Botanical Briefs: Neem Oil (Azadirachta indica).

Azadirachta indica, commonly known as neem, has many uses as a natural remedy. We review and discuss the pharmacologic, biologic, and medicinal properties of neem in disease management. We also report a rare clinical case of a 77-year-old man who presented with a hypopigmented rash on the lower back, bilateral flanks, and buttocks after 6 months of repeated application of neem oil to treat persistent arthritis and lower back pain.

Beneficial effects of Lactobacillus rhamnosus hsryfm 1301 fermented milk on rats with nonalcoholic fatty liver disease.

A growing stream of research suggests that probiotic fermented milk has a good effect on nonalcoholic fatty liver disease. This work aimed to study the beneficial effects of Lactobacillus rhamnosus hsryfm 1301 fermented milk (fermented milk) on rats with nonalcoholic fatty liver disease induced by a high-fat diet. The results showed that the body weight and the serum levels of total cholesterol, total glyceride, low-density lipoprotein, alanine transaminase, aspartate aminotransferase, free fatty acid, and reactive oxygen species were significantly increased in rats fed a high-fat diet (M) for 8 wk, whereas high-density lipoprotein cholesterol and superoxide dismutase were significantly decreased. However, the body weight and the serum levels of total cholesterol, total glyceride, alanine transaminase, aspartate aminotransferase, free fatty acid, reactive oxygen species, interleukin-8, tumor necrosis factor-α, and interleukin-6 were significantly decreased with fermented milk (T) for 8 wk, and the number of fat vacuoles in hepatocytes was lower than that in the M group. There were significant differences in 19 metabolites in serum between the M group and the C group (administration of nonfermented milk) and in 17 metabolites between the T group and the M group. The contents of 7 different metabolites, glycine, glycerophosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, thioetheramide-PC, d-aspartic acid, oleic acid, and l-glutamate, were significantly increased in the M group rat serum, and l-palmitoyl carnitine, N6-methyl-l-lysine, thymine, and 2-oxadipic acid were significantly decreased. In the T group rat serum, the contents of 8 different metabolites-1-O-(cis-9-octadecenyl)-2-O-acetyl-sn-glycero-3-phosphocholine, acetylcarnitine, glycine, glycerophosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, d-aspartic acid, oleic acid, and l-glutamate were significantly decreased, whereas creatinine and thymine were significantly increased. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that 50 metabolic pathways were enriched in the M/C group and T/M group rat serum, of which 12 metabolic pathways were significantly different, mainly distributed in lipid metabolism, amino acid, and endocrine system metabolic pathways. Fermented milk ameliorated inflammation, oxygenation, and hepatocyte injury by regulating lipid metabolism, amino acid metabolic pathways, and related metabolites in the serum of rats with nonalcoholic fatty liver disease.

Performance and gut health status of broilers fed diets supplemented with two graded levels of a monoglyceride blend.

The present study was undertaken to evaluate the efficacy of 2 graded levels (0.03 and 0.05% of diet) of a monoglyceride blend containing butyric, caprylic, and capric acids in broilers' diet for optimizing gut structure and animal growth performance. For this purpose, a total of 210, one-day-old male Ross 308 broiler chicks were randomly allocated to 3 experimental treatments using 7 replicates each and 10 birds/replicate. The treatment groups involved supplementation of blend of short and medium chain fatty acids at the level of 0, 0.03, and 0.05% of the diet for 42 d. The incorporation of mixes of monoglycerides into broilers' diet linearly improved BWG between d 0 and 21 (P = 0.034). At the end of trial, however, no significant changes were observed in performance indexes (BWG, FI, FCR). Jejunal morphometric parameters (villus height, crypt depth, and their ratio) remained unaltered with the monoglyceride supplementation on d 21. The results further showed that monoglycerides supplementation increased the goblet cell counts along the jejunal villi (P = 0.034) and crypt regions (P = 0.022), as well as it effectively modulated the mRNA abundances of tight junction protein (ZO-1, P = 0.033) and nutrient transporters (SGLT, PePT1; P = 0.005, 0.023, respectively) in the jejunum. Moreover, the downregulation in mRNA abundance of TNFα (P = 0.030) was observed with the monoglyceride supplementation. The SCFAs analysis of cecal contents showed no notable differences with monoglyceride blend supplementation when compared to the unsupplemented group. Collectively, high goblet cell numbers in the jejunum along with downregulation of the mRNA abundances of pro-inflammatory cytokines, upregulation of tight junction proteins, and nutrient transporters showed favorable responses of low doses of monoglycerides blend in broiler feeding. Further studies should be conducted in different rearing conditions to examine the effectiveness of such low levels of a monoglyceride blend in the modulation of gut structure, its functionality and animal performance.

Butyrate Glycerides Protect against Intestinal Inflammation and Barrier Dysfunction in Mice.

This study investigates the attenuating effects of butyrate glycerides (BG) on intestinal inflammatory responses and barrier dysfunction induced by LPS stimulation. An initial dose-response test was carried out to identify the optimal dose of BG for further testing. The mice were given intragastric administration of BG at different doses followed by lipopolysaccharide (LPS) intraperitoneal injection. The small intestinal morphology and cytokine mRNA expression were measured. With 1.5 g/kg BW BG administration, it was possible to alleviate the injury of duodenal morphology, attenuate ileum villus height reduction and promote IL-10 mRNA expression. Therefore, the optimal dosage of 1.5 g/kg BW BG was selected for the main experiment. The ultrastructure image of jejunum and ileum epithelial cells, mRNA expression, the level of cytokine and immunofluorescence in the ileum were analyzed. The results showed that BG maintain the ileac brush border, tight junction structures and protein expression. BG attenuated the increased inflammatory cytokines, TLR4 and JNK mRNA expression. Taken together, 1.5 g/kg BW BG administration maintained intestinal barrier function and reduced intestinal and body inflammation responses induced by LPS in mice. The mechanism by which BG alleviated intestinal inflammatory response and maintained intestinal barrier function may be related to the JNK signaling pathway.

Oudemansiella raphanipies Polysaccharides Improve Lipid Metabolism Disorders in Murine High-Fat Diet-Induced Non-Alcoholic Fatty Liver Disease.

Oudemansiella raphanipies, also called "Edible Queen," is a mushroom that possesses antioxidant, anti-inflammatory, anti-bacterial, anti-tumor and immunity-enhancing properties. The present study aimed to assess the effect of O. raphanipies-derived polysaccharide (ORPS) on the progression of nonalcoholic fatty liver disease (NAFLD) in mice. We studied the structure of ORPS-1 by high-performance gel permeation chromatography (HPGPC), ion chromatography-mass spectrometry (GC-MS), and Fourier transform-infrared spectroscopy (FT-IR). ORPS-1 mainly comprised galactose, fucose, glucose, mannose, and xylose, following an 18:6:6:4:1 molar ratio. In addition, the therapeutic effect as well as a potential mechanism of ORPS-1 in the treatment of high-fat diet (HFD)-induced NAFLD were investigated. The results showed that ORPS-1 improved liver function, ameliorated liver steatosis, and reduced lipid droplet accumulation in HFD mice. A metabolomics approach with GC-MS was utilized to evaluate liver improvement by ORPS-1 treatment. Principal component analysis showed that liver metabolic profiling was significantly altered by HFD feeding or treatment with an intermediate dose of ORPS-1 in mice compared with that of control mice. By investigating the metabolic pathways with identified biomarkers, various pathways such as steroid biosynthesis, valine, leucine, and isoleucine biosynthesis, glycerol phospholipid metabolism, glyceride metabolism, and arginine and proline metabolism in HFD mice were observed to be significantly influenced by ORPS-1 treatment. The results indicate ORPS-1 metabolic effects on liver tissues, provide methods for assessing the molecular impact of ORPS-1 on NAFLD, and suggest the potential mechanism underlying its health benefits.

Paraquat exposure induces Parkinsonism by altering lipid profile and evoking neuroinflammation in the midbrain.

Paraquat (PQ) is the most widely used herbicide in the world and a well-known potent neurotoxin for humans. PQ exposure has been linked to increase the risk of Parkinson's disease (PD). However, the mechanism underlying its neurotoxic effects in PD pathogenesis is unclear. In our present study, C57BL/6J mice treated with PQ manifested severe motor deficits indicated by the significant reductions in suspension score, latency to fall from rotarod, and grip strength at 8 weeks after PQ exposure. Pathological hallmarks of Parkinsonism in the midbrain such as dopaminergic neuron loss, increased α-synuclein protein, and dysregulated PD-related genes were observed. Non-targeted lipidome analysis demonstrated that PQ exposure alters lipid profile and abundance, increases pro-inflammatory lipids.27 significantly altered subclasses of lipids belonged to 6 different lipid categories. Glycerophospholipids, sphingolipids, and glycerides were the most abundant lipids. Abundance of pro-inflammatory lipids such as Cer, LPC, LPS, and LPI was significantly increased in the midbrain. mRNA expressions of genes regulating ceramide biosynthesis in the midbrain were markedly up-regulated. Moreover, PQ exposure increased serum pro-inflammatory cytokines and provoked neuroinflammation in the midbrain. Pro-inflammatory lipids and cytokines in the midbrain were positively correlated with motor deficits. PQ poisoning in humans significantly also elevated serum pro-inflammatory cytokines and induced an intense systemic inflammation. In summary, we presented initial investigations of PQ induced molecular events related to the PD pathogenesis, capturing aspects of disturbed lipid metabolism, neuroinflammation, impairment of dopaminergic neurons in the midbrain, and an intense systemic inflammation. These neurotoxic effects of PQ exposure may mechanistically contribute to the pathogenesis of PQ induced Parkinsonism. Results of this study also strongly support the hypothesis that ever-increasing prevalence of Parkinson's disease is etiologically linked to the health risk of exposure to neurotoxic environmental pollutants.

Co-culturing fungus Penicillium citrinum and strain Citrobacter freundii improved nitrate removal and carbon utilization by promoting glyceride metabolism.

Exploring the interaction between denitrifying microbial species is significant for improving denitrification performance. In this study, the effects of co-culturing fungus Penicillium citrinum and strain Citrobacter freundii on denitrification were investigated. Results showed that the maximum nitrate removal and carbon utilization in co-culture were 68.0 and 14.1 mg·L-1·d-1, respectively. The total adenosine triphosphatase activity was increased to 9.87 U‧mg-1 protein in co-culture, and nicotinamide adenine dinucleotide production was 1.7-2.3 times that of monoculture, attributing to increased carbon utilization. Further metabolomics and membrane permeability assay revealed that co-culture increased the metabolism of glycerides, thereby enhancing the membrane permeability of strain Citrobacter freundii and promoting the transmembrane transport of nitrate and glucose, which boosted nitrate reductase activity and nicotinamide adenine dinucleotide production in turn. In summary, co-culturing promoted carbon utilization and enhanced substrate removal efficiency through the metabolism of glycerides, which provided a strategy to enhance denitrification performance in wastewater treatment.

In vitro antibacterial activity of nimbolide against Helicobacter pylori.

ETHNOPHARMACOLOGICAL RELEVANCE: Nimbolide is one of hundreds of phytochemicals that have been identified within the neem tree (Azadirachta indica A. Juss). As an evergreen tree native to the Indian subcontinent, components of the neem tree have been used for millennia in traditional medicine to treat dental, gastrointestinal, urinary tract, and blood-related ailments, ulcers, headaches, heartburn, and diabetes. In modern times, natural oils and extracts from the neem tree have been found to have activities against a variety of microorganisms, including human pathogens. AIM OF THE STUDY: Helicobacter pylori, a prevalent gastric pathogen, shows increasing levels of antibiotic resistance. Thus, there is an increasing demand for novel therapeutics to treat chronic infections. The in vitro activity of neem oil extract against H. pylori was previously characterized and found to be bactericidal. Given the numerous phytochemicals found in neem oil extract, the present study was designed to define and characterize specific compounds showing bactericidal activity against H. pylori. MATERIALS AND METHODS: Azadirachtin, gedunin, and nimbolide, which are all common in neem extracts, were tested for antimicrobial activity; the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for nine strains of H. pylori. The specific properties of nimbolide were further characterized against H. pylori strain G27. Bactericidal kinetics, reversibility, effectiveness at low pH, and activity under bacteriostatic conditions were examined. The hemolytic activity of nimbolide was also measured. Finally, neem oil extract and nimbolide effectiveness against H. pylori biofilms were examined in comparison to common antibiotics used to treat H. pylori infection. RESULTS: Nimbolide, but not azadirachtin or gedunin, was effective against H. pylori; MICs and MBCs against the nine tested strains ranged between 1.25-5 µg/mL and 2.5-10 µg/mL, respectively. Additionally, neem oil extract and nimbolide were both effective against H. pylori biofilms. Nimbolide exhibited no significant hemolytic activity at biologically relevant concentrations. The bactericidal activity of nimbolide was time- and dose-dependent, independent of active H. pylori growth, and synergistic with low pH. Furthermore, nimbolide-mediated H. pylori cell death was irreversible after exposure to high nimbolide concentrations (80 µg/mL, after 2 h of exposure time and 40 µg/mL after 8 h of exposure). CONCLUSIONS: Nimbolide has significant bactericidal activity against H. pylori, killing both free living bacterial cells as well as cells within a biofilm. Furthermore, the lack of hemolytic activity, synergistic activity at low pH and bactericidal properties even against bacteria in a state of growth arrest are all ideal pharmacological and biologically relevant properties for a potential new agent. This study underscores the potential of neem oil extract or nimbolide to be used as a future treatment for H. pylori infection.

ABHD6 Controls Amphetamine-Stimulated Hyperlocomotion: Involvement of CB1 Receptors.

Introduction: Activation of cannabinoid 1 receptors (CB1Rs) by endocannabinoids (eCBs) is controlled by both eCB production and eCB inactivation. Accordingly, inhibition of eCB hydrolyzing enzymes, monoacylglycerol lipase (MAGL) and α/ß-hydrolase domain containing 6 (ABHD6), enhances eCB accumulation and CB1R activation. It is known that inhibition of MAGL regulates select CB1R-dependent behaviors in mice, including locomotor behaviors and their modulation by psychostimulants, but much less is known about the effect of inhibiting ABHD6 activity on such behaviors. Methods: We report a new mouse line that carries a genetic deletion of Abhd6 and evaluated its effect on spontaneous locomotion measured in a home cage monitoring system, motor coordination measured on a Rotarod, and amphetamine-stimulated hyperlocomotion and amphetamine sensitization (AS) measured in an open-field chamber. Results: ABHD6 knockout (KO) mice reached adulthood without exhibiting overt behavioral impairment, and we measured only mild reduction in spontaneous locomotion and motor coordination in adult ABHD6 KO mice compared to wild-type (WT) mice. Significantly, amphetamine-stimulated hyperlocomotion was enhanced by twofold in ABHD6 KO mice compared to WT mice and yet ABHD6 KO mice expressed AS to the same extent as WT mice. A twofold increase in amphetamine-stimulated hyperlocomotion was also measured in ABHD6 heterozygote mice and in WT mice treated with the ABHD6 inhibitor KT-182. It is known that amphetamine-stimulated hyperlocomotion is not affected by the CB1R antagonist, SR141617, and we discovered that the enhanced amphetamine-stimulated hyperlocomotion resulting from ABHD6 inhibition is blocked by SR141617. Conclusions: Our study suggests that ABHD6 controls amphetamine-stimulated hyperlocomotion by a mechanistic switch to a CB1R-dependent mechanism.

Virucidal effect of monogalactosyl diacylglyceride from a green microalga, Coccomyxa sp. KJ, against clinical isolates of SARS-CoV-2 as assessed by a plaque assay.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) and is capable of human-to-human transmission and rapid global spread. Thus, the establishment of high-quality viral detection and quantification methods, and the development of anti-SARS-CoV-2 agents are critical. METHODS: Here, we present the rapid detection of infectious SARS-CoV-2 particles using a plaque assay with 0.5% agarose-ME (Medium Electroosmosis) as an overlay medium. RESULTS: The plaques were capable of detecting the virus within 36-40 h post-infection. In addition, we showed that a monogalactosyl diacylglyceride isolated from a microalga (Coccomyxa sp. KJ) could inactivate the clinical isolates of SARS-CoV-2 in a time- and concentration-dependent manner. CONCLUSIONS: These results would allow rapid quantification of the infectious virus titers and help develop more potent virucidal agents against SARS-CoV-2.

Distigmasterol-Modified Acylglycerols as New Structured Lipids-Synthesis, Identification and Cytotoxicity.

Plant sterols, also referred as phytosterols, have been known as bioactive compounds which have cholesterol-lowering properties in human blood. It has been established that a diet rich in plant sterols or their esters alleviates cardiovascular diseases (CVD), and also may inhibit breast, colon and lung carcinogenesis. Phytosterols, in their free and esterified forms, are prone to thermo-oxidative degradation, where time and temperature affect the level of degradation. Looking for new derivatives of phytosterols with high thermo-oxidative stability for application in foods, our idea was to obtain novel structured acylglycerols in which two fatty acid parts are replaced by stigmasterol residues. In this work, asymmetric (1,2- and 2,3-) distigmasterol-modified acylglycerols (dStigMAs) were synthesized by the covalent attachment of stigmasterol residues to sn-1 and sn-2 or sn-2 and sn-3 positions of 3-palmitoyl-sn-glycerol or 1-oleoyl-sn-glycerol, respectively, using a succinate or carbonate linker. The chemical structures of the synthesized compounds were identified by NMR, HR-MS, and IR data. Moreover, the cytotoxicity of the obtained compounds was determined. The dStigMAs possessing a carbonate linker showed potent cytotoxicity to cells isolated from the small intestine and colon epithelium and liver, whereas the opposite results were obtained for compounds containing a succinate linker.

New Pyridone-Based Derivatives as Cannabinoid Receptor Type 2 Agonists.

The activation of the human cannabinoid receptor type II (CB2R) is known to mediate analgesic and anti-inflammatory processes without the central adverse effects related to cannabinoid receptor type I (CB1R). In this work we describe the synthesis and evaluation of a novel series of N-aryl-2-pyridone-3-carboxamide derivatives tested as human cannabinoid receptor type II (CB2R) agonists. Different cycloalkanes linked to the N-aryl pyridone by an amide group displayed CB2R agonist activity as determined by intracellular [cAMP] levels. The most promising compound 8d exhibited a non-toxic profile and similar potency (EC50 = 112 nM) to endogenous agonists Anandamide (AEA) and 2-Arachidonoylglycerol (2-AG) providing new information for the development of small molecules activating CB2R. Molecular docking studies showed a binding pose consistent with two structurally different agonists WIN-55212-2 and AM12033 and suggested structural requirements on the pyridone substituents that can satisfy the orthosteric pocket and induce an agonist response. Our results provide additional evidence to support the 2-pyridone ring as a suitable scaffold for the design of CB2R agonists and represent a starting point for further optimization and development of novel compounds for the treatment of pain and inflammation.

Orphan Nuclear Receptor ERRγ Is a Transcriptional Regulator of CB1 Receptor-Mediated TFR2 Gene Expression in Hepatocytes.

Orphan nuclear receptor estrogen-related receptor γ (ERRγ) is an important transcription factor modulating gene transcription involved in endocrine control of liver metabolism. Transferrin receptor 2 (TFR2), a carrier protein for transferrin, is involved in hepatic iron overload in alcoholic liver disease (ALD). However, TFR2 gene transcriptional regulation in hepatocytes remains largely unknown. In this study, we described a detailed molecular mechanism of hepatic TFR2 gene expression involving ERRγ in response to an endocannabinoid 2-arachidonoylglycerol (2-AG). Treatment with 2-AG and arachidonyl-2'-chloroethylamide, a selective cannabinoid receptor type 1 (CB1) receptor agonist, increased ERRγ and TFR2 expression in hepatocytes. Overexpression of ERRγ was sufficient to induce TFR2 expression in both human and mouse hepatocytes. In addition, ERRγ knockdown significantly decreased 2-AG or alcohol-mediated TFR2 gene expression in cultured hepatocytes and mouse livers. Finally, deletion and mutation analysis of the TFR2 gene promoter demonstrated that ERRγ directly modulated TFR2 gene transcription via binding to an ERR-response element. This was further confirmed by chromatin immunoprecipitation assay. Taken together, these results reveal a previously unrecognized role of ERRγ in the transcriptional regulation of TFR2 gene expression in response to alcohol.

Protective role of endocannabinoid signaling in an animal model of haloperidol-induced tardive dyskinesia.

Tardive dyskinesia (TD) is a side effect associated with the long-term use of certain antipsychotics. Considering the modulatory role of the endocannabinoid system upon dopaminergic neurotransmission, the present study tested the hypothesis that increasing endocannabinoid (anandamide and 2-arachidonoylglycerol) levels attenuates haloperidol-induced TD (vacuous chewing movements, VCMs) in male Wistar rats. The animals received administration of chronic haloperidol (38 mg/kg; 29 days) followed by acute FAAH (URB597, 0.1-0.5 mg/kg) or MAGL (JZL184, 1-10 mg/kg) inhibitors before VCM quantification. The underlying mechanisms were evaluated by pre-treatments with a CB1 receptor antagonist (AM251, 1 mg/kg) or a TRPV1 channel blocker (SB366791, 1 mg/kg). Moreover, CB1 receptor expression was evaluated in the striatum of high-VCM animals. As expected, haloperidol induced VCMs only in a subset of rats. Either FAAH or MAGL inhibition reduced VCMs. These effects were prevented by CB1 receptor antagonism, but not by TRPV1 blockage. Remarkably, CB1 receptor expression was increased high-VCM rats, with a positive correlation between the levels of CB1 expression and the number of VCMs. In conclusion, increasing endocannabinoid levels results in CB1 receptor-mediated protection against haloperidol-induced TD in rats. The increased CB1 receptor expression after chronic haloperidol treatment suggests a counter-regulatory protective mechanism.

Angiotensin II-Induced Cardiac Effects Are Modulated by Endocannabinoid-Mediated CB1 Receptor Activation.

Angiotensin II (Ang II) has various cardiac effects and causes vasoconstriction. Ang II activates the type-1 angiotensin receptor-Gq/11 signaling pathway resulting in the release of 2-arachidonoylglycerol (2-AG). We aimed to investigate whether cardiac Ang II effects are modulated by 2-AG-release and to identify the role of type-1 cannabinoid receptors (CB1R) in these effects. Expression of CB1R in rat cardiac tissue was confirmed by immunohistochemistry. To characterize short-term Ang II effects, increasing concentrations of Ang II (10-9-10-7 M); whereas to assess tachyphylaxis, repeated infusions of Ang II (10-7 M) were administered to isolated Langendorff-perfused rat hearts. Ang II infusions caused a decrease in coronary flow and ventricular inotropy, which was more pronounced during the first administration. CB agonist 2-AG and WIN55,212-2 administration to the perfusate enhanced coronary flow. The flow-reducing effect of Ang II was moderated in the presence of CB1R blocker O2050 and diacylglycerol-lipase inhibitor Orlistat. Our findings indicate that Ang II-induced cardiac effects are modulated by simultaneous CB1R-activation, most likely due to 2-AG-release during Ang II signalling. In this combined effect, the response to 2-AG via cardiac CB1R may counteract the positive inotropic effect of Ang II, which may decrease metabolic demand and augment Ang II-induced coronary vasoconstriction.

Endocannabinoids increase human adipose stem cell differentiation and growth factor secretion in vitro.

Adipose stem cells (ASCs) possess the capacity to proliferate, to differentiate into various cells types, and they are able to secrete growth factors. These characteristics are supposed to contribute to their potential for regenerative medicine approaches. In order to advance the therapeutic effects of ASCs, different modulatory procedures have been examined. In this context, the endocannabinoid system (ECS) represents an interesting possibility, since the increased availability of cannabinoids and the underlying molecular pathways of the ECS are of relevance for the development of new regenerative strategies. The effects of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) were investigated on ASC metabolic activity, quantified by PrestoBlue conversion, and cell numbers, evaluated by crystal violet staining. enzyme-linked immunosorbent assay (ELISA) measures were performed to determine cytokine release, and differentiation was assessed by specific labeling techniques. AEA increased the metabolic activity, while 2-AG decreased it in a concentration dependent manner. AEA significantly enhanced OilRed O staining after adipogenic differentiation by over 100%, and both compounds significantly increased cresolphthalein staining after osteogenic differentiation. By contrast, they did not affect sphere diameter or safranin O staining after chondrogenic differentiation. Both substances significantly increased the release of insulin-like growth factor-1 and hepatocyte growth factor, while only AEA enhanced transforming growth factor-ß secretion. The results demonstrated that stimulating the ECS exerted significant effects on the biology of ASCs. Exposure to endocannabinoids modulated viability, induced release of regenerative growth factors, and promoted adipogenic and osteogenic differentiation. Our findings could be of specific relevance in ASC based therapies for regenerative medicine.

Effects of α-glyceryl monolaurate on growth, immune function, volatile fatty acids, and gut microbiota in broiler chickens.

This study was conducted to determine the effects of dietary addition of α-glyceryl monolaurate (α-GML) on growth performance, immune function, volatile fatty acids production and cecal microbiota in broiler chickens. A total of 480 1-day-old yellow-feathered broilers were randomly assigned in equal numbers to 4 dietary treatments: basal diet (NCO) or supplementations with 30 mg/kg bacitracin (ANT), 500 mg/kg α-GML, or 1,000 mg/kg α-GML (GML2). And, each treatment contained 8 replicates with 15 chickens per replicate. After supplementation with α-GML, the total BW gain and average daily weight gain of broilers increased significantly (P < 0.05) compared with the broilers on the NCO diet. Moreover, compared with the NCO group, higher levels of immune globulin M and immune globulin Y were observed in both GML groups and the ANT group. Concentrations of acetate, propionate, butyrate, valerate, and isovalerate in GML2 were significantly higher (P < 0.05) than those in the NCO group on day 28. However, acetate, propionate, valerate, and isovalerate concentrations were reduced to significantly (P < 0.05) lower than those in the NCO group on day 56. The abundance and diversity of microbiota were found to be improved in broilers that were supplemented with GML, using operational taxonomic unit and diversity analyses. Furthermore, the GML treatments increased favorable microbiota, particularly acid-producing bacteria, on day 28 and, also, reduced opportunistic pathogens, such as Alistipes tidjanibacter and Bacteroides dorei by day 56. These results suggest that α-GML supplementation modulates cecal microbiota and broiler immunity and improves volatile fatty acid levels during the early growth stages of broilers.

The immunosuppressive effect of the endocannabinoid system on the inflammatory phenotypes of macrophages and mesenchymal stromal cells: a comparative study.

BACKGROUND: The inflammatory sequence is the first phase of wound healing. Macrophages (MPhs) and mesenchymal stromal cells (MSCs) respond to an inflammatory microenvironment by adapting their functional activity, which polarizes them into the pro-inflammatory phenotypes M1 and MSC1. Prolongation of the inflammatory phase results in the formation of chronic wounds. The endocannabinoid system (ECS) possesses immunomodulatory properties that may impede this cellular phenotypic switch. METHODS: We investigated the immunosuppressive influence of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) on the M1 and MSC1 cytokine secretion. Lipopolysaccharides (LPS) were used as inflammagen to stimulate MPhs and MSCs. Both inflammatory phenotypes were co-exposed to AEA or 2-AG, the specific cannabinoid receptor CB2 agonist JWH-133 served as reference. The inflammatory responses were detected by CD80/163 immuno-labelling and by ELISA measures of secreted IL-6, IL-8, MIF, TNF-α, TGF-ß, and VEGF. RESULTS: M1 cells were found positive for CD80 expression and secreted less IL-6 and IL-8 than MSC1 cells, while both cell types produced similar amounts of MIF. TNF-α release was increased by M1, and growth factors were secreted by MSC1, only. Cannabinoid receptor ligands efficiently decreased the inflammatory response of M1, while their impact was less pronounced in MSC1. CONCLUSIONS: The ECS down-regulated the inflammatory responses of MPhs and MSCs by decreasing the cytokine release upon LPS treatment, while CB2 appeared to be of particular importance. Hence, stimulating the ECS by manipulation of endo- or use of exogenous cannabinoids in vivo may constitute a potent therapeutic option against inflammatory disorders.

Endocannabinoid modulation of dopamine release during reward seeking, interval timing, and avoidance.

Endocannabinoids (eCBs) are neuromodulators that influence a wide range of neural systems and behaviors. In the current review, we describe our recent research showing how eCBs, particularly 2-arachidonoylglycerol (2-AG), concurrently shape mesolimbic dopamine (DA) release and associated behavior. We will restrict our discussion by emphasizing three distinct behaviors: reward seeking, interval timing, and active avoidance. During reward seeking we find that 2-AG is necessary to observe cue-evoked DA release events that are thought to represent the value of a rewarding outcome. We then describe data showing that 2-AG modulates unique patterns of DA release and behavior observed under conditions of periodic reinforcement. These data are discussed within the context of interval timing and adjunctive behavior. eCB modulation of DA release is also implicated in defensive behavior, including the avoidance of harm. As in reward seeking, our data suggest that the concentration of DA that is evoked by a warning signal can represent the value of an avoidance outcome. And, disrupting eCB signaling concomitantly reduces the concentration of the avoidance value signal and active avoidance. Disruptions in reward seeking, interval timing, and defensive behavior are commonly observed in a variety of movement disorders (e.g., Parkinson's and Huntington's disease) and disorders of motivation (e.g., addiction). We believe our data on eCB-DA interactions have implications for the development of novel pharmacotherapies to treat these disorders. Thus, we conclude by discussing how eCB pharmacology might be harnessed to treat disorders of movement and motivation.

Monitoring Cannabinoid CB2 -Receptor Mediated cAMP Dynamics by FRET-Based Live Cell Imaging.

G-protein coupled cannabinoid CB2 receptor signaling and function is primarily mediated by its inhibitory effect on adenylate cyclase. The visualization and monitoring of agonist dependent dynamic 3',5'-cyclic adenosine monophosphate (cAMP) signaling at the single cell level is still missing for CB2 receptors. This paper presents an application of a live cell imaging while using a Förster resonance energy transfer (FRET)-based biosensor, Epac1-camps, for quantification of cAMP. We established HEK293 cells stably co-expressing human CB2 and Epac1-camps and quantified cAMP responses upon Forskolin pre-stimulation, followed by treatment with the CB2 ligands JWH-133, HU308, ß-caryophyllene, or 2-arachidonoylglycerol. We could identify cells showing either an agonist dependent CB2-response as expected, cells displaying no response, and cells with constitutive receptor activity. In Epac1-CB2-HEK293 responder cells, the terpenoid ß-caryophyllene significantly modified the cAMP response through CB2. For all of the tested ligands, a relatively high proportion of cells with constitutively active CB2 receptors was identified. Our method enabled the visualization of intracellular dynamic cAMP responses to the stimuli at single cell level, providing insights into the nature of heterologous CB2 expression systems that contributes to the understanding of Gαi-mediated G-Protein coupled receptor (GPCR) signaling in living cells and opens up possibilities for future investigations of endogenous CB2 responses.