Role of Spinal Cholecystokinin Octapeptide, Nociceptin/Orphanin FQ, and Hemokinin-1 in Diabetic Allodynia.

A complication of diabetes is neuropathic pain, which is difficult to control with medication. We have confirmed that neuropathic pain due to mechanical allodynia in diabetic mice is mediated by a characteristic neuropeptide in the spinal cord. We evaluated the strength of mechanical allodynia in mice using von Frey filaments. When mice were intravenously injected with streptozotocin, mechanical allodynia appeared 3 days later. Antibodies of representative neuropeptides were intrathecally (i.t.) administered to allodynia-induced mice 7 days after the intravenous administration of streptozotocin, and allodynia was reduced by anti-cholecystokinin octapeptide antibodies, anti-nociceptin/orphanin FQ antibodies, and anti-hemokinin-1 antibodies. In contrast, i.t.-administered anti-substance P antibodies, anti-somatostatin antibodies, and anti-angiotensin II antibodies did not affect streptozotocin-induced diabetic allodynia mice. Mechanical allodynia was attenuated by the i.t. administration of CCK-B receptor antagonists and ORL-1 receptor antagonists. The mRNA level of CCK-B receptors in streptozotocin-induced diabetic allodynia mice increased in the spinal cord, but not in the dorsal root ganglion. These results indicate that diabetic allodynia is caused by cholecystokinin octapeptide, nociceptin/orphanin FQ, and hemokinin-1 released from primary afferent neurons in the spinal cord that transmit pain to the brain via the spinal dorsal horn.

Liangxue Tongyu prescription attenuates neuroinflammation by increasing cholecystokinin octapeptide in acute intracerebral hemorrhage rats.

Inflammatory reactions after acute intracerebral hemorrhage (AICH) contribute significantly to a poor prognosis. Liangxue Tongyu Prescription (LTP) has been proven to be clinically effective in treating AICH. Numerous studies have shown that LTP suppresses brain inflammatory damage in AICH, while the internal mechanisms underlying its action remain unclear. The aim of this study was to verify the anti-inflammatory effects of LTP on an AICH rat model and investigate the potential mechanisms. The AICH rat models were created by injecting autologous blood into the right caudate nucleus. LTP markedly decreased cerebral hematoma and brain water content and recovered from neurological deficits. Meanwhile, LTP prevented microglial activation and reduced the inflammatory reaction caused by pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6). Notably, the expression of cholecystokinin octapeptide (CCK-8) in the brain and intestine was increased by LTP or CCK-8 treatment. LTP further suppressed nuclear factor kappa B (NF-κB) in the brains of rats with AICH. Moreover, LTP increased the protein and mRNA expression of Occludin and Claudin-1 in the intestine and decreased the levels of lipopolysaccharide (LPS) and diamine oxidase (DAO) in serum. Furthermore, the results showed that LTP increased the protein and mRNA expression of Claudin-5 and zonula occludens-1 (ZO-1) in the brain. CCK-8 receptor antagonists increased the expression of NF-κB and the concentration of pro-inflammatory cytokines. These findings suggested that LTP attenuated neuroinflammation by increasing CCK-8 in the brain and intestine, and its mechanism might be related to alterations in the gut-brain axis (GBA).

Single-chain Fragment variable antibody targeting cholecystokinin-B receptor for pain reduction.

The cholecystokinin B receptor and its neuropeptide ligand are upregulated in chronic neuropathic pain models. Single-chain Fragment variable antibodies were generated as preferred non-opioid targeting therapy blocking the cholecystokinin B receptor to inhibit chronic neuropathic pain models in vivo and in vitro. Engineered antibodies of this type feature binding activity similar to monoclonal antibodies but with stronger affinity and increased tissue penetrability due to their smaller size. More importantly, single-chain Fragment variable antibodies have promising biotherapeutic applications for both nervous and immune systems, now recognized as interactive in chronic pain. A mouse single-chain Fragment variable antibody library recognizing a fifteen amino acid extracellular peptide fragment of the cholecystokinin B receptor was generated from immunized spleens. Ribosome display, a powerful cell-free technology, was applied for recombinant antibody selection. Antibodies with higher affinity, stability, solubility, and binding specificity for cholecystokinin B not A receptor were selected and optimized for in vivo and in vitro efficacy. A single dose of the lead candidate reduced mechanical and cold hypersensitivity in two rodent models of neuropathic pain for at least seven weeks. Continuing efficacy was evident with either intraperitoneal or intranasal dosing. Likewise, the lead single-chain Fragment variable antibody totally prevented development of anxiety- and depression-like behaviors and cognitive deficits typical in the models. Reduction of neuronal firing frequency was evident in trigeminal ganglia primary neuronal cultures treated in vitro with the cholecystokinin B receptor antibody. Immunofluorescent staining intensity in the trigeminal neuron primary cultures was significantly reduced incrementally after overnight binding with increasingly higher dilutions of the single-chain Fragment variable antibody. While it is reported that single-chain Fragment variable antibodies are removed systemically within 2-6 h, Western blot evidence indicates the His-tag marker remained after 7 weeks in the trigeminal ganglia and in the dorsolateral medulla, providing evidence of brain and ganglia penetrance known to be compromised in overactivated states. This project showcases the in vivo efficacy of our lead single-chain Fragment variable antibody indicating its potential for development as a non-opioid, non-addictive therapeutic intervention for chronic pain. Importantly, studies by others have indicated treatments with cholecystokinin B receptor antagonists suppress maintenance and reactivation of morphine dependence in place preference tests while lowering tolerance and dose requirements. Our future studies remain to address these potential benefits that may accompany the cholecystokinin B receptor biological therapy. Both chronic sciatic and orofacial pain can be unrelenting and excruciating, reducing quality of life as well as diminishing physical and mental function. An effective non-opiate, non-addictive therapy with potential to significantly reduce chronic neuropathic pain long term is greatly needed.

Cholecystokinin octapeptide improves hippocampal glutamatergic synaptogenesis and postoperative cognition by inhibiting induction of A1 reactive astrocytes in aged mice.

AIMS: Delayed neurocognitive recovery (dNCR) is a common postoperative complication in geriatric surgical patients for which there is no efficacious therapy. Cholecystokinin octapeptide (CCK-8), an immunomodulatory peptide, regulates memory and learning. Here, we explored the effects and mechanism of action of CCK-8 on dNCR. METHODS: We applied laparotomy to establish a model of dNCR in aged mice. Morris water maze and fear conditioning tests were used to evaluate cognition. Immunofluorescence was used to detect the density of CCK-8, A1 reactive astrocytes, glutamatergic synapses, and activation of microglia in the hippocampus. Quantitative PCR was performed to determine mRNA levels of synapse-associated factors. A1 reactive astrocytes, activated microglia, and glutamatergic synapse-associated protein levels in the hippocampus were assessed by western blotting. RESULTS: Administration of CCK-8 suppressed the activation of microglia, the induction of A1 reactive astrocytes, and the expression of tumor necrosis factor alpha, complement 1q, and interleukin 1 alpha in the hippocampus. Furthermore, it promoted glutamatergic synaptogenesis and neurocognitive recovery in aged dNCR model mice. CONCLUSION: Our findings indicated that CCK-8 alleviated cognitive impairment and promoted glutamatergic synaptogenesis by inhibiting the induction of A1 reactive astrocytes and the activation of microglia. CCK-8 is, therefore, a potential therapeutic target for dNCR.

Inhibitory Effect of CCK-8 on Methamphetamine-Induced Apoptosis.

OBJECTIVES: To investigate the inhibitory effect of cholecystokinin octapeptide (CCK-8) binding to cholecystokinin 2 receptor (CCK2R) on methamphetamine (METH)-induced neuronal apoptosis, and to explore the signal transduction mechanism of ß-arrestin 2 in CCK-8 inhibiting METH-induced neuronal apoptosis. METHODS: SH-SY5Y cell line was cultured, and HEK293-CCK1R and HEK293-CCK2R cell line were constructed by lentivirus transfection. Small interfering RNA (siRNA) was used to knockdown the expression of ß-arrestin 2. Annexin Ⅴ-FITC/PI staining and flow cytometry were used to detect the apoptotic rate of cells, and Western blotting was used to detect the expression of apoptosis-related proteins. RESULTS: The apoptosis of SH-SY5Y cells was induced by 1 mmol/L and 2 mmol/L METH treatment, the number of nuclear fragmentation and pyknotic cells was significantly increased, and the expression of apoptosis-related proteins Bax and cleaved caspase-3 were increased. CCK-8 pre-treatment at the dose of 0.1 mmol/L and 1 mmol/L significantly reversed METH-induced apoptosis in SH-SY5Y cells, and inhibited cell nuclear fragmentation, pyknosis and the changes of apoptosis-related proteins induced by METH. In lentivirus transfected HEK293-CCK1R and HEK293-CCK2R cells, the results revealed that CCK-8 had no significant effect on METH-induced changes of apoptosis-related proteins in HEK293-CCK1R cells, but it could inhibit the expression level of apoptosis-related proteins in HEK293-CCK2R cells induced by METH. The inhibitory effect of CCK-8 on METH-induced apoptosis was blocked by the knockdown of ß-arrestin 2 expression in SH-SY5Y cells. CONCLUSIONS: CCK-8 can bind to CCK2R and exert an inhibitory effect on METH-induced apoptosis by activating the ß-arrestin 2 signal.

Inhibitory Effect of CCK-8 on Methamphetamine-Induced Apoptosis / 法医学杂志

OBJECTIVES@#To investigate the inhibitory effect of cholecystokinin octapeptide (CCK-8) binding to cholecystokinin 2 receptor (CCK2R) on methamphetamine (METH)-induced neuronal apoptosis, and to explore the signal transduction mechanism of β-arrestin 2 in CCK-8 inhibiting METH-induced neuronal apoptosis.@*METHODS@#SH-SY5Y cell line was cultured, and HEK293-CCK1R and HEK293-CCK2R cell line were constructed by lentivirus transfection. Small interfering RNA (siRNA) was used to knockdown the expression of β-arrestin 2. Annexin Ⅴ-FITC/PI staining and flow cytometry were used to detect the apoptotic rate of cells, and Western blotting was used to detect the expression of apoptosis-related proteins.@*RESULTS@#The apoptosis of SH-SY5Y cells was induced by 1 mmol/L and 2 mmol/L METH treatment, the number of nuclear fragmentation and pyknotic cells was significantly increased, and the expression of apoptosis-related proteins Bax and cleaved caspase-3 were increased. CCK-8 pre-treatment at the dose of 0.1 mmol/L and 1 mmol/L significantly reversed METH-induced apoptosis in SH-SY5Y cells, and inhibited cell nuclear fragmentation, pyknosis and the changes of apoptosis-related proteins induced by METH. In lentivirus transfected HEK293-CCK1R and HEK293-CCK2R cells, the results revealed that CCK-8 had no significant effect on METH-induced changes of apoptosis-related proteins in HEK293-CCK1R cells, but it could inhibit the expression level of apoptosis-related proteins in HEK293-CCK2R cells induced by METH. The inhibitory effect of CCK-8 on METH-induced apoptosis was blocked by the knockdown of β-arrestin 2 expression in SH-SY5Y cells.@*CONCLUSIONS@#CCK-8 can bind to CCK2R and exert an inhibitory effect on METH-induced apoptosis by activating the β-arrestin 2 signal.

Cholecystokinin octapeptide reduces myocardial fibrosis and improves cardiac remodeling in post myocardial infarction rats.

BACKGROUND/AIMS: Myocardial infarction (MI) increases myocardial fibrosis (MF) and subsequent cardiac remodeling. Cholecystokinin octapeptide (CCK-8) is expressed in cardiomyocytes and plays an important role in cardiovascular regulation. In this study, we intend to use a rat model of myocardial infarction to evaluate the effects of CCK-8 on myocardial fibrosis and cardiac remodeling. METHODS: Male Sprague-Dawley rats were separated into 3 groups: sham operation, MI + NaCl, and MI + CCK-8. All rats were subjected to left coronary artery ligation to induce MI or sham operation and then treated with CCK-8 or saline for 28 days. After 4 weeks, echocardiography was performed to assess cardiac function and myocardial fibrosis was evaluated using H&E and Masson's Trichrome-stained sections. The levels of BNP, CCK-8 in the plasma of all rats were detected by ELISA; RNA sequencing (RNA-seq) analysis was also adapted to detect differentially expressed genes in myocardial tissues of each group. Myocardial expression of fibrosis markers was analyzed by western blotting, immunohistochemistry and qRT-PCR. RESULTS: CCK-8 was demonstrated to improve left ventricular function and results of H&E staining, Masson's trichrome staining, immunohistochemistry and western blotting showed that CCK-8 attenuated MF. Gene expression profiles of the left ventricles were analysed by RNA-seq and validated by qRT-PCR. Cardiac fibrosis genes were downregulated by CCK-8 in the left ventricle. SIGNIFICANCE: CCK-8 can alleviate fibrosis in the noninfarcted regions and delay the left ventricular remodeling and the progress of heart failure in a MI rat model.

Protective effect of cholecystokinin octapeptide on angiotensin II-induced apoptosis in H9c2 cardiomyoblast cells.

Cholecystokinin (CCK) and its receptors are expressed in mammalian cardiomyocytes and are involved in cardiovascular system regulation; however, the exact effect and underlying mechanism of CCK in cardiomyocyte apoptosis remain to be elucidated. We examined whether sulfated CCK octapeptide (CCK-8) protects H9c2 cardiomyoblast cells against angiotensin II (Ang II)-induced apoptosis. The H9c2 cardiomyoblasts were subjected to Ang II with or without CCK-8 and the viability and apoptotic rate were detected using a Cell Counting Kit-8 assay, Hoechst 33342 staining, terminal deoxyribonucleotide transferase-mediated nick-end labeling assays, and flow cytometry. In addition, specific antiapoptotic mechanisms of CCK-8 were investigated using specific CCK1 (Devazepide) or CCK2 (L365260) receptor antagonists, or the PI3K inhibitor LY294002. The expression of CCK, CCK1 receptor, CCK2 receptor, Akt, p-Akt, Bad, p-Bad, Bax, Bcl-2, and caspase-3 were detected by Western blot analysis and real-time polymerase chain reaction. We found that CCK and its receptor messenger RNA (mRNA) and protein are expressed in H9c2 cardiomyoblasts. Ang II-induced increased levels of CCK mRNA and protein expression and decreased levels of CCK1 receptor protein and mRNA. Pretreatment of CCK-8 attenuated Ang II-induced cell toxicity and apoptosis. In addition, pretreatment of H9c2 cells with CCK-8 markedly induced expression of p-Akt, p-bad, and Bcl-2 and decreased the expression levels of Bax and caspase-3. The protective effects of CCK-8 were partly abolished by Devazepide or LY294002. Our results suggest that CCK-8 protects H9c2 cardiomyoblasts from Ang II-induced apoptosis partly via activation of the CCK1 receptor and the phosphatidyqinositol-3 kinase/protein kinase B (PI3K/Akt) signaling pathway.

Cholecystokinin octapeptide inhibits the inflammatory response and improves neurological outcome in a porcine model of cardiopulmonary resuscitation.

Previous studies have demonstrated that cholecystokinin octapeptide (CCK8) induces hypothermia and inhibits the systemic inflammatory response in septic shock in rat and murine models. The present study aimed to ascertain whether CCK8 induced hypothermia and improved the neurological outcomes in a porcine model of cardiopulmonary resuscitation (CPR). Ventricular fibrillation was induced and left untreated for 10 min in 12 male Bama miniature pigs. Defibrillation was attempted after 5 min of CPR. At 5 min following resuscitation, the pigs were randomized and equally assigned into the CCK8 or the control group. CCK8 was continuously infused for 1 h at a dose of 44.4 µg/kg/h and a rate of 20 ml/h in the CCK8 group. Body temperature, hemodynamic measurements and post-resuscitation myocardial function were monitored in the first 4 h following CPR. Neuron specific enzyme (NSE), S100B protein, tumor necrosis factor (TNF)-α and interleukin (IL)-6 were measured at baseline and 4, 12 and 24 h following resuscitation. The neurological deficient score (NDS) was recorded and cerebral samples were collected for terminal deoxynucleotidyl-transferase-mediated dUTP nick end labelling assay and integrated optical density (IOD) analysis at 24 h following CPR. The results revealed that hypothermia was not induced by CCK8; however, post-resuscitation NSE, S100B, IL-6 and TNF-α were significantly decreased, and NDS and IOD were significantly improved in the CCK8 group compared with the control group (P<0.05). The present study revealed that in a porcine model of CPR, CCK8 does not induce hypothermia, but inhibits the inflammatory response and significantly improves neurological outcomes.

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.

Effects of CCK-8 and Cystathionine γ-Lyase/Hydrogen Sulfide System on Acute Lung Injury in Rats.

Acute lung injury (ALI) is mainly characterized by diffusive injuries to lung epithelium and increased permeability of alveolar-capillary membranes caused by various factors, which leads to pulmonary edema and pulmonary closure. Lipopolysaccharide (LPS), which is the main component of the cell wall of gram-negative bacteria, is one of the most important factors causing pulmonary infection and ALI. More and more reports have indicated that hydrogen sulfide (H2S) is closely correlated with ALI and has anti-inflammation function, while the specific mechanism needs further investigation. Cholecystokinin-octapeptide (CCK-8), which is an important endogenous functional fragment belonging to CCK family, participates in anti-inflammatory and anti-endotoxic shock (ES). Whether CCK-8 plays important roles in curing ALI also needs further investigation. Herein, we concluded that CCK-8 alleviated the ALI induced by LPS via regulating the catalytic activity of cystathionine γ-lyase (CSE) and the formation of H2S. This work provides new medicine-designed target for clinical doctor to prevent and cure ALI.

Effect of CCK-8 on expression of MMPs/TIMP-1 in TNF-α-induced RSC-364 / 中国药理学通报

Aim To observe the influence of CCK-8 on expression of MMPs/TIMP-1 in TNF-α-induced rat fibroblast-like synovial cell line RSC-364.Methods The secretion levels of MMP-1, MMP-3, MMP-9 and TIMP-1 were determined using ELISA;MMP-3 and MMP-9 mRNA expressions were detected by RT-PCR.Results MMP-3 and MMP-9 could not be examined in RSC-364 incubated with CCK-8 and unstimulated RSC-364, which was able to product a little MMP-1, TIMP-1 and express even less MMP-3,-9 mRNA.CCK-8 inhibited the increase in MMP-1, MMP-3, MMP-9 secretion and MMP-3,-9 mRNA expression in TNF-α-induced RSC-364.TIMP-1 production was also increased in TNF-α-induced RSC-364.CCK-8 had no effect on TIMP-1 production in TNF-α-induced RSC-364, but was able to reduce the ratios of MMP-1, MMP-3, MMP-9 to TIMP-1.Conclusion The inhibitory effect of CCK-8 on MMPs activity may be related to the decrease of MMPs mRNA expression, MMPs secretion and the ratios of MMPs to TIMP-1 in TNF-α-induced RSC-364, which indicates that CCK-8 might be a possible regulator in the pathogenesis of rheumatoid arthritis.

Cholecystokinin octapeptide antagonizes apoptosis in human retinal pigment epithelial cells.

Although cholecystokinin octapeptide-8 is important for neurological function, its neuroprotective properties remain unclear. We speculated that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against oxidative injury. In this study, retinal pigment epithelial cells were treated with peroxynitrite to induce oxidative stress. Peroxynitrite triggered apoptosis in these cells, and increased the expression of Fas-associated death domain, Bax, caspa-se-8 and Bcl-2. These changes were suppressed by treatment with cholecystokinin octapeptide-8. These results suggest that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against apoptosis induced by peroxynitrite.

Cholecystokinin octapeptide induces endogenous opioid-dependent anxiolytic effects in morphine-withdrawal rats.

Cholecystokinin octapeptide (CCK-8), a brain-gut peptide, plays an important role in several opioid addictive behaviors. We previously reported that CCK-8 attenuated the expression and reinstatement of morphine-induced conditioned place preference. The possible effects of CCK-8 on the negative affective components of drug abstinence are not clear. There are no studies evaluating the effect of CCK-8 on emotional symptoms, such as anxiety, in morphine-withdrawal animals. We investigated the effects of CCK-8 on the anxiety-like behavior in morphine-withdrawal rats using an elevated plus-maze. Morphine withdrawal elicited time-dependent anxiety-like behaviors with peak effects on day 10 (5 days after induction of morphine dependence). Treatment with CCK-8 (0.1 and 1 µg, i.c.v.) blocked this anxiety in a dose-dependent fashion. A CCK1 receptor antagonist (L-364,718, 10 µg, i.c.v.) blocked the effect of CCK-8. Mu-opioid receptor antagonism with CTAP (10 µg, i.c.v.) decreased the 'anxiolytic' effect. CCK-8 inhibited anxiety-like behaviors in morphine-withdrawal rats by up-regulating endogenous opioids via the CCK1 receptor in rats. This study clearly identifies a distinct function of CCK-8 and a potential medication target of central CCK1 receptors for drugs aimed at ameliorating drug addiction.

Roles of sphincter of Oddi motility and serum vasoactive intestinal peptide, gastrin and cholecystokinin octapeptide.

AIM: To investigate roles of sphincter of Oddi (SO) motility played in pigment gallbladder stone formation in model of guinea pigs. METHODS: Thirty-four adult male Hartley guinea pigs were divided randomly into two groups: the control group and pigment stone group. The pigment stone group was divided into 4 subgroups with 6 guinea pigs each according to time of sacrifice, and were fed a pigment lithogenic diet and sacrificed after 3, 6, 9 and 12 wk. SO manometry and recording of myoelectric activity of the guinea pigs were obtained by multifunctional physiograph at each stage. Serum vasoactive intestinal peptide (VIP), gastrin and cholecystokinin octapeptide (CCK-8) were detected at each stage in the process of pigment gallbladder stone formation by enzyme-linked immunosorbent assay. RESULTS: The incidence of pigment gallstone formation was 0%, 0%, 16.7% and 66.7% in the 3-, 6-, 9- and 12-wk group, respectively. The frequency of myoelectric activity decreased in the 3-wk group. The amplitude of myoelectric activity had a tendency to decrease but not significantly. The frequency of the SO decreased significantly in the 9-wk group. The SO basal pressure and common bile duct pressure increased in the 12-wk group (25.19 ± 7.77 mmHg vs 40.56 ± 11.81 mmHg, 22.35 ± 7.60 mmHg vs 38.51 ± 11.57 mmHg, P < 0.05). Serum VIP was significantly elevated in the 6- and 12-wk groups and serum CCK-8 was decreased significantly in the 12-wk group. CONCLUSION: Pigment gallstone-causing diet may induce SO dysfunction. The tension of the SO increased. The disturbance in SO motility may play a role in pigment gallstone formation, and changes in serum VIP and CCK-8 may be important causes of SO dysfunction.

Cholecystokinin-octapeptide restored morphine-induced hippocampal long-term potentiation impairment in rats.

Cholecystokinin-octapeptide (CCK-8), which is a typical brain-gut peptide, exerts a wide range of biological activities on the central nervous system. We have previously reported that CCK-8 significantly alleviated morphine-induced amnesia and reversed spine density decreases in the CA1 region of the hippocampus in morphine-treated animals. Here, we investigated the effects of CCK-8 on long-term potentiation (LTP) in the lateral perforant path (LPP)-granule cell synapse of rat dentate gyrus (DG) in acute saline or morphine-treated rats. Population spikes (PS), which were evoked by stimulation of the LPP, were recorded in the DG region. Acute morphine (30mg/kg, s.c.) treatment significantly attenuated hippocampal LTP and CCK-8 (1µg, i.c.v.) restored the amplitude of PS that was attenuated by morphine injection. Furthermore, microinjection of CCK-8 (0.1 and 1µg, i.c.v.) also significantly augmented hippocampal LTP in saline-treated (1ml/kg, s.c.) rats. Pre-treatment of the CCK2 receptor antagonist L-365,260 (10µg, i.c.v) reversed the effects of CCK-8, but the CCK1 receptor antagonist L-364,718 (10µg, i.c.v) did not. The present results demonstrate that CCK-8 attenuates the effect of morphine on hippocampal LTP through CCK2 receptors and suggest an ameliorative function of CCK-8 on morphine-induced memory impairment.

Effects of CCK-8 and its receptor antagonists on expression of CREB and pCREB in prefrontal cortex and hippocampus of morphine withdrawal rats / 中国病理生理杂志

AIM:To observe the effects of cholecystokinin octapeptide (CCK-8) and its receptor antagonists on cAMP response element binding protein ( CREB) and phosphorylated CREB ( pCREB) expression in frontal cortex and hippocampus of morphine withdrawal rats , which aim to explore the post-receptor mechanism through which CCK-8 regu-lates morphine withdrawal .METHODS: After the morphine dependence and naloxone-precipitated withdrawal animal models were established, the effects of CCK-8, L-364718 (CCK1 receptor antagonist) and LY-288513 (CCK2 receptor an-tagonist) pretreatment on CREB and pCREB expression in frontal cortex and hippocampus were observed by Western blot -ting and immunohistochemistry .RESULTS:In rat frontal cortex neuron , CREB was expressed in both cytoplasm and nu-cleus, but pCREB was only highly expressed in the nucleus .In the pyramidal cell layer of hippocampal CA 1 region, CREB showed high expression in the cytoplasm and low expression in the nucleus , while pCREB was only expressed in the nu-cleus.No obvious change of CREB was observed after either chronic morphine treatment or naloxone withdrawal .The pCREB expression was increased after chronic morphine treatment and further increased after naloxone withdrawal .Com-pared with the withdrawal group , chronic pretreatment with CCK-8, L-364718 and LY-288513 had no effect on CREB expression in the frontal cortex , but obviously decreased the pCREB expression .In the hippocampus , pretreatment with L-364718 and LY-288513 decreased CREB and pCREB expression , but only the pCREB expression was decreased after CCK-8 treatment.CONCLUSION:CCK-8 and CCK receptor antagonists may alleviate morphine withdrawal symptoms by regulating CREB , with specificity in different brain regions .

Effects of cholecystokinin-8 on morphine-induced spatial reference memory impairment in mice.

Acute and chronic exposure to opiate drugs impaired various types of memory processes. To date, there is no preventive treatment for opiate-induced memory impairment and the related mechanism is still unclear. CCK-8 is the most potent endogenous anti-opioid peptide and has been shown to exert memory-enhancing effect, but the effect of CCK-8 on morphine-induced memory impairment has not been reported. By using Morris water maze, we found that escape latency to the hidden platform in navigation test was not influenced, but performance in the probe test was seriously poor in morphine dependency mice. Amnesia induced by chronic morphine treatment was significantly alleviated by pre-treatment with CCK-8 (0.01, 0.1 and 1 µg, i.c.v.), and CCK-8 (0.1 and 1 µg, i.c.v.) treatment alone could improve performance in either navigation or probe test. Furthermore, Golgi-Cox staining analysis revealed that pre-treatment with CCK-8 (1 µg, i.c.v.) reversed spine density decreased in CA1 region of hippocampus in morphine dependency mice, and CCK-8 (1 µg, i.c.v.) alone obviously increased spine density in CA1. Our findings conclude spine density change in CA1 region of hippocampus may be the structural plasticity mechanism which is responsible for enhancing effect of CCK-8 on spatial reference memory. Therefore, CCK-8 could effectively improve memory impairment in morphine dependency mice.

Mechanisms of cholecystokinin-induced calcium mobilization in gastric antral interstitial cells of Cajal.

AIM: To investigate the effect of sulfated cholecystokinin-8 (CCK-8S) on calcium mobilization in cultured murine gastric antral interstitial cells of Cajal (ICC) and its possible mechanisms. METHODS: ICC were isolated from the gastric antrum of mice and cultured. Immunofluorescence staining with a monoclonal antibody for c-Kit was used to identify ICC. The responsiveness of ICC to CCK-8S was measured using Fluo-3/AM based digital microfluorimetric measurement of intracellular Ca(2+) concentration ([Ca(2+)]i). A confocal laser scanning microscope was used to monitor [Ca(2+)]i changes. The selective CCK(1) receptor antagonist lorglumide, the intracellular Ca(2+)-ATPase inhibitor thapsigargin, the type III inositol 1,4,5-triphosphate (InsP(3)) receptor blocker xestospongin C and the L-type voltage-operated Ca(2+) channel inhibitor nifedipine were used to examine the mechanisms of [Ca(2+)]i elevation caused by CCK-8S. Immunoprecipitation and Western blotting were used to determine the regulatory effect of PKC on phosphorylation of type III InsP(3) receptor (InsP(3)R3) in ICC. Protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and inhibitor chelerythrine were used to assess the role of PKC in the CCK-8S-evoked [Ca(2+)]i increment of ICC. RESULTS: ICC were successfully isolated from the gastric antrum of mice and cultured. Cultured ICC were identified by immunofluorescence staining. When given 80 nmol/L or more than 80 nmol/L CCK-8S, the [Ca(2+)]i in ICC increased and 100 nmol/L CCK-8S significantly increased the mean [Ca(2+)]i by 59.30% ± 4.85% (P < 0.01). Pretreatment of ICC with 5 µmol/L lorglumide inhibited 100 nmol/L CCK-8S-induced [Ca(2+)]i increment from 59.30% ± 4.85% to 14.97% ± 9.05% (P < 0.01), suggesting a CCK(1)R-mediated event. Emptying of intracellular calcium stores by thapsigargin (5 µmol/L) prevented CCK-8S (100 nmol/L) from inducing a [Ca(2+)]i increase. Moreover, pretreatment with xestospongin C (1 µmol/L) could also abolish the CCK-8S-induced effect, indicating that Ca(2+) release from InsP(3)R-operated stores appeared to be a major mechanism responsible for CCK-8S-induced calcium mobilization in ICC. On the other hand, by removing extracellular calcium or blocking the L-type voltage-operated calcium channel with nifedipine, a smaller but significant rise in the [Ca(2+)]i could be still elicited by CCK-8S. These data suggest that the [Ca(2+)]i release is not stimulated or activated by the influx of extracellular Ca(2+) in ICC, but the influx of extracellular Ca(2+) can facilitate the [Ca(2+)]i increase evoked by CCK-8S. CCK-8S increased the phosphorylation of InsP(3)R3, which could be prevented by chelerythrine. Pretreatment with lorglumide (5 µmol/L) could significantly reduce the CCK-8S intensified phosphorylation of InsP(3)R3. In the positive control group, treatment of cells with PMA also resulted in an enhanced phosphorylation of InsP(3)R3. Pretreatment with various concentrations of PMA (10 nmol/L-10 µmol/L) apparently inhibited the effect of CCK-8S and the effect of 100 nmol/L PMA was most obvious. Likewise, the effect of CCK-8S was augmented by the pretreatment with chelerythrine (10 nmol/L-10 µmol/L) and 100 nmol/L chelerythrine exhibited the maximum effect. CONCLUSION: CCK-8S increases [Ca(2+)]i in ICC via the CCK(1) receptor. This effect depends on the release of InsP(3)R-operated Ca(2+) stores, which is negatively regulated by PKC-mediated phosphorylation of InsP(3)R3.

Peroxynitrite-induced expression of inducible nitric oxide synthase and activated apoptosis via nuclear factor-kappa B pathway in retinal pigment epithelial cells and antagonism of cholecystokinin octapeptide-8 in vitro.

AIM: To explore that if peroxynitrite induced the expression of inducible nitric oxide synthase (iNOS)via nuclear factor-kappa B (NF-κB) pathway in retinal pigment epithelial (RPE) cells and the antagonism of cholecystokinin octapeptide-8 (Melatonin, CCK-8) in vitro. METHODS: RPE cells were obtained from eyes of C57BL/6 mouse and divided into control, peroxynitrite and CCK-8 groups. Control group was treated with saline, peroxynitrite group was treated with peroxynitrite, and CCK-8 group was treated with CCK-8 after added with peroxynitrite. All changes were observered at 6, 12 and 24 hours after treatment. Gene array analysis, Reverse Transcription Polymerase Chain Reaction (RT-PCR) were used to determine the expression of inducible nitric oxide synthase (iNOS) mRNA in RPE cells. Western blotting was used to test the apoptosis of RPE cells. Immunofluorescent staining was used to determine the NF-κB pathway signal transduction. RESULTS: Compared to the control group, the expression of iNOS mRNA was up-regulated in peroxynitrite group and down-regulated in CCK-8 group with gene array analysis. Apoptosis was increased in peroxynitrite group and decreased in CCK-8 group with western blotting. The NF-κB pathway signal transduction was more and more stronger in the peroxynitrite group. But in CCK-8 group, little stronger could be observed at 12 hours, then weak at 24 hours with immunofluorescent staining (P<0.001). CONCLUSION: This study suggested that apoptosis of RPE cells was partly induced by peroxynitrite, which may be the new way of oxidative damage to the RPE cells. The NF-κB signal transduction may affect and reinforce apoptosis mediated by peroxynitrite. CCK-8 decreased apoptosis of RPE cells induced by peroxynitrite and is a potential agent for therapy of retinopathy. The mechanism of CCK-8 dealing with RPE cells may be related to its direct inhibition of the formation of iNOS to produce peroxynitrite and antagnism of damage of peroxynitrite to the RPE cells.