GSPE Pre-Treatment Exerts Long-Lasting Preventive Effects against Aging-Induced Changes in the Colonic Enterohormone Profile of Female Rats.

The impact that healthy aging can have on society has raised great interest in understanding aging mechanisms. However, the effects this biological process may have on the gastrointestinal tract (GIT) have not yet been fully described. Results in relation to changes observed in the enteroendocrine system along the GIT are controversial. Grape seed proanthocyanidin extracts (GSPE) have been shown to protect against several pathologies associated with aging. Based on previous results, we hypothesized that a GSPE pre-treatment could prevent the aging processes that affect the enteroendocrine system. To test this hypothesis, we treated 21-month-old female rats with GSPE for 10 days. Eleven weeks after the treatment, we analyzed the effects of GSPE by comparing these aged animals with young animals. Aging induced a greater endocrine response to stimulation in the upper GIT segments (cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1)), a decrease in the mRNA abundance of GLP-1, peptide YY (PYY) and chromogranin A (ChgA) in the colon, and an increase in colonic butyrate. GSPE-treated rats were protected against a decrease in enterohormone expression in the colon. This effect is not directly related to the abundance of microbiome or short-chain fatty acids (SCFA) at this location. GSPE may therefore be effective in preventing a decrease in the colonic abundance of enterohormone expression induced by aging.

Investigating the effect of Shenmai injection on cardiac electrophysiology and calcium signaling using human-induced pluripotent stem cell-derived cardiomyocytes.

Traditional Chinese medicine injection (TCMI) refers to the use of modern technology to make Chinese patent medicines in injectable forms, which shorten the onset time of the traditional Chinese medicine (TCM). Although there have been clinical cases in which Shenmai injection (SMI) was used to treat cardiovascular diseases (CVDs), there are no pharmacological experiments that investigate the efficacy of the drug in vitro or the underlying mechanisms. Aim of the study: We aimed to systemically evaluate the efficacy and investigate the mechanisms of SMI in modulating electrophysiology and calcium (Ca2+) signaling using a microelectrode array (MEA) and a genetically encoded Ca2+ indicator, GCaMP6s, respectively, in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Materials and methods: A MEA system was employed to record field potentials (FPs) in hiPSC-CMs. The QT interval is corrected by the RR interval, the reciprocal of the beating rate. GCaMP6s was used to measure Ca2+ signaling in hiPSC-CMs. Meanwhile, the transcriptome changes in hiPSC-CMs treated with 2% SMI were examined using RNAseq. In addition, the ingredients of SMI were investigated using liquid chromatography-mass spectrometry (LC-MS). Results: It was found that 0.5%, 1%, and 2% (v/v) SMIs could increase corrected QT (QTc) but did not change other FP parameters. GCaMP6s was successfully applied to measure the chronic function of SMI. The full width at half maximum (FWHM), rise time, and decay time significantly decreased after treatment with SMI for 1 h and 24 h, whereas an increased Ca2+ transient frequency was observed. Conclusions: We first used the Ca2+ indicator to measure the chronic effects of TCM. We found that SMI treatment can modulate electrophysiology and calcium signaling and regulate oxidative phosphorylation, cardiac muscle contraction, and the cell cycle pathway in hiPSC-CMs.

Neurogenic Hypotension and Bradycardia Modulated by Electroacupuncture in Hypothalamic Paraventricular Nucleus.

Electroacupuncture (EA) stimulates somatic median afferents underlying P5-6 acupoints and modulates parasympathoexcitatory reflex responses through central processing in the brainstem. Although decreases in blood pressure and heart rate by the neural-mediated Bezold-Jarisch reflex responses are modulated by EA through opioid actions in the nucleus tractus solitarius and nucleus ambiguus, the role of the hypothalamus is unclear. The hypothalamic paraventricular nucleus (PVN) is activated by sympathetic afferents and regulates sympathetic outflow and sympathoexcitatory cardiovascular responses. In addition, the PVN is activated by vagal afferents, but little is known about its regulation of cardiopulmonary inhibitory hemodynamic responses. We hypothesized that the PVN participates in the Bezold-Jarisch reflex responses and EA inhibits these cardiopulmonary responses through the PVN opioid system. Rats were anesthetized and ventilated, and their heart rate and blood pressures were monitored. Application of phenylbiguanide every 10 min close to the right atrium induced consistent depressor and bradycardia reflex responses. Unilateral microinjection of the depolarization blockade agent kainic acid or glutamate receptor antagonist kynurenic acid in the PVN reduced these reflex responses. In at least 70% of the rats, 30 min of bilateral EA at P5-6 acupoints reduced the depressor and bradycardia responses for at least 60 min. Blockade of the CCK-1 receptors converted the non-responders into EA-responders. Unilateral PVN-microinjection with naloxone reversed the EA inhibition. Vagal-evoked activity of the PVN cardiovascular neurons was reduced by 30 min EA (P5-6) through opioid receptor activation. These data indicate that PVN processes inhibitory cardiopulmonary reflexes and participates in EA-modulation of the neural-mediated vasodepression and bradycardia.

The hyperthermic effect of central cholecystokinin is mediated by the cyclooxygenase-2 pathway.

Cholecystokinin (CCK) increases core body temperature via CCK2 receptors when administered intracerebroventricularly (icv). The mechanisms of CCK-induced hyperthermia are unknown, and it is also unknown whether CCK contributes to the fever response to systemic inflammation. We studied the interaction between central CCK signaling and the cyclooxygenase (COX) pathway. Body temperature was measured in adult male Wistar rats pretreated with intraperitoneal infusion of the nonselective COX enzyme inhibitor metamizol (120 mg/kg) or a selective COX-2 inhibitor, meloxicam, or etoricoxib (10 mg/kg for both) and, 30 min later, treated with intracerebroventricular CCK (1.7 µg/kg). In separate experiments, CCK-induced neuronal activation (with and without COX inhibition) was studied in thermoregulation- and feeding-related nuclei with c-Fos immunohistochemistry. CCK increased body temperature by ∼0.4°C from 10 min postinfusion, which was attenuated by metamizol. CCK reduced the number of c-Fos-positive cells in the median preoptic area (by ∼70%) but increased it in the dorsal hypothalamic area and in the rostral raphe pallidus (by ∼50% in both); all these changes were completely blocked with metamizol. In contrast, CCK-induced satiety and neuronal activation in the ventromedial hypothalamus were not influenced by metamizol. CCK-induced hyperthermia was also completely blocked with both selective COX-2 inhibitors studied. Finally, the CCK2 receptor antagonist YM022 (10 µg/kg icv) attenuated the late phases of fever induced by bacterial lipopolysaccharide (10 µg/kg; intravenously). We conclude that centrally administered CCK causes hyperthermia through changes in the activity of "classical" thermoeffector pathways and that the activation of COX-2 is required for the development of this response.NEW & NOTEWORTHY An association between central cholecystokinin signaling and the cyclooxygenase-prostaglandin E pathway has been proposed but remained poorly understood. We show that the hyperthermic response to the central administration of cholecystokinin alters the neuronal activity within efferent thermoeffector pathways and that these effects are fully blocked by the inhibition of cyclooxygenase. We also show that the activation of cyclooxygenase-2 is required for the hyperthermic effect of cholecystokinin and that cholecystokinin is a modulator of endotoxin-induced fever.

[Effect of electroacupuncture of "biaoben acupoint combination" on central sensitivity of cholecystokinin in obese rats with insulin resistance].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "biaoben acupoint combination" on appetite, body fat, insulin sensitivity and central sensitivity of cholecystokinin (CCK) in obese rats with insulin resistance (IR), and to explore the mechanism of EA on improving obesity with insulin resistance. METHODS: Among the fifty 8-week-old healthy SPF male Wistar rats, 10 rats were randomly selected and fed with normal diet; after 8 weeks, 8 rats were randomly selected as a normal group. The remaining 40 rats were fed with high-fat diet to establish the model of obsesity IR; after 8 weeks, 24 rats with successful model of obsesity IR were randomly divided into a model group, an EA group and a sham EA group, 8 rats in each group. Eight weeks after model establishment, the rats in the EA group were intervened with EA at "Fenglong" (ST 40), "Zhongwan" (CV 12), "Guanyuan" (CV 4) and "Zusanli" (ST 36), with continuous wave, in frequency of 2 Hz, and current intensity of 1 mA, for 10 min each time. The rats in the sham EA group were intervened with EA at the points 5 mm next to the acupoints used in the EA group and no electricity was given; the sham EA was given for 10 min each time. Both the treatments were given once every other day for 8 weeks. The Lee's index and food intake were observed before the intervention as well as 2 weeks, 4 weeks, 6 weeks and 8 weeks into intervention; after the intervention, serum insulin (INS) and glucose infusion rate (GIR) were detected; serum cholecystokinin (CCK) level was detected by ELISA; c-fos expression in the area postrema (AP) and nucleus tractus solitarius (NTS) of medulla oblongata was detected by immunohistochemistry. RESULTS: Before the intervention as well as 2 weeks, 4 weeks, 6 weeks and 8 weeks into intervention, the Lee's index and food intake in the model group were higher than those in normal group (P<0.01). The Lee's index (6 weeks and 8 weeks into intervention) and food intake (4 weeks, 6 weeks and 8 weeks into intervention) in the EA group were lower than those in the model group and the sham EA group (P<0.05, P<0.01). After intervention, compared with the normal group, serum level of INS was increased (P<0.01), while GIR, serum CCK level, c-fos expression in AP and NTS in the model group were decreased (P<0.01, P<0.05). Compared with the model group and the sham EA group, serum level of INS in the EA group was decreased (P<0.01), and GIR, serum CCK level, c-fos expression in AP and NTS were increased (P<0.01, P<0.05). CONCLUSION: EA of "biaoben acupoint combination" could effectively reduce appetite, body fat and enhance insulin sensitivity in obese rats with IR. The mechanism may be related to the regulation of central sensitivity of CCK.

Effects of Flavanols on Enteroendocrine Secretion.

Some beneficial effects of grape seed proanthocyanidin extract (GSPE) can be explained by the modulation of enterohormone secretion. As GSPE comprises a combination of different molecules, the pure compounds that cause these effects need to be elucidated. The enterohormones and chemoreceptors present in the gastrointestinal tract differ between species, so if humans are to gain beneficial effects, species closer to humans-and humans themselves-must be used. We demonstrate that 100 mg/L of GSPE stimulates peptide YY (PYY) release, but not glucagon-like peptide 1 (GLP-1) release in the human colon. We used a pig ex vivo system that differentiates between apical and basolateral intestinal sides to analyse how apical stimulation with GSPE and its pure compounds affects the gastrointestinal tract. In pigs, apical GSPE treatment stimulates the basolateral release of PYY in the duodenum and colon and that of GLP-1 in the ascending, but not the descending colon. In the duodenum, luminal stimulation with procyanidin dimer B2 increased PYY secretion, but not CCK secretion, while catechin monomers (catechin/epicatechin) significantly increased CCK release, but not PYY release. The differential effects of GSPE and its pure compounds on enterohormone release at the same intestinal segment suggest that they act through chemosensors located apically and unevenly distributed along the gastrointestinal tract.

Effect of electroacupuncture of " acupoint combination" on central sensitivity of cholecystokinin in obese rats with insulin resistance / 中国针灸

OBJECTIVE@#To observe the effect of electroacupuncture (EA) of " acupoint combination" on appetite, body fat, insulin sensitivity and central sensitivity of cholecystokinin (CCK) in obese rats with insulin resistance (IR), and to explore the mechanism of EA on improving obesity with insulin resistance.@*METHODS@#Among the fifty 8-week-old healthy SPF male Wistar rats, 10 rats were randomly selected and fed with normal diet; after 8 weeks, 8 rats were randomly selected as a normal group. The remaining 40 rats were fed with high-fat diet to establish the model of obsesity IR; after 8 weeks, 24 rats with successful model of obsesity IR were randomly divided into a model group, an EA group and a sham EA group, 8 rats in each group. Eight weeks after model establishment, the rats in the EA group were intervened with EA at "Fenglong" (ST 40), "Zhongwan" (CV 12), "Guanyuan" (CV 4) and "Zusanli" (ST 36), with continuous wave, in frequency of 2 Hz, and current intensity of 1 mA, for 10 min each time. The rats in the sham EA group were intervened with EA at the points 5 mm next to the acupoints used in the EA group and no electricity was given; the sham EA was given for 10 min each time. Both the treatments were given once every other day for 8 weeks. The Lee's index and food intake were observed before the intervention as well as 2 weeks, 4 weeks, 6 weeks and 8 weeks into intervention; after the intervention, serum insulin (INS) and glucose infusion rate (GIR) were detected; serum cholecystokinin (CCK) level was detected by ELISA; c-fos expression in the area postrema (AP) and nucleus tractus solitarius (NTS) of medulla oblongata was detected by immunohistochemistry.@*RESULTS@#Before the intervention as well as 2 weeks, 4 weeks, 6 weeks and 8 weeks into intervention, the Lee's index and food intake in the model group were higher than those in normal group (<0.01). The Lee's index (6 weeks and 8 weeks into intervention) and food intake (4 weeks, 6 weeks and 8 weeks into intervention) in the EA group were lower than those in the model group and the sham EA group (<0.05, <0.01). After intervention, compared with the normal group, serum level of INS was increased (<0.01), while GIR, serum CCK level, c-fos expression in AP and NTS in the model group were decreased (<0.01, <0.05). Compared with the model group and the sham EA group, serum level of INS in the EA group was decreased (<0.01), and GIR, serum CCK level, c-fos expression in AP and NTS were increased (<0.01, <0.05).@*CONCLUSION@#EA of " acupoint combination" could effectively reduce appetite, body fat and enhance insulin sensitivity in obese rats with IR. The mechanism may be related to the regulation of central sensitivity of CCK.

A herbal premix containing Macrotyloma uniflorum, ginger, and whey curtails obesity in rats fed a high-fat diet by a novel mechanism.

The present study designed and evaluated a polyherbal premix comprising Macrotyloma uniflorum, whey protein, Zingiber officinale, and Mentha piperita. Animals were fed a high-fat diet (HFD) for 30 days and were daily administered the premix (1.5 g/kg) in milk (PM) and water (PW), aerobic exercise (AE), premix in milk and water along with AE (PMAE and PWAE), ferulic acid (100 mg/kg), and the reference drug fluoxetine (6 mg/kg). All treatments showed significant reduction in food intake, weight gain, abdominal circumference, and body mass index compared with their initial values. All treatments generated a faster peak of the satiety marker cholecystokinin compared with the HFD group and control groups; PMAE and PWAE exhibited sustained satiety. The HFD-elevated blood glucose levels were significantly attenuated on the 30th day by all treatments when compared with their 15th day and basal values; PMAE exhibited the best results. All treatments significantly attenuated the HFD-elevated serum insulin, homeostasis model assessment of insulin resistance, C-reactive protein, triglycerides, total cholesterol, very-low-density lipoprotein, and low-density lipoprotein levels and significantly restored the HFD-depleted high-density lipoprotein and adiponectin levels. HFD-elevated thiobarbituric acid reactive substances values were attenuated successfully and the HFD-depleted reduced glutathione, superoxide dismutase, and catalase levels were significantly restored by all treatments. The histological findings corroborated the biochemical results. Novelty The polyherbal premix brought about appetite regulation and induction of satiety to control obesity in HFD-fed rats through homeostasis of energy metabolism. The premix along with exercise is a complete way to combat obesity.

Cholecystokinin Receptor Antagonist Therapy Decreases Inflammation and Fibrosis in Chronic Pancreatitis.

BACKGROUND AND AIMS: Chronic pancreatitis is associated with recurrent inflammation, pain, fibrosis, and loss of exocrine and endocrine pancreatic function and risk of cancer. We hypothesized that activation of the CCK receptor contributes to pancreatitis and blockade of this pathway would improve chronic pancreatitis. METHODS: Two murine models were used to determine whether CCK receptor blockade with proglumide could prevent and reverse histologic and biochemical features of chronic pancreatitis: the 6-week repetitive chronic cerulein injection model and the modified 75% choline-deficient ethionine (CDE) diet. In the CDE-fed model, half the mice received water supplemented with proglumide, for 18 weeks. After chronic pancreatitis was established in the cerulein model, half the mice were treated with proglumide and half with water. Histology was scored in a blinded fashion for inflammation, fibrosis and acinar ductal metaplasia (ADM) and serum lipase levels were measured. RNA was extracted and examined for differentially expressed fibrosis genes. RESULTS: Proglumide therapy decreased pancreatic weight in the CDE diet study and the cerulein-induced chronic pancreatitis model. Fibrosis, inflammation, and ADM scores were significantly reduced in both models. Lipase values improved with proglumide but not in controls in both models. Proglumide decreased pancreas mRNA expression of amylase, collagen-4, and TGFßR2 gene expression by 44, 38, and 25%, respectively, compared to control mice. CONCLUSION: New strategies are needed to decreased inflammation and reduce fibrosis in chronic pancreatitis. CCK receptor antagonist therapy may improve chronic pancreatitis by reversing fibrosis and inflammation. The decrease in ADM may reduce the risk of the development of pancreatic cancer.

The Effect of Dihydroartemisinin on the Malignancy and Epithelial-Mesenchymal Transition of Gastric Cancer Cells.

OBJECTIVE: This study aimed to observe the effects of dihydroartemisinin (DHA) on the proliferation, apoptosis, invasion, migration, and epithelial-mesenchymal transition (EMT) of the human gastric cancer cell line SGC7901 cultured in vitro. METHODS: We applied varying concentrations of DHA to SGC7901 cells. Cell proliferation was measured using the cell counting kit-8 (CCK-8). Flow cytometry, Transwell invasion assay, and cell scratch assay were used to investigate the cells' apoptosis, invasion, and migration. Western blot was used to assess the expression levels of EMT markers E-cadhein and Vimentin, protein kinases Akt and phosphorylated AKT (p-AKT), and the cell transcription factor Snail. RESULTS: DHA can effectively inhibit the malignant proliferation of gastric cancer cells in a time- and dose-dependent manner. In this study, with longer incubation times and increased drug concentrations, the antiproliferation effect of DHA on SGC7901 cells increased gradually (P<0.05). In addition, with the increase of drug concentration, the expression levels of E-cadhein, an epithelial-mesenchymal transition marker, remarkably increased, whereas the protein expression levels of the mesenchymal markers Vimentin, Akt, p-Akt, and Snail significantly decreased (P<0.05). CONCLUSION: DHA can effectively inhibit the proliferation, invasion, and metastasis of the gastric cancer cell line SGC7901 and induce cancer cell apoptosis. DHA can also downregulate PI3K/AKT and Snail activities and inhibit the epithelial-mesenchymal transition of gastric cancer cells. The potential anticancer effects of DHA deserve further investigation.

Thymosin Beta 4 Is Involved in the Development of Electroacupuncture Tolerance.

Background: Electroacupuncture (EA) tolerance, a negative therapeutic effect, is a gradual decline in antinociception because of its repeated or prolonged use. This study aims to explore the role of thymosin beta 4 (Tß4), having neuro-protection properties, in EA tolerance (EAT). Methods: Rats were treated with EA once daily for eight consecutive days to establish EAT, effect of Tß4 on the development of EAT was determined through microinjection of Tß4 antibody and siRNA into the cerebroventricle. The mRNA and protein expression profiles of Tß4, opioid peptides (enkephalin, dynorphin and endorphin), and anti-opioid peptides (cholecystokinin octapeptide, CCK-8 and orphanin FQ, OFQ), and mu opioid receptor (MOR) and CCK B receptor (CCKBR) in the brain areas (hypothalamus, thalamus, cortex, midbrain and medulla) were characterized after Tß4 siRNA was administered. Results: Tß4 levels were increased at day 1, 4, and 8 and negatively correlated with the changes of tail flick latency in all areas. Tß4 antibody and siRNA postponed EAT. Tß4 siRNA caused decreased Tß4 levels in all areas, which resulted in increased enkephalin, dynorphin, endorphin and MOR levels in most measured areas during repeated EA, but unchanged OFQ, CCK-8, and CCKBR levels in most measured areas. Tß4 levels were negatively correlated with enkephalin, dynorphin, endorphin, or MOR levels in all areas except medulla, while positively correlated with OFQ and CCK-8 levels in some areas. Conclusion: These results confirmed Tß4 facilitates EAT probably through negatively changing endogenous opioid peptides and their receptors and positively influencing anti-opioid peptides in the central nervous system.

Seafood intake and the development of obesity, insulin resistance and type 2 diabetes.

We provide an overview of studies on seafood intake in relation to obesity, insulin resistance and type 2 diabetes. Overweight and obesity development is for most individuals the result of years of positive energy balance. Evidence from intervention trials and animal studies suggests that frequent intake of lean seafood, as compared with intake of terrestrial meats, reduces energy intake by 4-9 %, sufficient to prevent a positive energy balance and obesity. At equal energy intake, lean seafood reduces fasting and postprandial risk markers of insulin resistance, and improves insulin sensitivity in insulin-resistant adults. Energy restriction combined with intake of lean and fatty seafood seems to increase weight loss. Marine n-3 PUFA are probably of importance through n-3 PUFA-derived lipid mediators such as endocannabinoids and oxylipins, but other constituents of seafood such as the fish protein per se, trace elements or vitamins also seem to play a largely neglected role. A high intake of fatty seafood increases circulating levels of the insulin-sensitising hormone adiponectin. As compared with a high meat intake, high intake of seafood has been reported to reduce plasma levels of the hepatic acute-phase protein C-reactive protein level in some, but not all studies. More studies are needed to confirm the dietary effects on energy intake, obesity and insulin resistance. Future studies should be designed to elucidate the potential contribution of trace elements, vitamins and undesirables present in seafood, and we argue that stratification into responders and non-responders in randomised controlled trials may improve the understanding of health effects from intake of seafood.

Ginsenoside Rb2 inhibits epithelial-mesenchymal transition of colorectal cancer cells by suppressing TGF-ß/Smad signaling.

BACKGROUND: Treating colorectal cancer (CRC) continues to be a clinical challenge. Studies have shown that epithelial-mesenchymal transition (EMT) is a critical step in tumor progression and transforming growth factor-ß1 (TGF-ß1) signaling has been shown to play a crucial role in EMT. Here, we investigate the inhibition effect of Ginsenoside Rb2, main bioactive component of ginseng, in human colorectal cancer cells via TGF-ß1. PURPOSE: The current study aims to study the inhibitory effect of Ginsenoside Rb2 on HCT116 and SW620 cells and its anti-tumor mechanism. METHODS: Histomorphological analysis and western blot analysis were performed to evaluate expression of TGF-ß1 in human cancerous colon samples and the adjacent normal samples. The docking simulation assay were performed to explore the potential mode of binding of Ginsenoside Rb2 to the TGF-ß1 protein. CCK8, adhesion and invasion assay were used to assess the effects of Ginsenoside Rb2 in HCT116 and SW620 cells. RT-PCR, Western blot and Immunohistochemical staining were employed to detect the TGF-ß1-related signaling pathways in the colon cancer cells and/or xenograft mice. RESULTS: The expression of TGF-ß1 in human cancerous colon samples was significantly increased compared with the adjacent normal samples. Ginsenoside Rb2 inhibit the growth, adhesion, EMT and metastasis of human colorectal cancer cells. The docking simulation assay confirmed that Ginsenoside Rb2 bound to the hydrophobic pocket of TGF-ß1, which partially overlaps with the binding sites on TGF-ß1, and thus disrupted TGF-ß1 dimerization. Western Blot analysis further confirmed that Ginsenoside Rb2 could inhibit the expression of TGF-ß1 in vitro and in vivo. Furthermore, Ginsenoside Rb2 could inhibit the expression of Smad4 and phosphorylated Smad2/3. CONCLUSION: Ginsenoside Rb2 could inhibit EMT of colorectal cancer cells through the TGF-ß1/Smad signaling, and might be a potential candidate for the treatment of colorectal cancer.

Effects of CCK-8 and GLP-1 on fatty acid sensing and food intake regulation in trout.

We hypothesize that cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1) are involved in the modulation of metabolic regulation of food intake by fatty acids in fish. Therefore, we assessed in rainbow trout (Oncorhynchus mykiss) the effects of intracerebroventricular treatment with 1 ng/g of CCK-8 and with 2 ng/g of GLP-1 on food intake, expression of neuropeptides involved in food intake control and the activity of fatty acid-sensing systems in hypothalamus and hindbrain. Food intake decreased up to 24 h post-treatment to 49.8-72.3% and 3.1-17.8% for CCK-8 and GLP-1, respectively. These anorectic responses are associated with changes in fatty acid metabolism and an activation of fatty acid-sensing mechanisms in the hypothalamus and hindbrain. These changes occurred in parallel with those in the expression of anorexigenic and orexigenic peptides. Moreover, we observed that the activation of fatty acid sensing and the enhanced anorectic potential elicited by CCK-8 and GLP-1 treatments occurred in parallel with the activation of mTOR and FoxO1 and the inhibition of AMPKα, BSX and CREB. The results are discussed in the context of metabolic regulation of food intake in fish.

Dissolution kinetics of Compound Danshen Tablets based on cell bioelectrical sensing effect / 中草药

Objective: Establishing the model of cell bioelectrical sensing effect of Compound Danshen Tablets to study its dissolution kinetics. Methods: By means of real-time cell-based assay, the in vitro dissolution of Compound Danshen Tablets can be investigated, and then the dissolution kinetics model can also be established. In addition, the result was compared and verified by UV-Vis. Results: The cell line with specific dependence on Compound Danshen Tablets was screened by CCK-8 experiment and RTCA experiment. The dissolution kinetics model of Compound Danshen Tablets based on RTCA technology was established, and the best fitting model was obtained: Weibull model ln{ln[1/(1-Q)] =1.071 4 lnt-3.736 7; Establish a dissolution kinetic model of Compound Danshen Tablets based on UV spectrophotometry to obtain the best fitting model, Weibull model ln{ln[1/(1-Q)]}=1.080 4 lnt-3.723 4; Comparing the two Weibull models, the RTCA fitted model worked better. Conclusion: The application of RTCA in the dissolution kinetics of traditional Chinese medicine compound solid preparations is feasible, Which provides new ideas for traditional Chinese medicines and the quality evaluation of traditional Chinese medicine compunds.

Celastrus Orbiculatus Extract Potentiates the Sensitivity of Cisplatin Via Caspase-Depenent Apoptosis in Gastric Cancer.

BACKGROUND: Cisplatin-based treatment often leads to therapeutic failure because the acquisition of cisplatin resistance. The combination of cisplatin with other agents has been recognized as a promising strategy to overcome cisplatin resistance. OBJECTIVE: Celastrus orbiculatus is a traditional Chinese medicine from Celastraceae family with multiple pharmacological activities. We previously found that the ethyl acetate extract of Celastrus orbiculatus (COE) exhibited significant antitumor activity in gastric cancer. Here, we asked whether COE could increase the sensitivity of cisplatin. METHODS: We use CCK8 assay to show synergistic cytotoxicity of COE and cisplatin. Then, PI single staining and FITC-Annexin V/PI double staining were used to observe apoptotic cells through flow cytometry. The proteins of caspase signaling pathway were examined by Western blotting. RESULTS: COE and cisplatin showed synergistic cytotoxicity in a dose-dependent manner in BGC 823 and SGC 7901 gastric cancer cells, and COE could increase the number of apoptotic cells upon cisplatin treatment in vitro. Moreover, our results indicated that COE could enhance cisplatin-induced activation of caspase-8 or caspase- 9/caspase-3/PARP1 signaling pathways. The xenograft study further confirmed that COE increased the sensitivity of cisplatin in vivo. CONCLUSION: Our findings provided new evidence that COE could increase the sensitivity of cisplatin on the antitumor effect.

The Expressing Patterns of Opioid Peptides, Anti-opioid Peptides and Their Receptors in the Central Nervous System Are Involved in Electroacupuncture Tolerance in Goats.

To investigate dynamic processes of enkephalin (ENK), cholecystokinin octapeptide (CCK-8), orphanin FQ (OFQ) and their receptors (µ opioid receptor, MOR; CCK B type receptor, CCKBR and opioid receptor-like 1 receptor, OPRL1) in the central nerve system (CNS) during electroacupuncture (EA) tolerance, EA of Sixty Hz was used to stimulate goats for 6 h. Pain threshold was measured using potassium iontophoresis. The expression levels of ENK, CCK-8, and OFQ and their receptors were determined with ELISA and qPCR, respectively. The results showed that the change rates of pain threshold in EA-treated goats decreased from 89.9 ± 11.7% at 0.5 h to -11.4 ± 8.9% at 6 h. EA induced the decreased ENK and increased CCK-8 and OFQ in the most measured nuclei. EA caused decreased preproenkephalin mRNAs in ACB, CAU, PVH, and PAG at 4 h, and decreased or unchanged MOR mRNAs at 2-6 h, but increased CCK mRNAs in CAU, PVT, PVH, PAG, and SCD at 4-12 h. Increased prepronociceptin mRNAs and fluctuated CCKBR and OPLR1 mRNAs were found in the most measured nuclei. ENK levels were positively correlated (p < 0.01) with the change rates of pain thresholds in the measured nuclei or areas while CCK-8 levels (or OFQ levels) were negatively correlated (p < 0.01) with the pain thresholds in CAU (or CAU and ACB). These results suggest that the development and recovery of EA tolerance may be associated with the specific expression patterns of opioid peptides, anti-opioid peptides and their receptors in the analgesia-related nuclei or areas.

In vitro anticancer properties of selected Eucalyptus species.

In spite of the recent advancements in oncology, the overall survival rate for pancreatic cancer has not improved over the last five decades. Eucalypts have been linked with cytotoxic and anticancer properties in various studies; however, there is very little scientific evidence that supports the direct role of eucalypts in the treatment of pancreatic cancer. This study assessed the anticancer properties of aqueous and ethanolic extracts of four Eucalyptus species using an MTT assay. The most promising extracts were further evaluated using a CCK-8 assay. Apoptotic studies were performed using a caspase 3/7 assay in MIA PaCa-2 cells. The aqueous extract of Eucalyptus microcorys leaf and the ethanolic extract of Eucalyptus microcorys fruit inhibited the growth of glioblastoma, neuroblastoma, lung and pancreatic cancer cells by more than 80% at 100 µg/mL. The E. microcorys and Eucalyptus saligna extracts showed lower GI50 values than the ethanolic Eucalyptus robusta extract in MIA PaCa-2 cells. Aqueous E. microcorys leaf and fruit extracts at 100 µg/mL exerted significantly higher cell growth inhibition in MIA PaCa-2 cells than other extracts (p < 0.05). Statistically similar IC50 values (p > 0.05) were observed in aqueous E. microcorys leaf (86.05 ± 4.75 µg/mL) and fruit (64.66 ± 15.97 µg/mL) and ethanolic E. microcorys leaf (79.30 ± 29.45 µg/mL) extracts in MIA PaCa-2 cells using the CCK-8 assay. Caspase 3/7-mediated apoptosis and morphological changes of cells were also witnessed in MIA PaCa-2 cells after 24 h of treatment with the extracts. This study highlighted the significance of E. microcorys as an important source of phytochemicals with efficacy against pancreatic cancer cells. Further studies are warranted to purify and structurally identify individual compounds and elucidate their mechanisms of action for the development of more potent and specific chemotherapeutic agents for pancreatic cancer.

Appetite regulating factors in dourado, Salminus brasiliensis: cDNA cloning and effects of fasting and feeding on gene expression.

The dourado, Salminus brasiliensis (Cuvier, 1816) is a freshwater piscivorous Characin native to South American rivers. Owing to the high quality of its flesh and its fast growth, it is the object of both capture fisheries and fish farming. However, very little is known about the endocrine regulation of feeding and metabolism of dourado. In this study, cDNAs for orexin, CART and CCK were isolated in dourado, and their mRNA tissue distributions examined. In order to assess the role of these peptides in the regulation of feeding of dourado, the effects of fasting and feeding on mRNA expression levels of orexin, CART and CCK in the brain as well as CCK in the intestine were assessed. Whereas orexin and CCK have widespread mRNA distributions in the brain and peripheral organs, CART seems to be mostly limited to the brain. Orexin brain expression increased with fasting and displayed periprandial changes, suggesting it is involved in both long- and short-term regulation of feeding and appetite. CART and CCK hypothalamic expressions were not affected by fasting, but displayed periprandial changes with post-feeding decreases, suggesting roles in short-term satiation. CCK expression in the anterior intestine was not affected by fasting and did not display periprandial changes. Overall, our results suggest that orexin, CART and CCK are involved in the physiology of feeding of dourado.

Preclinical pharmacokinetics, biodistribution, radiation dosimetry and toxicity studies required for regulatory approval of a phase I clinical trial with (111)In-CP04 in medullary thyroid carcinoma patients.

INTRODUCTION: From a series of radiolabelled cholecystokinin (CCK) and gastrin analogues, (111)In-CP04 ((111)In-DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2) was selected for further translation as a diagnostic radiopharmaceutical towards a first-in-man study in patients with medullary thyroid carcinoma (MTC). A freeze-dried kit formulation for multicentre application has been developed. We herein report on biosafety, in vivo stability, biodistribution and dosimetry aspects of (111)In-CP04 in animal models, essential for the regulatory approval of the clinical trial. MATERIALS AND METHODS: Acute and extended single dose toxicity of CP04 was tested in rodents, while the in vivo stability of (111)In-CP04 was assessed by HPLC analysis of mouse blood samples. The biodistribution of (111)In-CP04 prepared from a freeze-dried kit was studied in SCID mice bearing double A431-CCK2R(±) xenografts at 1, 4 and 24h pi. Further 4-h animal groups were either additionally treated with the plasma expander gelofusine or injected with (111)In-CP04 prepared by wet-labelling. Pharmacokinetics in healthy mice included the 30min, 1, 4, 24, 48 and 72h time points pi. Dosimetric calculations were based on extrapolation of mice data to humans adopting two scaling models. RESULTS: CP04 was well-tolerated by both mice and rats, with an LD50>178.5µg/kg body weight for mice and a NOAEL (no-observed-adverse-effect-level) of 89µg/kg body weight for rats. After labelling, (111)In-CP04 remained >70% intact in peripheral mouse blood at 5min pi. The uptake of (111)In-CP04 prepared from the freeze-dried kit and by wet-labelling were comparable in the A431-CCK2R(+)-xenografts (9.24±1.35%ID/g and 8.49±0.39%ID/g, respectively; P>0.05). Gelofusine-treated mice exhibited significantly reduced kidneys values (1.69±0.15%ID/g vs. 5.55±0.94%ID/g in controls, P<0.001). Dosimetry data revealed very comparable effective tumour doses for the two scaling models applied, of 0.045 and 0.044mSv/MBq. CONCLUSION: The present study has provided convincing toxicology, biodistribution and dosimetry data for prompt implementation of the freeze-dried kit formulation without or with gelofusine administration in a multicentre clinical trial in MTC patients.