The Role Of CCK-8 In Methamphetamine Induced Nerve Injury And Oxidative Stress Mechanism

Objective: Drug abuse, especially the abuse of new chemical synthetic drug methamphetamine, ecstasy and other amphetamines, is one of the medical and social problems to be solved all over the world. Methamphetamine(methamphetamine, METH), an amphetamine type stimulant, whose hydrochloride salt is colorless and transparent crystal, is commonly known as "ice". METH has been abused worldwide. The METH molecule is fat soluble to easily passthe blood-brain barrier, resulting in damage of the central nervous system above the brain stem. Long-term abuse of METH can induce obvious behavior, mental and cognitive dysfunction, such as mania, auditory hallucination, visual hallucination, anxiety, irritability, schizophrenia. Because the abusers are mostly teenagers, the social harm is especially serious. But at present, the mechanism of METH addiction and neurotoxic is not very clear, and there is no effective prevention and treatment.At present, people generally believed that METH can cause neurotoxicity via these classical pathway, such as oxidative stress, toxicity of excitatory amino acids and mitochondrial dysfunction. Studies have demonstrated that METH was neurotoxic to striatal DA terminals, as exemplified by a significant reduction in striatal DA, dopamine transporter(DAT) or DA receptor levels. Cholecystokinin is a 33 amino acid peptide and acts via CCK1 and CCK2 receptor subtypes. A number of biologically-active molecular variants were subsequently described, and the most abundant peptide present in the brain was shown to be Cholecystokinin octapeptide(CCK-8). CCK-8 is involved in the regulation of feeding, pain perception, learning and memory, and possibly in the pathogenesis of anxiety and psychosis. It has been shown to modulate the release of several neurotransmitters, such as DA and gamma-aminobutyric acid(GABA), and possibly acts as a neurotransmitter/modulator. And, some researches have shown that CCK-8 has anti-oxidative stress and anti- inflammatory effects. It also produced neuroprotective effects in neuronal injury models. These data suggest that CCK-8 exhibits pharmaco-therapeutic potential in the treatment of METH-induced neurotoxicity in CNS.Based on the above viewpoints, this study focuses on the effect of methamphetamine on the injury of PC12 cells, a clonal cell line derived from a pheochromocytoma of the rat adrenal medulla, as a neuronal cell model. And, we observe whether oxidative stress function in the injury, and explore the protective mechanism of CCK-8. The data would provide new ideas for the protective role of CCK-8 in nerve cell injury.Methods:1 RT-PCR was used for identification CCK1R(CCK1 receptor) and CCK2R(CCK2 receptor) on PC12 cell.2 Effects of CCK-8 and METH on PC12 cells. MTT experiment was used to observe the survival rate of PC12 cell after 0.1, 0.5, 1, 2, 4 m M METH treatment for 24 h. In addition, the survival rate after 0.1, 0.5, μM CCK-8 with/without 2m M METH treatment was evaluated.3 Effects of CCK-8 and METH on PC12 cells knocked down CCK1 R and CCK2 R. Ttransfection with RNAi was applied to knock down CCK1 R and CCK2 R on PC12 cell. The transfection result was validated by RT-PCR and agarose gel electrophoresis method. With MTT, we observed the effect of 2m M METH, 1 μM CCK-8 for 24 h on the PC12 cells.4 Effects of CCK-8 and METH on CCK1 R and CCK2 R overexpressed HEK293 cells. With MTT mothod, we observed the effect of 1, 2, 4, 6, 8, 10 m M METH or 0.1, 0.5, 1μM CCK-8 treatment for 24 h on HEK293 cell survival rate. The survival rate of CCK1 R and CCK2 R overexpressed HEK293 cells, with 4 m M METH or 0.1, 0.5, 1 μM CCK-8 or both of them treatment were dectected.5 Effects of CCK-8 and METH on the oxidative stress of PC12 cells. ROS fluorescence probe-DHE biochemistry kit was uesd to observe the effect of 1m M METH and 1 μM CCK-8 treatment separately and together on PC12 cells treated for 15 min, 30 min, 45 min, 60 min, to find the changes of ROS content of PC12 cells.MDA biochemical reagent kit was uesd to observe the effect of 1m M METH on PC12 cells treated for 5min, 15 min, 25 min, 30 min, and 1m M METH and 1μM CCK-8 treatment for 15 min alone and together on PC12.6 Effects of CCK-8 and METH treatment on intracellular P47 phox, gp91 phox and P47 phox protein levels in PC12 cells. The Western blot was used to observe protein levels of the Nox complex, consisted of P47 phox and gp91 phox, and phosphorylated P47phox(p-P47). Also, P47 phox, gp91 phox protein levels were detected after 1m M METH treatment for 2min, 4min, 6min, 8min, 10 min, and 1m M METH and 1 μM CCK-8 treatment for 4min alone and together in PC12 cells.7 Effects of METH and CCK-8 on P47 phox and gp91 phox interaction. The relationship between P47 phox and gp91 phox was studied after 1 m M METH and 1 μM CCK-8 treatment for 4min alone and together in the PC12 cells by Co-Immunoprecipitation method.8 The data were presented as Means±S.E.M and analyzed with one way ANOVA by SPSS17.0 statistical program,Student-Newman-Keuls do the comparison between groups. A level of P < 0.05 was supposed to be statistically significant.Results:1 PC12 cell receptor validation results suggested that CCK1 R and CCK2 R receptors were on PC12 cells.2 The change of the survival rate of PC12 cells: 0.1, 0.5, 1 μM CCK-8 treament alone had no effect on the survival rate of PC12 cells, compared with the normal control group(P>0.05). The viability of PC12 cells decreased significantly after 1, 2, 4 m M METH treament for 24 h(P<0.001), while the survival rate was significantly increased after 0.1, 0.5, 1 μM CCK-8 and 2m M METH co-treatment(P<0.001). Tip: METH could damage the PC12 cells,and CCK-8 could inhibite the METH-induced cell damage.3 The survival rate had no change in CCK1 R and CCK2 R i RNA transfected cell lines and overexpressed CCK1 R and CCK2 R HEK293 cells after treatment with 0.1, 0.5, 1 μM CCK-8, compared with untransfected cells(P>0.05). However, CCK-8 protection against METH induced cell damage decreased after CCK2 R knockdown(P<0.01), and increased after CCK2 R overexpressed(P<0.01). Meanwhile, neither CCK1 R knockdown nor CCK1 R overexpressed had any effect on CCK-8 protection against METH induced cell damage(P>0.05). Tip: CCK-8 functioned its protection aginst METH induced cell damaging through CCK2 R.4 The changes of ROS levels and MDA content in PC12 cells. In 1m M METH treatment group, ROS levels and MDA content were significantly increased, compared with normal control group(P<0.001). 1 μM CCK-8 inhibited METH induced ROS and MDA’s increase(P<0.001). Tip: oxidative stress invovled in METH induced cell injury. CCK-8 protected these cells from oxidative stress injury.5 Effects of CCK-8 and METH treatment on intracellular P47 phox, gp91 phox and P47 phox protein levels in PC12 cells The expression of p-P47 phox protein increased after METH treatment(P<0.001), while CCK-8 could reduce the increase(P<0.01). But expression of P47 phox, gp91 phox protein had no change. METH could promote formation of Nox complex(P<0.05), while CCK-8 reversed this action by CO immunoprecipitation method(P<0.05). Tip: METH trigged the P47 phox phosphorylation, promoted to the formation of Nox complexes, which induced cell oxidative stress injury. CCK-8 could downregulate this oxidative stress reaction through inhibiting formation of Nox complex.Conclusion: Methamphetamine could induce oxidative stress injury in PC12 cells. CCK-8 functioned its protection against METH induced cell damaging through CCK2 R.