Anti-Inflammatory, Wound Healing, and Anti-Diabetic Effects of Pure Active Compounds Present in the Ryudai Gold Variety of Curcuma longa.

Turmeric (Curcuma longa) contains curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC). Nevertheless, curcumin is the most researched active ingredient for its numerous pharmacological effects. We investigated the impact of these curcuminoids found in Ryudai gold, an approved cultivar of Curcuma longa, on wound healing, inflammation, and diabetes. Sub-planter injections of carrageenan induced acute paw inflammation in rats. The wound-healing ability of 1% curcuminoids was examined by making a 6 mm round wound on the shaved dorsum of the mice with a biopsy punch. A single intraperitoneal injection of streptozotocin (50 mg/kg) was used to induce diabetes in mice. Curcuminoids at a dose rate of 100 mg/kg body weight were used with feed and as a gastric gavage to treat diabetes and inflammation in experimental animals. Paw thickness was measured at 1, 3, and 6 h following carrageenan injection. After three hours, mean paw volume was 58% in carrageenan-injected mice, which was 35%, 37%, and 31% in the curcumin, DMC, and BDMC groups, respectively. Histopathology of the paw tissue demonstrated severe infiltration of inflammatory cells and thickening of the dermis, which were remarkably improved by the curcuminoids. The wound-healing abilities were significantly higher in the curcumin- (95.0%), DMC- (93.17%), and BDMC-treated (89.0%) groups, in comparison to that of the control (65.09%) group at day nine. There were no significant differences in wound-healing activity among the groups treated with 1% curcuminoids throughout the study. Streptozotocin-induced diabetes was characterized by an increased blood glucose (552.2 mg/dL) and decreased body weight (31.2 g), compared to that of the control rats (145.6 mg/dL and 46.8 g blood glucose and body weight, respectively). It also caused an increase in serum alanine aminotransferase (ALT; 44.2 U/L) and aspartate aminotransferase (AST; 55.8 U/L) compared to that of the control group (18.6 U/L and 20.1 U/L, respectively). Histopathological examination of the liver showed that diabetes caused hepatic cellular necrosis, congestion of the central vein, and parenchymatous degeneration. However, all three curcuminoids significantly decreased blood glucose levels, ALT, and AST and improved the histopathological score of the liver. These results evidenced that not only curcumin but also DMC and BDMC have potent anti-inflammatory, wound healing, and anti-diabetic efficacy, and the Ryudai gold variety of turmeric could be used as a functional food supplement.

Investigations of hemato-biochemical and histopathological parameters, and growth performance of walking catfish (Clarias batrachus) exposed to PET and LDPE microplastics.

Fish inhabiting various trophic levels are affected differently as the presence of microplastic (MP) in the water column and their ingestion by fish varies. Walking catfish (Clarias batrachus) inhabits the bottom of the water bodies. To understand the effects of MP, we exposed C. batrachus to two types of MP - polyethylene terephthalate (PET) and low-density polyethylene (LDPE) for 60 days. After exposure, hematological indices, mainly red blood cells and hemoglobin levels decreased, and white blood cells increased significantly compared to the control group (p < 0.05). A significant increase in the levels of blood urea and glucose was observed, and serum glutamic pyruvate transaminase and serum glutamyl oxaloacetic transaminase activity remained elevated (p < 0.05). Histopathological examination of the liver, kidney, intestine, and gills showed morphological alterations. Moreover, MP exposure caused growth retardation (p < 0.05) in C. batrachus. Widespread pollution of water bodies by MP may impose serious ecological risks to bottom-feeding fish in Bangladesh.

Photoperiodic light pulse induces ovarian development in the catfish, Mystus cavasius: Possible roles of dopamine and melatonin in the brain.

In the freshwater catfish, Mystus cavasius, locally known as gulsha, ovarian maturation is triggered by long-day conditions. Using dopaminergic neuronal activity in the brain, the purpose of this study was to identify the brain's detection of a nocturnal light pulse that induced ovarian development. Since direct inhibition of pituitary gonadotropin release is exerted by dopamine (DA), it may serve as a neuromodulator of photoperiodic stimulation in teleosts. We studied functional effects of photoperiodicity on dopaminergic rhythmicity in gulsha brain. Nocturnal illumination and Nanda-Hamner photocycles revealed that ovarian development is induced by a 1 h light pulse between zeitgeber time (ZT) 12 and 13. Daily fluctuations in DA, 3, 4-dihydroxyphenylacetic acid (DOPAC) and DOPAC/DA were observed under a 12L:12D photoperiod. Fish exhibited increased levels during the daytime and decreased levels at night. Rhythmic patterns of dopaminergic activity also showed clear circadian oscillations under constant light, but not constant dark conditions. After 7 days of exposure to long photoperiod (14L:10D), DA, DOPAC and DOPAC/DA in the brain at ZT12 and ZT16 were significantly higher than during a short photoperiod (10L:14D). Melatonin-containing water inhibited the release of DA and DOPAC 6 h and 24 h after treatment, respectively, and DOPAC/DA 6 h after treatment. This inhibition was blocked by the melatonin receptor antagonist, luzindole. These results suggest that a 1 h nocturnal light pulse induces ovarian development through alteration of dopaminergic neuronal excitability in the brain, via oscillation in melatonin triggered by photic stimuli, which may interfere with the reproductive endocrine axis in gulsha.

Ninjin'yoeito Targets Distinct Ca2+ Channels to Activate Ghrelin-Responsive vs. Unresponsive NPY Neurons in the Arcuate Nucleus.

Appetite loss or anorexia substantially deteriorates quality of life in various diseases, and stand upstream of frailty. Neuropeptide Y (NPY) in the hypothalamic arcuate nucleus (ARC) and ghrelin released from stomach are potent inducers of appetite. We previously reported that Ninjin'yoeito, a Japanese kampo medicine comprising twelve herbs, restores food intake, and body weight in cisplatin-treated anorectic mice. Furthermore, Ninjin'yoeito increased cytosolic Ca2+ concentration ([Ca2+]i) in not only ghrelin-responsive but ghrelin-unresponsive NPY neurons in ARC. The cellular lineage/differentiation of ghrelin-unresponsive neuron is less defined but might alter along with aging and diet. This study examined the occupancy of ghrelin-unresponsive neurons among ARC NPY neurons in adult mice fed normal chow, and explored the mechanisms underlying Ninjin'yoeito-induced [Ca2+]i increases in ghrelin-unresponsive vs. ghrelin-responsive NPY neurons. Single ARC neurons were subjected to [Ca2+]i measurement and subsequent immunostaining for NPY. Ghrelin failed to increase [Ca2+]i in 42% of ARC NPY neurons. Ninjin'yoeito (10 µg/ml)-induced increases in [Ca2+]i were abolished in Ca2+ free condition in ghrelin-responsive and ghrelin-unresponsive ARC NPY neurons. Ninjin'yoeito-induced [Ca2+]i increases were inhibited by N-type Ca2+ channel blocker ω-conotoxin in the majority (17 of 20), while by L-type Ca2+ channel blocker nitrendipine in the minority (2 of 23), of ghrelin-responsive neurons. In contrast, Ninjin'yoeito-induced [Ca2+]i increases were inhibited by nitrendipine in the majority (14 of 17), while by ω-conotoxin in the minority (8 of 24), of ghrelin-unresponsive neurons. These results indicate that ghrelin-unresponsive neurons occur substantially among NPY neurons of ARC in adult mice fed normal chow. Ninjin'yoeito preferentially target N-type and L-type Ca2+ channels in the majority of ghrelin-responsive and ghrelin-unresponsive neurons, respectively, to increase [Ca2+]i. We suggest ARC N- and L-type Ca2+ channels as potential targets for activating, respectively, ghrelin-responsive, and unresponsive NPY neurons to treat anorexia.

Ninjin-yoeito activates ghrelin-responsive and unresponsive NPY neurons in the arcuate nucleus and counteracts cisplatin-induced anorexia.

Reduced appetite or anorexia substantially deteriorates quality of life in various diseases including cancer, depression and heart failure. Furthermore, reduced appetite may stand upstream of sarcopenia and frailty. All these diseases are heavy burdens in the modern medicine and society. Therefore, the means that counteracts reduced appetite has been awaited, however, effective and well evidenced substance is not currently available. Ninjin-yoeito, a Japanese kampo medicine comprising twelve herbs has been used to treat anorexia. However, underlying mechanism is little known. Neuropeptide Y (NPY) and ghrelin are the most potent central and peripheral inducers of appetite, respectively. This study sought to determine whether Ninjin-yoeito influences NPY and/or ghrelin-responsive neurons in the hypothalamic arcuate nucleus (ARC), a feeding center. We isolated single neurons from ARC of mice and measured cytosolic Ca2+ concentration ([Ca2+]i) with fura-2 fluorescence imaging, followed by immunocytochemical identification of NPY neurons. Ninjin-yoeito (1-10 µg/ml) increased [Ca2+]i in ARC neurons, the majority (80%) of which was immunoreactive to NPY. One fraction of these Ninjin-yoeito-responsive NPY neurons also responded to ghrelin, while another fraction did not. Furthermore, oral administration of Ninjin-yoeito (1 g/kg/day) counteracted the reductions in food intake and body weight by cisplatin, an anti-cancer drug, in mice. These results demonstrate that Ninjin-yoeito directly targets both ghrelin-responsive and unresponsive NPY neurons in ARC and preserves food intake and body weight in cisplatin-treated anorectic mice. Ninjin-yoeito's signaling through ghrelin-responsive and ghrelin-unresponsive NPY pathways may provide strong mechanistic basis for this medicine for treating anorectic conditions associated with cancer, depression, heart failure, sarcopenia, frailty and aging.

Short-chain fatty acids suppress food intake by activating vagal afferent neurons.

Fermentable carbohydrates including dietary fibers and resistant starch produce short-chain fatty acids (SCFAs), including acetate, propionate and butyrate, through microbial fermentation in the intestine of rodents and humans. Consumption of fermentable carbohydrate and SCFAs suppress food intake, an effect involving the brain. However, their signaling pathway to the brain remains unclear. Vagal afferents serve to link intestinal information to the brain. In the present study, we explored possible role of vagal afferents in the anorexigenic effect of SCFAs. Intraperitoneal (ip) injection of three SCFA molecules (6 mmol/kg) suppressed food intake in fasted mice with the rank order of butyrate > propionate > acetate. The suppressions of feeding by butyrate, propionate and acetate were attenuated by vagotomy of hepatic branch and blunted by systemic treatment with capsaicin that denervates capsaicin-sensitive sensory nerves including vagal afferents. Ip injection of butyrate induced significant phosphorylation of extracellular-signal-regulated kinase 1/2, cellular activation markers, in nodose ganglia and their projection site, medial nucleus tractus solitaries. Moreover, butyrate directly interacted with single neurons isolated from nodose ganglia and induced intracellular Ca2+ signaling. The present results identify the vagal afferent as the novel pathway through which exogenous SCFAs execute the remote control of feeding behavior and possibly other brain functions. Vagal afferents might participate in suppression of feeding by intestine-born SCFAs.

GLP-1 release and vagal afferent activation mediate the beneficial metabolic and chronotherapeutic effects of D-allulose.

Overeating and arrhythmic feeding promote obesity and diabetes. Glucagon-like peptide-1 receptor (GLP-1R) agonists are effective anti-obesity drugs but their use is limited by side effects. Here we show that oral administration of the non-calorie sweetener, rare sugar D-allulose (D-psicose), induces GLP-1 release, activates vagal afferent signaling, reduces food intake and promotes glucose tolerance in healthy and obese-diabetic animal models. Subchronic D-allulose administered at the light period (LP) onset ameliorates LP-specific hyperphagia, visceral obesity, and glucose intolerance. These effects are blunted by vagotomy or pharmacological GLP-1R blockade, and by genetic inactivation of GLP-1R signaling in whole body or selectively in vagal afferents. Our results identify D-allulose as prominent GLP-1 releaser that acts via vagal afferents to restrict feeding and hyperglycemia. Furthermore, when administered in a time-specific manner, chronic D-allulose corrects arrhythmic overeating, obesity and diabetes, suggesting that chronotherapeutic modulation of vagal afferent GLP-1R signaling may aid in treating metabolic disorders.

Glucagon-like peptide-1 and insulin synergistically activate vagal afferent neurons.

Intestinal glucagon-like peptide-1 (GLP-1) and pancreatic insulin, released postprandially, commonly regulate glucose metabolism. Recent clinical experience indicates that the GLP-1R agonist and insulin in combination, compared to insulin alone, results in better glycemic and weight controls in type 2 diabetic patients. These observations suggest possible interactive effect of these hormones. These hormones, in addition to peripherally controlling glycemia, exert central regulation of food intake and glucose metabolism, the effect at least partly mediated by signaling to the brain via the vagal afferents. However, whether the vagal afferents are involved in the interactive effects of GLP-1 and insulin remains unknown. The present study explored possible cooperative effect of GLP-1 and insulin on vagal afferent neurons isolated from nodose ganglion (NG) of mice, while monitoring the neuronal activity by measuring cytosolic Ca2+ concentration ([Ca2+]i) with fura-2. GLP-1 at 10-8M increased [Ca2+]i in 8-11% of single NG neurons. GLP-1-induced [Ca2+]i increases were inhibited by GLP-1 receptor antagonist exendin (9-39). Majority (92%) of GLP-1-responseive NG neurons also responded to 10-7M insulin with [Ca2+]i increases. Both GLP-1 and insulin at lower concentration of 10-9M induced [Ca2+]i increases with smaller amplitude in lesser NG neuron population (4-7%). These hormones at 10-9M in combination recruited the unresponsive neurons to [Ca2+]i increases, and induced [Ca2+]i increases with greater amplitude in the responsive neurons. The results demonstrate that GLP-1 and insulin synergistically and additively activate vagal afferent neurons. This interaction may be linked to the postprandial functions mediated commonly by GLP-1 and insulin and in the beneficial outcome of the therapy with GLP-1 receptor agonist and insulin in combination.

Motilin Stimulates Gastric Acid Secretion in Coordination with Ghrelin in Suncus murinus.

Motilin and ghrelin constitute a peptide family, and these hormones are important for the regulation of gastrointestinal motility. In this study, we examined the effect of motilin and ghrelin on gastric acid secretion in anesthetized suncus (house musk shrew, Suncus murinus), a ghrelin- and motilin-producing mammal. We first established a gastric lumen-perfusion system in the suncus and confirmed that intravenous (i.v.) administration of histamine (1 mg/kg body weight) stimulated acid secretion. Motilin (0.1, 1.0, and 10 µg/kg BW) stimulated the acid output in a dose-dependent manner in suncus, whereas ghrelin (0.1, 1.0, and 10 µg/kg BW) alone did not induce acid output. Furthermore, in comparison with the vehicle administration, the co-administration of low-dose (1 µg/kg BW) motilin and ghrelin significantly stimulated gastric acid secretion, whereas either motilin (1 µg/kg BW) or ghrelin (1 µg/kg BW) alone did not significantly induce gastric acid secretion. This indicates an additive role of ghrelin in motilin-induced gastric acid secretion. We then investigated the pathways of motilin/motilin and ghrelin-stimulated acid secretion using receptor antagonists. Treatment with YM 022 (a CCK-B receptor antagonist) and atropine (a muscarinic acetylcholine receptor antagonist) had no effect on motilin or motilin-ghrelin co-administration-induced acid output. In contrast, famotidine (a histamine H2 receptor antagonist) completely inhibited motilin-stimulated acid secretion and co-administration of motilin and ghrelin induced gastric acid output. This is the first report demonstrating that motilin stimulates gastric secretion in mammals. Our results also suggest that motilin and co-administration of motilin and ghrelin stimulate gastric acid secretion via the histamine-mediated pathway in suncus.

Motilin stimulates pepsinogen secretion in Suncus murinus.

Motilin and ghrelin are gastrointestinal hormones that stimulate the migrating motor complex (MMC) of gastrointestinal motility during the fasting state. In this study, we examined the effect of motilin and ghrelin on pepsinogen secretion in anesthetized suncus (house musk shrew, Suncus murinus), a ghrelin- and motilin-producing mammal. By using a gastric lumen-perfusion system, we found that the intravenous administration of carbachol and motilin stimulated pepsinogen secretion, the latter in a dose-dependent manner, whereas ghrelin had no effect. We then investigated the pathways of motilin-induced pepsinogen secretion using acetylcholine receptor antagonists. Treatment with atropine, a muscarinic acetylcholine receptor antagonist, completely inhibited both carbachol and motilin-induced pepsinogen secretion. Motilin-induced pepsinogen secretion was observed in the vagotomized suncus. This is the first report demonstrating that motilin stimulates pepsinogen secretion, and suggest that this effect occurs through a cholinergic pathway in suncus.

Mechanism of ghrelin-induced gastric contractions in Suncus murinus (house musk shrew): involvement of intrinsic primary afferent neurons.

Here, we have reported that motilin can induce contractions in a dose-dependent manner in isolated Suncus murinus (house musk shrew) stomach. We have also shown that after pretreatment with a low dose of motilin (10(-10) M), ghrelin also induces gastric contractions at levels of 10(-10) M to 10(-7) M. However, the neural mechanism of ghrelin action in the stomach has not been fully revealed. In the present study, we studied the mechanism of ghrelin-induced contraction in vitro using a pharmacological method. The responses to ghrelin in the stomach were almost completely abolished by hexamethonium and were significantly suppressed by the administration of phentolamine, prazosin, ondansetron, and naloxone. Additionally, N-nitro-l-arginine methylester significantly potentiated the contractions. Importantly, the mucosa is essential for ghrelin-induced, but not motilin-induced, gastric contractions. To evaluate the involvement of intrinsic primary afferent neurons (IPANs), which are multiaxonal neurons that pass signals from the mucosa to the myenteric plexus, we examined the effect of the IPAN-related pathway on ghrelin-induced contractions and found that pretreatment with adenosine and tachykinergic receptor 3 antagonists (SR142801) significantly eliminated the contractions and GR113808 (5-hydroxytryptamine receptor 4 antagonist) almost completely eliminated it. The results indicate that ghrelin stimulates and modulates suncus gastric contractions through cholinergic, adrenergic, serotonergic, opioidergic neurons and nitric oxide synthases in the myenteric plexus. The mucosa is also important for ghrelin-induced gastric contractions, and IPANs may be the important interneurons that pass the signal from the mucosa to the myenteric plexus.