Effect And Mechanism Of CCK-8 On Methamphetamine-induced Neuronal Apoptosis

Background: Methamphetamine(METH)is a stimulant of the central nervous system,and its long-term abuse is liable to produce toxic effects on the central nervous system.Neuronal apoptosis is one of the mechanisms by which METH exerts its toxic effects.There is no effective treatment for METH-induced neurotoxicity.Cholecystokinin octapeptide(CCK-8)is a brain-gut peptide.Recent ten years' research in our laboratory has found that CCK-8 has antagonistic effects on METH-induced oxidative stress and neuroinflammation,and also has protective effects on morphine-induced neuronal apoptosis.The cholecystokinin(CCK)receptor is the central and peripheral target of CCK-8.There are two subtypes of CCK1 receptor(CCK1 receptor,CCK1R)and CCK2 receptor(CCK2 receptor,CCK2R).Our laboratory found that CCK1 R is a low-sensitivity receptor,and CCK2 R is a high-sensitivity receptor,that is,high-dose CCK-8 can activate CCK1 R,while low-dose CCK-8 can activate CCK2 R to play a biological role.Due to different types and properties of action of morphine and METH,whether CCK-8 and its CCK receptor subtypes play a protective role in METH-induced neuronal apoptosis has not been reported.CCK receptor belongs to G protein-coupled receptor(GPCR).?-arrestin can be used as a signal sensor of GPCR to play an anti-apoptotic or pro-apoptotic role.The mechanism of ?-arrestin2 in effects of CCK-8 on METH-induced neuronal apoptosis also has not been reported.Objective: This study aims to elucidate the protective effect and mechanism of CCK-8 on METH-induced SH-SH5 Y cell apoptosis,and further clarify whether ?-arrestin2 mediates bias agonism in the protective process.Understanding the mechanism of CCK-8 on neurotoxicity of METH will provide a new way to prevent and treat METH addiction and neurological damage.Methods: Annexin-V-FITC/PI flow cytometry and Hoechst33258 staining were used to detect the apoptotic rate and nuclear morphology of SH-SH5 Y cells.Western Blot was used to detect the changes of apoptosis-related proteins.Construction of the HEK293-CCK1 R cell line and HEK293-CCK2 R cell line by means of stable lentivirus transfection was used to investigate the role of CCK receptor in the intervention of CCK-8 on METH-induced apoptosis.In-vitro liposome transfection of small interfering RNA(siRNA)interference with ?-arrestin2 in SH-SH5 Y cells were used to further explore the downstream role of ?-arrestin2 in CCK receptor when CCK-8 exerts an anti-apoptotic effect.SPSS 21.0 software was used to analyze the data.The results were presented as mean ± standard error(mean ± S.E.M.).One-way ANOVA was adopted to compare the mean values between groups,and the least significant difference t test(the LSD-t test)was used for pairwise comparison,with P < 0.05 as statistical difference.Results:1.Flow cytometry showed that CCK-8 pretreatment reversed the increase of SH-SY5 Y cell apoptosis induced by METH.Hoechst33258 staining showed that CCK-8 pretreatment reduced the number of irregular dense stained nuclei compared with the group treated with METH alone.It is suggested that CCK-8 may protect METH-induced neuronal apoptosis at the cellular levels.2.METH induced up-regulation of the pro-apoptotic proteins Bax and Cleaved Caspase3 and increase of Bax/Bcl2 ratio in SH-SY5 Y cells.Pretreatment with CCK-8 before METH administration reversed the above-mentioned changes of SH-SY5 Y apoptotic protein induced by METH in varying degrees.It is suggested that CCK-8 may protect METH-induced neuronal apoptosis at the molecular levels.3.CCK-8 pretreatment had no effect on the expression of Bax,Cleaved Caspase3 and Bax/Bcl2 in HEK293-CCK1 R cell line induced by METH,but could reverse the up-regulated expression of Bax and Cleaved Caspase3 and increase of the Bax/Bcl2 ratio in HEK293-CCK2 R cell line induced by METH.These results suggest that CCK-8 may play a protective role in METH-induced neuronal apoptosis after binding to CCK2 R.4.In SH-SY5 Y cells with ?-arrestin2 interfered by siRNA,CCK-8 pretreatment had no significant effect on the expression of SH-SY5 Y cell apoptosis-related proteins induced by METH.These results suggest that ?-arrestin2 may be involved in protective effect of CCK-8 on METH-induced neuronal apoptosis pathway.Conclusions:1.CCK-8 protects SH-SY5 Y cells from METH-induced apoptosis.2.CCK-8 protects METH-induced neuronal apoptosis by binding to CCK-2R;?-arrestin2 may play a biased signaling role in the protective process.