerenic acid (IS) at 7.02 min. Selective and sensitive detection and quantification was carried out using MS/MS fragmentation of VA resp. acetoxyvalerenic acid (ACVA) giving a quasimolecular ion at m/z 233/84 (VA): CE-29, dwell time 300 ms. MRM m/z 291/249 (ACVA): CE-24, dwell time 300 ms.The triple quadrupol mass spectrometer operated with the following parameters: APCI neg., NC-5, CUR 10, GS1 30, GS2 18, TEM 400 °C, CAD 12, EP-11, DP-65, CXP-5, CEM 2100, DF 200. MRM 2.8t-test; for in vivo experiments, one-way ANOVA  was used. Statistical analysis was done with Origin software . P-values of <0.05 were accepted as statistically significant. All data are given as mean±S.E.M.Statistical significance of electrophysiological data was calculated using a paired Student 33.1IGABA modulation by VA-esters was less pronounced than by VA. This is shown in IGABA through α1β3γ2S GABAA receptors during co-application of GABA (EC3–7) and either VA, VA-ME, VA-EE, VA-PE or VA-POM.IGABA enhancement than VA-PE and VA-POM (max. IGABA potentiation (VA-ME 30 µM): 70±13% (n=5) and max. IGABA potentiation (VA-EE 30 µM): 52±15% (n=6) vs. max. IGABA potentiation (VA-PE 30 µM): 27±6% (n=5) and max. IGABA potentiation (VA-POM 30 µM): 21±6% (n=6). Compared to VA the modulation of IGABA at 30 µM was drastically reduced (from 6.2-fold (VA-ME) to 20.6-fold reduction (VA-POM)). At 1 µM none of the studied ester derivatives induced significant IGABA enhancement  or drug containing solutions at a dose of 3 mg/kg bodyweight.n=33) in the open arms (OA) of the EPM. An increase of time spent in the OA was observed upon application of VA-ME and VA-PE  Animals treated with VA-esters also covered significantly longer distances on the OA , while the other parameters did significantly not differ from control . No anticonvulsant effect was observed either at 15 or 60 min after VA application . The anticonvulsant effect persisted 30 min after treatment , however, VA-ME did not induce any significant effects on seizure threshold 60 min after application.Application of VA induced an increased threshold against pentylenetetrazole (PTZ)-induced seizures 30 min after application of VA . Seizure threshold was further significantly increased 90 min after application  and remained at the same level even 120 min after application . 150 min after application the seizure threshold of VA-EE-treated mice did not differ from the control . The seizure threshold elevation at this time point was even more pronounced than that of VA or the other derivatives (P<0.05). However, no statistically significant anticonvulsant effects could be detected at a later time point.As illustrated in n=7 vs. VA-POM: 48.4±2.1 mg/kg; n=4; P<0.05). No anticonvulsant activity, however, was observed 90 min after application  prevents anxiolytic activity of VA in mice. It was therefore concluded that anxiolysis occurs via neurons expressing β3 comprising GABAA receptors  see  which main vitro  their poin vitro B and D. Taken together, several VA-esters display similar or stronger in vivo activity than VA. The different time courses of anticonvulsant activity (e.g. fast onset of the VA-ME and long lasting effects of the VA-EE) may be beneficial for potential therapeutic use of this molecule. Future studies will show whether the increased lipophilicity of the esters will affect the oral bioavailability of VA.


Herbaleonuri), which was previously found highly cardioprotective, both in vitro and in vivo.Neuroinflammation mediated by overactivated microglia plays a key role in many neurodegenerative diseases, including Alzheimer’s disease (AD). In this study, we investigated for the first time the anti-neuroinflammatory effects and possible mechanisms of SCM-198  or β-amyloid1-40 (Aβ1-40) was applied to induce microglial overactivation. Proinflammatory mediators were measured and activations of NF-κB and mitogen-activated protein kinases’ (MAPKs) pathways were investigated. Further protective effect of SCM-198 was evaluated in microglia-neuron co-culture assay and Sprague-Dawley (SD) rats intrahippocampally-injected with Aβ1-40.For 1-40-induced neuronal death and lactate dehydrogenase (LDH) release in primary cortical neurons. For in vivo studies, SCM-198 significantly enhanced cognitive performances of rats 12 days after intrahippocampal injections of aged Aβ1-40 peptides in the Morris water maze (MWM), accompanied by less hippocampal microglial activation, decreased synaptophysin loss and phosphorylation of ERK and tau. Co-administration of donepezil and SCM-198 resulted in a slight cognitive improvement in SD rats 50 days after intrahippocampal injections of aged Aβ1-40 peptides as compared to only donepezil or SCM-198 treated group.SCM-198 reduced expressions of nitric oxide (NO), TNF-α, IL-1β and IL-6 possibly via, at least partially, inhibiting c-Jun N-terminal kinase (JNK) and NF-κB signaling pathways in microglia. Co-culture assay showed that activated microglia pretreated with SCM-198 led to less neuron loss and decreased phosphorylation of tau and extracellular signal-regulated kinase (ERK) in neurons. Besides, SCM-198 also directly protected against AβOur findings are the first to report that SCM-198 has considerable anti-neuroinflammatory effects on inhibiting microglial overactivation and might become a new potential drug candidate for AD therapy in the future. Alzheimer’s disease (AD), the most common form of dementia among the elderly, is a chronic progressive disease characterized by cerebral deposition of senile plaques composed of amyloid-β (Aβ) peptides, intraneuronal neurofibrillary tangles originating from hyperphosphorylation of tau protein, profound loss of neurons and neuroinflammation –3. Sincepostmortem brains from AD patients and animals, most reactive microglia are located around dense-core Aβ plaques and elevated proinflammatory factors are also found in those brains which reveal the negative impact of neuroinflammation on AD progression : 5′-TTCCATCCAGTTGCCTTCTTG-3′ and 5′-TATCCTCTGTGAAGTCTCCTCTC-3′IL-1β [GenBank:NM_008361]: 5′-ATCTCGCAGCAGCACATCAAC-3′ and 5′-TGTTCATCTCGGAGCCTGTAGT-3′TNF-α [GenBank:NM_013693]: 5′-CGTGGAACTGGCAGAAGAGG-3′ and 5′-TCAGTAGACAGAAGAGCGTGGT-3′iNOS [GenBank:NM_010927]: 5′-GGACGAGACGGATAGGCAGAGATT-3′ and 5′-AAGCCACTGACACTTCGCACAA-3′β-actin [GenBank:NM_007393]: 5′-CTATTGGCAACGAGCGGTTCC-3′ and 5′-CAGCACTGTGTTGGCATAGAGG-3′Total RNA was extracted from BV-2 cells using TRIzol reagent  according to the manufacturer’s instructions. One microgram of total RNA of each sample was reverse transcribed into cDNAs using the PrimeScript® RT Master Mix Perfect Real Time kit . The resulting cDNAs were amplified by using a SYBR® Premix Ex TaqTM kit in iQ™5 real-time PCR detection system  at 95°C for 30 seconds, 40 cycles at 94°C for 10 seconds and 60°C for 30 seconds, followed by 1 minute at 95°C, 1 minute at 60°C and finally 71 cycles at 60°C. Gene expressions of TNF-α, IL-1β, IL-6 and inducible nitric oxide synthase (iNOS) were analyzed with β-actin as an internal control. The primer sequences are listed below:g for 20 minutes at 4°C and protein concentrations were measured using a BCA-100 Protein Quantitative Analysis kit . For the analysis of NF-κB p65 translocation, nuclear proteins of BV-2 cells were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents  according to the manufacturer’s instructions. Equal amounts of proteins were separated by 10 to 12% SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes . After blocking with 5% skim milk at RT for 1 hour, membranes were incubated with: polyclonal rabbit anti-NF-кB p65, monoclonal rabbit anti-histone H3 (D1H2), monoclonal mouse anti-IкBα (L35A5), monoclonal rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), monoclonal rabbit anti-p44/42 MAPK (Erk1/2) (137 F5), monoclonal rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (D3F9), polyclonal rabbit anti-p38 MAPK, monoclonal rabbit anti-phospho-SAPK/JNK (Thr183/Tyr185) (81E11), monoclonal rabbit anti-SAPK/JNK (56G8), monoclonal mouse anti-phospho-Tau (Ser396) (PHF13), monoclonal mouse anti-Tau (Tau46), monoclonal rabbit anti-synaptophysin (D35E4), monoclonal rabbit anti-β-tubulin , polyclonal rabbit anti-phospho-Tau (Thr205), polyclonal rabbit anti-phospho-Tau (Ser404)  primary antibodies overnight at 4°C, followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies for 1 hour at RT. Blots were visualized using SuperSignal West Dura chemiluminescent substrate  in Alpha Imager Detection System  and images were analyzed by ImageJ software .Cells or rat hippocampus were lysed for 30 minutes on ice in radioimmunoprecipitation assay (RIPA)lysis buffer  supplemented with 1 mM phenyl methanesulfonyl fluoride, 1% phosphatase inhibitor cocktail 2 and 3. Supernatants were collected after centrifugation at 16,200 1–40 for 24 hours. Cell viability was measured by MTT assay according to an earlier protocol [3P04 and 0.2% N-(1-naphthyl) ethylenediaminedihydrochloride) and incubated for 15 minutes at RT. The absorbance was measured at 546 nm and NaNO2 (0 to 100 μM) was used as the standard. Morphological changes of primary microglia were observed using phase-contrast microscope  and quantified by radius ratio (radius ratio = maximum radius of cell/minimum radius of cell) using Image-Pro Plus 6.0 Analysis System . Primary cortical neurons were preincubated with or without 0.1 to 10 μM SCM-198 or DON for 2 hours and stimulated with 20 μM aged Aβ1-40 for 12 hours. Neuron viability was detected by SRB assay according to descriptions by Wai H Yu et al. [Microglial cells were preincubated with or without 0.1 to 10 μM SCM-198, IBU or MAPK inhibitors for 2 hours and stimulated with 1 μg/ml LPS for 24 hours or with 3 μM Aβprotocol . TNF-α au et al.  and lact1-40 for 30 minutes. Cells were fixed with 4% paraformaldehyde and blocked with 10% BSA for 1 hour at RT, then incubated with monoclonal rabbit NF-κB p65 (D14E12) antibody  overnight at 4°C, followed by stained with Alexa Fluor 488-conjugated goat anti-rabbit IgG  for 2 hours in dark at RT. The nuclear translocation of NF-кB p65 was captured using fluorescence or confocal microscope .BV-2 cells and primary microglia were pretreated with or without 1 μM SCM-198, 100 μM IBU or 20 μM DON and stimulated with 1 μg/ml LPS or 3 μM Aβ1-40 group , Aβ1-40 + SCM-198 15, 30, 60 mg/kg groups, DON group . Drugs were given by gavage seven days before surgery, followed by daily administration until the end of the behavioral tests. Animals were provided with ad libitum food and water, and housed 5 per cage in a specific pathogen-free environment with 12-hour light/dark cycle and constant temperature (22°C ± 1°C). Seven days after drug pretreatment, rats were anesthetized with 7% chloral hydrate and positioned in a stereotaxic frame . Two micrograms per liter of aggregated Aβ1-40  or vehicle  was bilaterally injected into the hippocampus at a rate of 0.5 μl/minute . Twelve days after surgery, Morris water maze (MWM) was applied to evaluate the spatial memory of the animals.Sixty male SD rats   were randomly divided into 6 groups: sham group, Aβ1-40 group , Aβ1-40 + SCM-198 60 mg/kg groups, Aβ1-40 + DON 1 mg/kg group and Aβ1-40 + SCM-198 60 mg/kg + 1 mg/kg group DON group. Fifty days after surgery, MWM was applied to evaluate the possible long-lasting effect of SCM-198 and co-administration of SCM-198 and DON. All animal experiments conformed to guidelines of Regulations of Experimental Animal Administration of PR China (14 November 1988) and were approved by the Animal Ethics Committee of Fudan University.For investigating whether SCM-198 could improve the effect of DON, which is at the moment a palliative drug used in clinical management of AD, 45 male SD rats   were randomly divided into 5 groups: sham group, AβAnimals were tested in the MWM for assessment of spatial reference memory in a room with constant temperature (23 ± 1°C) and humidity (around 50%). The apparatus consists of a transparent platform (9 cm in diameter) and a black Plexiglas circular pool (160 cm in diameter) which is equally divided into 4 quadrants. Spatial cues of different geometric shapes and colors were fixed on the upper wall of the pool and water temperature was kept at 21 ± 1°C. A four-day protocol was conducted: during the acquisition phase (from the first day to the third day), the platform was fixed in the middle of one quadrant (target quadrant) and submerged 1 to 2 cm below the water surface. Each rat was trained twice per day to find the invisible platform within one or two minutes randomly from two different starting points equidistant from the platform. The time for finding the platform was defined as escape latency. If animals failed to locate the hidden platform within two minutes, they were gently guided onto the platform and left there for ten seconds. On the fourth day, the platform was removed and each rat was given one minute to swim in the pool. Time spent in the target quadrant, swimming speed and paths was recorded and analyzed by the Doctor Mice software  .2O2  solution. After blocked with a solution containing 5% BSA, 5% goat serum and 0.1% NaN3, sections were incubated with goat polyclonal anti-iba-1 antibody  overnight at 4°C, then stained with MaxVision™ HRP-Polymer anti-Goat IHC Kit  for 15 minutes at RT. Microglia were visualized using a diaminobenzidine kit , and photographed by a phase-contrast microscope . Integrated optical density (IOD) of iba-1 expression was calculated using Image-Pro Plus 6.0 Analysis System .Rats were transcardially perfused with 0.9% saline followed by ice-cold 4% paraformaldehyde. The brains were fixed in the same fixative for 3 days and then in 30% sucrose for 4 days at 4°C. Thirty-micron coronal sections were made using a cryostat microtome . Sections were incubated in 0.3% Triton X-100 for 30 minutes, followed by 17 minutes in methanol/3% Hpost-hoc analysis using GraphPad Prism . Values of P < 0.05 were considered statistically significant.Data were expressed as mean ± SEM and analyzed either by one-way analysis of variance (ANOVA) or two-way repeated-measures ANOVA followed by Tukey’s in vitro. As nitric oxide (NO) and cytokines, such as TNF-α, IL-1β and IL-6, are indicators of inflammatory process, we first investigated the inhibitory effects of SCM-198 on NO and proinflammatory cytokine release induced by LPS or Aβ1-40 in microglia. Two-hour pretreatment of BV-2 cells with 1 to 10 μM SCM-198 or 100 μM IBU could significantly suppress upregulation of iNOS, TNF-α, IL-1β and IL-6 mRNA expressions  = 17.42, P < 0.0001, Figure F  = 6.42, P = 0.0001, Figure F  = 6.56, P = 0.0006, Figure F  = 10.27, P = 0.0002, Figure F  = 4.08, P = 0.0033, Figure F = 9.50, P = 0.0007, Figure F = 10.23, P = 0.0001, Figure F  = 15.59, P < 0.0001, Figure 1-40 doubled the production of TNF-α in BV-2 cells, which was effectively inhibited by 2-hour pretreatment of 1 to 10 μM SCM-198 or 20 μM DON  = 14.74, P < 0.0001, Figure 1-40 also increased NO and TNF-α productions, which could also be significantly inhibited by 0.1 to 10 μM SCM-198 or 20 μM DON  = 7.022, P < 0.0001, Figure F  = 6.177, P = 0.0002, Figure No obvious cytotoxicity was observed for 0.001 to 100 μM SCM-198 (data not shown). Possible anti-neuroinflammatory mechanisms of SCM-198 were studied mainly using BV-2 cells 1-40 stimulation, while pretreatment of 1 μM SCM-198 or IBU or DON in some extent helped to prevent this cellular transformation  = 48.66, P < 0.0001, Figure F  = 9.794, P < 0.0001, Figure Morphological studies Figure  showed tn  = 5.36, P = 0.0009, Figure F  = 2.52, P = 0.0305, Figure F  = 36.58, P < 0.0001, Figure F  = 26.17, P < 0.0001, Figure 1-40 could also mildly induce similar IкB-α degradation and MAPKs phosphorylation in BV-2 cells, and 30 minutes was chosen as the optimal time for LPS or Aβ1-40 stimulation. Two-hour pretreatment with SCM-198 could significantly inhibit JNK phosphorylation and IкB-α degradation, but not ERK and p38  = 5.47, P = 0.0018, Figure F  = 6.27, P = 0.0002, Figure F  = 7.63, P = 0.0002, Figure F  = 74.44, P < 0.0001, Figure F  = 6.585, P = 0.0003, Figure F  = 4.772, P = 0.0036, Figure F  = 7.959, P = 0.0004, Figure F  = 16.00, P < 0.0001, Figure F  = 10.42, P < 0.0001, Figure F  = 16.55, P < 0.0001, Figure 1-40 activated the NF-кB signalling pathway and induced p65 translocation into the nucleus in both BV-2 cells  degradation and phosphorylation of MAPKs Figure a, includF 7, 2 = 5.36, F 7, 2 = 5.36, F 7, 2 = 5.36, F 7, 2 = 5.36, F 7, 2 = 5.36, F  = 9.984, P = 0.0006, Figure F  = 4.27, P = 0.0026, Figure F  = 3.40, P = 0.0150, Figure F  = 5.599, P = 0.0069, Figure F  = 8.544, P < 0.0001, Figure 1-40-induced neuronal death  = 7.07, P = 0.0008, Figure F  = 23.41, P < 0.0001, Figure As SCM-198 could effectively inhibit microglial overactivation, we then ventured into how these ‘SCM-198- or IBU-pretreated’ microglia would interact with neurons. A co-culture system was applied to investigate whether SCM-198 could protect neurons indirectly via directly suppressing overactivated microglia. LPS-preactivated or ‘SCM-198- or IBU-pretreated’ BV-2 cells in inserts were washed twice with fresh DMEM medium to remove residual LPS, SCM-198 or IBU. These washed cells in inserts were then placed into wells containing primary neurons and were co-cultured for 24 hours. Between inserts and wells, there is a semi-permeable membrane (0.4-μm pore-size) which blocks the direct contact between neurons and microglia, but allows exchange of molecules. LPS-preactivated BV-2 cells caused a decrease in neuronal viability which was reversed by ‘SCM-198- or IBU-pretreated’ microglia and 1 μM SCM-198 turned out to be the optimal dose  = 2.06, P = 0.085; F  = 0.98, P = 0.440; F  = 1.11, P = 0.3668, respectively, Figure 1-40-treated group  = 4.70, P = 0.0013, Figure F  = 4.10, P = 0.0032; F  = 4.00, P = 0.0037, respectively, Figure F  = 5.44, P = 0.0004, Figure F  = 8.667, P < 0.0001) and trial effect  = 84.80, P < 0.0001). Body weight remains normal and no statistical differences were found in swimming speed of rats between groups throughout the experiment (data not shown). DON (1 mg/kg), a first-line inhibitor of acetylcholinesterase and now clinically used for AD treatment, was used as the positive control [To further explore neuroprotective effects of SCM-198 ure F 5, 5 = 8.6671-40 led to elevated ERK phosphorylation, NF-кB p65 activation, increased tau phosphorylation, and synaptophysin loss, which were significantly reversed by SCM-198 treatment in a dose-dependent manner, with 60 mg/kg as the optimal dose  = 4.44, P = 0.0045, Figure F  = 13.23, P < 0.0001, Figure F  = 6.93, P = 0.0001, Figure F  = 6.13, P = 0.0005, Figure 1-40 injections induced excessive microglial activation at and around the injection site and SCM-198 at 60 mg/kg and DON could attenuate this activation  = 22.04, P < 0.0001, Figure Intrahippocampal injections of Aβure F 5,2 = 4.44, ure F 5,2 = 4.44, ure F 5,2 = 4.44, ure F 5,2 = 4.44, ure F 5,2 = 4.44, ure F 5,2 = 4.44, 1-40-injected group showed more severe cognitive impairments in spatial reference memory as compared with that of rats of 12-day recovery from surgery. Even up to trial 8, rats of only the Aβ1-40-injected group still needed 37.3 seconds in average to find the invisible platform. No significant differences were observed from trial 1 to trail 4  = 1.292, P = 0.2895; F  = 2.078, P = 0.1018; F  = 2.40, P = 0.066; F  = 2.603, P = 0.0502, respectively, Figure F  = 4.517, P = 0.0042; F  = 6.299, P = 0.0005; F  = 9.255, P < 0.0001; F  = 12.75, P < 0.0001, respectively, Figure F  = 21.41, P < 0.0001) and trial effect  = 35.76, P < 0.0001). Body weight remains normal and no statistical differences were found in swimming speed of rats between groups throughout the experiment (data not shown). Time spent in the target quadrant was also assessed during probe trial (Day 5). Figure F  = 4.562, P = 0.004, Figure As described in the Material and Methods section, 45 male rats were pretreated with vehicle, 60 mg/kg SCM-198, 1 mg/kg DON or co-administrated with SCM-198 and DON for 7 days. Fifty days after surgery, rats of only the AβThe role neuroinflammation plays in the pathological development of AD still remains controversial today, as inflammation itself is an innate defense against both endogenous and exogenous insults under normal physiological conditions. In neurodegenerative diseases like AD, chronic long-lasting inflammation is considered, to a great extent, as the driving force or even initiator of the disease . During in vitro study. Our data obtained from both primary microglia and BV-2 cell line indicated considerable inhibitory effects of SCM-198 on overactivated microglia via suppressing proinflammatory cytokines and NO productions. Possible underlying mechanisms were demonstrated to be, at least partially, through the inhibitions of NF-кB and JNK pathways. Microglial phenotype transition from amoeboid back to ramified morphology was observed after SCM-198 treatment  . This means that 3 μM Aβ1-40 is sublethal for primary neurons while it could induce significant elevation of TNF-α in microglia. Besides, astrocytes seemed less sensitive to Aβ1-40 than microglia, as up to 3 times higher concentration of Aβ1-40 and 48 hour stimulation increased astrocytic NO and TNF-α productionsas compared to 3 μM Aβ1-40 and 24 hour stimulation in microglia . Activated downstream transcription factors subsequently enter the nuclei, bind to the promoter regions and initiate transcriptions of various proinflammatory mediators such as NO, TNF-α, IL-1β and so on ,33. In o1-40 as the in vivo inflammation inducer. Many literatures have stated that intrahippocampal injections of Aβ1-40 or Aβ1-42 could activate glial cells, elicit neuroinflammation and cause cognitive impairments in rodents [1-40 injections caused microglial activation, synaptophysin loss, elevated phosphorylation of tau, ERK and NF-кB p65, which were later significantly reversed by SCM-198. Our unpublished data obtained from HPLC analysis showed that SCM-198 administrated by gavage could be detected in rat brains, which could be considered as supportive evidence that SCM-198 could penetrate the blood-brain barrier and directly exert neuroprotective effects in CNS .As Aβ deposits, neurofibrillary tangles and dystrophic neurites are widely accepted hallmarks of AD. We herein used pre-aggregated Aβ rodents –39. In o1-40 toxicity or indirectly protect neurons against synaptophysin loss and elevations of p-tau, p-ERK and p-p65 of NF-кB via directly suppressing NF-кB and JNK pathways in activated microglia.Interestingly, SCM-198 inhibited NF-кB and JNK pathways in activated microglia, while it inhibited ERK and NF-кB pathways in co-cultured neurons and rat hippocampus. Possible explanations for the difference were as follows: 1) in the co-culture paradigm, neurons were directly stimulated by molecules released from pretreated microglia, but not directly by LPS and SCM-198, which were removed from the media before microglia/neuron co-culture; 2) Other studies have proved that activated microglia upregulated p-ERK with no change in total ERK in neurons and rodents’ brains and this elevation of p-ERK was accompanied by neuronal dysfunctions and cognitive impairments of animals –42; 3) Hin vitro and in vivo and therefore protected co-cultured neurons and improved overall cognitive performances of rats. Hence, our data may provide new insights into AD treatment with SCM-198 in the near future.Further investigations will be necessary to clarify how SCM-198 interacts with neurons and astrocytes. Several other transgenic AD models will be needed to further verify neuroprotective effects or unravel new potential mechanisms of SCM-198. Taken together, our study, for the first time, demonstrated that SCM-198 possessed considerable anti-neuroinflammatory effect both 1-40-injected SD rats, indicating its potential as a drug candidate for AD treatment in the future. SCM-198 may directly inhibit overactivated microglia, maintain their ramified morphology, decrease proinflammatory cytokines via NF-кB and JNK pathways and therefore indirectly protect co-cultured neurons. Besides, when directly applied to neurons, SCM-198 decreased neuronal death and LDH leakage caused by Aβ1-40 stimulation. In vivo Aβ1-40 injection caused impairments of spatial memory and microglial overactivation, which were reversed by SCM-198 at 30 mg/kg and 60 mg/kg. In the chronic rat AD model, co-administration of SCM-198 and DON resulted in better cognitive performances of rats in the MWM test, indicating that SCM-198 could not only be used independently for AD treatment in the future, but that it could be used as an adjuvant to improve the therapeutic effect of DON. Further investigations will be necessary to clarify how SCM-198 interacts with neurons and astrocytes. Several other transgenic AD models will be needed to further verify neuroprotective effects or unravel new potential mechanisms of SCM-198.In summary, this is the first time that SCM-198 was found to have considerable anti-inflammatory effects in microglia and in Aβ


Pasteurella multocida toxin (PMT), the major virulence factor responsible for zoonotic atrophic rhinitis, is a protein deamidase that activates the alpha subunit of heterotrimeric G proteins. Initial activation of G alpha-q-coupled phospholipase C-beta-1 signaling by PMT is followed by uncoupling of G alpha-q-dependent signaling, causing downregulation of downstream calcium and mitogenic signaling pathways. Here, we show that PMT decreases endogenous and exogenously expressed G alpha-q protein content in host cell plasma membranes and in detergent resistant membrane (DRM) fractions. This membrane depletion of G alpha-q protein was dependent upon the catalytic activity of PMT. Results indicate that PMT-modified G alpha-q redistributes within the host cell membrane from the DRM fraction into the soluble membrane and cytosolic fractions. In contrast, PMT had no affect on G alpha-s or G beta protein levels, which are not substrate targets of PMT. PMT also had no affect on G alpha-11 levels, even though G alpha-11 can serve as a substrate for deamidation by PMT, suggesting that membrane depletion of PMT-modified G-alpha-q has specificity. Pasteurella multocida toxin (PMT) is the major virulence factor responsible for atrophic rhinitis, pasteurellosis, and dermonecrosis caused by infection with toxigenic capsular type D and some A strains of Pasteurella multocida . PMT isiewed in . PMT is iewed in ,4,5, wheiewed in ). PMT acfamilies ,8,9,10 tq of the Gαq/11 family of G-proteins [2+ and activates mitogenic signaling pathways, leading to increased cellular proliferation and in some cell types cell differentiation [q/11 and Gα12/13 [s [i family [Physiologically, PMT stimulates phospholipase C β-1 (PLCβ1) signaling through its preferential activation of Gαproteins ,10. By dntiation ,12. PMT  Gα12/13 ,15,16, l Gα12/13 ,19,20,2112/13 [s ,22,23, ti family ).q-CPLCβ1 [i-AC [q-mediated G-protein-coupled inward rectifying K+ channels [i protein [Although PMT modulates numerous intracellular signaling pathways to dictate cellular fate by constitutive activation of the Gα proteins, the overall activation of downstream signaling is not sustained and is subsequently mitigated by an unknown mechanism of downregulation. PMT causes an initial strong activation of downstream calcium and mitogenic signaling, which is subsequently followed by an uncoupling of the signaling in the case of Gαq-CPLCβ1 ,21 and Gβ1 [i-AC . Moreovechannels ,26,27 an protein .q and G11 proteins, to their cognate agonist results in desensitization of the pathway due to a sequestration, modification or reduction in cellular levels of the receptor and/or the Gα subunit [It is well established that sustained exposure of many G-protein-coupled receptors (GPCR), including those coupled with G subunit ,32,33,34 subunit ,36,37,38 subunit . In eachq, Gα11, and Gαs proteins in HEK-293T cells after PMT treatment by determining the levels of the Gα protein in whole cell lysates, total membranes, detergent resistant membranes (DRMs), and the soluble membrane and cytosolic fractions. Results from our experiments indicate that treatment of HEK-293T cells with wildtype PMT, but not catalytically inactive mutant PMT (C1165S), stimulates the selective downregulation of Gαq, but not Gαs or Gα11, through redistribution of the Gαq protein from DRMs to soluble membrane and cytosolic fractions with overall loss of Gαq protein from the plasma membrane.In this study, we investigated the fate of the GαN-terminal (residues 1–506) or C-terminal (residues 486–1285) PMT fragments had no effect on cell morphology (data not shown).Recombinant, full-length PMT induced morphological effects in cultured HEK-293T cells, similar to that previously described for other cells . PMT cauq/11-PLCβ1 signaling: (1) PMT deamidation and activation of Gαq and/or Gα11 leads to degradation of the resulting constitutively active Gα subunit and loss of cellular levels of Gαq and/or Gα11; or (2) PMT deamidation and activation of Gαq and/or Gα11 leads to redistribution of the resulting constitutively active Gα subunit from DRMs to soluble membrane and/or cytosolic fractions. In agreement with a previous report [C-terminus of Gαq/11 showed that overnight treatment of the cells with PMT did not alter endogenous protein levels of Gαq/11 in total cell lysates  since many membrane signaling molecules are enriched in these membrane micro-domains.Because PMT acts directly on its Gα substrates and subsequent modulation of signaling is independent of agonist-receptor coupling to the G protein ,40,41, ws report , immunob lysates . This res subunits were found throughout the gradient in both higher density (#8–12) and lower density fractions (#4–7) from untreated control cells, the majority of the less abundant Gαq/11 subunits were localized mostly to the low-density, DRM-containing fraction (#5), as evidenced by its co-localization with flotillin-1, a known specific marker for DRMs [q/11, but not Gβ or Gαs, from the DRM-containing fraction #5. With this loss was a concomitant increase of Gαq/11 in the soluble membrane and cytosolic fractions (#8–12), suggesting that PMT modification and activation of Gαq/11 results in the release of active form of the Gαq/11 protein from DRMs. That PMT treatment did not significantly alter the levels of Gαs or Gβ in DRMs, as compared with flotillin-1, confirms previous findings, indicating that Gαs protein is not a target substrate of PMT.PMT-treated HEK-293T cells were lysed in the presence of detergent and the cellular contents were subjected to subcellular fractionation by separation using Optiprep density gradient centrifugation, followed by immunoblotting analysis of the gradient fractions. In the density gradient centrifugation profile, fractions #1–7 contain low-density lipid-rich membranes and vesicles, while fractions #8–12 contain high-density protein-rich soluble membrane and cytosolic fractions . As showfor DRMs . In contq/11 protein levels in cells were not discernably affected by exposure to PMT over 12 h, the Gαq/11 levels in DRMs were reduced by over 80% by 8–10 h in cells treated with 0.3 nM PMT . Taken together, PMT selectively induces the loss of Gαq/11 proteins from DRMs in a time and dose dependent manner, although this loss can be masked or overcome at later times or higher toxin concentrations by PMT-stimulation of new protein synthesis.A time course revealed that while overall endogenous Gα3 nM PMT a. This rf ~70 pM b. Althouq and Gα11, we expressed exogenously either Gαq or Gα11 in HEK-293T cells over the endogenous background and detected the proteins using the anti-Gαq/11 antibodies under conditions where the corresponding endogenous proteins were negligible  reduces the enzymatic deamidase activity by 1000-fold [q, and neither PMT nor PMT C1165S caused the depletion of Gα11, confirming that the enzymatic activity of PMT is necessary for the depletion of Gαq from the host cell membrane.Since PMT specifically induces the depletion of only Gα000-fold . As showq from total membrane samples, and the consequence of PMT action renders the G-proteins constitutively active, we wanted to explore whether or not constitutive activity of Gαq alone was sufficient to induce the depletion or exclusion of the activated Gαq protein from the plasma membrane. We transfected HEK-293T cells separately with Gαq, Gα11, and their corresponding constitutively active mutants, with known dominant-active mutations, Gαq Q209L and Gα11 Q209L, and with mutations equivalent to that introduced by PMT action, Gαq Q209E and Gα11 Q209E  Ca2+-dependent Cl− current, which involved activation of Gαq/11-PLCβ1 [2+ and mitogenic signaling was evidenced in mammalian cells through studies involving cell cycle analysis [q signaling by PMT was reminiscent of receptor-mediated downregulation of Gq-protein signaling that occurs upon prolonged exposure to agonist [q proteins from DRMs to soluble membrane fractions or the cytosol during agonist-stimulated signal transduction [q and/or Gα11, results in a similar uncoupling of the corresponding G-protein-signaling pathway.We previously showed that microinjection of PMT into voltage-clamped 11-PLCβ1 . In thisanalysis ,46. This agonist ,36,37,38sduction . This prq/11 proteins, PMT deamidation and subsequent activation of the Gαq/11 proteins may cause downregulation of Gq/11 signaling through depletion of Gαq/11 levels in DRMs. The results presented here show for the first time that PMT induces both dose- and time-dependent depletion of Gαq but not Gα11 from membrane lipid rafts in HEK-293T cells with concomitant shift of Gαq to soluble membrane fractions or cytosol. Indeed, there was no significant decrease in Gαq or Gα11 observed for any of the time points in the control experiments with no PMT treatment or for the Gα11 protein in the PMT-treated experiments; a transient decrease in Gαq protein levels was observed only in PMT-treated cells. We further showed that this PMT-induced depletion occurs within DRM fractions from cells expressing endogenous levels of Gαq/11 proteins, as well as whole membranes and DRM fractions from cells overexpressing wildtype Gαq, and in DRM fractions from cells overexpressing FLAG-tagged Gαq, but not Gα11 proteins. Moreover, this PMT-mediated membrane redistribution of Gαq was dependent on the deamidase activity of PMT since a catalytic mutant PMT C1165S did not cause membrane depletion of Gαq.We considered the possibility that although PMT might have little or no effect on total cellular content of Gαq subunit from the flotillin-1-enriched DRM-containing fraction #5 with a concomitant increase of Gαq protein in the more soluble fractions (#8–12) of the gradient. The effect of PMT was specific for Gαq, and not for other Gα subunits that are not targets of PMT, such as Gαs or Gβ. Moreover, PMT-treatment caused membrane depletion of Gαq and not Gα11. Gαq and Gα11 share very similar structural homology and sequence identity of over 90%, and Gα11 can also be deamidated by PMT in cells [C-terminus of both Gαq and Gα11. Thus, to distinguish the specificity of the PMT-mediated DRM and membrane depletion for Gαq vs. Gα11, we decided to use an overexpression approach, where we exogenously expressed either Gαq or Gα11 and detected the proteins using the anti-Gαq/11 antibodies. Under these conditions, endogenous levels of Gαq and Gα11 were not detectable in the resulting immunoblots.Specifically, when cellular contents of PMT treated cells were separated into fractions by OptiPrep or sucrose density gradient centrifugation, we found that PMT treatment resulted in the loss of Gαin cells ,8. The sq from DRMs suggests that the cellular Gαq levels in DRMs are replenished as a result of increased Gαq protein synthesis. Previous studies have shown that PMT induces protein synthesis and cellular proliferation in a cycloheximide-sensitive manner [11 under similar conditions. Alternatively, it is possible that the observed replenishment of Gαq in the DRMs may also be due to a redistribution of the preexisting or newly synthesized monomeric Gαq protein that is in the membrane. However, unlike in the case for DRMs, the loss of Gαq from total membranes did not appear to replenish with time, suggesting that PMT modification and activation of Gαq uncouples the signaling pathway in such a way as to interfere with the ability of newly synthesized Gαq to interact with membranes, presumably through preventing reassociation with Gβγ subunits and coupling to GCPRs. The relatively faster response times for depletion and replenishment of the exogenously expressed Gαq protein, compared to that observed for endogenous protein, was presumably due to the increased levels of Gαq protein available as a substrate for PMT and saturation of the signaling system from enhanced protein synthesis of Gαq encoded by the expression plasmid. Further, this PMT-mediated loss of Gαq from DRMs and membranes is apparently masked at the total cell level (whole cell lysates) through redistribution to soluble membrane fractions or cytosol, as evidenced from the results shown in q protein, which counters the overall PMT-mediated loss of Gαq protein from membranes.The finding that the continued exposure to PMT was neither able to sustain the loss of nor perpetuate further loss of Gαe manner ,48. Thisq Q209E or Gαq Q209L in HEK-293T cells resulted in similar morphological effects as PMT treatment, we explored whether exogenous expression of these constitutively active mutants would lead to their localization in the membrane and DRMs or would also result in their membrane depletion. Since we found that the presence of constitutively active Gαq mutant proteins alone was not sufficient to induce their depletion or exclusion from membranes, we next considered whether PMT treatment could induce the removal of pre-existing constitutively active Gαq from the cell plasma membrane. However, we found that PMT treatment had no effect on the membrane localization of exogenously expressed constitutively active Gαq mutant proteins.Since overexpression of Gαq is depleted from DRMs, our results clearly indicate the PMT catalytic activity is required. If PMT-mediated deamidation is required for depletion, and both Gαq and Gα11 can serve as substrates for deamidation by PMT [11 should also be depleted from DRMs. However, our results (11 is a very poor substrate for PMT and does not get deamidated to any significant extent during the time studied or that the as-yet-undefined factor(s) responsible for depletion do not recognize the PMT-deamidated Gα11 as they do the PMT-deamidated Gαq. These possibilities might be explored further under endogenous conditions by using antibodies that distinguish Gαq from Gα11 coupled with those that recognize the deamidated forms of the Gα proteins, such as those previously reported [Although the antibodies we used could not distinguish whether only the deamidated form of the Gαn by PMT , then PM results c indicatreported ,8.q protein from the membrane, but PMT might indirectly activate an unknown cellular component at the membrane or expedite a cellular process responsible for the downregulation of membrane-associated PMT-modified Gαq. This further suggests that the depletion mechanism of PMT-modified Gαq proteins from the membrane has specificity and that PMT-catalyzed deamidation and activation of the Gαq protein is the not the sole signal for initiating the downregulation process. Whether similar membrane depletion occurs for other Gα substrates of PMT once they are deamidated and activated remains to be determined.These results suggest that PMT does not directly deplete the modified Gαs , mouse monoclonal antibody against β tubulin , rabbit polyclonal antibodies against Gαq/11 , Gβ  and flotillin-1 , as well as horseradish peroxidase-conjugated donkey anti-goat IgG (sc-2020), goat anti-rabbit IgG (sc-2004), and goat anti-mouse IgG (sc-2005) antibodies, were purchased from Santa Cruz Biotechnology . Mouse monoclonal anti-FLAG antibody  and mammalian protease inhibitor cocktail was purchased from Sigma . All cell culture reagents were purchased from Life Technologies . Restriction enzymes and other molecular biology reagents were obtained from Roche Biochemicals  or New England Biolabs . OptiPrep was obtained from Axis-Shield PoCAS . SuperSignal West Pico Chemiluminescent Substrate was purchased from Pierce Biotechnology . Bio-Rad protein assay dye reagent was purchased from Bio-Rad Laboratories . Full-length recombinant PMT and catalytically inactive mutant PMT C1165S were cloned, expressed, purified, quantified, and assayed for biological activity, as previously described [Human embryonic kidney 293-T (HEK-293T) cells were obtained from the American Type Culture Collection (ATCC# CRL-3216). Goat polyclonal antibodies against Gαescribed .q, Gα11, Gαq Q209L, and Gα11 Q209L genes  [+ . For immunoblot detection, a FLAG-tag flanked at each end by a 6-residue linker sequence (SGGGGS) was introduced between residues 121 and 122 of Gαq and Gα11 by using the following primers:qGαGGTACCACCATGACTCTGGAGTCCAT-3′F1: 5′-GCTTqGαGATATCCGCCGCCGCCAGACACCTTCCCACATCAAC-3′R1: 5′-GAqGαGATATCCCGATACAAAGACGATGACGACAAGGGCGGAGGCGGC-3′F4: 5′-GAqGαF5: 5′-GACGACAAGGGCGGAGGCGGCTCTGCTTTTGAGAATCCATATGTAG-3′qGαCTCGAGTTAGACCAGATTGTACTC-3′R2: 5′-GA11GαGGTACCACCATGACTCTGGAGTC-3′F1: 5′-GCTT11GαGATATCCGCCGCCGCCGGACACCTTCTCCACGTCCAC-3′R1: 5′-GA11GαF3: 5′-GACGACAAGGGCGGAGGCGGCTCTACCTTCGAGCATCAGTACGTC-3′11GαCTCGAGTCAGACCAGGTTGTAC-3′R2: 5′-CTAGADNA encoding human Gα Center)  was clonq and Gα11 was completed by PCR cloning using the following primers:qGαF1: 5′-CGATGTAGGCGGCGAAAGGTC-3′qGαR1: 5′-GACCTTTCGCCGCCTACATCG-3′qGαF2: 5′-TGTCGTAACAACTCCGCCCCA-3′qGαR2: 5′-ACACCTACTCAGACAATGCGATGCA-3′11GαF1: 5′-GTGGGCGGCGAGCGGTCGG-3′11GαR1: 5′-CCGACCGCTCGCCGCCCAC-3′11GαF2: 5′-TGTCGTAACAACTCCGCCCCA-3′11GαR2: 5′-ACACCTACTCAGACAATGCGATGCA-3′. All subcloning procedures were confirmed by using restriction enzyme and DNA sequencing analysis. Generation of the Q209E mutants of Gαl-glutamine and 10% (v/v) bovine growth serum at 37 °C and 5% CO2. Cells grown to 60%–80% confluence (~4 × 106 per 100-mm dish) were transiently transfected for 24 h with mammalian expression vectors: pcDNA3.1 (empty vector), pcDNA3.1/Gαq, pcDNA3.1/Gα11, pcDNA3.1/Gαq Q209E, pcDNA3.1/Gα11 Q209E, pcDNA3.1/Gαq Q209L, or pcDNA3.1/Gα11 Q209L, using LipofectAMINE 2000 reagent (Invitrogen), according to the manufacturer’s instructions, or using the CalPhos Mammalian Transfection protocol .HEK-293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 mM dithiothreitol, and 10% glycerol) to generate whole cell lysates. Cellular fractionation was performed essentially, as described previously [g for 5 min, and the post-nuclear supernatant was fractionated into membrane pellets and supernatants by centrifugation at 4 °C and 18,000× g for 30 min. The membrane pellets were resuspended in 0.5 mL of lysis buffer A. Membrane proteins were quantified using the Bio-Rad protein assay. Samples containing 10 µg of total membrane proteins were resolved by SDS-PAGE and analyzed by immunoblotting, as described below.HEK-293T cells, transfected as described above, were treated with or without the indicated amounts of PMT for the indicated times, washed with 1× cold phosphate buffered saline, scraped and transferred into a microcentrifuge tube , then lysed by 10 passages through a 27-gauge needle in 0.5 mL of an ice-cold lysis buffer A  and captured on autoradiographic film. Image analysis and quantification of protein bands was performed using NIH ImageJ  and Microsoft Excel software .2, 0.18 mM CaCl2, and 5.6 mM glucose). Cells were collected and washed twice with the ice-cold Tyrode’s solution, then centrifuged at 1000× g for 10 min. The pellet was solubilized in 1 mL of lysis buffer B . The cell were lysed via sonication, then incubated at 4 °C for 1 h with frequent agitation. Cell lysates (1 mL) were mixed with 3 mL of OptiPrep or sucrose solution  in a 12-mL centrifuge tube, and the mixture was overlaid first with 4 mL of 35% OptiPrep/sucrose solution diluted in STE buffer  and then 4 mL of 5% OptiPrep/sucrose solution. Ultracentrifugation was performed at 200,000× g and 4 °C for 20 h using a Beckman SW40Ti rotor  Fractions of 1.0 mL each were collected from the top of the gradient. Total protein content of each fraction was quantified using Bio-Rad protein assay. The distribution of various proteins in the fractions was assessed by SDS-PAGE and immunoblotting, as described above.DRMs were prepared by density gradient centrifugation with OptiPrep or sucrose, as indicated, using a modification of previously described methods ,43. All p values were determined using the unpaired two-tailed Student’s t-test. For All data are presented as the mean ± standard deviation (SD) from at least three independent experiments, or as indicated. The


In addition, there is a slight twist in the bicyclic core, with a dihedral angle of 2.39 (4)° between the thienyl and pyridine rings. The conformation of the carbamoyl moiety is partially determined by an intramolecular N—H⋯O hydrogen bond. In the crystal, complementary N—H⋯O hydrogen bonds form dimers which are then associated into chains parallel to the c axis through O—H⋯N hydrogen bonds involving the water molecule of crystallization. Electron density associated with an additional solvent molecule of partial occupancy and disordered about a twofold axis was removed with the SQUEEZE procedure in PLATON [Spek .In the title molecule, CSpek 2015. Acta Cr In addition there is a slight twist in the bicyclic core with a dihedral angle of 2.39 (4)° between the thienyl and pyridine rings. The conformation of the carbamoyl moiety is partially determined by an intramolecular N2—H2A⋯O1 hydrogen bond  1 − x, y, z] form dimers which are then associated into chains parallel to the c axis through O4—H4A⋯N3 and O4—H4B⋯N2ii  hydrogen bonds involving the water molecules of crystallization acetamide (10 mmol) in absolute ethanol (25 ml) containing sodium ethoxide (0.3 g) on a steam bath for 30 mins. The product that formed on cooling was collected by filtration and recrystallized from ethanol 95% as yellow needles. Yield (73%); m.p. IR (KBr) ν = 3500, 3350, , 1701 , 1638  cm−1. 1H NMR (DMSO-d6): 9.41 , 7.73–7.75 , 7.64–7.66 , 7.55–7.56 , 7.38–7.44 , 7.13–7.15 , 6.81–6.85 .The title compound was prepared by heating equimolar quantities of ethyl 3-cyano-1,2-dihydro-6-methyl-4-(2-phenylethenyl)-2-thioxopyridine-5-carboxylate and chloro global, I. DOI: 10.1107/S2056989016001341/rz5183Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016001341/rz5183Isup3.cmlSupporting information file. DOI: 1448789CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: