Immobilization of M3 Muscarinic Receptor to Rapidly Analyze Drug-Protein Interactions and Bioactive Components in a Natural Plant.

Despite its increasing application in pursing potential ligands, the capacity of receptor affinity chromatography is greatly challenged as most current research studies lack a comprehensive characterization of the ligand-receptor interaction, particularly when simultaneously determining their binding thermodynamics and kinetics. This work developed an immobilized M3 muscarinic receptor (M3R) affinity column by fixing M3R on amino polystyrene microspheres via the interaction of a 6-chlorohexanoic acid linker with haloalkane dehalogenase. The efficiency of the immobilized M3R was tested by characterizing the binding thermodynamics and kinetics of three known drugs to immobilized M3R using a frontal analysis and the peak profiling method, as well as by analyzing the bioactive compounds in Daturae Flos (DF) extract. The data showed that the immobilized M3R demonstrated good specificity, stability, and competence for analyzing drug-protein interactions. The association constants of (-)-scopolamine hydrochloride, atropine sulfate, and pilocarpine to M3R were determined to be (2.39 ± 0.03) × 104, (3.71 ± 0.03) × 104, and (2.73 ± 0.04) × 104 M-1, respectively, with dissociation rate constants of 27.47 ± 0.65, 14.28 ± 0.17, and 10.70 ± 0.35 min-1, respectively. Hyoscyamine and scopolamine were verified as the bioactive compounds that bind to M3R in the DF extract. Our results suggest that the immobilized M3R method was capable of determining drug-protein binding parameters and probing specific ligands in a natural plant, thus enhancing the effectiveness of receptor affinity chromatography in diverse stages of drug discovery.

Exploring the Target and Mechanism of Radix Paeoniae Alba on Sjogren's Syndrome.

BACKGROUND: Radix Paeoniae Alba is a traditional Chinese herbal medicine. It can accelerate salivary secretion and alleviate the dry mouth of patients with Sjogren's syndrome (SS). Although it is widely used in clinical treatment, its target and mechanism remain unclear. OBJECTIVE: This study aims to analyze the main components of Radix Paeoniae Alba, explore the target genes, and propose the possible mechanism for Radix Paeoniae Alba's acceleration of salivary secretion. METHODS: The main active components and potential targets of Radix Paeoniae Alba were searched through the TCMSP database. Efforts were made to search for the related genes of Sjogren's syndrome in OMIM and GeneCards databases. Cytoscape v3.8.0 software was used to link target genes of active components and key genes of the disease. The software Autodock vina1.1.2. was adopted to simulate the interaction between active components and target genes. Human submandibular gland (HSG) cells were used in vitro experiments to verify the results of our analysis. RESULTS: ß-Sitosterol, the main component of Radix Paeoniae Alba, may intervene in the disease through CHRM3. Molecular docking shows ß-Sitosterol has a high affinity with CHRM3, and the interaction between CHRM3 and ß-Sitosterol is the basis of biological activity. The in vitro experiments showed that ß-Sitosterol could significantly up-regulate the mRNA and protein expression levels of both CHRM3 and secretion-related genes in HSG cells. CONCLUSION: Our study shows that the chemical components of Radix Paeoniae Alba have a positive effect on the related mechanism of salivary secretion. We found that ß-Sitosterol can promote the expression of CHRM3, stimulate salivary secretion, treat Sjogren's syndrome and potentially improve its prognosis.

An in silico and in vitro integrated analysis method to reveal the curative mechanisms and pharmacodynamic substances of Bufei granule on chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a common respiratory disease with high disability and mortality. Clinical studies have shown that the Traditional Chinese Medicine Bufei Granule (BFG) has conspicuous effects on relieving cough and improving lung function in patients with COPD and has a reliable effect on the treatment of COPD, whereas the therapeutic mechanism is vague. In the present study, the latent bronchodilators and mechanism of BFG in the treatment of COPD were discussed through the method of network pharmacology. Then, the molecular docking and molecular dynamics simulation were performed to calculate the binding efficacy of corresponding compounds in BFG to muscarinic receptor. Finally, the effects of BFG on bronchial smooth muscle were validated by in vitro experiments. The network pharmacology results manifested the anti-COPD effect of BFG was mainly realized via restraining airway smooth muscle contraction, activating cAMP pathways and relieving oxidative stress. The results of molecular docking and molecular dynamics simulation showed alpinetin could bind to cholinergic receptor muscarinic 3. The in vitro experiment verified both BFG and alpinetin could inhibit the levels of CHRM3 and acetylcholine and could be potential bronchodilators for treating COPD. This study provides an integrating network pharmacology method for understanding the therapeutic mechanisms of traditional Chinese medicine, as well as a new strategy for developing natural medicines for treating COPD.

Mechanistic insight of the potential of geraniol against Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) as a neurodegenerative disease occupies 3/5-4/5 cases among patients with dementia, yet its pathogenetic mechanism remains unclear. Geraniol, on the other hand, is a well-known extract from essential oils of aromatic plants and has been proven that it has outstanding neuroprotective effects as well as ameliorating influence in memory impairment. Therefore, the present study aims to elucidate the potential of geraniol against AD by network pharmacology-based approach combined with molecular modeling study. MATERIALS AND METHODS: Firstly, we evaluated the druggability of geraniol by ADME method. Then, we obtained the geraniol targets and AD-related targets from multiple open data sources. Afterward, we calculated the intersection through a Venn diagram to find common targets, and via Panther classification system to categorize them. In order to gain a macroscopic understanding of these common targets, we carried out GO terms and KEGG pathways enrichment analyses, according to which we constructed a compound-target-pathway-disease network. In addition, we built a preliminary PPI network which was further analyzed both functionally and topologically. Consequently, five hub targets were sorted out. Finally, we conducted molecular docking and molecular dynamic simulation to validate our findings. RESULTS: In the present study, the pharmacological properties of geraniol were assessed according to ADME and Lipinski's rule, which demonstrate promising druggability. Then, from 10,972 AD-related targets and 33 geraniol targets, 29 common targets were identified, among which 38.1% of them are metabolite interconversion enzymes, 23.8% are protein modifying enzymes, 33.3% are transmembrane receptors, and the rest are transporters. Enrichment analyses hint that geraniol is involved in cholinergic synapse, serotonergic synapse, and neuroactive ligand-receptor interaction. We also built a preliminary PPI network to investigate the interplay between these targets and their extensive interactions. Then, by functionally clustering the preliminary PPI network, we gained a cluster of proteins which formed a subnetwork with score of 8.476, and 22 nodes. Its results of GO terms and KEGG pathways enrichment analyses once again suggests that geraniol actively participates in cholinergic synapse, serotonergic synapse, and neuroactive ligand-receptor interaction, which are believed to be strongly associated with AD pathogenesis. Besides, topological analyses of the preliminary PPI network helped find 5 hub targets (i.e., CHRM3, PRKCA, PRKCD, JAK1, JAK2). To verify their interaction with geraniol molecule, we conducted molecular docking, and found that CHRM3 possesses the highest affinity in binding, indicating that geraniol molecules are closely bound to each hub target, and CHRM3 may serve as a key target of geraniol against AD. It was then further confirmed by molecular dynamic simulation, the result of which supports our hypothesis. CONCLUSION: The present study shares a mechanistic insight of the potential of geraniol against AD, giving a reference to future experimental studies.

Activation of cholinergic pathway induced vasodilation in rat aorta using aqueous and methanolic leaf extracts of Gynura procumbens.

Gynura procumbens (GP) is a herbal medicinal plant of South-East Asian origin, popularly recognised as 'Sambung nyawa'. The plant has been used traditionally to treat various diseases including hypertension. The anti-hypertensive activity of this plant has also been scientifically proven both in vivo and in vitro yet the investigation on its mechanisms of actions remains limited. Our previous study has demonstrated the vasodilatory action of both aqueous and methanol GP extracts possibly via activation of the cholinergic pathway and that kaempferol 3-O-rutinoside is the active ingredient responsible in mediating this effect. Hence, in this study we further confirm the involvement of the cholinergic pathway by using several pharmacological interventions, focusing on the downstream mechanism of this pathway. Our results showed that in the presence of endothelium, GP extracts induced vasodilation via activation of the muscarinic M3 receptors. However, in the absence of endothelium, GP mediated vasodilation possibly via stimulation of other muscarinic receptors and/or involvement of nicotinic receptors, a speculation that needs further investigations. GP-induced relaxation was markedly inhibited by nitric oxide (NO) blocker, L-NAME, suggesting that GP elicited ACh endothelium-dependent relaxation by producing NO in rat aortic rings. In conclusion, these data demonstrate that the vasodilatory effect of GP extracts appears to be mediated via cholinergic pathway.

Spirulina platensis Consumption Prevents Obesity and Improves the Deleterious Effects on Intestinal Reactivity in Rats Fed a Hypercaloric Diet.

The consumption of hypercaloric diets is related to the development of obesity, favoring the etiology of gastrointestinal disorders. In this context, Spirulina platensis (SP), some blue-green algae with antioxidant action, appears as a potential therapeutic alternative to prevent obesity and associated intestinal disorders. Thus, the present study is aimed at evaluating the deleterious effects of the hypercaloric diet on the contractile and relaxing reactivity of the ileum of rats, as well as the possible preventive mechanisms of dietary supplementation with SP. Wistar rats were divided into three groups: fed a standard diet (SD), a hypercaloric diet (HCD), and/or supplemented with 25 mg/kg SP (HCD + SP25) for 8 weeks. The hypercaloric diet was effective in promoting obesity in rats, as well as decreasing potency and ileal relaxing and contractile efficacy. In contrast, dietary supplementation with SP was able to prevent some of the parameters of experimental obesity. In addition, SP prevented the reduction of intestinal reactivity, possibly due to a positive modulation of voltage-gated calcium channels (CaV) and negative regulation of muscarinic receptors (M3). Thus, food supplementation with Spirulina platensis becomes a promising alternative in the prevention of gastrointestinal diseases induced and/or aggravated by obesity.

Vasorelaxant property of Plectranthus vettiveroides root essential oil and its possible mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Plectranthus vettiveroides (Jacob) N.P. Singh & B.D. Sharma is a traditional medicinal plant used in Siddha System of Medicine and its aromatic root is used to reduce the elevated blood pressure. AIM: The aim of the present study was to study vasorelaxant property of the root essential oil nanoemulsion (EON) of P. vettiveroides. METHODS: The EON was formulated to enhance the solubility and bioavailability and characterized. The preliminary screening was performed by treating the EON with aortic rings pre-contracted with phenylephrine (1 µM) and potassium chloride (80 mM). The role of K⁺ channels in EON induced vasorelaxation was investigated by pre-incubating the aortic rings with different K⁺ channel inhibitors namely, glibenclamide (a non-specific ATP sensitive K⁺ channel blocker, 10 µM), TEA (a Ca2⁺ activated non-selective K⁺ channel blocker, 10-2 M), 4-AP (a voltage-activated K⁺ channel blocker, 10-3 M) and barium chloride (inward rectifier K⁺ channel blocker, 1 mM). The involvement of extracellular Ca2+ was performed by adding cumulative dose of extracellular calcium in the presence and absence of EON and the concentration-response curve (CRC) obtained is compared. Similarly, the role of nitric oxide synthase, muscarinic and prostacyclin receptors on EON induced vasorelaxation were evaluated by pre-incubating the aortic rings with their inhibitors and the CRC obtained in the presence and absence of inhibitor were compared. RESULTS: The GC-MS and GC-FID analyses of the root essential oil revealed the presence of 62 volatile compounds. The EON exhibited significant vasorelaxant effect through nitric oxide-mediated pathway, G-protein coupled muscarinic (M3) receptor pathway, involvement of K+ channels (KATP, KIR, KCa), and blocking of the calcium influx by receptor-operated calcium channel. CONCLUSION: It is concluded that the root essential oil of P. vettiveroides is possessing marked vasorelaxant property. The multiple mechanisms of action of the essential oil of P. vettiveroides make it a potential source of antihypertensive drug.

Evaluation of QSAR Equations for Virtual Screening.

Quantitative Structure Activity Relationship (QSAR) models can inform on the correlation between activities and structure-based molecular descriptors. This information is important for the understanding of the factors that govern molecular properties and for designing new compounds with favorable properties. Due to the large number of calculate-able descriptors and consequently, the much larger number of descriptors combinations, the derivation of QSAR models could be treated as an optimization problem. For continuous responses, metrics which are typically being optimized in this process are related to model performances on the training set, for example, R2 and QCV2. Similar metrics, calculated on an external set of data (e.g., QF1/F2/F32), are used to evaluate the performances of the final models. A common theme of these metrics is that they are context -" ignorant". In this work we propose that QSAR models should be evaluated based on their intended usage. More specifically, we argue that QSAR models developed for Virtual Screening (VS) should be derived and evaluated using a virtual screening-aware metric, e.g., an enrichment-based metric. To demonstrate this point, we have developed 21 Multiple Linear Regression (MLR) models for seven targets (three models per target), evaluated them first on validation sets and subsequently tested their performances on two additional test sets constructed to mimic small-scale virtual screening campaigns. As expected, we found no correlation between model performances evaluated by "classical" metrics, e.g., R2 and QF1/F2/F32 and the number of active compounds picked by the models from within a pool of random compounds. In particular, in some cases models with favorable R2 and/or QF1/F2/F32 values were unable to pick a single active compound from within the pool whereas in other cases, models with poor R2 and/or QF1/F2/F32 values performed well in the context of virtual screening. We also found no significant correlation between the number of active compounds correctly identified by the models in the training, validation and test sets. Next, we have developed a new algorithm for the derivation of MLR models by optimizing an enrichment-based metric and tested its performances on the same datasets. We found that the best models derived in this manner showed, in most cases, much more consistent results across the training, validation and test sets and outperformed the corresponding MLR models in most virtual screening tests. Finally, we demonstrated that when tested as binary classifiers, models derived for the same targets by the new algorithm outperformed Random Forest (RF) and Support Vector Machine (SVM)-based models across training/validation/test sets, in most cases. We attribute the better performances of the Enrichment Optimizer Algorithm (EOA) models in VS to better handling of inactive random compounds. Optimizing an enrichment-based metric is therefore a promising strategy for the derivation of QSAR models for classification and virtual screening.

Mechanism of cAMP-PKA Signaling Pathway Mediated by Shaoyao Gancao Decoction () on Regulation of Aquaporin 5 and Muscarinic Receptor 3 Levels in Sjögren's Syndrome.

OBJECTIVE: To investigate the mechanism of cAMP-PKA signaling pathway mediated by Chinese medicine formula Shaoyao Gancao Decoction (, SGD) on the regulation of aquaporin 5 (AQP5) and muscarinic receptor 3 (M3R) levels in Sjögren's syndrome (SS). METHODS: Of the 30 mice, 5 were randomly selected as control, and others were used for creating SS model. After successful modeling, mice were randomly divided into 5 groups (n=5 per group) and intragastrically administered with saline (8 mL/kg), pilocarpine (1.4 mg/kg), or low, medium and high doses SGD (0.14, 0.21, 0.35 g/kg Radix paeoniae with 0.01 g/kg Radix glycyrrhizae, respectively) for 6 weeks. Human labial gland acinar cells were treated with pilocarpine or varying doses of SGD with saline as the placebo. Hematoxylin and eosin staining was used to observe the histopathological changes of the submandibular glands of mice. The serum levels of anti-SS antigen A (SS-A), anti-SS antigen B (SS-B), M3R, and α-fodrin in submandibular glands of mice were measured by enzyme-linked immunosorbent assay. Immunofluorescence staining was used to observe the spatial localization of AQP5 and M3R in acinar cells. Reverse transcriptase polymerase chain reaction and Western blot were used to detect the expressions of PKA, cAMP, Epac1, AQP5, M3R, nuclear factor kappa-B (NF-κB), and tumor necrosis factor (TNF)-α in submandibular gland tissues and cells of each group. RESULTS: Compared to normal mice, body weight, 5-min salivary secretion, 30-min secretion of tears and breakup time of tear film of model mice decreased at 1-6 weeks after immunization (all P<0.05), whereas water intake increased (all P<0.05). In the model group, glands of the submandibular glands showed atrophy, accompanied by acini of different sizes, decreased numbers and loose arrangement, with catheter dilatation and different degrees of lymphocyte infiltration. Conditions of mice in SGD groups were improved. The positive expression of AQP5 and M3R were higher in the acinar cells treated with all doses SGD compared to the normal group; serum levels of SS-A, SS-B, and α-fodrin were lower, and that of M3R was higher in all doses SGD treated animals than the model or pilocarpine treated ones (all P<0.05). Compared to the model and pilocarpine groups, the mRNA and protein levels of NF-κB and TNF-α were lower in mice or cells treated with medium or high-dose SGD (all P<0.05), while those of PKA, Epac1, AQP5 and M3R were higher (all P<0.05). CONCLUSION: SGD can improve symptoms of SS by regulating the cAMP-PKA signaling pathway and increasing AQP5 and M3R levels.

Therapeutic effects and modulatory mechanism of Alpiniae oxyphyllae Fructus in chronic intermittent hypoxia induced enuresis in rats.

OBJECTIVE: The objective of this study was to explore the effect of Alpiniae oxyphyllae Fructus (AOF) on a rat model of chronic intermittent hypoxia (CIH)-induced enuresis. Findings of this study may help identify therapeutic targets in children with nocturnal enuresis (NE). METHODS: Female rats were randomly divided into a control group (saline gavage, 4 weeks of normal air), CIH group (saline gavage, 4 weeks of CIH), and AOF group (AOF gavage, 4 weeks of CIH). The variables measured in this study included water intake, urine output, bladder leak point pressure (BLPP), malondialdehyde (MDA) levels, and superoxide dismutase (SOD) activity. The expression levels of the purinergic P2X3 receptor, muscarinic M3 receptor, and ß3-adrenergic receptor (ß3-AR) in the bladder were also measured. The bladder was subjected to haematoxylin and eosin (HE) and Weigert staining, and histological changes were observed under a light microscope to evaluate the morphological changes in the bladder in each group. RESULTS: Compared with the control group, urine output was increased, and the BLPP was decreased in the CIH group, but AOF administration decreased urine output and increased BLPP. In addition, the serum MDA level increased and the SOD activity decreased in the CIH group compared with the control group. Administration of AOF decreased the MDA level and increased the SOD activity. Additionally, compared with the control group, HE and Weigert staining in the CIH group showed that the bladder detrusor muscle bundles were disordered and loose, some muscle bundles were broken, the content of collagen fibres in the gap was reduced, and the gap was significantly widened. However, following the administration of AOF, the bladder detrusor muscle bundles were neatly arranged, and the content of collagen fibres in the gap was increased. Furthermore, compared with the control group, the purinergic P2X3 receptor and muscarinic M3 receptor were expressed at higher levels, and ß3-AR was expressed at lower levels in the CIH group, but AOF administration decreased the expression of the purinergic P2X3 receptor and muscarinic M3 receptor and increased the expression of the ß3-AR. CONCLUSIONS: AOF improves enuresis by inhibiting oxidative stress and regulating the expression of the purinergic P2X3 receptor, muscarinic M3 receptor, and ß3 adrenergic receptor.

The Traditional Medicine Banhasasim-Tang Depolarizes Pacemaker Potentials of Cultured Interstitial Cells of Cajal through M3 Muscarinic and 5-HT3 Receptors in Murine Small Intestine.

BACKGROUND: Banhasasim-tang (BHSST) is a classic herbal formulation in traditional Chinese medicine widely used for gastrointestinal (GI) tract motility disorder. We investigated the effects of BHSST on the pacemaker potentials of cultured interstitial cells of Cajal (ICCs) in small intestine in vitro and its effects on GI motor functions in vivo. METHODS: We isolated ICCs from the small intestines and recorded pacemaker potentials in cultured ICCs with the whole-cell patch-clamp configuration in vitro. Intestinal transit rates (ITR%) were investigated in normal mice and GI motility dysfunction (GMD) mouse models in vivo. RESULTS: BHSST (20-50 mg/mL) depolarized pacemaker potentials and decreased their amplitudes in a concentration-dependent manner. Pretreatment with methoctramine (a muscarinic M2 receptor antagonist) did not inhibit BHSST-induced pacemaker potential depolarization. However, when we applied 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP; a muscarinic M3 receptor antagonist), BHSST-induced effects were blocked. Pretreatment with Y25130 (a 5-HT3 receptor antagonist) blocked BHSST-induced effects in ICCs. In addition, when we applied 4-DAMP and Y25130 together, BHSST-induced effects were completely blocked. Pretreatment with Ca2+-free solution or thapsigargin inhibited BHSST-induced effects. Moreover, BHSST blocked both the transient receptor potential melastatin (TRPM) 7 and voltage-sensitive calcium-activated chloride (anoctamin-1, ANO1) channels. In normal mice, ITR% values were significantly increased by BHSST in a dose-dependent manner. The ITR% of GMD mice was significantly reduced relative to those of normal mice, which were significantly reversed by BHSST in a dose-dependent manner. CONCLUSION: These results suggested that BHSST depolarizes the pacemaker potentials of ICCs in a dose-dependent manner through the M3 and 5-HT3 receptors via internal and external Ca2+-dependent and TRPM7- and ANO1-independent pathways in vitro. Moreover, BHSST increased ITR% in vivo in normal mice and GMD mouse models. Taken together, the results of this study showed that BHSST had the potential for development as a prokinetic agent in GI motility function.

Acacetin-induced cell apoptosis in head and neck squamous cell carcinoma cells: Evidence for the role of muscarinic M3 receptor.

Aacacetin, a plant flavone has shown antitumor efficacy recently. However, its associated mechanisms are poorly known. We hypothesized that the muscarinic M3 receptor (M3 R), which is highly expressed in some cancer tissue, is related to the antitumor effect of acacetin in head and neck squamous cell carcinoma (HNSCC) cells. Our results showed that 12.5- to 200-µM acacetin inhibited cell viability in dose- and time-dependent manners in HNSCC cells, but a relative higher concentration was needed for oral adenoid cystic carcinoma cells. M3 R expression level was higher in HNSCC cells than that in adenoid cystic carcinoma cells. Flow cytometry and electron microscopy confirmed acacetin-induced cell apoptosis in 22B cells, a HNSCC cell line. Acacetin promoted mitochondrial cytochrome c release and caspase 9, 3 processing. Knocking down of M3 R expression by specific siRNA significantly prevented the acacetin-induced cell viability damage, cell apoptosis, and caspase 3 activation. Besides, M3 R was also involved in acacetin-induced elevation of reactive oxygen species and intracellular calcium ([Ca2+ ]i ). These data indicate that acacetin-induced cell apoptosis in HNSCC cells may through M3 R related calcium signaling and caspase 3 activation. Acacetin is a potent natural antitumor reagent especially for the tumor cells, which highly expressed M3 R.

I-Tiao-Gung extract through its active component daidzin improves cyclophosphamide-induced bladder dysfunction in rat model.

AIMS: We explored the therapeutic potential of intragastric administration traditional Chinese medicine Glycine tomentella Hayata (I-Tiao-Gung, ITG) extract and its active component Daidzin on cyclophosphamide (CYP)-induced cystitis and bladder hyperactivity in rats. METHODS: Female Wistar rats were divided into control, CYP (200 mg/kg), CYP + ITG (1.17 g/kg/day), CYP + Daidzin (12.5 mg/kg/day), and 1 week of ITG preconditioning with CYP (ITG + CYP) groups. We determined the trans cystometrogram associated with external urethral sphincter electromyogram, and the expression of M2 and M3 muscarinic and P2 × 2 and P2 × 3 purinergic receptors by Western blot in these animals. RESULTS: ITG extract contains 1.07% of Daidzin and 0.77% of Daidzein by high-performance liquid chromatography. Daidzin was more efficient than Daidzein in scavenging H2 O2 activity by a chemiluminescence analyzer. CYP induced higher frequency, shorter intercontraction interval, lower maximal voiding pressure, lower threshold pressure, and Phase-2 emptying contraction with a depressed external urethral sphincter electromyogram activity, and hemorrhagic cystitis in the bladders. The altered parameters by CYP were significantly improved in CYP + ITG, CYP + Daidzin, and ITG + CYP groups. The P2 × 2 and P2 × 3 expressions were significantly upregulated in CYP group, but were depressed in CYP + ITG, CYP + Daidzin, and ITG + CYP groups. The M2 expression was not significantly different among these five groups. The M3 expression was significantly upregulated in CYP group, but was significantly depressed in CYP + ITG, CYP + Daidzin, and ITG + CYP groups. CONCLUSIONS: These data suggest that ITG extract through its active component Daidzin effectively improved CYP-induced cystitis by the action of restoring Phase 2 activity and inhibiting the expressions of P2 × 2, P2 × 3, and M3 receptors.

CHRM3 rs2165870 polymorphism is independently associated with postoperative nausea and vomiting, but combined prophylaxis is effective.

BACKGROUND: Clinical risk factors for postoperative nausea and vomiting (PONV) are evaluated with the Apfel score, however patients with low Apfel scores still experience PONV suggesting a genetic predisposition. PONV risk associates with specific M3 muscarinic acetylcholine receptor (CHRM3) rs 2165870 polymorphism. We investigated whether the Apfel score and this genetic variation independently contribute to PONV risk and whether prophylaxis reduces PONV in patients with low Apfel score but at high genetic risk. METHODS: In a prospective, controlled study, 454 subjects undergoing elective surgery were genotyped for rs2165870 and its association with PONV was investigated with log-binomial regression analysis. Subjects were randomised to receive acustimulation/dexamethasone, acustimulation/vehicle, sham acustimulation/dexamethasone, or sham acustimulation/vehicle to investigate their effects on PONV risk. RESULTS: Early PONV occurred in 37% of subjects. The rs2165870 genotype distribution was GG in 191, GA in 207, and AA in 56 subjects. The CHRM3 polymorphism was associated with a relative risk (RR) of 1.5 for GA vs GG [95% confidence interval (CI): 1.1-1.9; P=0.003] and 1.6 for AA vs GG (95% CI: 1.1-2.2; P=0.009) genotypes to develop PONV, and this was independent from the Apfel score (RR per Apfel point: 1.3, 95% CI: 1.2-1.5; P<0.0001). While dexamethasone and acustimulation each reduced the PONV risk by 30% in AA genotype carriers with low Apfel score, combined therapy reduced the risk by 86% (P=0.015). CONCLUSIONS: The CHRM3 polymorphism and the Apfel score independently predict PONV susceptibility. Dexamethasone/acustimulation should be considered in patients with low Apfel score but at high genetic risk. CLINICAL TRIAL REGISTRATION: DRKS00005664.

[Impacts on urodynamic parameters and the protein expressions of M2 and M3 receptors of detrusor in the rats of detrusor hyperreflexia treated with ginger-salt-isolated moxibustion at "Shenque" (CV 8)].

OBJECTIVE: To discuss the effects on detrusor hyperreflexia treated with ginger-salt-isolated moxibustion at "Shenque" (CV 8) and its mechanism. METHODS: Thirty female adult SD rats were selected. The model of detrusor hyperreflexia was prepared with complete spinal transection at T9, of which, 20 rats were randomized into a model group (10 rats) and a moxibustion group (10 rats). A sham-operation group (10 rats) was set up for sham-spinal transection. In the moxibustion group, when urine incontinence occurred (about in 2 weeks of modeling), the ginger-salt-isolated moxibustion at "Shenque" (CV 8) was given, 3 moxa cones each time, once a day, continuously for 7 days. After treatment, in each group, the urodynamic parameters were determined, after which, the bladder detrusor was collected. Western blot was used to determine the protein expressions of M2 and M3 receptors. RESULTS: Compared with the sham-operation group, the micturition interval was shortened apparently (P<0.01); the maximal bladder pressure was increased apparently (P<0.01); the protein expression of M2 receptor in the detrusor was increased significantly (P<0.05) and that of M3 receptor had no apparent change (P>0.05) in the rats of the model group. Compared with the model group, the micturition interval was longer apparently (P<0.01), the maximal bladder pressure was reduced apparently (P<0.01), the protein expression of M2 receptor in the detrusor was reduced significantly (P<0.05) and that of M3 receptor had no apparent change (P>0.05) in the rats of the moxibustion group.Compared with the sham-operation group, the results of the above indicators were not different significantly in the moxibustion group (all P>0.05). CONCLUSIONS: The ginger-salt-isolated moxibustion at "Shenque" (CV 8) suppresses the overactive bladder in the rat with spinal transection and its effect mechanism is possibly relevant with reducing the protein expression of detrusor M2 and inhibiting the excessive contraction of the detrusor.

Magnolia Officinalis Bark Extract Induces Depolarization of Pacemaker Potentials Through M2 and M3 Muscarinic Receptors in Cultured Murine Small Intestine Interstitial Cells of Cajal.

BACKGROUND: Magnolia officinalis Rehder and EH Wilson (M. officinalis) are traditional Chinese medicines widely used for gastrointestinal (GI) tract motility disorder in Asian countries. We investigated the effects of an ethanol extract of M. officinalis (MOE) on the pacemaker potentials of cultured interstitial cells of Cajal (ICCs) in vitro and its effects on GI motor functions in vivo. METHODS: We isolated ICCs from small intestines, and the whole-cell patch-clamp configuration was used to record the pacemaker potentials in cultured ICCs in vitro. Both gastric emptying (GE) and intestinal transit rates (ITRs) were investigated in normal and GI motility dysfunction (GMD) mice models in vivo. RESULTS: MOE depolarized ICC pacemaker potentials dose-dependently. Pretreatment with methoctramine (a muscarinic M2 receptor antagonist) and 4-DAMP (a muscarinic M3 receptor antagonist) inhibited the effects of MOE on the pacemaker potential relative to treatment with MOE alone. In addition, MOE depolarized pacemaker potentials after pretreatment with Y25130 (a 5-HT3 receptor antagonist), GR113808 (a 5-HT4 receptor antagonist) or SB269970 (a 5-HT7 receptor antagonist). However, pretreatment with RS39604 (a 5-HT4 receptor antagonist) blocked MOE-induced pacemaker potential depolarizations. Intracellular GDPßS inhibited MOE-induced pacemaker potential depolarization, as did pretreatment with Ca2+ free solution or thapsigargin. In normal mice, the GE and ITR values were significantly and dose-dependently increased by MOE. In loperamide-and cisplatin-induced GE delay models, MOE administration reversed the GE deficits. The ITRs of the GMD mice were significantly reduced relative to those of normal mice, which were significantly and dose-dependently reversed by MOE. CONCLUSION: These results suggest that MOE dose-dependently depolarizes ICCs pacemaker potentials through M2 and M3 receptors via internal and external Ca2+ regulation through G protein pathways in vitro. Moreover, MOE increased GE and ITRs in vivo in normal and GMD mouse models. Taken together, the results of this study show that MOE have the potential for development as a gastroprokinetic agent in GI motility function.

Genome-Wide Interaction Study of Omega-3 PUFAs and Other Fatty Acids on Inflammatory Biomarkers of Cardiovascular Health in the Framingham Heart Study.

Numerous genetic loci have been identified as being associated with circulating fatty acid (FA) levels and/or inflammatory biomarkers of cardiovascular health (e.g., C-reactive protein). Recently, using red blood cell (RBC) FA data from the Framingham Offspring Study, we conducted a genome-wide association study of over 2.5 million single nucleotide polymorphisms (SNPs) and 22 RBC FAs (and associated ratios), including the four Omega-3 FAs (ALA, DHA, DPA, and EPA). Our analyses identified numerous causal loci. In this manuscript, we investigate the extent to which polyunsaturated fatty acid (PUFA) levels moderate the relationship of genetics to cardiovascular health biomarkers using a genome-wide interaction study approach. In particular, we test for possible gene-FA interactions on 9 inflammatory biomarkers, with 2.5 million SNPs and 12 FAs, including all Omega-3 PUFAs. We identified eighteen novel loci, including loci which demonstrate strong evidence of modifying the impact of heritable genetics on biomarker levels, and subsequently cardiovascular health. The identified genes provide increased clarity on the biological functioning and role of Omega-3 PUFAs, as well as other common fatty acids, in cardiovascular health, and suggest numerous candidate loci for future replication and biological characterization.

Contribution of muscarinic receptors to in vitro and in vivo effects of Ruscus extract.

The objectives of this study were to evaluate, in vitro and in vivo, the contribution of muscarinic receptors to the effects of Ruscus extract. Ruscus extract was tested in competition binding experiments at recombinant human muscarinic receptors, heterologous expressed in Chinese Hamster Ovary (CHO) cells and in cellular assays measuring Ca2+ liberation and activator protein-1 (AP-1) reporter gene activation. The impact of muscarinic blockade on prolonged treatment outcome was evaluated using the hamster cheek pouch (HCP) microcirculation examining macromolecular permeability increase induced by histamine or ischemia/reperfusion (I/R), mean arteriolar and venular diameters, functional capillary density and I/R-induced leukocyte rolling and sticking. Ruscus extract exhibited affinities for muscarinic receptor subtypes at a range of 50-100µg/ml and behaved as partial agonist at human recombinant M1 and M3 receptors for Ca2+ liberation, confirmed in an AP-1 reporter gene assay. In the HCP model, topical application of atropine completely or partially blocked Ruscus extract-induced reductions of histamine- and I/R-induced increases of macromolecular permeability and leukocyte-endothelium interaction. Our results showed that Ruscus extract in vitro binds and activates different subtypes of muscarinic receptors and in vivo its anti-inflammatory effects are, at least partially, mediated via muscarinic receptors.

Discovery of new muscarinic acetylcholine receptor antagonists from Scopolia tangutica.

Scopolia tangutica (S. tangutica) is a traditional Chinese medicinal plant used for antispasmodics, anesthesia, analgesia and sedation. Its pharmacological activities are mostly associated with the antagonistic activity at muscarinic acetylcholine receptors (mAchRs) of several known alkaloids such as atropine and scopolamine. With our recent identification of four hydroxycinnamic acid amides from S. tangutica, we hypothesized that this plant may contain previously unidentified alkaloids that may also contribute to its in vivo effect. Herein, we used a bioassay-guided multi-dimension separation strategy to discover novel mAchR antagonists from S. tangutica. The core of this approach is to use label-free cell phenotypic assay to first identify active fractions, and then to guide purification of active ligands. Besides four tropanes and six cinnamic acid amides that have been previously isolated from S. tangutica, we recently identified two new tropanes, one new cinnamic acid amide, and nine other compounds. Six tropane compounds purified from S. tangutica for the first time were confirmed to be competitive antagonists of muscarinic receptor 3 (M3), including the two new ones 8 and 12 with IC50 values of 1.97 µM and 4.47 µM, respectively. Furthermore, the cinnamic acid amide 17 displayed 15-fold selectivity for M1 over M3 receptors. These findings will be useful in designing lead compounds for mAchRs and elucidating mechanisms of action of S. tangutica.

Muscarinic antagonists in early stage clinical development for the treatment of asthma.

INTRODUCTION: Parasympathetic neurons utilize the neurotransmitter acetylcholine to modulate and constrict airway smooth muscles at the muscarinic acetylcholine receptor. Inhaled agents that antagonize the muscarinic (M) acetylcholine receptor, particularly airway M3 receptors, have increasing data supporting use in persistent asthma. Areas covered: Use of inhaled long-acting muscarinic antagonists (LAMA) in asthma is explored. The LAMA tiotropium is approved for maintenance in symptomatic asthma patients despite the use of inhaled corticosteroids (ICS), leukotriene receptor antagonists (LTRA) and/or long-acting beta2 agonists (LABA). LAMA agents currently approved for chronic obstructive pulmonary disease (COPD) include tiotropium, glycopyrrolate/glycopyrronium, umeclidinium and aclidinium. These agents are reviewed for their pharmacological differences and clinical trials in asthma. Expert opinion: Current guidelines place inhaled LAMAs as adjunctive maintenance therapy in symptomatic asthma not controlled by an ICS and/or a LTRA. LAMA agents will play an increasing role in moderate to severe symptomatic asthma patients. Additional LAMA agents are likely to seek a maintenance indication perhaps as a combined inhaler with an ICS or with an ICS and a LABA. These fixed-dose combination inhalers are being tested in COPD and asthma patients. Once-a-day dosing of inhaled LAMA agents in severe asthma patients will likely become the future standard.