Anti-depressant effect of curcumin-loaded guanidine-chitosan thermo-sensitive hydrogel by nasal delivery.

Curcumin, a polyphenol compound extracted from the roots of turmeric plants, possesses anti-depressant effect by regulating the levels of neuroendocrine immunological factors. The purpose of this study was to investigate the anti-depressant effect of curcumin through nasal delivery. The results of phase solubility, Fourier transform infrared spectra, Differential scanning calorimetry, X-ray powder diffractometry and 1H NMR spectra assays showed that curcumin/hydroxypropyl-ß-cyclodextrin complex had been obtained. The viscosity of hydrogel increased rapidly at the temperature range of 29-30 °C through the test of rheological property of Guanidine-Chitosan thermo-sensitive hydrogel. And the hydrogel had good mucoadhesion properties. The cumulative release rate of curcumin was 55% in 10 h in vitro drug release test. Curcumin-loaded (14.6, 29.2, or 58.4 µg/kg) thermo-sensitive hydrogel could reduce the immobility time of mice in force swimming test and tail suspension test, while could not increase the independent behavioral activity of mice. In addition, curcumin-loaded (14.6, 29.2, or 58.4 µg/kg) thermo-sensitive hydrogel could increase the concentration of Norepinephrine, Dopamine, 5-Hydroxytryptamine and their metabolites in hippocampus and striatum. In conclusion, thermo-sensitive hydrogel delivery system can be seen as a promising formulation of curcumin for the treatment of depression through nasal delivery.

Identification and analysis of the reactive metabolites related to the hepatotoxicity of safrole.

1. Safrole is the main component of the volatile oil in Xixin, which has a strong antifungal effect. However, safrole has been shown to be associated with the development of hepatocellular carcinoma. Methylenedioxyphenyl and allyl-benzene substructures of safrole may cause a mechanism-based inhibition (MBI) of CYP450 enzymes (CYPs) and produce reactive metabolites (RMs), resulting in inhibition of enzyme activity and toxic effects. 2. Based on the experiments of CYPs cocktail screening, glutathione (GSH) capture and the IC50 data, we found that safrole had an inhibitory effect on CYP1A2. The test of enzyme activity recovery when adding GSH may help to verify the MBI of safrole. 3. Two metabolites, 1,2-dihydroxy-4-allylbenzene (M1) and 1'-hydroxy safrole (M2) could be captured by GSH. The ultra performance liquid chromatography - tandem mass spectrometer (UPLC-MS/MS) method was used to identify the RMs through a detailed characterization of the safrole cleavage processes and the GSH-M1 adduct. The RMs identified are quinone and its tautomer. Thus, preliminary conclusion can be obtained that safrole is a mechanism-based inhibitor of CYP1A2. 4. The cleavage process of the GSH-M1/M2 adduct was analyzed in further detail. We believe the safrole hepatotoxicity mechanism is related to the RMs mediated by CYP1A2. This work provides important information on predicting in vivo drug induced liver injury.

AFLP Genome Scanning Reveals Divergent Selection in Natural Populations of Liriodendron chinense (Magnoliaceae) along a Latitudinal Transect.

Understanding adaptive genetic variation and its relation to environmental factors are important for understanding how plants adapt to climate change and for managing genetic resources. Genome scans for the loci exhibiting either notably high or low levels of population differentiation (outlier loci) provide one means of identifying genomic regions possibly associated with convergent or divergent selection. In this study, we combined Amplified Fragment Length Polymorphism (AFLP) genome scan and environmental association analysis to test for signals of natural selection in natural populations of Liriodendron chinense (Chinese Tulip Tree; Magnoliaceae) along a latitudinal transect. We genotyped 276 individuals from 11 populations of L. chinense using 987 AFLP markers. Both frequency-based (Dfdist and BayeScan) and correlation-based (MLM) methods were applied to detect outlier loci. Our analyses recovered both neutral and potentially adaptive genetic differentiation among populations of L. chinense. We found moderate genetic diversity within populations and high genetic differentiation among populations with reduced genetic diversity toward the periphery of the species ranges. Nine AFLP marker loci showed evidence of being outliers for population differentiation for both detection methods. Of these, six were strongly associated with at least one climate factor. Temperature, precipitation, and radiation were found to be three important factors influencing local adaptation of L. chinense. The outlier AFLP loci are likely not the target of natural selection, but the neighboring genes of these loci might be involved in local adaptation. Hence, these candidates should be validated by further studies.

Histamine Increases Neuronal Excitability and Sensitivity of the Lateral Vestibular Nucleus and Promotes Motor Behaviors via HCN Channel Coupled to H2 Receptor.

Histamine and histamine receptors in the central nervous system actively participate in the modulation of motor control. In clinic, histamine-related agents have traditionally been used to treat vestibular disorders. Immunohistochemical studies have revealed a distribution of histaminergic afferents in the brainstem vestibular nuclei, including the lateral vestibular nucleus (LVN), which is critical for adjustment of muscle tone and vestibular reflexes. However, the mechanisms underlying the effect of histamine on LVN neurons and the role of histamine and histaminergic afferents in the LVN in motor control are still largely unknown. Here, we show that histamine, in cellular and molecular levels, elicits the LVN neurons of rats an excitatory response, which is co-mediated by the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and K+ channels linked to H2 receptors. Blockage of HCN channels coupled to H2 receptors decreases LVN neuronal sensitivity and changes their dynamic properties. Furthermore, in behavioral level, microinjection of histamine into bilateral LVNs significantly promotes motor performances of rats on both accelerating rota-rod and balance beam. This promotion is mimicked by selective H2 receptor agonist dimaprit, and blocked by selective H2 receptor antagonist ranitidine. More importantly, blockage of HCN channels to suppress endogenous histaminergic inputs in the LVN considerably attenuates motor balance and coordination, indicating a promotion role of hypothalamo-vestibular histaminergic circuit in motor control. All these results demonstrate that histamine H2 receptors and their coupled HCN channels mediate the histamine-induced increase in excitability and sensitivity of LVN neurons and contribute to the histaminergic improvement of the LVN-related motor behaviors. The findings suggest that histamine and the histaminergic afferents may directly modulate LVN neurons and play a critical role in the central vestibular-mediated motor reflexes and behaviors.

Mechanism-based inhibition of CYPs and RMs-induced hepatoxicity by rutaecarpine.

1. Rutaecarpine, a quinolone alkaloid isolated from the unripe fruit of Evodia rutaecarpa, is one of the main active components used in a variety of clinical applications, including the treatment of hypertension and arrhythmia. However, its hepatotoxicity has also been reported in recent years. 2. Reactive metabolites (RMs) play a vital role in drug-induced liver injury. Rutaecarpine has a secondary amine structure that may be activated to RMs. The aim of the study was to investigate the inhibition of rutaecarpine on CYPs and explore the possible relationship between RMs and potential hepatotoxicity. 3. A cell counting kit-8 cytotoxicity assay indicated that rutaecarpine can decrease the primary rat hepatocyte viability, increase lactate dehydrogenase and reactive oxygen species, reduce JC-1, and cause cell stress and membrane damage. The indexes were significantly restored by adding ABT, an inhibitor of CYPs. A cocktail assay showed that CYP1A2, CYP2C9, CYP2C19, CYP2E1 and CYP3A4 can be inhibited by rutaecarpine in human liver microsomes. The IC50 values of CYP1A2 with and without NADPH were 2.2 and 7.4 µM, respectively, which presented a 3.3 shift. The results from a metabolic assay indicated that three mono-hydroxylated metabolites and two di-hydroxylated metabolites were identified and two GSH conjugates were also trapped. 4. Rutaecarpine can inhibit the activities of CYPs and exhibit a potential mechanism-based inhibition on CYP1A2. RMs may cause herb-drug interactions, providing important information for predicting drug-induced hepatotoxicity.

Identification and characterization of reactive metabolites in myristicin-mediated mechanism-based inhibition of CYP1A2.

Myristicin belongs to the methylenedioxyphenyl or allyl-benzene family of compounds, which are found widely in plants of the Umbelliferae family, such as parsley and carrot. Myristicin is also the major active component in the essential oils of mace and nutmeg. However, this compound can cause adverse reactions, particularly when taken inappropriately or in overdoses. One important source of toxicity of natural products arises from their metabolic biotransformations into reactive metabolites. Myristicin contains a methylenedioxyphenyl substructure, and this specific structural feature may allow compounds to cause a mechanism-based inhibition of cytochrome P450 enzymes and produce reactive metabolites. Therefore, the aim of this work was to identify whether the role of myristicin in CYP enzyme inhibition is mechanism-based inhibition and to gain further information regarding the structure of the resulting reactive metabolites. CYP cocktail assays showed that myristicin most significantly inhibits CYP1A2 among five CYP enzymes (CYP1A2, CYP2D6, CYP2E1, CYP3A4 and CYP2C19) from human liver microsomes. The 3.21-fold IC50 shift value of CYP1A2 indicates that myristicin may be a mechanism-based inhibitor of CYP1A2. Next, reduced glutathione was shown to block the inhibition of CYP1A2, indicating that myristicin utilized a mechanism-based inhibition. Phase I metabolism assays identified two metabolites, 5-allyl-1-methoxy-2,3-dihydroxybenzene (M1) and 1'-hydroxymyristicin or 2',3'-epoxy-myristicin (M2). Reduced glutathione capturing assays captured the glutathione-M1 adduct, and the reactive metabolites were identified using UPLC-MS(2) as a quinone and its tautomer. Thus, it was concluded that myristicin is a mechanism-based inhibitor of CYP1A2, and the reactive metabolites are quinone tautomers. Additionally, the cleavage process of the glutathione-M1 adduct was analyzed in further detail. This study provides additional information on the metabolic mechanism of myristicin inhibition and improves risk evaluation for this compound.

Reactive metabolite activation by CYP2C19-mediated rhein hepatotoxicity.

1. Rhein, an active ingredient in the root of rhubarb, is used for its beneficial effects in a variety of clinical applications including the treatment of osteoarthritis and diabetic nephropathy. However, its hepatotoxicity has been reported in recent years. Rhein belongs to the conjugate structure which could be activated to reactive metabolites (RMs) inducing side-effects. This study is to explore the relationship between RMs and hepatotoxicity. 2. Based on the early detection of RMs, we have established a series of key technologies to research rhein hepatotoxicity mechanism: IC50 shift experiments and reduced glutathione (GSH) trapping experiment are adopted to identify RMs. The model of low activity of CYP450 enzymes (CYPs) in primary rat hepatocyte is constructed to analyze the relationship between the primary metabolic enzyme and hepatotoxicity of rhein better. 3. The IC50 shift value for CYP2C19 is 1.989, it suggests that CYP2C19 could activate rhein to RM. The structure of RM is epoxide intermediate. Besides, it is found that CYP2C19 is a primary metabolic enzyme for rhein. In the cytotoxicity assay, it is reported that rhein could cause mitochondrial dysfunction. Furthermore, mitochondrial membrane potential (Δψm) and AST levels could be restored by adding inhibitor of CYP2C19 together with rhein, which further shows that CYP2C19 could mediate the hepatotoxicity of rhein. 4. We put forward the possible mechanism that reactive metabolite activation by CYP2C19 mediated rhein hepatotoxicity, it provides important information on predicting in vivo drug-induced liver injury (DILI).

Two three-dimensional lanthanide frameworks exhibiting luminescence increases upon dehydration and novel water layer involving in situ decarboxylation.

Two three-dimensional polymeric Tb(III) and Yb(III) frameworks, namely, {[Tb3(Hptc)(ptc)(pdc)(H2O)6]·2H2O}n (1) and {[Yb2(ptc)(ox)(H2O)5]·7H2O}n (2) (H4ptc = pyridine-2,3,5,6-tetracarboxylic acid, H2pdc = pyridine-3,5-dicarboxylic acid, ox = oxalate), have been synthesized by a hydrothermal method and characterized by infrared spectra, elemental analysis, powder X-ray diffraction, thermogravimetric analysis, and single-crystal X-ray diffraction. Framework 1 shows an interesting three-dimensional (8,8)-connected net with a Schläfli symbol of (3(3)·4(18)·5(5)·6(2))2(3(6)·4(14)·5(7)·6), while 2 exhibits an unusual (4,8)-connected sqc21 net with a Schläfli symbol of (3(2)·4(2)·5(2))(3(4)·4(8)·5(12)·6(4)). Luminescence studies of 1 reveal that the luminescence intensity increases when the framework is dehydrated.

[Effects of E23K polymorphism in KCNJ11 gene on membrane current].

OBJECTIVE: E23K polymorphism in KCNJ11 gene is associated with cardiovascular disease and diabetes. In order to explore the mechanism of E23K correlation to related diseases, the effect of E23K polymorphism in KCNJ11 gene on membrane current was investigated. METHODS: The exon of KCNJ11 was obtained by PCR amplification and the G-->A mutation was completed by overlap extension PCR. The sequences of KCNJ11 exon contained 23E or 23K was inserted into pcDNA3.1/CT-GFP vector respectively. The recombinant plasmid, pcDNA3.1-KCNJ11(E) and pcDNA3.1-KCNJ11(K), were transfected into HEK293T cells by lipofectamine and the membrane current density was determined by whole-cell patch clamp technique. RESULTS: 1,173 bp sequences of KCNJ11 gene's exon were amplified by PCR and the recombinant expression plasmid, pcDNA3.1-KCNJ11(E) and pcDNA3.1-KCNJ11(K), were constructed successful. Positive and negative currents were detected in HEK293T cells transfected with difference plasmid by whole-cell patch clamp technique. Results showed that the reversed voltage was 50mV. The current in HEK293T cells with pcDNA3.1-KCNJ11(E) was significantly greater than that with pcDNA3.1-KCNJ11(K) (P < 0.05, n = 10). CONCLUSION: The polymorphism of E23K in exon of KCNJ11 gene changed the membrane currents in HEK293T cells. It could be an experiment support for the possible mechanism between the locus and related diseases.

Characterization of 39 novel EST-SSR markers for Liriodendron tulipifera and cross-species amplification in L. chinense (Magnoliaceae).

PREMISE OF THE STUDY: A novel set of simple sequence repeat (SSR) markers were developed and characterized from the expressed sequence tag (EST) database of Liriodendron tulipifera for application in population genetic studies of Liriodendron. METHODS AND RESULTS: Thirty-nine polymorphic EST-SSR loci were identified among 27 individuals sampled from a cultivated population of L. tulipifera. The number of alleles per locus ranged from three to 18. The average observed heterozygosity and expected heterozygosity were 0.684 and 0.778, respectively. Of the 39 loci, 32 showed interspecific transferability and polymorphism in a related species, L. chinense. The number of alleles per locus ranged from two to 11, and the average observed heterozygosity and expected heterozygosity were 0.475 and 0.736, respectively. CONCLUSIONS: The developed EST-SSR markers will be useful for investigating adaptive genetic differentiation in Liriodendron.

Chloroplast microsatellite markers in Liriodendron tulipifera (Magnoliaceae) and cross-species amplification in L. chinense.

PREMISE OF THE STUDY: A set of cpSSR markers were developed for the tree genus Liriodendron L. to investigate population genetic structure and phylogeographic history. METHODS AND RESULTS: Primers were designed directly from the chloroplast genome sequences of Liriodendron tulipifera. Among the 55 cpSSR markers tested, 11 polymorphic markers were identified in L. tulipifera. The number of alleles in the population tested ranged from two to five, and the unbiased haploid diversity per locus ranged from 0.074 to 0.644. Eighteen primer pairs generated polymorphic amplification in L. chinense. The number of alleles per locus ranged from two to seven, and the unbiased haploid diversity per locus was from 0.250 to 0.964. CONCLUSIONS: cpSSR markers developed here will be useful for phylogeography and population genetics studies of Liriodendron.