Histamine bidirectionally regulates the intrinsic excitability of parvalbumin-positive neurons in the lateral globus pallidus and promotes motor behaviour.

BACKGROUND AND PURPOSE: Parvalbumin (PV)-positive neurons are a type of neuron in the lateral globus pallidus (LGP) which plays an important role in motor control. The present study investigated the effect of histamine on LGPPV neurons and motor behaviour. EXPERIMENTAL APPROACH: Histamine levels in LGP as well as its histaminergic innervation were determined through brain stimulation, microdialysis, anterograde tracing and immunostaining. Mechanisms of histamine action were detected by immunostaining, single-cell qPCR, whole-cell patch-clamp recording, optogenetic stimulation and CRISPR/Cas9 gene-editing techniques. The effect of histamine on motor behaviour was detected by animal behavioural tests. KEY RESULTS: A direct histaminergic innervation in LGP from the tuberomammillary nucleus (TMN) and a histamine-induced increase in the intrinsic excitability of LGPPV neurons were determined by pharmacological blockade or by genetic knockout of the histamine H1 receptor (H1 R)-coupled TWIK-related potassium channel-1 (TREK-1) and the small-conductance calcium-activated potassium channel (SK3), as well as by activation or overexpression of the histamine H2 receptor (H2 R)-coupled hyperpolarization-activated cyclic nucleotide-gated channel (HCN2). Histamine negatively regulated the STN → LGPGlu transmission in LGPPV neurons via the histamine H3 receptor (H3 R), whereas blockage or knockout of H3 R increased the intrinsic excitability of LGPPV neurons. CONCLUSIONS AND IMPLICATIONS: Our results indicated that the endogenous histaminergic innervation in the LGP can bidirectionally promote motor control by increasing the intrinsic excitability of LGPPV neurons through postsynaptic H1 R and H2 R, albeit its action was negatively regulated by the presynaptic H3 R, thereby suggesting possible role of histamine in motor deficits manifested in Parkinson's disease (PD).

Receptor and Ionic Mechanism of Histamine on Mouse Dorsolateral Striatal Neurons.

The dorsolateral striatum (DLS) is the critical neural substrate that plays a role in motor control and motor learning. Our past study revealed a direct histaminergic projection from the tuberomammillary nucleus (TMN) of the hypothalamus to the rat striatum. However, the afferent of histaminergic fibers in the mouse DLS, the effect of histamine on DLS neurons, and the underlying receptor and ionic mechanisms remain unclear. Here, we demonstrated a direct histaminergic innervation from the TMN in the mouse DLS, and histamine excited both the direct-pathway spiny projection neurons (d-SPNs) and the indirect-pathway spiny projection neurons (i-SPNs) of DLS via activation of postsynaptic H1R and H2R, albeit activation of presynaptic H3R suppressed neuronal activity by inhibiting glutamatergic synaptic transmission on d-SPNs and i-SPNs in DLS. Moreover, sodium-calcium exchanger 3 (NCX3), potassium-leak channels linked to H1R, and hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) coupled to H2R co-mediated the excitatory effect induced by histamine on d-SPNs and i-SPNs in DLS. These results demonstrated the pre- and postsynaptic receptors and their downstream multiple ionic mechanisms underlying the inhibitory and excitatory effects of histamine on d-SPNs and i-SPNs in DLS, suggesting a potential modulatory effect of the central histaminergic system on the DLS as well as its related motor control and motor learning.

Diversity-oriented synthesis of marine sponge derived hyrtioreticulins and their anti-inflammatory activities.

Diversity-oriented synthesis is aimed to increase the chemical diversity of target natural products for extensive biological activity evaluation. Indole ring is an important functional group in a large number of drugs and other biologically active agents, and indole-containing natural products have been frequently isolated from marine sources in recent years. In this paper, a series of indole-containing marine natural hyrtioreticulin derivatives, including 19 new ones, were designed, synthesized through a key Pictet-Spengler reaction, and evaluated for their inflammation related activity. Compound 13b displayed the most promising activity by inhibiting TNF-α cytokine release with an inhibitory rate of 92% at a concentration of 20 µmol·L-1. A preliminary structure-activity relationship analysis was also discussed. This research may throw light on the discovery of marine indole alkaloid derived anti-inflammatory drug leads.

The biodegradation of cefuroxime, cefotaxime and cefpirome by the synthetic consortium with probiotic Bacillus clausii and investigation of their potential biodegradation pathways.

Cephalosporin residues in the environment are a great concern, but bioremediation options do exist. Bacillus clausii T reached a removal rate of 100% within 8 h when challenged with a mixture of cefuroxime (CFX), cefotaxime (CTX), and cefpirome (CPR). The co-culture of B. clausii T and B. clausii O/C displayed a higher removal efficiency for the mixture of CFX, CTX and CPR than a pure culture of B. clausii O/C. B. clausii T alleviated the biotoxicity of CFX and CPR. What's more, the biotoxicity of for CFX and CPR transformation products released by the co-culture of B. clausii T and B. clausii O/C was lower than that in pure cultures. Real-time PCR was applied to detect the changes in the expression levels of the relevant antibiotic-resistance genes of B. clausii T during CFX and CPR degradation. The results indicated that CFX and CPR enhanced the expression of the ß-lactamase gene bcl1. Hydrolysis, deacetylation and decarboxylation are likely the major mechanisms of CTX biodegradation by B. clausii. These results demonstrate that B. clausii T is a promising strain for the bioremediation of environmental contamination by CFX, CTX, and CPR.

The application of SVR model in the improvement of QbD: a case study of the extraction of podophyllotoxin.

In order to make a further optimization of process design via increasing the stability of design space, we brought in the model of Support Vector Regression (SVR). In this work, the extraction of podophyllotoxin was researched as a case study based on Quality by Design (QbD). We compared the fitting effect of SVR and the most used quadratic polynomial model (QPM) in QbD, and an analysis was made between the two design spaces obtained by SVR and QPM. As a result, the SVR stayed ahead of QPM in prediction accuracy, the stability of model and the generalization ability. The introduction of SVR into QbD made the extraction process of podophyllotoxin well designed and easier to control. The better fitting effect of SVR improved the application effect of QbD and the universal applicability of SVR, especially for non-linear, complicated and weak-regularity problems, widened the application field of QbD.

[Extraction Optimization of Rhizome of Curcuma longa by Response Surface Methodology and Support Vector Regression].

OBJECTIVE: To optimize the optimal microwave-assisted extraction method of curcuminoids from Curcuma longa. METHODS: On the base of single factor experiment, the ethanol concentration, the ratio of liquid to solid and the microwave time were selected for further optimization. Support Vector Regression (SVR) and Central Composite Design-Response Surface Methodology (CCD) algorithm were utilized to design and establish models respectively, while Particle Swarm Optimization (PSO) was introduced to optimize the parameters of SVR models and to search optimal points of models. The evaluation indicator, the sum of curcumin, demethoxycurcumin and bisdemethoxycurcumin by HPLC, were used. RESULTS: The optimal parameters of microwave-assisted extraction were as follows: ethanol concentration of 69%, ratio of liquid to solid of 21 : 1, microwave time of 55 s. On those conditions, the sum of three curcuminoids was 28.97 mg/g (per gram of rhizomes powder). CONCLUSION: Both the CCD model and the SVR model were credible, for they have predicted the similar process condition and the deviation of yield were less than 1.2%.

[Chromatographic Fingerprinting Study of Zhenyuan Granules Dry Extract by HPLC-DAD and HPLC-MS/MS].

OBJECTIVE: To establish a novel, accurate and valid fingerprint method of Zhenyuan granules dry extract by using HPLC-DAD method, to study herbs belonging of fingerprint peaks and to identify some of the chromatographic peaks by HPLC-MS/MS analysis, for providing the basis for scientific evaluation of the quality. METHODS: The sample solutions were analyzed by an Agilent SB C18 (250 mm x 4.6 mm, 5 µm) column, and gradiently eluted with acetonitrile (containing 0.1% formic acid) and aqueous phase (containing 0.1% formic acid) as the mobile phase. The flow rates were 1.2 mL/min (0~70 min) and 0.8 mL/min (70~150 min); the column temperature was 30 °C; and the detection wavelength was 254 nm. RESULTS: 40 peaks were selected as fingerprint peaks under the optimal chromatographic condition, and the similarity coefficients of 10 batches of Zhenyuan granules dry extract were all greater than 0.98. 27 peaks were tentatively identified with reference to literature data based on their mass spectrometry. CONCLUSION: The chromatographic fingerprint of Zhenyuan granules is proved to be a reliable method for comprehensive quality control and assessment.

Feature selection for the identification of antitumor compounds in the alcohol total extracts of Curcuma longa.

Antitumor activity has been reported for turmeric, the dried rhizome of Curcuma longa. This study proposes a new feature selection method for the identification of the antitumor compounds in turmeric total extracts. The chemical composition of turmeric total extracts was analyzed by gas chromatography-mass spectrometry (21 ingredients) and high-performance liquid chromatography-mass spectrometry (22 ingredients), and their cytotoxicity was detected through an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay against HeLa cells. A support vector machine for regression and a generalized regression neural network were used to research the composition-activity relationship and were later combined with the mean impact value to identify the antitumor compounds. The results showed that six volatile constituents (three terpenes and three ketones) and seven nonvolatile constituents (five curcuminoids and two unknown ingredients) with high absolute mean impact values exhibited a significant correlation with the cytotoxicity against HeLa cells. With the exception of the two unknown ingredients, the identified 11 constituents have been reported to exhibit cytotoxicity. This finding indicates that the feature selection method may be a supplementary tool for the identification of active compounds from herbs.

A novel approach to active compounds identification based on support vector regression model and mean impact value.

Traditionally, active compounds were discovered from natural product extracts by bioassay-guided fractionation, which was with high cost and low efficiency. A well-trained support vector regression model based on mean impact value was used to identify lead active compounds on inhibiting the proliferation of the HeLa cells in curcuminoids from Curcuma longa L. Eight constituents possessing the high absolute mean impact value were identified to have significant cytotoxicity, and the cytotoxic effect of these constituents was partly confirmed by subsequent MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays and previous reports. In the dosage range of 0.2-211.2, 0.1-140.2, 0.2-149.9 µm, 50% inhibiting concentrations (IC50 ) of curcumin, demethoxycurcumin, and bisdemethoxycurcumin were 26.99 ± 1.11, 19.90 ± 1.22, and 35.51 ± 7.29 µm, respectively. It was demonstrated that our method could successfully identify lead active compounds in curcuminoids from Curcuma longa L. prior to bioassay-guided separation. The use of a support vector regression model combined with mean impact value analysis could provide an efficient and economical approach for drug discovery from natural products.

Identification of antitumor constituents in curcuminoids from Curcuma longa L. based on the composition-activity relationship.

Using orthogonal partial least squares (OPLS), based on the Simca-p11.5 software, and canonical correlation analysis (CCA), performed on MatLab r2010 software, the correlation between curcuminoids extracted from Curcuma longa L. and the antitumor activity on HeLa cells was investigated to identify the significantly active constituents. Fingerprints from 31 batches of curcuminoids from C. longa L. were established using high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS), and a total of 26 selected characteristic peaks were quantitatively analyzed. Afterward, the antitumor activities of the curcuminoids on HeLa cells were measured using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. We found that 13 of the curcuminoids (peaks 9, 18, 14, 8, 16, 17, 24, 12, 4, 13, 10, 20 and 11) were significantly correlated with antitumor activity via a Loadings plot and VIP (variable importance in projection) in OPLS and a correlation coefficient in CCA. These results support a method for the discovery of antitumor active constituents.

Adrenoreceptor-coupled signal-transduction mechanisms mediating lymphocyte apoptosis induced by endogenous catecholamines.

Our previous work has shown that lymphocytes synthesize catecholamines (CAs) and the endogenous CAs accelerate apoptosis of concanavalin A (Con A)-activated lymphocyte. Here, we explored the involvement of adrenoreceptors (ARs) and signal molecules coupled to the ARs in the endogenous CA-mediated modulation of lymphocyte apoptosis. Pargyline, an inhibitor of CA degradation, up-regulated the expression of cleaved caspase-3 protein and increased the number of apoptotic cells. Antagonists of alpha(1)-ARs and beta(2)-ARs, not antagonists of alpha(2)-ARs or beta(1)-ARs, blocked these effects of pargyline. The facilitating effects of pargyline on lymphocyte apoptosis were mimicked by activators of adenylate cyclase and PKC, but reversed by inhibitors of PKA, PLC and PKC. Pargyline-stimulated CREB activation and Smac/DIABLO expression were prevented by the inhibitors of PKA, PLC and PKC. These results imply that endogenous CA-induced lymphocyte apoptosis is mediated by cAMP-PKA- and PLC-PKC-linked CREB-Smac/DIABLO pathways coupled with alpha(1)-ARs and beta(2)-ARs.

[Effect of the endogenous catecholamines synthesized by lymphocytes on T cell proliferation].

AIM: To provide further evidence for the synthesis of catecholamines (CAs) in lymphocytes and to investigate the effect of the endogenous CAs synthesized by lymphocytes on function of the lymphocytes themselves and the receptor mechanisms involved in the effect. METHODS: RT-PCR was performed to detect the expression of TH mRNA in the lymphocytes from the mesenteric lymph nodes of rats. Different concentrations of pargyline, an inhibitor of monoamine oxydase, and antagonists of alpha1-, alpha2-, beta1-, and beta2-adrenergic receptor (AR) were added to the lymphocyte cultures, and then proliferative response of the lymphocytes to mitogen concanavalin A (Con A) were measured via methyl-thiazole-tetrazolium (MTT) assay. RESULTS: The lymphocytes could express TH mRNA, and the expression of TH mRNA was significantly higher in the Con A-activated lymphocytes than in the resting ones. The treatment of pargyline of 10(-6) and 10(-5) mol/L (not 10(-7) mol/L) notably attenuated Con A-induced lymphocyte proliferation. Beta2-AR antagonist ICI118551 (10(-7) and 10(-6) mol/L) completely blocked, but alpha1-AR antagonist corynanthine and alpha2-AR antagonist yohimbine (10(-7) and 10(-6) mol/L) partly blocked the suppressive effect of pargyline on the Con A-induced lymphocyte proliferation. Nevertheless, atenolol, an antagonist of beta1-AR, had no blocking effect on pargyline inhibition of lymphocyte proliferation. CONCLUSION: Lymphocytes have the ability to synthesize CAs and the ability is enhanced in the activated lymphocytes. The endogenous CAs synthesized by lymphocytes can inhibit T cell proliferation and the inhibition of T cells by the CAs is mediated predominantly by beta2-AR on the lymphocytes.

[Stability of physical state on compound hawthorn dropping pills].

OBJECTIVE: To evaluate the stability of physical state with accelerate test and dropping in process before and after on compound hawthorn dropping pills. METHOD: Scanning electron microscope, TG-DTA, FT-IR and XRD were used. RESULT: The active components presented amorphous, tiny crystal and molecular state in dropping pills, and it had no obvious reaction between PEG 4000 and active components. With time prolonging, a little of active components changed from amorphous state to tiny crystal or molecular state. CONCLUSION: Solid dispersion improved the stability and dissolution of compound hawthorn dropping pills.

Effect of endogenous catecholamines on apoptosis of Con A-activated lymphocytes of rats.

Our previous studies show that lymphocytes express tyrosine hydroxylase (TH) and synthesize catecholamines (CAs) including dopamine, epinephrine and norepinephrine, and that the lymphocytes-derived endogenous CAs affect function of lymphocytes via autocrine/paracrine pathways. Over recent years, induction of apoptosis has been suggested to be a possible mechanism underlying the endogenous CAs-mediated lymphocyte proliferation, differentiation and activation. However, direct effect of the lymphocytes-synthesized CAs on lymphocyte apoptosis is less known. In the present study, TH inhibitor alpha-methyl-p-tyrosine (alpha-MT) and monoamine oxydase inhibitor pargyline were employed to block the synthesis and degradation of CAs in lymphocytes activated by concanavalin A (Con A). Apoptotic cells and apoptosis-related genes and proteins, Bax, Bcl-2, Fas, Fas-Ligand (FasL) and caspase-3, were examined in the lymphocytes treated with alpha-MT or pargyline by means of Annexin V/propidium iodide (PI) staining, real-time PCR and Western blot analyses, respectively. The treatment with alpha-MT of 10(-6) M and 10(-5) M (not 10(-7) M) notably reduced intracellular and supernatant DA, E and NE of the Con A-activated lymphocytes in a dose-dependent manner, and correspondingly, the treatment induced a remarkable decrease of apoptotic lymphocytes but not necrotic cells. The expression of Bax, Fas, FasL and caspase-3 mRNAs and proteins was significantly inhibited in the Con A-activated lymphocytes after the cells were treated with alpha-MT of 10(-6) M and 10(-5) M; but the expression of Bcl-2 mRNA and protein was dramatically increased by the alpha-MT treatment. Contrarily, the treatment with pargyline of 10(-6) M and 10(-5) M (not 10(-7) M) evidently increased the intracellular and supernatant DA, E and NE contents of the Con A-activated lymphocytes in a dose-dependent manner, and meanwhile, it caused a striking increase of apoptotic lymphocytes but not necrotic cells. The expression of Bax, Fas, FasL and caspase-3 mRNAs and proteins in the Con A-stimulated lymphocytes was remarkably enhanced by the treatment with pargyline of 10(-6) M and 10(-5) M, but the expression of Bcl-2 mRNA and protein was notably attenuated by the pargyline treatment. These results imply that endogenous CAs synthesized and secreted by lymphocytes accelerate lymphocyte apoptosis by altering fine balance between the expression of antiapoptotic and proapoptotic markers at transcriptional and translational levels, and suggest that both the death receptor pathway and the mitochondrial pathway are involved in the endogenous CAs-induced apoptosis.

Immunoregulatory role of endogenous catecholamines synthesized by immune cells.

It has been well known that catecholamines (CAs) in the body, including norepinephrine (NE), epinephrine (E) and dopamine (DA), are synthesized and secreted by neurons and endocrine cells and mainly modulate visceral activities such as cardiovascular, respiratory and digestive functions. The studies over the past nearly 30 years have shown that CAs can also regulate immune function. The immunomodulation of CAs is generally considered as a role mediating the regulation of nervous and endocrine systems. However, recent studies reveal that immune cells can also synthesize CAs, which is an update of traditional concept. A classical metabolic pathway of CAs shared by the nervous and endocrine systems is present in the immune cells, i.e., the immunocytes have the enzymes for synthesis of CAs [e.g. tyrosine hydroxylase (TH)] and the enzymes for degradation of CAs [e.g. monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT)]. The endogenous CAs synthesized by immune cells can regulate many immune functions, including cellular proliferation, differentiation, apoptosis and cytokine production. These roles of the endogenous CAs may be mediated by an autocrine/paracrine pathway via relevant receptors on the immunocytes and intracellular cAMP. Intracellular oxidative mechanism may also be involved in immunoregulation of endogenous CAs in immune cells. In addition, some metabolic abnormalities of CAs in the immune cells probably induce some autoimmune diseases, such as multiple sclerosis (MS) and rheumatoid arthritis. These findings not only provide evidence for the new concept that the immune system is possible to become the third CA system other than the nervous and endocrine systems, but also extend our comprehension on functional significance of the endogenous CAs synthesized by immune cells.

[Effect of acetylcholine on the cytotoxicity of natural killer cells].

AIM: To investigate the effect of acetylcholine (ACh) on the cytotoxicity of natural killer (NK) cells and to explore the receptor mechanisms involved in the effect. METHODS: The effector cells (i. e. NK cells) from the spleens of rats were collected and cultured with the target cells (Yac-1 cells). The various concentrations of ACh, cholinergic receptor agonists or antagonists were added to the cultures, respectively according to distinct experimental purposes. Lactate dehydrogenase (LDH) release assay was used to evaluate NK cell cytotoxicity. RESULTS: NK-cell-mediated lysis of Yac-1 lymphoma cells was reduced by 10(-10) - 10(-6) mol/L ACh. The inhibitory effect of ACh on NK cell cytotoxicity was mimicked by pilocarpine, an agonist of muscarinic receptor, and by nicotine, an agonist of nicotinic receptor, at all applied concentrations (10(-10) - 10(-6) mol/L). Muscarinic receptor antagonist atropine blocked the inhibitory effect of ACh on the cytotoxicity of NK cells. Nevertheless, tubocurarine, an antagonist of nicotinic receptor, had no blocking effect on the suppression of NK cell cytotoxicity by ACh. CONCLUSION: ACh results in an inhibition of the cytotoxicity of NK cells, and this inhibition is realized mainly through M and N1 cholinergic receptor.

Effect of catecholamines on IL-2 production and NK cytotoxicity of rats in vitro.

AIM: To explore effects of exogenous and endogenous catecholamines on function of lymphocytes and primary mechanisms mediating the effects. METHODS: Splenocytes of rats were exposed to norepinephrine (NE), alpha- or beta-adrenoceptor antagonists plus NE, or alpha-methyl-p-tyrosine (alpha-MT), and then concanavalin A (Con A)-induced interleukin-2 (IL-2) production and natural killer (NK) cell cytotoxicity were determined by MTT assay and LDH assay, respectively. RESULTS: Optical density (OD) values of NE-treated groups, which reflected IL-2 production, were 0.63, 0.61, and 0.60, respectively for 10(-10), 10(-9), and 10(-8) mol/L NE. They were all significantly reduced in comparison with control value of 0.68 (P<0.01). The effect of NE was blocked by either phentolamine (an alpha-adrenoceptor antagonist) or propanolol (a beta-adrenoceptor antagonist). OD values of alpha-MT, an inhibitor of tyrosine hydroxylase, at doses of 10(-10), 10(-9), and 10(-8) mol/L respectively were 0.71, 0.71, and 0.69, which were all notably higher than that of control (0.65, P<0.01). NK cytotoxicity was markedly attenuated by both NE and alpha-MT at the three doses mentioned above (17.69 %, 17.06 %, and 16.89 % versus 25.18 % for NE; 18.85 %, 18.44 %, and 17.04 % versus 23.22 % for alpha-MT; all P<0.01). The suppression of NK cytotoxicity by NE was prevented by propranolol but not by phentolamine. CONCLUSION: Exogenous NE exerts a suppressive action in modulating functions of T and NK cells, with the former via both alpha- and beta-adrenoceptor mediated mechanisms and the later mainly through beta-adrenoceptors. Endogenous catecholamines synthesized by lymphocytes have also an autoregulatory effect on the lymphocytes themselves.

Inhibitor studies of isopentenyl pyrophosphate biosynthesis in suspension cultures of the yew Taxus chinensis var. mairei.

Some metabolic inhibitors, mevastatin (MVS), chlorocholine chloride (CCC), sodium pyrophosphate (NaPP) and D,L-glyceraldehyde (DLG), were respectively added into the suspension cultures of the yew Taxus chinensis var. mairei at the late phase of cell growth to study isopentenyl pyrophosphate (IPP) biosynthesis. The content of total taxanes decreased in the cases of MVS, NaPP and DLG addition, regardless of the inhibitor concentration, whereas it increased in the case of CCC addition. It was thus presumed that a mevalonate pathway might exist in IPP biosynthesis at the late growth phase of Taxus cells and that some IPP might transfer from the cytoplasm to the plastids. This presumption was also confirmed by analysing the effects of the inhibitors on taxane content. It was further demonstrated that IPP in the biosynthesis of taxol, baccatin III and 10-deacetylbaccatin III might transfer from the cytoplasm to the plastids, whereas the translocation of IPP might be not involved in cephalomannine biosynthesis. Furthermore, in combination with the results of previous studies, it is very probable that different IPP biosynthesis pathways exist during different growth phases in Taxus cells.