Alterations in expression of α1-adrenergic receptors possibly are involved in prevention of age-associated apoptosis in rat hippocampus by treadmill exercise.

OBJECTIVES: Exercise is assumed to attenuate age-related neuronal apoptosis, but the detailed mechanism(s) is not fully understood. α1-Adrenergic receptors (ARs) can either trigger or suppress apoptosis, therefore, here we determined the impact of treadmill exercise on the expression of the apoptosis regulatory proteins as well as α1-AR subtypes α1A- and α1B-ARs, in order to elucidate a possible association between apoptosis and the hippocampal expression of α1-ARs in aged male rats. METHODS: Twenty-one male Wistar rats were divided into 3 groups (n=7): young control, aged sedentary, and aged + exercise. Western blot for α1A- and α1B-ARs as well as pro-(Bax and p53) and anti-apoptotic (Bcl2) proteins was conducted. An 8-week regular moderate-intensity treadmill exercise intervention was carried out in exercise group. RESULTS: In aged rats, α1A-AR expression in the hippocampus was significantly increased, and exercise markedly prevented this event. While α1B-AR expression was no altered with aging, a marked reduction in α1B-AR level was detected in exercise group when compared to aged group. Furthermore, pro-apoptotic protein levels of Bax and p53 were upregulated and anti-apoptotic protein Bcl2 was downregulated in the aging hippocampus, but could be reversed by treadmill exercise. In the present research, exercise-induced reduction in α1A- and α1B-ARs was associated with an obvious downregulation of Bax/Bcl2 ratio in aged rats, suggesting that exercise may inhibit apoptosis through regulating α1-ARs, particularly α1A-AR. CONCLUSIONS: Our study suggests that manipulations attenuating α1-AR activity, including nonselective α1-adrenergic antagonists, may protect against hippocampal neurodegeneration in aging brains.

Smilax china Polyphenols Stimulate Browning via [Formula: see text]3-Adrenergic Receptor/AMP-Activated Protein Kinase [Formula: see text] Signaling Pathway in 3T3-L1 Adipocytes.

The aim of this study is to investigate the molecular mechanism of Smilax china L. polyphenols (SCLPs) in enhancing lipid metabolism and stimulating browning to reduce lipid accumulation in 3T3-L1 adipocytes. SCLP treatment obviously decreased lipid content in a dose-dependent manner (10-40 µg/mL) in adipocytes. SCLP treatment cooperated with noradrenalin to increase lipolysis. SCLPs reduced the gene expressions of C/EBP[Formula: see text] and Ap2 and enhanced the expressions of ACO, CPT, pHSL/HSL, ATGL, and PKA in adipocytes. Furthermore, SCLPs increased mRNA and protein expressions of brown adipocyte-specific factors (UCP-1, PRDM16, PGC-1α, and PPARγ) and mRNA expressions of beige adipocyte-specific markers (CD137, Tbx1, and Tmem26) in 3T3-L1 adipocytes, as well as mitochondrial biogenesis genes (Nrf1 and Tfam). In addition, according to the immunofluorescence staining, the mitochondria number was increased by SCLP. Moreover, ß3-AR or AMPK agonist synergistic SCLPs enhanced the expressions of UCP-1, PRDM16, and PGC-1α. While ß3-AR or AMPK antagonist significantly decreased the expressions of these brown adipocyte-specific factors, SCLP treatment inhibited the effect of antagonist to improve the expression of UCP-1, PRDM16, and PGC-1α. These results indicated that SCLPs may regulate lipid metabolism and stimulate browning via the ß3-AR/AMPKα signaling pathway. Thus, SCLPs likely have potential therapeutic effects on obesity.

Zeaxanthin ameliorates obesity by activating the ß3-adrenergic receptor to stimulate inguinal fat thermogenesis and modulating the gut microbiota.

The stimulation of fat thermogenesis and modulation of the gut microbiota are promising therapeutic strategies against obesity. Zeaxanthin (ZEA), a carotenoid plant pigment, has been shown to prevent various diseases; however, the therapeutic mechanism for obesity remains unclear. Herein, whether ZEA improves obesity by activating the ß3-adrenergic receptor (ß3-AR) to stimulate white adipose tissue (WAT) thermogenesis and modulating the gut microbiota was investigated. C57BL6/N mice were fed a high-fat diet (HFD) supplemented with ZEA for 22 weeks. ZEA treatment reduced body weight, fat weight, adipocyte hypertrophy, liver weight, and lipid deposition, and improved dyslipidaemia, serum GPT, GOT, leptin, and irisin levels, glucose intolerance, and insulin resistance in HFD-fed mice. Mechanistically, ZEA treatment induced the expression of ß3-AR and thermogenic factors, such as PRDM16, PGC-1α, and UCP1, in inguinal WAT (iWAT) and brown adipose tissue. ZEA treatment stimulated iWAT thermogenesis through the synergistic cooperation of key organelles, which manifested as an increased expression of lipid droplet degradation factors (ATGL, CGI-58 and pHSL), mitochondrial biogenesis factors (Sirt1, Nrf2, Tfam, Nampt and Cyt-C), peroxisomal biogenesis factors (Pex16, Pex19 and Pmp70), and ß-oxidation factors (Cpt1, Cpt2, Acadm and Acox1). The thermogenic effect of ZEA was abolished by ß3-AR antagonist (SR59230A) treatment. Additionally, dietary supplementation with ZEA reversed gut microbiota dysbiosis by regulating the abundance of Firmicutes, Clostridia, Proteobacteria, and Desulfovibrio, which were associated with the thermogenesis- and obesity-associated indices by Spearman's correlation analysis. Functional analysis of the gut microbiota indicated that ZEA treatment significantly enriched the lipid metabolism pathways. These results demonstrate that ZEA is a promising multi-target functional food for the treatment of obesity by activating ß3-AR to stimulate iWAT thermogenesis, and modulating the gut microbiota.

Berries and Their Polyphenols as a Potential Therapy for Coronary Microvascular Dysfunction: A Mini-Review.

Ischemia with no obstructive coronary artery disease (INOCA) is a common diagnosis with a higher prevalence in women compared to men. Despite the absence of obstructive coronary artery disease and no structural heart disease, INOCA is associated with major adverse cardiovascular outcomes as well a significant contributor to angina and related disability. A major feature of INOCA is coronary microvascular dysfunction (CMD), which can be detected by non-invasive imaging and invasive coronary physiology assessments in humans. CMD is associated with epicardial endothelial-dependent and -independent dysfunction, diffuse atherosclerosis, and left-ventricular hypertrophy, all of which lead to insufficient blood flow to the myocardium. Inflammatory and oxidative stress signaling, upregulation of the renin-angiotensin-aldosterone system and adrenergic receptor signaling are major drivers of CMD. Treatment of CMD centers around addressing cardiovascular risk factors; however, there are limited treatment options for those who do not respond to traditional anti-anginal therapies. In this review, we highlight the ability of berry-derived polyphenols to modulate those pathways. The evidence supports the need for future clinical trials to investigate the effectiveness of berries and their polyphenols in the treatment of CMD in INOCA patients.

Relationship between cardiotonic activity of Fuzi (Radix Aconiti Lateralis Preparata) and its fingerprint determined by liquid chromatography-mass spectrometry.

OBJECTIVE: To investigate the relationship between the cardiotonic activity of Fuzi (Radix Aconiti Lateralis Preparata, RALP) and its fingerprint determined by liquid chromatography-mass spectrometry (LC-MS). METHODS: First, the fingerprints of six processed products of RALP were established by high performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS) followed by analysis of the principal component of the relative peak area of its common peaks. Next, the scores of the first five principal components were used as input for an artificial neural network (ANN). Additionally, the therapeutic effect of RALP was assessed by measuring the hemodynamic indexes of heart failure model rats. Subsequently, fluorescence semi-quantitative polymerase chain reaction and an enzyme-linked immunosorbent assay kit were used to determine the effects of RALP-processed products on the serum levels of noradrenaline (NA), angiotensin-Ⅰ (Ang-Ⅰ), and the expression of ß-norepinephrine receptor mRNA (ß-NRm) in the rat cardiac tissues. P < 0.05 was used as the output of the ANN. Finally, a network was constructed to display the relationship between the LC-MS fingerprints and the cardiotonic activity of the RALP-processed products. RESULTS: Several types of RALPs can improve diastolic function, systolic function and heart rate. On the basis of the findings from the principal component analysis (PCA) of 16 common peaks of fingerprints of six RALP-processed products, it was revealed that the first five principal components may include 100% of the information of the original data. As observed from the multilayer perceptron neural network analysis, principal component 4 presented with the strongest effects on serum levels of NA and Ang-Ⅰ in rats, while principal component 1 exerted the greatest effect on ß-NRm expression in cardiac tissue. CONCLUSION: The key findings obtained from this study indicated that the network constructed by the PCA-ANN may predict pharmacodynamic effects of the main ingredients of Traditional Chinese Medicine (TCM). This method may serve as a new approach to identify the relationship between LC-MS fingerprints and the pharmacodynamic effects of TCM ingredients.

Nicotinamide Riboside Enhances In Vitro Beta-adrenergic Brown Adipose Tissue Activity in Humans.

CONTEXT: Elevating nicotinamide adenine dinucleotide (NAD+) levels systemically improves metabolic health, which can be accomplished via nicotinamide riboside (NR). Previously, it was demonstrated that NR supplementation in high-fat-diet (HFD)-fed mice decreased weight gain, normalized glucose metabolism, and enhanced cold tolerance. OBJECTIVE: Because brown adipose tissue (BAT) is a major source of thermogenesis, we hypothesize that NR stimulates BAT in mice and humans. DESIGN AND INTERVENTION: HFD-fed C56BL/6J mice were supplemented with 400 mg/kg/day NR for 4 weeks and subsequently exposed to cold. In vitro primary adipocytes derived from human BAT biopsies were pretreated with 50 µM or 500 µM NR before measuring mitochondrial uncoupling. Human volunteers (45-65 years; body mass index, 27-35 kg/m2) were supplemented with 1000 mg/day NR for 6 weeks to determine whether BAT activity increased, as measured by [18F]FDG uptake via positron emission tomography-computed tomography (randomized, double blinded, placebo-controlled, crossover study with NR supplementation). RESULTS: NR supplementation in HFD-fed mice decreased adipocyte cell size in BAT. Cold exposure further decreased adipocyte cell size on top of that achieved by NR alone independent of ex vivo lipolysis. In adipocytes derived from human BAT, NR enhanced in vitro norepinephrine-stimulated mitochondrial uncoupling. However, NR supplementation in human volunteers did not alter BAT activity or cold-induced thermogenesis. CONCLUSIONS: NR stimulates in vitro human BAT but not in vivo BAT in humans. Our research demonstrates the need for further translational research to better understand the differences in NAD+ metabolism in mouse and human.

Grain-sized moxibustion promotes NK cell antitumour immunity by inhibiting adrenergic signalling in non-small cell lung cancer.

Lung cancer is the leading cause of cancer-related death worldwide, and non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer diagnoses. As an ancient therapy, moxibustion has been used to treat cancer-related symptoms in clinical practice. However, its antitumour effect on NSCLC remains largely unexplored. In the present study, a Lewis lung cancer (LLC) xenograft tumour model was established, and grain-sized moxibustion (gMoxi) was performed at the acupoint of Zusanli (ST36). Flow cytometry and RNA sequencing (RNA-Seq) were used to access the immune cell phenotype, cytotoxicity and gene expression. PK136, propranolol and epinephrine were used for natural killer (NK) cell depletion, ß-adrenoceptor blockade and activation, respectively. Results showed that gMoxi significantly inhibited LLC tumour growth. Moreover, gMoxi significantly increased the proportion, infiltration and activation of NK cells, whereas it did not affect CD4+ and CD8+ T cells. NK cell depletion reversed gMoxi-mediated tumour regression. LLC tumour RNA-Seq indicated that these effects might be related to the inhibition of adrenergic signalling. Surely, ß-blocker propranolol clearly inhibited LLC tumour growth and promoted NK cells, and gMoxi no longer increased tumour regression and promoted NK cells after propranolol treatment. Epinephrine could inhibit NK cell activity, and gMoxi significantly inhibited tumour growth and promoted NK cells after epinephrine treatment. These results demonstrated that gMoxi could promote NK cell antitumour immunity by inhibiting adrenergic signalling, suggesting that gMoxi could be used as a promising therapeutic regimen for the treatment of NSCLC, and it had a great potential in NK cell-based cancer immunotherapy.

Evaluating kratom alkaloids using PHASE.

Kratom is a botanical substance that is marketed and promoted in the US for pharmaceutical opioid indications despite having no US Food and Drug Administration approved uses. Kratom contains over forty alkaloids including two partial agonists at the mu opioid receptor, mitragynine and 7-hydroxymitragynine, that have been subjected to the FDA's scientific and medical evaluation. However, pharmacological and toxicological data for the remaining alkaloids are limited. Therefore, we applied the Public Health Assessment via Structural Evaluation (PHASE) protocol to generate in silico binding profiles for 25 kratom alkaloids to facilitate the risk evaluation of kratom. PHASE demonstrates that kratom alkaloids share structural features with controlled opioids, indicates that several alkaloids bind to the opioid, adrenergic, and serotonin receptors, and suggests that mitragynine and 7-hydroxymitragynine are the strongest binders at the mu opioid receptor. Subsequently, the in silico binding profiles of a subset of the alkaloids were experimentally verified at the opioid, adrenergic, and serotonin receptors using radioligand binding assays. The verified binding profiles demonstrate the ability of PHASE to identify potential safety signals and provide a tool for prioritizing experimental evaluation of high-risk compounds.

Antiulcer and Antioxidant Activity of a Lectin from Mucuna pruriens Seeds on Ethanol- induced Gastropathy: Involvement of Alpha-2 Adrenoceptors and Prostaglandins.

ETHNOPHARMACOLOGICAL RELEVANCE: Mucuna pruriens (Mp) belongs to Leguminosae family, it is native of tropical regions and used to treat several maladies such as urinary, neurological, and menstruation disorders, constipation, edema, fever, tuberculosis, ulcers, diabetes, arthritis, dysentery, and cardiovascular diseases. Mp seeds are rich in bioactive compounds, for instance, lectins, a heterogeneous group of proteins and glycoproteins with a potential role as therapeutic tools for several conditions, including gastric disorders. This study investigated the acute toxicity, gastroprotective, and antioxidant activities of a lectin from Mucuna pruriens seeds (MpLec) on ethanol-induced gastropathy model in mice. MATERIAL AND METHODS: Mice received MpLec (5 or 10 mg/kg; i.v.) and were observed for acute toxicity signs; in another experimental series, mice were pre-treated with MpLec (0.001; 0.01 or 0.1 mg/kg, i.v.), ranitidine (80 mg/kg, p.o.), or saline (0.3 mL/30g, i.v.) before ethanol 99.9% (0.2 mL/animal, p.o.), and euthanized 30 min after ethanol challenge. Macroscopic and microscopic gastric aspects, biochemical parameters (tissue hemoglobin levels, iron-induced lipid peroxidation, GSH content, SOD activity, and gastric mucosal PGE2) were measured. Additionally, pharmacological tools (yohimbine, indomethacin, naloxone, L-NAME) were opportunely used to clarify MpLec gastroprotective mechanisms of action. RESULTS: No toxicity signs nor death were observed at acute toxicity tests. MpLec reduced ethanol-induced gastric damage, edema, and hemorrhagic patches formation, as well as decreased lipid peroxidation, SOD activity, and increased GSH content. Yohimbine and indomethacin prevented MpLec effects, suggesting the involvement of alpha-2 adrenoceptors and prostaglandins in the MpLec-mediated effects. CONCLUSION: MpLec does not present toxicity signs and shows gastroprotective and antioxidant activities via alpha-2 adrenoceptors and prostaglandins in the ethanol-induced gastropathy model.

Effect of N-methyltyramine on the regulation of adrenergic receptors via enzymatic epinephrine synthesis for the treatment of gastrointestinal disorders.

BACKGROUND: Citri Reticulatae Pericarpium (CRP), Aurantii Fructus Immaturus (AFI) and Aurantii Fructus (AF) are all important Citrus species used in traditional Chinese medicines (TCMs) for the treatment of gastrointestinal disorders. Although they have been used since ancient times and are still in use today, the mechanistic basis for their regulation of adrenergic receptors (ARs) is still not clear. PURPOSE: In this study, we aimed to determine the active components and mechanisms of action of CRP, AFI and AF in treating gastrointestinal disorders related to ARs. METHODS: First, the phenethylamine alkaloid components of CRP, AFI and AF were identified and compared across 30 samples of three Citrus species by UPLC-Q/TOF-MS in combination with content difference analysis. Second, the effect of the main active alkaloid component on AR-based gastrointestinal disorders was investigated by an in vivo small intestinal propulsive test and an in vitro relaxing small intestinal smooth muscle activity test. The mechanism of AR regulation of the active alkaloid was further studied by evaluating its effect on relaxing small intestinal smooth muscle in the presence of an inhibitor. Lastly, the enzymes, which played an important role in epinephrine synthesis and AR regulation, were detected by immunohistochemistry. RESULTS: Three phenethylamine AR regulators (N-methyltyramine, synephrine and hordenine) in CRP, AFI and AF were characterized. It was found that N-methyltyramine could relax mouse small intestinal smooth muscle and inhibit small intestinal propulsion. The effect of N-methyltyramine on relaxing small intestinal smooth muscle could be inhibited by a-methyl-l-tyrosine. The enzymes related epinephrine synthesis and AR function were found in the mouse small intestine. The biotransformation process that converts N-methyltyramine to epinephrine was determined. CONCLUSION: The treatment of gastrointestinal disorders of CRP, AFI and AF is associated with their alkaloid component N-methyltyramine via the regulation of ARs, and the mechanism is considered to be the biotransformation of N-methyltyramine to epinephrine by serial synthase, which takes place at the nerves cells in small intestine.

Discovery of ß2- adrenoceptor agonists in Curcuma zedoaria Rosc using label-free cell phenotypic assay combined with two-dimensional liquid chromatography.

Traditional Chinese Medicines (TCMs) have been widely used in clinical practice, and provided a rich source for discovering new drug leads. However, efficient identification of active molecules responsible for the therapeutic effects of complex TCMs is still highly challenging. Here, we combined label-free cell phenotypic assay with two dimensional liquid chromatography (2DLC) to identify potential ß2-adrenoceptor (ß2-AR) agonists related to anti-asthmatic effect of Curcuma zedoaria Rosc (C.zedoaria), a commonly used TCM. The ethyl acetate extract of C.zedoaria was first fractionated into 26 fractions. Label-free cell phenotypic profiling was then used to locate the active sites. Orthogonal second-dimensional (D2) separation was performed on two fractions displaying agonistic effect at the ß2-AR, combined with screening of the D2 fractions to track the activity. Finally, this approach led to the isolation of three known diarylheptanoids, among which diarylheptanoid b exhibited the most potent agonistic activity with an EC50 value of 5.93 µM. This result was further demonstrated through the chemical synthesis of diarylheptanoid b. It is the first time to discover that diarylheptanoids could activate the ß2-AR, which may be responsible for the anti-asthmatic effect of C.zedoaria observed traditionally and in clinical application. This study also demonstrates the potential of this integrated strategy for identifying active ingredients and determining the basis of therapeutic materials in complex TCMs.

Maytenus erythroxylon Reissek (Celastraceae) ethanol extract presents antidiarrheal activity via antimotility and antisecretory mechanisms.

AIM: To investigate the acute toxicity, phytochemical profile, antidiarrheal activity and mechanisms of action of Maytenus erythroxylon (M. erythroxylon) ethanol extract. METHODS: A castor oil-induced diarrhea model was used to evaluate antidiarrheal activity. Intestinal transit and gastric emptying protocols were used to evaluate a possible antimotility effect. KATP channels, nitric oxide, presynaptic α2-adrenergic and tissue adrenergic receptors were investigated to uncover antimotility mechanisms of action and castor oil-induced enteropooling to elucidate antisecretory mechanisms. RESULTS: All tested doses of the extract (62.5, 125, 250 and 500 mg/kg) possessed antidiarrheal activity, with a significant decrease of the evacuation index. This activity is possibly related to a reduced gastric emptying (125, 250 and 500 mg/kg) and to a decreased percentage of intestinal transit for all tested doses. That last effect seems to be modulated by nitric oxide, KATP channels and tissue adrenergic receptors. Besides, the extract also presented antisecretory effect due to a decrease of intestinal fluid accumulation. CONCLUSION: The antidiarrheal effect of M. erythroxylon found in this study involves antimotility and antisecretory mechanisms that may be attributed to the chemical compounds found in this species: saponins, flavonoids, tannins, triterpenes and steroids.

Effects of liver depression and psychological stress on human uterine leiomyoma cells by an AR-cAMP-PKA signal transduction pathway.

OBJECTIVE: Based on the emotional theory of Traditional Chinese Medicine, and combined with the modern medicine theory of psychological stress, a research model of human uterine leiomyoma cells (ULM) was cultured in vitro to determine the effectiveness of adrenergic receptor (AR) agonists in human ULM cell growth. In addition, we studied the functional influence of "liver depression and psychological stress theory" on fibroid formation by intervening in the AR-cAMP-PKA signaling pathway. The intention was to establish a new method to prevent and cure fibroids through "liver depression and psychological stress theory" and provide an experimental basis for the Traditional Chinese Medicine emotional theory. MATERIALS AND METHODS: Primary human ULM cells were enriched by collagenase digestion. Immunohistochemistry and hematoxylin and eosin (HE) staining were used for cytological identification. Using this model, we studied intervention using specific AR agonists on ULM cells to observe the influence of "liver depression and psychological stress theory" on estrogen receptor (ER), progesterone receptor (PR), vascular endothelial growth factor (VEGF) and fibroblast growth factors (FGF). RESULTS: Norepinephrine (NE) and epinephrine (E) are adrenergic receptor agonists. They promoted ULM cell proliferation and increased the levels of ER, PR, VEGF and FGF. In contrast, isoproterenol (ISO) inhibited ULM cell proliferation and decreased the levels of ER, PR, VEGF and FGF. The protein expression of cAMP and PKA in ULM cells was reduced and the levels of ER, PR, VEGF and FGF were increased when co-treatment with the α-AR blocker (phentolamine). The ß-AR blocker (metoprolol) displayed an opposite effect. CONCLUSIONS: AR agonists modulated ER, PR, VEGF and FGF levels in ULM cells in an AR-cAMP-PKA-dependent signaling pathways to influence fibroid occurrence and development.

Morphological correlates of sex differences in acoustic startle response and prepulse inhibition through projections from locus coeruleus to cochlear root neurons.

The noradrenergic locus coeruleus (LC) plays an important role in the promotion and maintenance of arousal and alertness. Our group recently described coerulean projections to cochlear root neurons (CRNs), the first relay of the primary acoustic startle reflex (ASR) circuit. However, the role of the LC in the ASR and its modulation, prepulse inhibition (PPI), is not clear. In this study, we damaged LC neurons and fibers using a highly selective neurotoxin, DSP-4, and then assessed ASR and PPI in male and female rats. Our results showed that ASR amplitude was higher in males at 14 days after DSP-4 injection when compared to pre-administration values and those in the male control group. Such modifications in ASR amplitude did not occur in DSP-4-injected females, which exhibited ASR amplitude within the range of control values. PPI differences between males and females seen in controls were not observed in DSP-4-injected rats for any interstimulus interval tested. DSP-4 injection did not affect ASR and PPI latencies in either the male or the female groups, showing values that were consistent with the sex-related variability observed in control rats. Furthermore, we studied the noradrenergic receptor system in the cochlear nerve root using gene expression analysis. When compared to controls, DSP-4-injected males showed higher levels of expression in all adrenoceptor subtypes; however, DSP-4-injected females showed varied effects depending on the receptor type, with either up-, downregulations, or maintenance of expression levels. Lastly, we determined noradrenaline levels in CRNs and other LC-targeted areas using HPLC assays, and these results correlated with behavioral and adrenoceptor expression changes post DSP-4 injection. Our study supports the participation of LC in ASR and PPI, and contributes toward a better understanding of sex-related differences observed in somatosensory gating paradigms.

Dexmedetomidine relieves formaldehyde-induced pain in rats through both α2 adrenoceptor and imidazoline receptor.

Dexmedetomidine (DEX) is a highly selective α2 adrenergic receptor agonist. In this study, we investigated the analgesic effect and the underlying mechanisms of DEX on inflammatory visceral pain in rats. Twenty-five male Sprague Dawley (SD) rats were randomly divided into 5 groups, including control, sham, low dose DEX, medium dose DEX and high dose DEX group. Pain was induced with 10% formalin and scored every 15min till 2 h-post the induction. Hematoxylin-eosin (HE) staining was used to evaluate the toxicity of DEX on spinal cord neurons. Acetycholine (Ach) and noradrenaline (NA) levels were determined by using ELISA method. The expressions of natural nitric oxide synthase (nNOS), protein kinase γ (PKCγ) and protease-activated receptor 2 (PAR2) were determined by using western blot. DEX treatment relieved formaldehyde-induced pain in rats in a dose-dependent manner. Furthermore, DEX showed little neuro-toxicity on the spinal cord neurons, even at the highest dosage used in our study. Ach level was significantly increased in Sham group compared with control group. DEX treatment decreased NA levels and increased Ach levels in the incubation medium of spinal cord sections. Western blot analysis showed that the expression of nNOS, PKCγ and PAR2 was significantly decreased in DEX group compared with Sham group, whereas these effects of DEX on nNOS, PKCγ and PAR2 were blocked by both yohimbine and idazoxan, indicating that the analgesic effect of DEX is mediated by both α2 adrenergic receptor and imidazoline receptor. Yohimbine and idazoxan treatment significantly enhanced pain scores compared to DEX group, and which antagonizes the effects DEX. In conclusion, our study demonstrated that DEX could inhibit formaldehyde-induced pain by inhibiting nNOS, PKCγ and PAR2 expression through α2 adrenergic receptor and imidazoline receptor.

The hemostatic effect study of Cirsium setosum on regulating α1-ARs via mediating norepinephrine synthesis by enzyme catalysis.

BACKGROUND: Cirsium setosum (CS) is the aboveground part of Cephalanoplos segetum Kitam. Although it has been used as a hemostatic treatment for thousands of years and is still in use today, the mechanism of CS on regulating ARs is still not clear. PURPOSE: In this study, we aimed to clarify the mechanism of CS on regulating ARs. METHODS: We developed a simple method based on UPLC/Q-TOF MS combined adrenergic receptor dual-luciferase reporter assay systems for the rapid determination of active constituents in CS. The mechanism of tyramine, the main active component for regulating ARs, was further investigated by an in vitro norepinephrine biotransformation test and in vivo vaso activity tests. RESULTS: Two phenethylamine ARs regulators (tyramine and N-methyltyramine) in CS were characterized, and it was found that tyramine could induce vasoconstriction via regulation of α1-ARs by mediating norepinephrine synthesis. CONCLUSION: The hemostatic effect of CS is associated with tyramine and N-methyltyramine, via regulation of α1-ARs, and the mechanism of tyramine is related to mediating norepinephrine synthesis by enzyme catalysis.

Frontline Science: D1 dopaminergic receptor signaling activates the AMPK-bioenergetic pathway in macrophages and alveolar epithelial cells and reduces endotoxin-induced ALI.

Catecholamines, including ß-adrenergic and dopaminergic neurotransmitters, have an essential role in regulating the "fight or flight" reflex and also affects immune cell proinflammatory action. However, little is known about whether catecholamines prevent dysfunction of metabolic pathways associated with inflammatory organ injury, including development of acute lung injury (ALI). We hypothesize that selected catecholamines may reduce metabolic alterations in LPS-stimulated macrophages and in the lungs of mice subjected to endotoxin-induced ALI, a situation characterized by diminished activity of AMP-activated protein kinase (AMPK). We found that activation of the dopamine 1 receptor (D1R) with fenoldopam, but not stimulation of adrenergic receptors with norepinephrine, resulted in a robust activation of AMPK in peritoneal macrophages, human monocytes, or alveolar epithelial cells (AECs). Such AMPK activation was mediated by a phospholipase C (PLC)-dependent mechanism. Unlike norepinephrine, D1R activation also prevented Thr172-AMPK dephosphorylation and kinase inactivation in LPS-treated macrophages. Furthermore, we show that a culture of AECs with either fenoldopam or the AMPK activator metformin effectively diminished IL-1ß-induced release of adverse paracrine signaling, which promotes the macrophage proinflammatory response. In vivo, fenoldopam reduced the severity of LPS-induced ALI, including development of pulmonary edema, lung permeability, and production of inflammatory cytokines TNF-α, MIP-2, or KC and HMGB1. Fenoldopam also prevented AMPK dephosphorylation in the lungs of LPS-treated mice and prevented loss of mitochondrial complexes NDUFB8 (complex I) and ATP synthase (complex V). Collectively, these results suggest that dopamine is coupled to AMPK activation, which provides a substantial anti-inflammatory and bioenergetic advantage and reduces the severity of endotoxin-induced ALI.

[Effect of Manual Acupuncture Intervention on Levels of 5-HTT, 5-HT1AR, NEα2R of Brain Tissues in Rats with Post-stroke Depression].

OBJECTIVE: To observe the effect of manual acupuncture intervention on serotonin transporter (5-HTT), 5-HT 1 A receptor (5-HT1AR), norepinephrine (NE) alpha receptor 2 (NEα2R) protein of brain tissues in rats with post-stroke depression (PSD). METHODS: Forty SD rats were randomly divided into normal group, model group, medication group and acupuncture group, with 10 rats in each. The PSD model was established by the combination of middle cerebral artery occlusion and chronic non- predictable mild stress. Rats of the medication group were treated by gavage of fluoxetine (2 mg/kg, once daily for 21 days), and those of the acupuncture group treated by manual acupuncture stimulation of "Baihui"(GV 20), "Fengfu" (GV 16), bilateral"Shenmen" (HT 7) and "Taichong" (LR 3) for 20 min, once a day for 21 days, with one day's interval between each every 7 days. The animals' neurological deficit was detected by Zea Longa's scaling, and the depression status assessed by sugar solution consumption, and the locomotor activity (crossing and rearing times in 5 min) detected by open-field tests. The contents of 5-HT, NE, acetylcholine (ACh), gamma aminobutyric acid (GABA), and glutamate (Glu) of the cerebral cortex were detected using high performance liquid chromatography (HPLC) and expression levels of 5-HTT, 5-HT1AR, NEα2R proteins of the hippocampus, raphe nuclei and locus coeruleus(LC) tissues detected using western blotting (EB), respectively. RESULTS: After modeling, the neurological deficit score was significantly increased, while the sugar solution consumption and the locomotor activities (crossi-ng and rearing times in 5 min) were notably decreased (P<0.05, P<0.001). Findings of HPLC and WB indicated that after modeling, the cerebrocortical 5-HT and NE contents and 5-HTT protein expression levels in the hippocampus, raphe nucleus and LC were obviously decreased and the cortical ACh, GABA and Glu contents as well as the expression of 5-HT1AR and NEα2R proteins in the hippocampus, raphe nucleus and LC considerably increased in the model group (P<0.01). Following acupuncture intervention and medication, the increased neurological deficit score, cortical ACh, GABA and Glu contents, and the expression le-vels of 5-HT1AR and NEα2R proteins in the hippocampus, rahpe nucleus and LC, as well as the decreased sugar solution consumption, crossing and rearing times of open field tests, cortical 5-HT and NE contents, as well as cortical 5-HT protein expression were all reversed in both acupuncture and medication groups (P<0.05, P<0.01). No significant differences were found between the acupuncture and medication groups in all the above-mentioned indexes (P>0.05). CONCLUSIONS: Acupuncture can improve the locomotor function changes in stroke rats with depression after stroke, which may be associated with its effects in suppressing the up-regulated cortical ACh, GABA, Glu contents, the expression levels of 5-HT1AR and NEα2R proteins in the hippocampus, raphe nucleus and LC, and the down-regulated 5-HT and NE contents in the cerebral cortex and 5-HTT protein expression levels in the hippocampus, raphe nucleus and LC.

Natural Diterpenes from Coffee, Cafestol, and Kahweol Induce Peripheral Antinoceception by Adrenergic System Interaction.

Cafestol and kahweol are diterpenes found only in the non-saponified lipid fraction of coffee. They are released during boiling and retained in the filtration process. Previous studies have shown peripheral antinociception induced by endogenous opioid peptides released by these diterpenes. Considering that the activation of the opioid system leads to a noradrenaline release, the aim of this study was to verify the participation of the noradrenergic system in the peripheral antinociception induced by cafestol and kahweol. Hyperalgesia was induced by an intraplantar injection of prostaglandin E2 (2 µg). Cafestol or kahweol (80 µg/paw) were administered locally into the right hindpaw alone, and after the agents α 2-adrenoceptor antagonist yohimbine (5, 10 and 20 µg/paw), α 2 A-adrenoceptor antagonist BRL 44 408 (40 µg/paw), α 2B-adrenoceptor antagonist imiloxan (40 µg/paw), α 2 C-adrenoceptor antagonist rauwolscine (10, 15 and 20 µg/paw), α 2D-adrenoceptor antagonist RX 821 002 (40 µg/paw), α 1-adrenoceptor antagonist prazosin (0.5, 1 and 2 µg/paw), or ß-adrenoceptor antagonist propranolol (150, 300 and 600 ng/paw), respectively. Noradrenaline reuptake inhibitor reboxetine (30 µg/paw) was administered prior to cafestol or kahweol low dose (40 µg/paw) and guanetidine 3 days prior to the experiment (30 mg/kg, once a day), depleting the noradrenaline storage. Intraplantar injection of cafestol or kahweol (80 µg/paw) induced a peripheral antinociception against hyperalgesia induced by PGE2. This effect was reversed by intraplantar injections of yohimbine, rauwolscine, prazosin and propranolol. Reboxetine injection intensified the antinociceptive effect of cafestol or kahweol low-dose, and guanethidine reversed almost 70 % of the cafestol or kahweol-induced peripheral antinociception. This study gives evidence that the noradrenergic system participates in cafestol and kahweol-induced peripheral antinociception with the release of endogenous noradrenaline.

Predator Stress-Induced CRF Release Causes Enduring Sensitization of Basolateral Amygdala Norepinephrine Systems that Promote PTSD-Like Startle Abnormalities.

The neurobiology of post-traumatic stress disorder (PTSD) remains unclear. Intense stress promotes PTSD, which has been associated with exaggerated startle and deficient sensorimotor gating. Here, we examined the long-term sequelae of a rodent model of traumatic stress (repeated predator exposure) on amygdala systems that modulate startle and prepulse inhibition (PPI), an operational measure of sensorimotor gating. We show in rodents that repeated psychogenic stress (predator) induces long-lasting sensitization of basolateral amygdala (BLA) noradrenergic (NE) receptors (α1) via a corticotropin-releasing factor receptor 1 (CRF-R1)-dependent mechanism, and that these CRF1 and NE α1 receptors are highly colocalized on presumptive excitatory output projection neurons of the BLA. A profile identical to that seen with predator exposure was produced in nonstressed rats by intra-BLA infusions of CRF (200 ng/0.5 µl), but not by repeated NE infusions (20 µg/0.5 µl). Infusions into the adjacent central nucleus of amygdala had no effect. Importantly, the predator stress- or CRF-induced sensitization of BLA manifested as heightened startle and PPI deficits in response to subsequent subthreshold NE system challenges (with intra-BLA infusions of 0.3 µg/0.5 µl NE), up to 1 month after stress. This profile of effects closely resembles aspects of PTSD. Hence, we reveal a discrete neural pathway mediating the enhancement of NE system function seen in PTSD, and we offer a model for characterizing potential new treatments that may work by modulating this BLA circuitry. SIGNIFICANCE STATEMENT: The present findings reveal a novel and discrete neural substrate that could underlie certain core deficits (startle and prepulse inhibition) that are observed in post-traumatic stress disorder (PTSD). It is shown here that repeated exposure to a rodent model of traumatic stress (predator exposure) produces a long-lasting sensitization of basolateral amygdala noradrenergic substrates [via a corticotropin-releasing factor (CRF)-dependent mechanism] that regulate startle, which is exaggerated in PTSD. Moreover, it is demonstrated that the sensitized noradrenergic receptors colocalize with CRF1 receptors on output projection neurons of the basolateral amygdala. Hence, this stress-induced sensitization of noradrenergic receptors on basolateral nucleus efferents has wide-ranging implications for the numerous deleterious sequelae of trauma exposure that are seen in multiple psychiatric illnesses, including PTSD.