| Drug abuse is an extremely serious and growing global problem. Psychostimulant drugs including Methamphetamine(methamphetamine, METH), MDMA and other new types of chemical synthetic drugs become very popular in our country. METH is commonly known as "ice". METH is a lipophilic compound, which is easily through the blood-brain barrier, resulting in serious damage to the central nervous system stem. Repeated administration of METH is known to induce dopaminergic neurotoxicity in rodents and non-human primates, by producing long-term depletion of dopamine(DA) and its metabolite, 3,4-dihydroxyphenylacetic acid(DOPAC), as well as reducing the density of DA transporter(DAT) in the striatum. Long-term abuse of METH may result in memory loss, aggression, psychotic symptoms and potential heart and brain damage. However, the precise mechanisms by which METH elicits neurotoxic effects are still being elucidated.Recently, the role of the immune system in methamphetamine’s neurotoxic effects has been examined in detail. A number of molecular and cellular mechanisms are triggered following exposure of cells or animals to methamphetamine, and the cascade of events from exposure to neurotoxicity involves cellular components from receptors to immune system activation and inflammation, to energy metabolism. A single high dose of METH has been shown to induce IL-6 and TNF-a in the striatum and hippocampus of mice and IL-1b in the hypothalamus of rats. However, the specific molecular mechanism involved in the increased expression of these proinflammatory cytokines is still unknown. Microglial cells, the resident macrophages of the brain, play a crucial role in the innate immune response and are the first line of defense against microbial invasion and injury to the central nervous system. Microglia is involved in regulation of numerous pro- and anti-inflammatory cytokines. Increased inflammatory markers released from microglia are associated with a variety of CNS complications such as Alzheimer’s and Parkinson’s disease. Furthermore, microglial activation has been shown to be critical in the regulation of the rewarding effects induced by drugs of abuse.It is generally accepted that METH induces oxidative stress, neroinflmmtion which can increase proinflammatory cytokines by increasing the activities of transcription factors such as nuclear factor-Kappa B NF-kB, activator protein-1(AP-1) and the c AMP-response element-binding protein(CREB). NF-kB is a critical transcription factor for inflammatory mediator induction. The specific blockage of NF-kB activity or knockout of its gene in microglia suppresses production of the inflammatory cascade against LPS stimulation. Activating NF-kB is necessary to induce the i NOS and TNF-a genes. Activating NF-kB stimulates the expression of proinflammatory mediators. Activation of NF-kB occurs via phosphorylation of its endogenous inhibitor I-kB-aresulting in the release and nuclear translocation of active NF-kB.Cholecystokinin(CCK), a gut-brain peptide, exerts a wide range of biological activities in the gastrointestinal tract and central nervous system(CNS). It was initially isolated from the porcine duodenum as a 33 amino acid peptide and acts via the CCK1 and CCK2 receptor subtypes. CCK-8 is involved in the regulation of feeding, pain perception, and learning and memory and possibly in the pathogenesis of anxiety and psychosis. It modulates the release of several neurotransmitters, such as DA and gamma-aminobutyric acid. Loonam TM et al. found that CCK regulated neurochemical responses to METH in the striatum. Additionally, there are reports on the role of CCK in DA-mediated behaviors, with different subregions of the NAc involved in different effects on DA-mediated locomotor activity. Moreover, our previous results have shown that CCK-8 has anti-oxidative stress and anti-inflammatory effects. In addition, it produced neuroprotective effects in neuronal injury models. Moreover, many studies indicated that CCK-8 showed anti-inflammatory effect in different aspects. CCK plays a novel protective role in diabetic kidney through antiinflammatory actions on macrophage. However, there is no report on the effect of CCK-8 in neuroinflammation. These data show that CCK-8 exhibits a pharmaco-therapeutic potential for treating METH-induced neurotoxicity in CNS.The present study aimed to evaluate the effects of CCK-8 on METH-induced behavioral changes, including hyperlocomotion, behavioral sensitization, and stereotypical behavior; the effects of CCK-8 on high doses of METH-induced hyperthermia and dopamine neurotoxicity; the effects of CCK-8 on METH-induced microglial activition and pro-inflammatory cytokines upregulation in vivo and vitro; and also the cross-talk between PKA/PKC pathway mediated by CCK receptors and NF-kB pathway. Part I Protective effects of cholecystokinin-8 on methamphetamine-induced behavioral changes and dopaminergic neurodegeneration in mice.Objective: The present study aimed to evaluate the effects of CCK-8 on METH-induced behavioral changes, including hyperlocomotion, behavioral sensitization induced by low dose of METH(1 mg/kg), and stereotypical behavior induced by high dose of METH(10 mg/kg); the effects of CCK-8 on high doses of METH-induced hyperthermia and dopamine neurotoxicity were investigated;Methods:1 The mice were pretreated with CCK-8(0, 0.01, 0.1 mg/mouse) 15 min before the METH administration. After the METH injection, the mice were put into the test chambers to record their locomotor activity. Each locomotion test lasted 30 min.2 First, the mice were placed in the locomotion test apparatus for 30 min on days 1-3 to eliminate the baseline locomotor activity. The mice were injected with METH(1, 2 mg/kg, i.p.) once daily for the next 7 days to induce behavioral sensitization(development period), followed by a washout period for sensitization, which lasted 6 days. Next, sensitization was induced(day 14), where the mice were challenged with METH(1, 2 mg/kg, i.p.) and the locomotion was recorded for 30 min. Second, we examine the effects of CCK-8 on the development and expression of behavioral sensitization, CCK-8(0.001, 0.01, 0.1 μg, i.c.v.) was co-administered with METH(1 mg/kg) in the development or the expression period. Saline or CCK-8 was given 15 min before the METH injection. Last, we investigated whether CCK-8 injection alone could trigger behavioral sensitization, the mice received saline or CCK-8 on schedule.3 We tested the effects of CCK-8 on stereotypic behavior induced by a high dose of METH in mice. The stereotypic behaviors were scored as described by Sams-Dodd. To assess the dose-response relationship of METH-induced stereotypic behavior, the mice were administered four METH injections(3-h interval) at different doses(0, 3, 10, 20, and 40 mg/kg, i.p.). Stereotypic behavior was assessed 1 h after the last injection. Furthermore, the effects of CCK-8(1 mg, i.c.v.) on stereotypic behavior induced by METH(3, 10 mg/kg) were investigated. Additionally, the effects of CCK-8 on specific behaviors of mice, including the Sams-Dodd scoring method, were examined. Four CCK-8(1 mg, i.c.v.) microinjections were administered to the mice at 3-h intervals before testing.4 Effects of CCK-8 on induced hyperthermia by high doses of METH(10 mg/kg × 4, 3-h intervals) were observed. Rectal temperature was measured as follows: 30 min before the first injection and 1 h, 4 h, 7 h, 10 h, and 24 h after the first injection of METH.5 Immunocytochemistry and Western blot were poeformed to evaluate the effect of CCK-8 on decreased of tyrosine hydroxylase(TH) and dopamine transporter(DAT) in the striatum and and TH in the substantia nigra.Results:1 Pretreatment with CCK-8(0.01 and 0.1 μg) attenuates the hyperlocomotion induced by a low dose of METH(1 mg/kg) in mice.2 METH(1 and 2 mg/kg) induced behavioral sensitization in mice. CCK-8 pretreatment alone had no effect on locomotion in mice, but administration of CCK-8(0, 0.001, 0.01, and 0.1 μg) before injecting METH in the development or expression period effectively reduced the total distance traveled in the locomotion test in dose dependent manner.3 METH increased the total scores of stereotypic behaviors in a dose-dependent manner at doses of 3, 10, 20, and 40 mg/kg. Furthermore, treatment with CCK-8(1 mg x 4, 3-h intervals, i.c.v.) alone did not affect the total scores of stereotypic behavior in mice, but Pretreatment with 1 mg CCK-8 inhibited the increase in scores induced by 3 mg/kg METH.However, 1 mg CCK-8 pretreatment showed no effect on the increase in total scores induced by 10 mg/kg METH treatment.4 Pretreatment with 1 mg CCK-8 before METH administration significantly inhibited the increased rectal temperatures induced by METH,and CCK-8 alone did not affect rectal temperature in mice.5 Pretreatment with 1 mg CCK-8 before METH administration reverses METH-induced striatal TH and DAT decreases, and also reverses nigral TH.Summary: Our study demonstrates that pretreatment with CCK-8 inhibits changes such as hyperlocomotion, the development and expression of behavioral sensitization, and dopaminergic neurotoxicity typically induced by repeated exposure to METH. These observations indicate that CCK-8 could be a potential therapeutic agent for the treatment of multiple symptoms associated with METH abuse. Part II Cholecystokinin-8 attenuated METH-induced neuroinflamition in vivo and vitro.Objective: The present study aimed to evaluate the effects of CCK-8 on METH-induced microglial activition and pro-inflammatory cytokines upregulation in vivo and vitro.Methods:1 Effects of CCK-8 on neuroinflamition induced by high doses of METH(10 mg/kg × 4, 3-h intervals) were observed. Saline or CCK-8 are preinjected 15 min.Twenty-four hours after the first METH or saline injection, the mice were killed and the brains were harvested.CBA mouse inflammation kit was used to estimate the production of cytokines/chemokines in striatum. Immunocytochemistry was performed to observe the microglial activation in striatum, prefrontal cortex and hippocampus.2 Flow cytometry and immunofluorescence were performed to test the expression of Iba1 in N9 microglial cells.3 Realtime-PCR and immunofluorescence were performed to estimate the expresion of CCK1 R and CCK2 R in N9 microglial cells.4 MTT assay was performed to measure the effect of CCK-8 on the decreased cell viability induced by METH exposure.5 Flow cytometry was performed to evaluate the effects of CCK-8 on METH-induced increased mean fluorescence intensity of FITC-Iba1 in N9 microglial cells.6 Realtime-PCR was performed to estimate the effect of CCK-8 on METH-induced overexpression of cytokines/chemokines in in N9 microglial cells.7 BD Cytometric Bead Array(CBA) mouse inflammation kit was used to estimate the effect of CCK-8 on overproduction of cytokines/chemokines by METH exposure in N9 microglial cells.Results:1 Repeateted administration of METH(10 mg/kg × 4, 3-h intervals) results in microglial activation in the mouse striatum, prefrontal cortex and hippocampus region at 24 h post dosing. The term ‘‘activated microglial cells’ ’ was formally introduced when it became apparent that intrinsic(resident) microglia are capable of up-regulating certain molecules, including several that are not normally expressed in the CNS, Such chameleon-like behaviour illustrates the now well-recognized phenotypic and functional plasticity of microglia. The number of microglia is increased. CCK-8 showed no effect on microglia, but pre-injected with 1 mg CCK-8 inhibited METH-induced microglial activation in the mouse striatum, prefrontal cortex and hippocampus.Repeateted administration of METH(10 mg/kg × 4, 3-h intervals), results in enhanced production of protein for a variety of proinflammatory cytokines and chemokines in the mouse striatum at 24 h post dosing, including MCP-1, IL-6 and TNF-a; and decreased production of IL-10. Pre-injected with 1 mg CCK-8 inhibited the effects of METH. METH or CCK-8 showed no effect on striatal IL-12p70 and IFN-g because of low expression.2 It has been previously suggested that activated microglia express different proteins and surface markers. Of these, Iba1 has the greatest biological significance. Because increased expression of Iba1 is a typical feature of microglial activation, we assessed the expression of Iba1 in N9 cells by FACS and confocal microscopy. Results showed that Iba1 were expressed in N9 microglial cells. A similar pattern was observed with immunolocalization and confocal microscopy, all the N9 microglial cells stainned by antibody Iba1, immunofluorescence image proved that Iba1 labeled with red fluorescence preferred to stay in N9 microglial cells.3 We assessed the expression of CCK1 R and CCK2 R in N9 cells by Real-time PCR and confocal microscopy. Results showed CCK1 R and CCK2 R were all expressed in N9 microglial cells.4 METH significantly decreased cell viability in a concentration dependent manne when treated with 0, 0.5, 1, 2 and 4 m M METH for 24 h. CCK-8 at concentrations of 0.5 and 1mM significantly increased the cell viability of 1 mm METH-treated N9 microglial cells. CCK-8 at concentrations 0, 0.1, 0.5 and 1mM had no effect on the cell viability of microglial cells.5 Effect of METH on Iba1 expression in N9 microglial cells were tested by Flow cytometry analysis. LPS 1mg/m L and METH 1m M was found to significantly increase Iba1 expression, clearly shows increases in Iba1 expression by N9 cells 6 h after LPS and METH exposure. In contrast, no increase in Iba1 expression was observed in the CCK-8 exposure control groups. 1 mM CCK-8 inhibited METH-induced increases in Iba1 expression by N9 cells.6 We investigated the ability of METH to induce the expression of pro-inflammatory cytokine genes that play central roles in various inflammatory diseases: IL-1b, IL-6, TNF-a, MCP-1, IL-10, IL-12p70 and IFN-g. Cells were exposed to 1 mm METH for 0, 1, 3, 6 and 12 h.The m RNA levels of these genes were determined by RT-PCR. The results showed that the expression of these cytokines was most elevated at 3 h after METH treatment.Based on this result, METH-induced pro-inflammatory cytokine expression was tested subsequently at 3 h to examine further effects of METH on cytokine expression. To test the concentration–response relationship of METH-activated N9 microglia, cells were treated with METH(0, 0.5, 1, and 2 mm) or1mg/m L LPS for 3 h. Both LPS and METH at 1 m M significantly increased the expression of IL-1b, IL-6, TNF-a and MCP-1m RNAs.We further investigated whether CCK-8 could attenuate the METH induced increase in pro-inflammatory cytokines. Co-treatment with CCK-8(0, 0.1, 0.5 and 1 mM) significantly counteracted the IL-1b, IL-6, TNF-a and MCP-1 m RNA overexpression induced by 1 m M METH. Furthermore, high concentration of CCK-8(1 mM) showed no effects on IL-1b, IL-6, TNF-a and MCP-1 m RNA overexpression.7 we measured levels of IL-1b, IL-6, TNF-a and MCP-1 production, in cell culture medium supernatants at 24 h after METH(0, 1, 0.1, 0.01 and 0.001 m M) exposure in N9 microglial cells. METH 1mM exposure significantly induced over production of IL-6 and TNF-a. Based on this result, we further investigated whether CCK-8 could attenuate the METH induced increase in over production of IL-6 and TNF-a. Co-treatment with CCK-8 1 mM significantly counteracted the IL-6 and TNF-a over production in cell culture medium supernatants induced by 1 mM METH. Furthermore, CCK-8(1 mM) showed no effects IL-6 and TNF-a over production in N9 cell culture medium supernatants at 24 h.Summary: CCK-8 attenuated METH-induced microglial acttivation and pro-inflammatory cytokines upregulation in vivo and vitro.CCK receptors were expressed in N9 microglial cells. CCK-8 also attenuated METH-induced decreased cell viability.Part III The role of signalinng mediated by CCK Receptors in NF-kB activation and pro-inflammatory responses of METH-stimulated N9 microglial cellsObjective: The present study aimed to evaluate the effects of CCK-8 on METH-induced activation of NF-kB pathway, and also the cross talk between PKA/PKC pathway mediated by CCK receptors and NF-kB pathway.Methods:1 Western Blot was performed to evaluate METH exposure induced activation of NF-kB pathway in N9 microglial cells. The protein samples were harvested at the indicated times(0, 5, 15, 30, 60 and 90 min) after METH exposure in N9 cells. Western blot analysis shows NF-kB signaling pathway activation with antibodies against phospho-NF-kB p65(Ser536), NF-kB p65, phospho-I kB-a(Ser32) and IkB-a.2 Western Blot was performed to evaluate the effects of CCK-8 on METH- induced activation of NF-kB pathway in N9 microglial cells. N9 microglial cells were treated with METH 1m M and different concentration of CCK-8(0, 0.1, 0.5 and 1 mM) for 30 min. Moreover, N9 microglial cells were stimulated with METH for indicated time periods(0, 5, 15 and 30 min) with or without CCK-8(1 μM) treatment. Western blot analysis shows NF-kB signaling pathway with antibodies against phospho-NF-kB p65(Ser536), NF-kB p65, phospho-I kB-a(Ser32) and IkB-a.3 Western Blot was performed to evaluate the effect of CCK receptors antagonist on inhibiting effects of CCK-8 on activaton of NF-kB pathway induced by METH exposure in N9 microglial cells. BD Cytometric Bead Array(CBA) mouse inflammation kit was used to estimate the effect of CCK receptors antagonist on inhibiting effects of CCK-8 on overproduction of IL-6 and TNF-a induced by METH exposure in N9 microglial cells.4 Western Blot was performed to evaluate the effect of c AMP and PLC inhibitor on inhibiting effects of CCK-8 on activaton of NF-kB pathway induced by METH exposure in N9 microglial cells. BD Cytometric Bead Array(CBA) mouse inflammation kit was used to estimate c AMP and PLC inhibitor on inhibiting effects of CCK-8 on overproduction of IL-6 and TNF-a Part III The role of signalinng mediated by CCK Receptors in NF-kB induced by METH exposure in N9 microglial cells.Results:1 The level of phosphorylated of NF-kB pathway was highly increased after METH treatment 5, 15, and 30 min, including phosphorylated NF-kB p65 and I-kB in cytoplasm, also phosphorylated NF-kB p65 and total NF-kB p65 in nucleus. Moreover, NF-kB p65 translocated from cytoplasm to nucleus after METH treatment in N9 microglial cells. The levels of NF-kB p65 protein increased in the nucleus but decreased in the cytoplasm of METH-stimulated N9 cells at 30 min after METH treatment, indicating that METH induced the translocation of NF-kB p65 protein from cytosol to the nucleus.2 The level of phosphorylated of NF-kB pathway was highly increased by METH and was diminished by CCK-8(0.1, 0.5 and 1 mM) in dose dependent manner; N9 microglial cells were stimulated with METH for indicated time periods(0, 5, 15 and 30 min) with or without CCK-8(1 μM) treatment. CCK-8 inhibited the increased fold of phosphorylated NF-kB p65 and I-kB; CCK-8 shows no effect on phosphorylation of NF-kB signaling pathway in N9 microglial cells.3 Blocking CCK2 R rereversed the inhibiting effects of CCK-8 on activaton of NF-kB and overproduction of IL-6 and TNF-a induced by METH exposure in N9 microglial cells, but not blocking CCK1 R. However, CCK1 R antagonist can inhibit overproduction of IL-6 and TNF-a induced by METH exposure in N9 microglial cells.4 Inhibiting of c AMP or PLC showed no effect on on activaton of NF-kB and overproduction of IL-6 and TNF-a induced by METH exposure in N9 microglial cells. However, c AMP or PLC inhibitor decreased the overproduction TNF-a induced by METH exposure in N9 microglial cells, especially the PLC inhibitor.Summary: CCK-8 attenuated METH-induced upregulaition of IL-6 and TNF-a via NF-kB signaling pathway, which had a crosstalk with PKA/PKC pathway mediated by CCK receptors.Conclusions: The present study observed the effect of exogenous CCK-8 on METH-induced behavioral changes, including hyperlocomotion, behavioral sensitization, and stereotypical behavior; the effects of CCK-8 on high doses of METH-induced hyperthermia and dopamine neurotoxicity were investigated; the effects of CCK-8 on METH-induced microglial activition and pro-inflammatory cytokines upregulation in vivo and vitro; and also the cross talk between PKA/PKC pathway mediated by CCK receptors and NF-kB pathway. We can reach the conclusion as follows.1 pretreatment with CCK-8 inhibits changes such as hyperlocomotion, the development and expression of behavioral sensitization, and dopaminergic neurotoxicity typically induced by repeated exposure to METH.2 CCK receptors were expressed in N9 microglial cells.CCK-8 attenuated METH-induced microglial acttivation and pro-inflammatory cytokines upregulation in vivo and vitro in vivo and vitro. CCK-8 also attenuated METH-induced decreased cell viability.3 CCK-8 attenuated METH-induced upregulaition of IL-6 and TNF-a via NF-kB signaling pathway, which had a crosstalk with PKA/PKC pathway mediated by CCK receptors. |
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