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Objective To investigate the effect of ginsenoside Rg3 on mouse neural stem cells (NSCs) proliferation in vitro．Methods Fetal cortices of embryonic 14d (E14) C57BL/6 mouse were isolated for culturing primary NSCs．After being passaged twice， NSCs specific antibodies， Nestin and Sox2 were identified by immunocyto chemistry staining. CCK-8 assay and BrdU labelling assay were used to measure NSCs proliferation in response to different ginsenoside Rg3 concentrations (20 nM，50 nM，250 nMand 500 nM) . Results Primary and passaged NSCs were successfully cultured and almost all the cells were both Nestin and Sox2 positive NSCs， which indicated high purity of NSCs. BrdU labelling assay showed the group of 50 nM Rg3 had the highest relative ratio of BrdU positive percentage， and the difference with other groups was statistically significant (P<0.01) ． CCK-8 assay also showed 50 nM Rg3 group had the highest OD value， which was significantly higher than the control group (P<0.01)，20 nMgroup (P<0.05) and 500 nMgroup (P<0.01)， while other groups had no significant difference between each other(P>0.05) . Conclusion 50 nM ginsenoside Rg3 could significantly promote mouse NSCs proliferation in vitro. [ABSTRACT FROM AUTHOR]

For the development of potential anti-prostate cancer agents, 24 kinds of novel naftopidil-based arylpiperazine derivatives have been synthesized and characterized by spectroscopic methods. Their antitumor activities were evaluated against several classical prostate cancer cell lines including PC-3, LNCaP, and DU145. Among all the compounds, 9 , 13 , 17 , 21 and 27 showed strong cytotoxic activities against DU145 cells (IC 50  < 1 μM). Further testing confirmed that compound 17 inhibited the growth of DU145 cells by inducing cell cycle arrest at G0/G1 phase. Besides, antagonistic activities of compounds ( 9 , 13 , 17 , 21 and 27 ) towards a 1 -ARs (α 1A , α 1B , and α 1D ) were further evaluated using dual-luciferase reporter assays, and the compounds 13 and 17 exhibited better a 1 -ARs subtype selectivity. The structure–activity relationship (SAR) of these developed arylpiperazine derivatives was rationally discussed. Taken together, these results suggested that further development of such compounds may be of great interest. [ABSTRACT FROM AUTHOR]

BACKGROUND: Methylene blue is used as a developer to identify intervertebral disc degeneration in the transforaminal endoscopic surgery. However, many scholars have indicated that methylene blue can accelerate the degeneration process, whilst foreign researches have reported that it may play therapeutic effect on degenerative intervertebral discs under acidic conditions due to its acidophily. Therefore, whether methylene blue holds toxic effect on the disc remains controversial. OBJECTIVE: To determine whether methylene blue exerts toxic effect on nucleus pulposus cells by cell counting-kit 8 (CCK-8) assay. METHODS: The discarded nucleus pulposus from two patients with intervertebral disc herniation were selected. After digestion, nucleus pulposus cells were extracted and cultured until proliferated to 80% of the medium. Then, the cells were digested to make cell suspensions, divided into six groups and inoculated into the 96-well plates: blank control (only the medium, CCK-8 solution), control group (only medium, cells and CCK-8 solution), and the other groups were cultured with 1%, 2%, 3%, and 4% methylene blue, respectively. The absorbance values were measured by CCK-8 assay at 24, 48, 72 and 96 hours after incubation to calculate the cell viability, and the color change was observed. RESULTS AND CONCLUSION: The color in the control group was the deepest, and the color became lighter with the concentration of methylene blue increasing. The cell viability was the highest in the control group, and it was decreased with the concentration of methylene blue increasing. Thus, methylene blue may exert toxic effect on human nucleus pulposus cells. [ABSTRACT FROM AUTHOR]

A series of novel arylpiperazine derivatives was synthesized. The in vitro cytotoxic activities of all synthesized compounds against three human prostate cancer cell lines (PC-3, LNCaP, and DU145) were evaluated by a CCK-8 assay. Compounds 9 and 15 exhibited strong cytotoxic activities against LNCaP cells (IC50 < 5 μM), and compound 8 (IC50 = 8.25 μM) possessed the most potent activity against DU145 cells. However, these compounds also exhibited cytotoxicity towards human epithelial prostate normal cells RWPE-1. The structure–activity relationship (SAR) of these arylpiperazine derivatives was also discussed based on the obtained experimental data.

Prostate cancer is a major public health problem worldwide. For the development of potential anti-prostate cancer agents, a series of novel arylpiperazine derivatives containing the saccharin moiety based on previous studies was designed, synthesized, and evaluated in prostate (PC-3, LNCaP, and DU145) cancer cell lines for their anticancer activities. The majority of the compounds exhibited excellent selective activity for the tested cancer cells. Compounds 4 and 12 exhibited strong cytotoxic activities against DU145 cells (half maximal inhibitory concentration (IC50) < 2 μM). The structure-activity relationship (SAR) of these arylpiperazine derivatives was also discussed based on the obtained experimental data. This work provides a potential lead compound for anticancer agent development focusing on prostate cancer therapy. [ABSTRACT FROM AUTHOR]

Background: CCK1 (cholecystokinin receptor 1) and protein lysine acetylation were associated with several cancers, respectively. However, whether they are involved in the alternation of gallbladder carcinoma is unknown. Aims: This study investigated the characteristics of CCK1 and protein lysine acetylation in GBC-SD cells and carcinoma of gallbladder. Methods: The expression and localization of CCK1 were detected by western blot analysis and indirect immunofluorescence. GBC-SD cells were treated with CCK-8 and CCK-8+CCK1 inhibitor. The protein lysine acetylation from cells, as well as tissues of gallbladder carcinoma, was examined by western blotting. Results: CCK1 receptor was expressed and localized in the GBC-SD cells. The synthetic octapeptide of CCK (CCK-8) could accelerate the lysine acetylation of a subset of proteins in dose-dependent manners in GBC-SD cells. Further investigation demonstrated that the specific inhibitor (CR1409) of CCK1 receptor could attenuate the CCK8-induced increase of protein lysine acetylation. In addition, we revealed that the rise of CCK1 receptor expression is associated with the increase of protein lysine acetylation in tissues from carcinoma of gallbladder. Conclusions: CCK might regulate protein lysine acetylation via CCK1 receptor. [ABSTRACT FROM AUTHOR]

BACKGROUND: Under ischemia/hypoxia microenvironment, very low survival rate of transplanted bone marrow mesenchymal stem cells (BMSCs) in the host limits its efficacy in the treatment of acute myocardial infarction. OBJECTIVE: To explore the tolerance of human heme oxygenase 1 (hHO-1) gene modified rat BMSCs to ischemia/hypoxia injury. METHODS: The rat BMSCs were transfected with hHO-1 recombinant adenovirus. Western blot assay was used to determinate the optimal time of hHO-1 protein expression. hHO-1 modified rat BMSCs were cultured in hypoxia and serum-free conditions that simulated ischemia/hypoxia microenvironment in vivo. Cell counting kit-8 and trypan blue staining were performed to detect the survival rate of BMSCs at 12, 24, 48, 72 hours after culture under the ischemia/hypoxia microenvironment. Flow cytometry was used to detect apoptosis in BMSCs at 24 hours after culture under the ischemia/hypoxia microenvironment. RESULTS AND CONCLUSION: The expression of hHO-1 protein was highest at 4 days after transfection. Under the ischemia/hypoxia microenvironment for 12, 24, 48, 72 hours, the survival rates of transfected BMSCs were significantly higher as compared with the untransfected cells (P < 0.05), shown by the cell counting kit-8 and trypan blue staining. In addition, the results from flow cytometry showed that there was a higher survival rate of transfected BMSCs than untransfected cells at 24 hours of culture under the ischemia/hypoxia microenvironment (P < 0.05). To conclude, hHO-1 modified rat BMSCs have stronger tolerance to the ischemia/hypoxia microenvironment. [ABSTRACT FROM AUTHOR]

Lenalidomide is a type of immunomodulatory agent with anti-tumor activity by mainly expressed in the anti-angiogenesis. In order to enhance the pharmacological activity of Lenalidomide, a series of Lenalidomide derivatives were designed as tumor angiogenesis inhibitors. The potential anti-angiogenesis targets of Lenalidomide derivatives were virtual screened on Auto-Dock 4.0 by using reverse docking method. The six target proteins, such as vascular endothelial growth factor receptor, epidermal growth factor receptor, fibroblast growth factor receptor, BCR-ABL tyrosine kinase, p38 mitogen activated protein kinase and metal protein kinase, were chosen as the targets. The Lenalidomide derivatives were synthesized by alkylated, acylated or sulfonylated Lenalidomide and verified by the 1 H NMR, 13 C NMR and LC–MS. Their anti-cancer activities were detected by using CCK-8 in the esophageal carcinoma cell line EC9706. The results indicate that the inhibitory activities of Lenalidomide derivatives were higher than that of Lenalidomide. [ABSTRACT FROM AUTHOR]

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 GI 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 IC 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. [ABSTRACT FROM AUTHOR]

BACKGROUND: Macropore morphology of a composite scaffold prepared by the three-dimensional printing technique is of great importance in determining the physicochemical and biological properties of tissue engineering scaffolds. OBJECTIVE: To fabricate strontium-containing mesoporous (Sr-MBG) bioactive glass (PCL) scaffolds by the three-dimensional printing technique, and to explore the effect of these scaffolds on MC3T3-E1 proliferation and osteogenic differentiation, thereby to find out the optimal macropore morphology. METHODS: Sr-MBG/PCL composite scaffolds were fabricated by the three-dimensional printing technique. The angles between fibrous latitudes and longitudes were set to 45°, 60° and 90°. Then the proliferation and alkaline phosphatase activity of MC3T3-E1 cells on the scaffolds were tested. RESULTS AND CONCLUSION: Cell counting kit-8 results showed that MC3T3-E1 cells could proliferate on all the three kinds of scaffolds. The proliferation rate of MC3T3-E1 cells on the 45° Sr-MBG/PCL scaffolds was just slightly higher than that on the 60° and 90° Sr-MBG/PCL scaffolds at days 1 and 4 (P > 0.05), but there was a significant increase at day 7 (P < 0.05). The 45° Sr-MBG/PCL scaffolds exhibited a significant increase in alkaline phosphatase activity of MC3T3-E1 cells compared to the 60° and 90° Sr-MBG/PCL scaffolds at day 14 (P < 0.05), while there was no significant difference among three groups at day 21 (P > 0.05). These results indicate that the 45° Sr-MBG/PCL scaffold is more suitable to promote the proliferation and osteogenic differentiation of the MC3T3 cells than the 60° and 90° Sr-MBG/PCL scaffolds. [ABSTRACT FROM AUTHOR]

A growing body of evidence suggests that anesthetics impact the outcome of patients with cancer after surgical intervention. However, the optimal dose and underlying mechanisms of co-administered anesthetics in lung tumor therapy have been poorly studied. Here, we aimed to investigate the role of combined anesthetics propofol, sufentanil, and rocuronium in treating lung cancer using an orthogonal experimental design and to explore the optimal combination of anesthetics. First, we evaluated the effects of the three anesthetics on the proliferation and invasion of A-549 cells using Cell Counting Kit 8 and Transwell migration and invasion assays. Subsequently, we applied the orthogonal experimental design (OED) method to screen the appropriate concentrations of the combined anesthetics with the most effective antitumor activity. We found that all three agents inhibited the proliferation of A-549 cells in a dose- and time-dependent manner when applied individually or in combination, with the highest differences in the magnitude of inhibition occurring 24 h after combined drug exposure. The optimal combination of the three anesthetics that achieved the strongest reduction in cell viability was 1.4 µmol/L propofol, 2 nmol/L sufentanil, and 7.83 µmol/L rocuronium. This optimal 3-drug combination produced a more beneficial result at 24 h than either single drug. Our results provide a theoretical basis for improving the efficacy of lung tumor treatment and optimizing anesthetic strategies., Competing Interests: The authors declare there are no competing interests., (©2023 Tan et al.)

As a powerful addictive psychostimulant drug, coupled with its neurotoxicity, methamphetamine (METH) abuse may lead to long-lasting abnormalities in brain structure and function. We found that pretreatment of cholecystokinin-8 (CCK-8) inhibited METH-induced brain cellular dopaminergic (DA) damage in the striatum and substantia nigra, and related behavioural deficits and hyperthermia. However, the mechanism of CCK-8 action on METH-induced toxicity is not clear. The aim of this study was to explore whether the possible protective effect of CCK-8 on METH-induced neurotoxicity involved anti-oxidative stress mechanisms. The subtypes of CCK receptors mediating the regulatory action of CCK-8 were also investigated. The present results revealed that CCK-8 dose-dependently inhibited METH-induced cytotoxic effect by activating the CCK2 receptor subtype in PC12 cells and CCK2 receptor stable transfected-HEK293 cells. Pre-treatment of CCK-8 before METH stimulation significantly attenuated the generation of reactive oxygen species and NADPH oxidase activation in PC12 cells. In conclusion, our study demonstrated a protective effect of CCK-8 on METH-induced neurotoxicity in vitro and suggested that a possible mechanism of this action was dependent on the activation of the CCK2 receptor to reduce the neurotoxicity and oxidative stress induced by METH stimulation. [ABSTRACT FROM AUTHOR]

Food intake occurs in bouts or meals, and numerous meal-generated signals have been identified that act to limit the size of ongoing meals. Hormones such as cholecystokinin (CCK) are secreted from the intestine as ingested food is being processed, and in addition to aiding the digestive process, they provide a signal to the brain that contributes to satiation, limiting the size of the meal. The potency of CCK to elicit satiation is enhanced by elevated levels of adiposity signals such as insulin. In the present experiments we asked whether CCK and insulin interact at the level of the blood-brain barrier (BBB). We first isolated rat brain capillary endothelial cells that comprise the BBB and found that they express the mRNA for both the CCK1R and the insulin receptor, providing a basis for a possible interaction. We then administered insulin intraperitoneally to another group of rats and 15 min later administered CCK-8 intraperitoneally to half of those rats. After another 15 min, CSF and blood samples were obtained and assayed for immunoreactive insulin. Plasma insulin was comparably elevated above baseline in both the CCK-8 and control groups, indicating that the CCK had no effect on circulating insulin levels given these parameters. In contrast, rats administered CCK had CSF-insulin levels that were more than twice as high as those of control rats. We conclude that circulating CCK greatly facilitates the transport of insulin into the brain, likely by acting directly at the BBB. These findings imply that in circumstances in which the plasma levels of both CCK and insulin are elevated, such as during and soon after meals, satiation is likely to be due, in part, to this newly-discovered synergy between CCK and insulin. [ABSTRACT FROM AUTHOR]

Peripheral anorectic hormones, such as glucagon-like peptide (GLP)-1, cholecystokinin (CCK)-8 and leptin, suppress food intake. The newly-identified anorectic neuropeptide, nesfatin-1, is synthesised in both peripheral tissues and the central nervous system, particularly by various nuclei in the hypothalamus and brainstem. In the present study, we examined the effects of i.p. administration of GLP-1 and CCK-8 and co-administrations of GLP-1 and leptin at subthreshold doses as confirmed by measurement of food intake, on nesfatin-1-immunoreactive (-IR) neurones in the hypothalamus and brainstem of rats by Fos immunohistochemistry. Intraperitoneal administration of GLP-1 (100 µg/kg) caused significant increases in the number of nesfatin-1-IR neurones expressing Fos-immunoreactivity in the supraoptic nucleus (SON), the area postrema (AP) and the nucleus tractus solitarii (NTS) but not in the paraventricular nucleus (PVN), the arcuate nucleus (ARC) or the lateral hypothalamic area (LHA). On the other hand, i.p. administration of CCK-8 (50 µg/kg) resulted in marked increases in the number of nesfatin-1-IR neurones expressing Fos-immunoreactivity in the SON, PVN, AP and NTS but not in the ARC or LHA. No differences in the percentage of nesfatin-1-IR neurones expressing Fos-immunoreactivity in the nuclei of the hypothalamus and brainstem were observed between rats treated with saline, GLP-1 (33 µg/kg) or leptin. However, co-administration of GLP-1 (33 µg/kg) and leptin resulted in significant increases in the number of nesfatin-1-IR neurones expressing Fos-immunoreactivity in the AP and the NTS. Furthermore, decreased food intake induced by GLP-1, CCK-8 and leptin was attenuated significantly by pretreatment with i.c.v. administration of antisense nesfatin-1. These results indicate that nesfatin-1-expressing neurones in the brainstem may play an important role in sensing peripheral levels of GLP-1 and leptin in addition to CCK-8, and also suppress food intake in rats. [ABSTRACT FROM AUTHOR]

Objective: To investigate how microRNA-190 (miR-190) regulates FOXP2 genes in gastric cancer (GC) cell line SGC7901. Methods: We identified that miR-190 could target FOXP2 genes by using dual luciferase enzyme assay. Precursor fragment transfection of miR-190 was performed with GC cell line SGC7901 and human gastric mucosal cell line GES-1. miR-190 expression was detected by reverse transcription-polymerase chain reaction (RT-PCR) and FOXP2 protein expression was measured by Western blotting. Results: FOXP2-3'-untranslated region (UTR) in miR-190 transfection group was significantly decreased as compared with other groups. There were no significant differences in fluorescence signals of FOXP2mut-3'-UTR in each group. Therefore, it was assumed that miR-190 can target FOXP2 genes. Through RT-PCR verification, it was observed that the expression level of miR-190 was significantly higher in GC cell line SGC7901 than in human gastric mucosa cell line GES-1 after transfection with miR-190 mimics. The expression level of miR-190 was significantly higher in GES-1 cells than in SGC7901 cells after transfection with miR-190 inhibitors. Western blotting results showed the expression level of FOXP2 was significantly lower in GC cell line SGC7901 than in GES-1 cells. Compared with blank, mimics control, and inhibitors control groups, the miR-190 mimics group showed significantly enhanced proliferation, migration, and invasion abilities, while miR-190 inhibitors group showed decreased abilities toward proliferation, migration, and invasion (P<0.05). The transcription level of miR-190 and the expression level of FOXP2 in tumor tissues and adjacent normal tissues in GC patients were verified to be consistent with those of cell line experiments. Conclusion: Upregulation of miR-190 can lead to downregulation of FOXP2 protein expression. miR-190 may serve as a potential target for GC diagnosis. [ABSTRACT FROM AUTHOR]

This study aimed to determine the effects of different concentrations of soluble beta-amyloid 25–35 (Aβ25–35) on cell viability, calcium overload, and PI3K-p85 expression in cultured cortical rat neurons. Primary cultured cerebral cortical neurons of newborn rats were divided randomly into six groups. Five groups were treated with soluble Aβ25–35 at concentrations of 10 nmol/L, 100 nmol/L, 1 μmol/L, 10 μmol/L, or 30 μmol/L. Cell Counting Kit-8 staining was used to measure cell viability, laser-scanning confocal imaging was used to detect changes in intracellular free calcium concentration, and western blot assay was used to measure neuronal PI3K-p85 expression. Soluble Aβ25–35 was found to reduce cell viability and induce calcium overload in primary cultured rat cerebral cortical neurons, in a concentration-dependent manner. At certain concentrations, soluble Aβ25–35 also increased neuronal PI3K-p85 expression. These findings reveal that soluble Aβ25–35 reduces the viability of cultured cerebral cortical rat neurons. The neurotoxicity mechanism may involve calcium overload and disruption of insulin signal transduction pathways. [ABSTRACT FROM AUTHOR]

Pests have been subjected to heavy selection pressure, and the development of resistance to pyrethroid has been recorded. It is extremely valuable to identify the resistance genes that are relevant for pest control. In our previous studies, we reported that UBL40 is the deltamethrin resistance-associated gene. UBL40 is cleaved by specific endopeptidases to release UB and L40. Whether UB or L40 participates in deltamethrin resistance requires further study. In this study, quantitative real-time PCR was applied to reveal that UB and L40 were both overexpressed in Drosophila kc cells after deltamethrin stimulation. To investigate the roles of UB and L40 further, RNA interferences (RNAi) and cell transfections were utilized. UB and L40 knockdown both significantly reduced the level of resistance of RNAi-treated cells after 48 h, and the overexpressions of UB and L40 in Drosophila kc cells conferred a degree of protection against deltamethrin. These results represent the first evidence that UB and L40 both play roles in the regulation of deltamethrin resistance and that this study could help us to elucidate the resistance mechanisms and identify new target genes associated with resistance. [ABSTRACT FROM AUTHOR]

Background: Accumulating evidence indicates that the occurrence and development of tumors are related to the activation of oncogenes and the inactivation of tumor suppressor genes caused by epigenetic mechanisms. However, the function of serine protease 2 (PRSS2) in gastric cancer (GC) is still unknown. Our study aimed to find a regulation network involved in GC., Methods: The mRNA data (GSE158662 and GSE194261) of GC and normal tissues were downloaded from the Gene Expression Omnibus (GEO) dataset. Differential expression analysis was performed using R software, and Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was conducted by using Xiantao software. Besides, we used Quantitative Real-time PCR (qPCR) to verify our conclusions. After gene knockdown, cell migration and CCK-8 experiment were carried out to detect the effect of gene on cell proliferation and invasion., Results: Totally, 412 differentially expressed genes (DEGs) were identified from GSE158662 and 94 DEGs were identified from GSE196261. Km-plot database results indicated that PRSS2 exhibited high diagnosis worth for GC. Gene functional annotation enrichment analysis revealed that these hub mRNAs were mainly taken part in the process of tumorigenesis and development. Besides, vitro experiments showed that down-regulation of PRSS2 gene reduced the proliferation and invasion ability of GC cells., Conclusions: Our results indicated that PRSS2 may play vital roles in the carcinogenesis and progression of GC and can be potential biomarkers for patients with GC.

Objective: This study was to investigate the clinical significance of miR-551b-5p and SETD2 in thyroid cancers (TC) and their effects on the biological function of TC cells., Methods: The expression level of miR-551b-5p and SETD2 in tumor/nontumor tissues and TC cell lines was measured by quantitative real-time polymerase chain reaction (RT-qPCR). Subsequently, the relationship between miR-551b-5p or SETD2 expression and the clinicopathological feature was detected by Chi-square analysis. Kaplan-Meier and multivariate Cox regression analyses were used to assess their prognostic values. Finally, the regulatory effects of miR-551b-5p and SETD2 on the proliferation, migration and invasion ability of TC cells were detected by CCK-8 and Transwell assays., Results: Compared with non-tumor groups, the expression of miR-551b-5p was significantly increased in patients' tissues and TC cell lines, while SETD2 mRNA expression was decreased. Patients with up-regulated miR-551b-5p or downregulated SETD2 mRNA in TC showed more positive lymph node metastasis and advanced TNM stage. High miR-551b-5p expression level and low SETD2 mRNA level were related to poor survival rate. miR-551b-5p and SETD2 might be potential prognostic biomarkers for TC. miR-551b-5p knockdown can inhibit cell proliferation, migration and invasion by targeting SETD2., Conclusion: miR-551b-5p and SETD2 may be valuable prognostic biomarkers and new therapeutic targets for TC., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background Retinal ganglion cells (RGCs) are commonly experienced optic nerve diseases including glaucoma-induced injury that results in decrease of cell survival. However, the underlying mechanism remains to be elaborated. This present study was to focus on the miR-187 and Transforming growth factor-β (TGF-β) signal and investigated their roles in RGCs apoptosis and proliferation. Methods RGC-5 retinal ganglion cell line was chose in present study and subjected to miR-187 mimic or inhibitor transfection. Cell apoptosis was evaluated using flow cytometry-based Annexin V-PI assay. Cell proliferation was examined using CCK-8. Protein levels of Smad2/3/7 were determined using western blotting. Results miR-187 negatively regulated cell survival via inhibiting cell apoptosis and promoting cell proliferation. We observed that alteration expression of miR-187 is closely related to phosphorylation levels of Smad2 and Smad3. This correlation is associated with down-regulation of Smad7 induced by miR-187 via targeting Smad7 3’-UTR. From result of co-transfection of Smad7-plasmid and miR-187 mimic or siSmad7 and miR-187 inhibitor, we concluded that cell proliferation and apoptosis was mediated by miR-187/Smad7 axis. Conclusion In summary, cell internal signal transduction, miR-187 regulating Smad7 expression, plays a vital role in retinal ganglion cell survival. [ABSTRACT FROM AUTHOR]

Cholecystokinin (CCK) is a well-known gut hormone that shows anorexigenic effects via action at peripheral and central receptors. CCK is also widely distributed throughout the mammalian brain and appears to function as a neurotransmitter and neuromodulator. The area postrema is one of the circumventricular organs, located on the dorsal surface of the medulla oblongata at the caudal end of the fourth ventricle. Blood vessels in the area postrema lack a blood brain barrier, offering specific central neural elements unique access to circulating substances. Immunohistochemical studies show CCK-A receptors in the area postrema, and we reported CCK-sensitive area postrema neurons. However, the receptive mechanism of CCK in area postrema neurons still remains unexplained. We investigated the responses of area postrema neurons to agonists and antagonists of CCK receptors using whole cell and perforated patch-clamp recordings in rat brain slices. The application of CCK-8 elicited excitatory responses, such as increases in the frequency of mEPSCs (miniature excitatory postsynaptic currents), a shift toward larger amplitude mEPSCs, and increases in the frequency of action potentials. These changes were found mostly in cells not displaying the hyperpolarization-activated cation current (Ih), except for small excitatory changes in a minority of Ih-positive neurons. Tonic inward currents or an inhibitory response to CCK-8 were never seen. Analysis of the amplitude of mEPSCs before and after the administration of CCK-8 indicated the responses mediated via the presynaptic receptors. The effect of CCK-8 was abolished in the presence of CNQX (AMPA type glutamate receptor antagonist). In the presence of lorglumide (a selective CCK-A receptor antagonist), CCK-8-induced excitatory responses were inhibited. No cells responded to the administration of non-sulfated CCK-8 (CCK-8NS, a selective CCK-B receptor agonist). We conclude that CCK-8 exerts its action via presynaptic CCK-A receptors to facilitate glutamate release onto Ih-negative area postrema cells. [ABSTRACT FROM AUTHOR]

Fructus Ligustri Lucidi , fruits of Ligustrum lucidum Ait. (Oleaceae), has the effects of tonifying the liver and the kidney and strengthening the bones and muscles. In ancient times, Fructus Ligustri Lucidi can be prepared in ethanol or in water. Some active compounds have been found in Fructus Ligustri Lucidi , like Oleanolic acid and Ursolic acid, and Ursolic acid were proved to have osteogenic effects. Methods and results To prove that Fructus Ligustri Lucidi water extract have osteogenic effects on MC3T3-E1 cells and how these effects work, we used CCK-8 (cell counting kit-8), ELISA (enzyme-linked immunosorbent assay), FQ-PCR (realtime fluorescence quantitative PCR) and western blot assays. After treatment with Fructus Ligustri Lucidi for 48 h, 72 h, 96 h, the cell viability was marked increased, on concentration-dependently and time-dependently pattern. High and low concentrations of Fructus Ligustri Lucidi promoted differentiation of cells. Fructus Ligustri Lucidi could up-regulate OPG and RANKL protein in supernatant at 48 h and 72 h except for highest concentration (10 −1 mg/ml). Fructus Ligustri Lucidi promote OPG and RANKL mRNA expression at 48 h and 72 h, while the level of promoting at 72 was higher than 48 h. 10 −5 mg/ml of Fructus Ligustri Lucidi up-regulates OPG protein expression and down-regulates RANKL protein expression. After treatment with Fructus Ligustri Lucidi water extract, inhibitors, Fructus Ligustri Lucidi water extract with inhibitors for 72 h, inhibitors PD 98059, SB 203580, SP600125 and LY 294002 showed Fructus Ligustri Lucidi -induced cell proliferation and the leakage of OPG proteins effects. Fructus Ligustri Lucidi promoted the protein levels of ERK, p-ERK, p-JNK, p38, pp38, AKT and p-AKT, and inhibited the protein levels of JNK. Conclusions Fructus Ligustri Lucidi water extract promoted cell proliferation and differentiation, mRNA and protein expression of OPG and RANKL on MC3T3-E1 cells. The effects of cell proliferation and leakage of OPG related to MAPK and AKT signaling pathways in different ways. [ABSTRACT FROM AUTHOR]

We investigated whether pretreatment with the neuropeptide cholecystokinin-8 affected methamphetamine (METH)-induced behavioral changes and dopaminergic neurodegeneration in male C57/BL6 mice. CCK-8 pretreatment alone had no effect on locomotion and stereotypic behavior and could not induce behavioral sensitization; however, it attenuated, in a dose-dependent manner, hyperlocomotion and behavioral sensitization induced by a low dose of METH (1 mg/kg). CCK-8 attenuated METH-induced stereotypic behavior at a dose of 3 mg/kg but not at 10 mg/kg. CCK-8 pretreatment attenuated METH (10 mg/kg)-induced hyperthermia, the decrease of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the striatum, and TH in the substantia nigra. CCK-8 alone had no effect on rectal temperature, TH and DAT expression in the nigrostriatal region. In conclusion, our study demonstrated that pretreatment with CCK-8 inhibited changes typically induced by repeated exposure to METH, such as hyperlocomotion, behavioral sensitization, stereotypic behavior, and dopaminergic neurotoxicity. These findings make CCK-8 a potential therapeutic agent for the treatment of multiple symptoms associated with METH abuse. [ABSTRACT FROM AUTHOR]

Background The viability of periodontal ligament fibroblasts ( PDLF) can affect the long-term prognosis of replanted avulsed teeth. When immediate replantation of an avulsed tooth is not possible, the cells should be incubated in a physiological storage medium instantly to maintain their biological activity. The ability of different storage media to preserve PDLF viability has been previously evaluated. However, few studies have showed the effect of temperature on the viability of PDLF cultured with various storage media in vitro. Material and Methods This study was designed to measure PDLF activity by CCK-8 assay to compare the effectiveness at 4, 22 (room temperature), and 37°C under various storage media. Results Statistical analysis demonstrated that tap water, saline, and saliva decreased cell viability as the storage temperature increased. But the temperature played only a minor role on cell viability when cells were incubated in Hank's balanced salt solution ( HBSS), Dubelco's modified Eagle's medium ( DMEM), or milk. Conclusions Within the parameters of this study, it seems that room temperature is adequate for storing the avulsed teeth in HBSS, DMEM, or milk in the extra-alveolar period. [ABSTRACT FROM AUTHOR]

Graphene oxide (GO) is a hotspot, especially in the field of biomedical. However, the clinical application of GO is still faces a lot of challenges. In order to improve the solubility and biocompatibility of GO, polyethylene glycol (PEG) was grafted on the surface of graphene oxide by amide reaction. PEGylated graphene oxide (PEG-GO) was characterized using Fourier transform infrared spectroscopy (FTIR). The stability of PEG-GO detected in different solutions. Raji cell was selected as a lymphoma cell model to study the cytotoxicity of PEG-GO. Cell viability was detected using the Cell Counting Kit-8 assay. Cells were treated with different concentrations (10-100 µg/mL) of PEG-GO at different time points (6, 12, and 24 h). The FTIR spectrum of PEG-GO indicated that polyethylene glycol was successfully grafted onto GO. PEG-GO had excellent stability in all solutions. Cells treated with PEG-GO (10-100 µg/mL) for 24 hours had survival rates were over 80%. These results demonstrate that PEG-GO had an excellent dispersion in biological solutions and the toxicity of PEG-GO to lymphoma cells was low. The paper may provide cytological evidence for the application of PEG-GO in medicine. [ABSTRACT FROM AUTHOR]

The interaction between oral and gastric signals is an important part of food intake regulation. Previous studies suggest that bypassing oral stimulation diminishes the suppression of hunger and increases gastric emptying rate. However, the role of appetite hormones, like cholecystokinin-8 and ghrelin, in this process is still unclear. Our objective was to determine the contributions of gastric and oral stimulation to subsequent appetite and hormone responses and their effect on ad libitum intake. Fourteen healthy male subjects (age 24.6 ± 3.8y, BMI 22.3 ± 1.6 kg/m 2 ) completed a randomized, single-blinded, cross-over experiment with 3 treatment-sessions: 1) Stomach distention: naso-gastric infusion of 500 mL/0 kJ water, 2) Stomach distention with caloric content: naso-gastric infusion of 500 mL/1770 kJ chocolate milk, and 3) Stomach distention with caloric content and oral exposure: oral administration of 500 mL/1770 kJ chocolate milk. Changes in appetite ratings and plasma glucose, insulin, cholecystokinin-8, and active and total ghrelin concentrations were measured at fixed time-points up to 30 min after infusion or oral administration. Subsequently, subjects consumed an ad libitum buffet meal. Oral administration reduced appetite ratings more than both naso-gastric infusions ( P < 0.0001). Gastric infusion of a caloric load increased insulin and cholecystokinin-8 and decreased total ghrelin concentrations more than ingestion (all P < 0.0001). No differences in active ghrelin response were observed between conditions. Ad libitum intake did not differ between oral and gastric administration of chocolate milk ( P > 0.05). Thus, gastric infusion of nutrients induces greater appetite hormone responses than ingestion does. These data provide novel and additional evidence that bypassing oral stimulation not only affects the appetite profile but also increases anorexigenic hormone responses, probably driven in part by faster gastric emptying. This confirms the idea that learned associations between sensory characteristics and associated metabolic consequences serve to adapt hormone responses to nutrient content. These findings underscore the importance of oral stimulation in the regulation of food intake. [ABSTRACT FROM AUTHOR]

The pathogenesis of acute pancreatitis (AP) is still poorly understood. Thus, a reliable pharmacological therapy is currently lacking. In recent years, an impairment of the energy metabolism of pancreatic acinar cells, caused by Ca2+-mediated depolarization of the inner mitochondrial membrane and a decreased ATP supply, has been implicated as an important pathological event. In this study, we investigated whether quercetin exerts protection against mitochondrial dysfunction. Following treatment with or without quercetin, rat pancreatic acinar cells were stimulated with supramaximal cholecystokinin-8 (CCK). CCK caused a decrease in the mitochondrial membrane potential (MMP) and ATP concentration, whereas the mitochondrial dehydrogenase activity was significantly increased. Quercetin treatment before CCK application exerted no protection on MMP but increased ATP to a normal level, leading to a continuous decrease in the dehydrogenase activity. The protective effect of quercetin on mitochondrial function was accompanied by a reduction in CCK-induced changes to the cell membrane. Concerning the molecular mechanism underlying the protective effect of quercetin, an increased AMP/ATP ratio suggests that the AMP-activated protein kinase system may be activated. In addition, quercetin strongly inhibited CCK-induced trypsin activity. The results indicate that the use of quercetin may be a therapeutic strategy for reducing the severity of AP. [ABSTRACT FROM AUTHOR]

The survivin ( svv) gene is a newly discovered member of the inhibitors of apoptosis gene family. In recent years, svv has been confirmed to have an anti-apoptosis function and to play a critical role in cell division. We identified a survivin-like gene in the silkworm, Bombyx mori ( Bm- svv). In this study, to gain insight into its function, a baculovirus expression system was used to express the Bm- svv gene in insect cell lines. The recombinant viruses were then used as a vector to transform insect cells, and cell activity was determined using the Cell Counting Kit-8 (CCK-8), which is usually employed for detecting mammalian cell number. The results indicated that the Bm- svv gene plays a role in the cell growth arrest or apoptosis induced by viruses. Furthermore, the CCK-8 kit is effective in determining the activity of insect cells. [ABSTRACT FROM AUTHOR]

【目的】体外培养系统性红斑狼疮(SLE)皮损部位角质形成细胞(KC),观察其细胞学特性｡【方法】 原代培养SLE皮损和正常皮肤的角质形成细胞,倒置显微镜观察细胞的形态特点,免疫荧光法进行角蛋白检测并计算细胞纯度;两步消化法纯化细胞;CCK-8(cell counting Kit-8)评价细胞的增殖情况,绘制生长曲线｡流式细胞仪观察细胞的生长周期和凋亡情况｡【结果】使用K-SFM(serum-free keratinocyte medium)无血清培养基能成功体外培养SLE皮损的角质形成细胞,呈铺路石样｡结合人工纯化,能达到 > 95%的纯度;与正常对照相比,SLE皮损角质形成细胞进入指数生长期较迟(7 d vs. 4 d),但进入后增长迅速,S期比例为: (40.68 ± 1.81)% vs. (34.43 ± 1.24)%(P < 0.05);凋亡率高:(6.08 ± 0.72)% vs. (4.32 ± 0.44)%(P < 0.05)｡【结论】 SLE皮损角质形成细胞培养难度大,细胞的生物学行为存在异常,可能为SLE发病的独立､始发因素,其致病机制值得深入研究和探讨｡

• Parkinson's disease is the most common neurodegenerative disorder secondary. • This study aimed to explore the role of circTLK1 in PD pathology. • Targeting circTLK1 may contribute to improving PD therapy. Parkinson's disease (PD) is a neurodegenerative disorder that is featured by the elevated loss of substantia nigra pars compacta dopaminergic neurons and the disruption of motor functions. Aberrant expression of circular RNAs (circRNAs) is correlated with neurodegenerative diseases. This study aimed to explore the role of circTLK1 in PD pathology. MPTP-stimulated in vivo PD mouse model and MPP + and rotenone-induced in vitro PD model were established to investigate the function of circTLK1/miR-26a-5p/DAPK1 axis during dopaminergic neuron injury. The motor function of mice was evaluated by using the Rotarod test. Brain tissue damage was checked by hematoxylin and eosin, TdT-mediated dUTP-biotin nick end labeling. Cell viability, apoptosis, and cytotoxicity were evaluated by cell counting kit 8 (CCK-8), flow cytometry, and LDH activity. The interaction between circTLK1 and miR-26a-5p as well as miR-26a-5p and DAPK1 was detected by luciferase reporter assay. The expression of circTLK1 was notably elevated in in vitro and in vivo PD models. Knockdown of circTLK1 significantly improved cell viability, suppressed apoptosis and cytotoxicity, whereas inhibition of miR-16a-5p and overexpression of DAPK1 abolished these effects. MiR-26a-5p acts as a sponge of DAPK1 to mediate circTLK1 functions. Luciferase reporter gene assay confirmed the interaction between circTLK1 and miR-26a-5p as well as miR-26a-5p and DAPK1. Depletion of circTLK1 mitigates dopaminergic neuron injury in vitro and in vivo , via releasing miR-26a-5p to target DAPK1 expression. Targeting circTLK1 may contribute to improving PD therapy. [ABSTRACT FROM AUTHOR]

Simple Summary: MicroRNAs (miRNAs) play important roles in the development of skeletal muscle. Here, a comparative analysis of four skeletal muscle transcriptomes of cattle, rat, goat, and pig showed that miR-24-3p may conservatively regulate muscle development. There is a large proportion of the target genes of miR-24-3p shared by cattle, rat, goat, and pig. GO (Gene ontology) and KEGG (A Kyoto Encyclopedia of Genes and Genomes) enrichment analysis showed that these genes are enriched in multiple cell functions and signal pathways that are closely related to muscle development. In rat and cattle, a double luciferase test showed that three shared target genes, WNT4 (Wnt Family Member 4), CAMK2B (Calcium/Calmodulin Dependent Protein Kinase II Beta), TCF7 (Transcription Factor 7) were targeted by mmu-miR-24-3p and bta-miR-24-3p. The three shared target genes (WNT4, CAMK2B, and TCF7) were all targets in the Wnt signaling pathway, which plays an important role in muscle proliferation in rat and cattle. The shared target gene (CAMK2B) rat was increased significantly after the inhibition of miR-24-3p in rat and cattle. This study will be a good foundation by which to improve our understanding of the functions of miRNAs in the regulation of muscle development. Skeletal muscle plays an important role in the growth and development of meat animals. MicroRNAs (miRNAs) can participate in the regulation of muscle development-related functions; however, there have been few reports on whether there are related miRNAs that conservatively regulate muscle development among different species. In this study, the miRNA transcriptome sequencing data of the muscle tissue of cattle, rat, goat, and pig showed that miR-24-3p may conservatively regulate muscle development in these species. Furthermore, mmu-miR-24-3p can positively regulate C2C12 cell proliferation and apoptosis by regulating key proliferation and apoptosis genes in muscle development, which was verified by CCK-8 and RT-qPCR. Bta-miR-24-3p can also positively regulate the proliferation and apoptosis of bovine muscle primary cells by regulating key proliferation and apoptosis genes in the process of muscle development, as verified by CCK-8 and RT-qPCR. The target genes of miR-24-3p in cattle, rat, goat, and pig, which include a large proportion of target genes shared among the four species, are enriched in multiple cell functions and signal pathways that are closely related to muscle development, as revealed by GO and KEGG enrichment analysis. A double luciferase test showed that the shared target genes WNT4, CAMK2B, and TCF7 were targeted by mmu-miR-24-3p in rat and bta-miR-24-3p in cattle. These three shared target genes WNT4, CAMK2B, and TCF7 are involved in the Wnt signaling pathway, which showed that miR-24-3p plays an important role in rat and cattle. The shared target gene (CAMK2B) in rat and cattle increased significantly after the inhibition of miR-24-3p by RT-qPCR. The findings of this study contribute to a better understanding of the role of miR-24-3p in the regulation of muscle development. [ABSTRACT FROM AUTHOR]

Abstract: In isolated guinea-pig ileum (GPI), the κ-opioid acute withdrawal response is under the control of several neuronal signaling systems, including the μ-opioid, the A1-adenosine and the CB1 receptors, which are involved in the inhibitory control of the κ-withdrawal response. After κ-opioid system stimulation, indirect activation of μ-opioid, A1-adenosine and CB1 systems is prevented by the peptide cholecystokinin-8 (CCk-8). In the present study, we have investigated whether the NOP system is also involved in the regulation of the acute κ-withdrawal response. Interestingly, we found that in GPI preparation, the NOP system is not indirectly activated by the κ-opioid receptor stimulation, but instead this system is able by itself to directly regulate the acute κ-withdrawal response. Specifically, our results clearly highlight first the existence of an endogenous tone of the NOP system in GPI, and second that it behaves as a functional anti-opioid system. We also found that, the NOP receptor system is involved in the regulation of the CCk-8-induced contracture intensity, only when in the presence of the κ-opioid receptor stimulation. This effect seems to be regulated by an activation threshold mechanism. In conclusion, the NOP system could act as neuromodulatory system, whose action is strictly related to the modulation of both excitatory and inhibitory neurotransmitters released in GPI enteric nervous system. [Copyright &y& Elsevier]

Abstract: Cholecystokinin, a neuroactive peptide functioning as a neurotransmitter and neuromodulator in the central nervous system, mediates a number of processes and is implicated in neurological and psychiatric disorders such as Parkinson''s disease, anxiety and schizophrenia. Striatum is one of the brain structures with the highest concentrations of CCK in the brain, rich in CCK receptors as well. The physiological effect of CCK on cholinergic interneurons, which are the major interneurons in striatum and the modulatory interactions which exist between dopamine, acetylcholine and cholecystokinin in this brain structure are still unclear. We studied the effect of cholecystokinin octapeptide (CCK-8) on the release of acetylcholine (ACh) from striatal slices of the rat brain. CCK-8 (0.01–0.1μM) showed no statistically significant effect on the basal but enhanced dose-dependently the electrically (2Hz)-evoked release of [3H]ACh. When slices were preperfused with 100μM sulpiride, a selective dopamine D2 receptor antagonist, the CCK-8 (0.01μM) effect on electrically stimulated ACh release was increased nearly 2-fold. A similar increase was observed after depletion of endogenous dopamine (DA) from nigro-striatal dopaminergic neurons with 6-hydroxydopamine (6-OHDA) (2× 250μg/animal, i.c.v.). Furthermore in the presence of dopamine (100μM) or apomorphine (10μM), the prototypical DA receptor agonist, CCK-8 (0.01μM) failed to enhance the stimulation-evoked release of [3H]ACh. The D2 receptor agonist quinpirol (1μM) abolished the CCK-8 effect on electrically stimulated ACh release as well. The increase in electrically induced [3H]ACh release produced by 0.01μM CCK-8 was antagonized by d,l loxiglumide (CR 1505), 10μM, a non-peptide CCK-A receptor antagonist and by Suc-Tyr-(OSO3)-Met-Gly-Trp-Met-Asp-β-phenethyl-amide (GE-410), 1μM, a peptide CCK-A receptor antagonist. The antagonistic effect of GE-410 on the CCK-8-potentiated, electrically induced release of [3H]ACh was studied in striatum for the first time. CAM 1028 (10μM), a CCK-B receptor antagonist, also prevented the potentiating effect of CCK-8 (0.01μM) on electrically stimulated release of [3H]ACh. The presented results indicate that (i) CCK-8 is capable of increasing ACh elicited by field electrical stimulation in striatum; (ii) CCK-8 is more effective in its ACh-stimulating effect when dopaminergic activity in striatum is blocked i.e. CCK-8-facilitated release of electrically induced ACh from cholinergic interneurons in the striatum is under the inhibitory control of the tonic activity of dopamine from the nigrostriatal pathway; (iii) the enhancing effect of CCK-8 on electrically evoked ACh release is mediated through both CCK-A and CCK-B cholecystokinin receptors located most likely on the cell bodies of cholinergic interneurons in striatum. [Copyright &y& Elsevier]

Abstract: In earlier studies it has been found that rats respond to intracerebroventricular (i.c.v.) injection of cholecystokinin-octapeptide (CCK-8) with a febrile response characterized by rises of heat production and core temperature together with tail-skin vasoconstriction mediated by CCK2 receptors. Biotelemetric investigations of the same species have additionally shown that CCK-induced fever is accompanied by decreased locomotor activity. Similar data for mice have not been reported so far. In the present studies C57BL/6 mice were infused i.c.v. for 3 days with CCK-8 to see effects on body core temperature, locomotor activity, food intake and body weight. Biotelemetric monitoring disclosed a rise in daylight core temperature and a fall of night-time locomotor activity both lasting beyond the time of i.c.v. infusions. Food intake was suppressed only during infusion, while a significant decrease of body weight was sustained after the end of CCK-8 infusion. It is concluded that similar to rats mice also respond to i.c.v. infusion of CCK-8 with a fever-like (regulated) hyperthermia and some components of sickness behavior as measured by biotelemetry, and thus a CCK-mediated mechanism may contribute to fever genesis also in mice. [Copyright &y& Elsevier]

Abstract: Camostat mesilate (or mesylate) releases endogenous cholecystokinin (CCK) or CCK-58, the only detectable endocrine form of CCK in the rat, and reduces cumulative food intake by activating CCK1 receptor. However, the literature lacks meal pattern analysis and an appropriate dose–response curve for this peptide. Therefore, the current study determines meal size (MS), intermeal interval (IMI) and satiety ratio (SR) by orogastric gavage of camostat (0, 12.5, 25, 50, 100, 200, 300, 400, 800mg/kg) and compares them to those previously reported by a single dose of CCK-8 (1nmol/kg, i.p), the most utilized form of CCK. We found that camostat (200, 300, 400 and 800mg/kg) and CCK-8 reduced cumulative food intake and the size of the first meal, but only camostat prolonged IMI and increased SR. There was no change in the duration of the first two meals or in rated behaviors such as feeding, grooming, standing and resting in response to camostat and CCK-8, but there was more resting during the IMI in response to camostat. This study provides meal pattern analysis and an appropriate dose–response curve for camostat and CCK-8. Camostat reduces food intake by decreasing MS and prolonging IMI, whereas CCK-8 reduces food intake by reducing only meal size. [Copyright &y& Elsevier]

Abstract: The Kv4 potassium channel α subunits, Kv4.1, Kv4.2, and Kv4.3, determine some of the fundamental physiological properties of neurons in the CNS. Kv4 subunits are associated with auxiliary β-subunits, such as the potassium channel interacting proteins (KChIP1 - 4), which are thought to regulate the trafficking and gating of native Kv4 potassium channels. Intriguingly, KChIP1 is thought to show cell type-selective expression in GABA-ergic inhibitory interneurons, while other β-subunits (KChIP2-4) are associated with principal glutamatergic neurons. However, nothing is known about the expression of Kv4 family α- and β-subunits in specific interneurons populations in the BLA. Here, we have used immunofluorescence, co-immunoprecipitation, and Western Blotting to determine the relative expression of KChIP1 in the different interneuron subtypes within the BLA, and its co-localization with one or more of the Kv4 α subunits. We show that all three α-subunits of Kv4 potassium channel are found in rat BLA neurons, and that the immunoreactivity of KChIP1 closely resembles that of Kv4.3. Indeed, Kv4.3 showed almost complete co-localization with KChIP1 in the soma and dendrites of a distinct subpopulation of BLA neurons. Dual-immunofluorescence studies revealed this to be in BLA interneurons immunoreactive for parvalbumin, cholecystokin-8, and somatostatin. Finally, co-immunoprecipitation studies showed that KChIP1 was associated with all three Kv4 α subunits. Together our results suggest that KChIP1 is selectively expressed in BLA interneurons where it may function to regulate the activity of A-type potassium channels. Hence, KChIP1 might be considered as a cell type-specific regulator of GABAergic inhibitory circuits in the BLA. [ABSTRACT FROM AUTHOR]

As an important cellular energy regulation kinase, AMP-activated protein kinase (AMPK) has been demonstrated as a key molecule in the development of tolerance to nutrient starvation. Activation of AMPK includes the phosphorylation of Thr172 of the α-subunit. Nerve growth factor (NGF) was originally isolated for its ability to stimulate both survival and differentiation in peripheral neurons, but many investigations have shown that the NGF also plays an important role in survival, growth and invasion of many human cancers. In this study, we used CCK-8 cell viability assay to find that NGF could facilitate the viability of HeLa cells following glucose deprivation while not in glucose-normal control groups. This effect of NGF-induced viability promotion to glucose starvation can be suppressed by Compound C, a specific inhibitor of AMPK. Meanwhile, western blot analysis showed that AMPKα1/α2 Thr172 phosphorylation level in HeLa cells was up-regulated after NGF treatment under glucose starvation, and Compound C was able to reduce the AMPKα1/α2 Thr172 phosphorylation level which was up-regulated by NGF in HeLa cells. Taken together, these results indicate that AMP-activated protein kinase supports the NGF-induced viability of human HeLa cells to glucose starvation. [ABSTRACT FROM AUTHOR]

PURPOSE: To investigate the cytotoxicity in vitro of three kinds of dental ceramic alloys on L929 mouse fibroblasts. METHODS: CCK-8 assay was a quick method to study the cell relative proliferation rate or cytotoxicity. L929 mouse fibroblasts were used to detect the cell relative proliferation rate of the three kinds of dental ceramic alloys (NiCr,CoCr and golden alloys containing 58% of gold) by CCK-8 assay. The DNA damage of L929 mouse fibroblasts was determined by single cell gel electrophoresis (SCGE). The data was statistically analyzed with SAS 9.0 software package. RESULTS: The relative growth rate induced by NiCr, CoCr and golden dental ceramic alloys was (75.9510±7.6244)%, (84.8920±8.2660)% and (88.5420+12.3611)%, respectively. NiCr ceramic alloys caused the most marked cytotoxicity(P< 0.05), and there was no difference between CoCr ceramic alloys and 58% golden ceramic alloys(P>0.05). The cytotoxicity of the three kinds of metal ceramic alloys was all grade 1, i.e. mild cytotoxicity. The DNA damage of L929 mouse fibroblasts induced by NiCr ceramic alloys was most serious (P<0.05). There was no difference in the DNA damage between CoCr ceramic alloys and 58% golden ceramic alloys (P>0.05). CONCLUSIONS: NiCr ceramic alloys can suppress the proliferation of the L929 mouse fibroblasts and promote DNA damage. While CoCr ceramic alloys which are similar to 58% golden ceramic alloys show less cytotoxicity than NiCr ceramic alloys. CoCr or 58% golden ceramic alloys should be used as crown and bridge for patients instead of NiCr ceramic alloy clinically. Supported by National Natural Science Foundation of China(Grant No.30970726). [ABSTRACT FROM AUTHOR]

Abstract: The proteosome inhibitor bortezomib is used in the treatment of patients with multiple myeloma. Proteosomes are responsible for the degradation of I-κB, the inhibitory protein of transcription factor nuclear factor kappa B (Nf-κB). The heat shock protein (HSP) inducing effect of bortezomib is also documented. The aim of our work was to test the anti-inflammatory effect of bortezomib in cholecystokinin-octapeptide (CCK-8)-induced acute pancreatitis. Male Wistar rats were divided into three groups (n =8 in each). Group P received an i.p. injection of physiological saline (p.s.) 60 min. before the induction of acute pancreatitis by three hourly s.c. injections of 100 µg/kg CCK-8. Group BP received 1 mg/kg bortezomib dissolved in p.s. 1 h previous to pancreatitis induction. Group C was treated with the vehicle (p.s.). Animals were exsanguinated 4 h after the last injection of CCK-8. Bortezomib pre-treatment significantly reduced the pancreatic weight/body weight ratio, and improved the histology by decreasing the extent of vacuolization and infiltration. Bortezomib pre-treatment inhibited I-κBβ degradation, and induced the synthesis of HSP72. The results confirmed the anti-inflammatory effect of bortezomib in acute experimental pancreatitis. This effect of the drug is presumably mediated by the inhibition of Nf-κB activation and induction of HSP synthesis. [Copyright &y& Elsevier]

Abstract: In previous studies we showed that higher viscosity resulted in lower ad libitum intake and that eating rate is an important factor. In this study we aimed to explore the effect of viscosity on the gastro-intestinal hormones ghrelin, CCK-8 and GLP-1. Thirty-two subjects (22±2 y, BMI 21.9±2.2 kg/m2) participated in this cross-over study. Subjects received a fixed amount of a chocolate flavored milk-based liquid or semi-solid product similar in energy density and macronutrient composition. Before intake and 15, 30, 60 and 90 min thereafter, appetite was rated and blood was drawn to determine glucose, CCK-8, active ghrelin, desacyl ghrelin and GLP-1 concentrations. After the last blood withdrawal, subjects were offered a chocolate cake meal to consume ad libitum. In the appetite ratings we observed a small effect showing that the semi-solid product is apparently considered as more satisfying than the liquid. There was a significant product effect for fullness (p 0.03), desire to eat (p 0.04), appetite something sweet (p 0.002) and prospective consumption (p 0.0009). We observed no clear effect of viscosity on gastro-intestinal hormones. Only for desacyl ghrelin there was a significant product effect (p 0.004). Concentrations were consistently higher after intake of the semi-solid product. Ad libitum intake of the chocolate cake was 102±55 g after the liquid and 96±46 g after the semi-solid product (ns). The results of our study show a similar response of the gastro-intestinal hormones CCK-8, ghrelin and GLP-1 after a fixed preload of a liquid and semi-solid product similar in energy- and macronutrient composition. [Copyright &y& Elsevier]

Abstract: It has been shown in the rat that endogenous cholecystokinin (CCK), released in response to the non-nutrient trypsin inhibitor camostat, reduces food intake at meals and increases Fos-like immunoreactivity (Fos-LI; a marker for neuronal activation) in the dorsal vagal complex (DVC) of the hindbrain but not the myenteric plexus of the duodenum and jejunum. Experiment 1: We examined Fos-LI in the myenteric and the submucosal plexuses of the gut in response to orogastric gavage of camostat in rats. As we reported previously, camostat failed to increase Fos-LI in the myenteric plexus. We show here that camostat increased Fos-LI in the submucosal plexus of the duodenum and jejunum. Camostat also increased Fos-LI in the DVC. Experiment 2: Pretreatment with devazepide, a specific CCK1 receptor antagonist abolished camostat-induced Fos-LI in the submucosal plexus and the DVC. Experiment 3: Bilateral subdiaphragmatic vagotomy reduced camostat-induced Fos-LI in the submucosal plexus approximately 40% and abolished it in the DVC. Conclusions: Activation of the submucosal plexus by cholecystokinin at the CCK1 receptor accompanies stimulation of the dorsal vagal complex of the hindbrain and inhibition of food intake. Unlike the submucosal plexus, activation of the myenteric plexus is not necessary for cholecystokinin''s influence on the dorsal vagal complex and food intake. The lack of activation in the myenteric plexus after camostat stimulation, in contrast to nutrient releasers of CCK such as oleate, suggests that intestinal stimulants can either release different amounts of CCK or cause release of CCK from I cells with different molecular forms of CCK. This would suggest that CCK-8 is released by camostat and is not able to travel to the myenteric plexus while a more stable form of CCK such as CCK-58 can travel to this site that is further away from the I cell. [Copyright &y& Elsevier]

Introduction: Several cholecystokinin (CCK) forms have been detected in plasma, but most studies on food intake investigated the effects of CCK-8 only. Recently, it has been demonstrated that CCK-58 is the only endocrine-active form of CCK in rats. Methods: CCK-58 was synthesized with a peptide synthesizer using FMOC chemistry and CCK-58 effects on food intake were compared to CCK-8 in rats. Results: Both CCK-58 and CCK-8 inhibited food intake in a dose-dependent manner and were equally potent at 30 min. CCK-58 showed a prolonged inhibition of food intake compared to CCK8 at the higher dose tested (7 nmol/kg), inhibiting food intake also at 60 min, and cumulative food intake was inhibited for up to 210 min by CCK-58. Conclusions: CCK-58 has the same potency in inhibiting food intake as CCK-8 in rats, but inhibits food intake longer. This might be due to its tertiary structure resulting in a delayed plasma degradation or a prolonged binding at the CCK receptor. As CCK-58 is the major CCK form in the gut wall and possibly in the circulating blood in humans, the effects of CCK on food intake might have been underestimated in the past. [ABSTRACT FROM AUTHOR]

Abstract: Cholecystokinin octapeptide (CCK-8) is a physiological antagonist of endogenous opioids in the central nervous system (CNS). Our previous work has shown that CCK-8 plays an important role in the development of tolerance to morphine analgesia and electroacupuncture (EA) analgesia in the rat. The present studies were designed to examine whether the CCKB receptor is involved in the modulation of EA analgesia and the development of EA tolerance in mice. The latency to flick the tail in the radiant heat was used as index to assess the efficacy of EA analgesia. Subcutaneous (s.c.) injection of the CCKB receptor antagonist L365,260 produced a dose-dependent (0.125–2.0mg/kg) potentiation of the analgesia induced by 100Hz EA, with a maximal effect occurred at 0.5mg/kg. In addition, L365,260 (0.5mg/kg) significantly reversed chronic tolerance to 100Hz EA in mice. These results suggest that the CCKB receptor might play a role in the tonic inhibition of 100Hz EA-induced analgesia and in the mediation of chronic tolerance to 100Hz EA in mice. The results opened a way for further investigation of the function of CCK-8 in pain modulation using inbred strains of mice. [Copyright &y& Elsevier]

Abstract: In our previous studies, we demonstrated that intraperitoneal (i.p.) injections with the neurotransmitter/neuromodulatory peptide Cholecystokinin-8 (CCK-8) stimulate the synthesis of the neurotrophin nerve growth factor (NGF) resulting in the structural and functional recovery of neuronal damage. This neurotrophin-mediated neuroprotective action of CCK-8 has opened a new perspective for a better understanding of the CCK neurobiological and pharmacological properties. To explore the possible beneficial effects of the CCK-induced increase of neurotrophin availability in brain, we compared the effects of i.p. CCK-8 in healthy rats and in a chemical kindling model using a subconvulsive dose of pentylenetetrazol (PTZ). Behavioural changes were monitored during treatment and classified according to a six-point scale. After 3 weeks of treatment (12 trials), the PTZ group of rats manifested generalized clonic–tonic seizures (Class 5 behaviour). For this reason, this time point was chosen to compare the effects of CCK-8 treatment on the expression of NGF, the brain derived neurotrophin factor (BDNF) and their receptors in the septum and hippocampus. We found that repeated i.p. injections with CCK-8 in adult rats result in: (1) an increase of NGF and BDNF protein and mRNA levels in the septum and hippocampus; (2) a down-regulation of TrkA and p75NTR and an up-regulation of TrkB; (3) reduced susceptibility to develop chemical kindling; (4) recovery of the PTZ-induced changes in the expression of neurotrophin receptors in the septal and hippocampal tissues. This data clearly indicates that CCK-induced variation of neurotrophin synthesis in brain is able to influence the susceptibility to develop seizures in adult rats most probably by counteracting the progressive neuronal dysfunction and/or damage. [Copyright &y& Elsevier]

Abstract: Objective: To illustrate the existence of bile regurgitation under stress condition, and explore the possible effects and related mechanism of changes of plasma cholecystokinin octapeptide (CCK-8) and intragastric pH on stress-induced bile regurgitation in rats. Methods: (1) Changes in plasma CCK-8 and gastric bile concentration were respectively measured by using radioimmunoassay (RIA) method while simultaneously calculating gastric ulcer index (UI) and intragastric pH; (2) Each isolated gastric strips were suspended in a tissue chamber to record the contractile responses by polyphysiograph; (3) The responsiveness of gastric smooth muscle cells (SMCs) to sulfated cholecystokinin octapeptide (CCK-8S) were examined using fura-2-loaded microfluorimetric measurement of intracellular calcium concentration ([Ca2+]i); (4) The current of L-type calcium channels (I CaL) of SMCs were recorded by patch clamp techniques. Results: (1) Compared with the normal control group, plasma CCK-8 and gastric bile concentration significantly increased during stress (p <0.01) and both simultaneously reached the peak at the time point of 2 h after stress; UI and intragastric pH apparently increased (p <0.01); (2) Significant changes to CCK-8S were found in the mean contractile amplitude and frequency of circular muscle (CM) and longitudinal muscle (LM) of gastric antrum and pylorus; (3) CCK-8S-evoked significant increase in [Ca2+]i (p <0.01) could be suppressed by CCK-A receptor (CCK-AR) antagonist; whereas a small but significant increase was still elicited by CCK-8S under condition of the removal of extracellular calcium or by given nifidipine; (4) CCK-8S-intensified calcium current (I CaL) apparently inhibited by respective administration of nifidipine, Ca2+-ATPase inhibitors or calcium dependent chloride channel (I Cl–Ca) blocker (p <0.01). Conclusion: Gastric mucosal damage induced by bile regurgitation is closely connected with gastric antrum and pylorus dysmotility evoked by CCK-8 during the stress. CCK-8S-evoked [Ca2+]i increase in gastric antrum and pylorus SMC depends on the release of intracellular calcium stores which activates L-type voltage-dependent calcium channels (VDCC) through the activation of calcium dependent chloride channels. [Copyright &y& Elsevier]

Abstract: The possible pronociceptive role of peripheral cholecystokinin (CCK-8) as well as CCKA and CCKB receptors in diabetic rats was assessed. Subcutaneous injection of 0.5% formalin induced a greater nociceptive behavior in diabetic than in non-diabetic rats. Moreover, local peripheral injection of CCK-8 (0.1–100μg) significantly increased 0.5% formalin-induced nociceptive activity in diabetic, but not in non-diabetic, rats. This effect was restricted to the formalin-injected paw as administration of CCK-8 into the contralateral paw was ineffective. Local peripheral administration of CCK-8, in the absence of formalin injection, produced a low level of, but significant increase in, flinching behavior in diabetic compared to non-diabetic rats. In addition, local peripheral administration of the non-selective CCK receptor antagonist proglumide (1–100μg), CCKA receptor antagonist lorglumide (0.1–100μg) or CCKB receptor antagonist CR-2945 (0.1–100μg), but not vehicle or contralateral administration of CCK receptor antagonists, significantly reduced 0.5% formalin-induced flinching in diabetic rats. CR-2945 was the most effective drug in this condition. These effects were not observed in non-diabetic rats. The local peripheral pronociceptive effect of CCK-8 (100μg) was significantly reduced by proglumide (100μg), lorglumide (100μg), and CR-2945 (100μg). Results suggest that diabetes-induced peripheral sensitization could be due to a local peripheral release of CCK-8, which in turn would act on CCKB, mainly but also in CCKA, receptors located on the primary afferent neurons. [Copyright &y& Elsevier]

Abstract: In the present study, we have investigated the effect of ethanol on amylase release in response to cholecystokinin octapeptide (CCK-8). We have also studied the effect of ethanol on cytosolic free Ca2+ concentration ([Ca2+]c) and reactive oxygen species (ROS) production by loading of cells with fura-2 and 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), respectively. Our results show that stimulation of pancreatic acinar cells with CCK-8 induced a dose-dependent amylase secretion, resulting in a maximum at 0.3nM of 19.39±2.73% of the total content of amylase. Treatment of pancreatic acini with ethanol did not induce any significant effect on amylase release at a wide range of concentrations (1–50mM). In contrast, incubation of cells with 50mM ethanol clearly reduced amylase release stimulated by CCK-8. The inhibitory effect of ethanol on CCK-8-induced amylase secretion was abolished by dithiothreitol, a sulfhydryl reducing agent. Ethanol induced an increase in [Ca2+]c resulting in a level higher than the prestimulation level both in the presence and in the absence of extracellular Ca2+. Additionally, ethanol led to an increase in fluorescence of CM-H2DCFDA, reflecting an increase in oxidation. A decrease in oxidation was observed in the absence of extracellular Ca2+ and in the presence of ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid. Similarly, when the cells were challenged in the presence of the intracellular Ca2+ chelator 1,2-Bis(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and in the absence of extracellular Ca2+, the responses to ethanol were reduced, although not completely inhibited. Taken together, our results suggest that ethanol induces generation of ROS by a Ca2+-dependent mechanism and reduces CCK-8-evoked amylase secretion in exocrine pancreatic cells. [Copyright &y& Elsevier]