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.
目的: 研究八肽胆囊收缩素（cholecystokinin octapeptide，CCK-8）与胆囊收缩素2受体（cholecystokinin 2 receptor，CCK2R）结合对甲基苯丙胺（methamphetamine，METH）诱导的神经元凋亡的抑制作用，并探讨β-arrestin 2在CCK-8抑制METH诱导神经元凋亡中的信号转导机制。方法: 培养SH-SY5Y细胞和慢病毒转染的HEK293-CCK1R和HEK293-CCK2R细胞，应用干扰小RNA（small interfering RNA，siRNA）敲减β-arrestin 2的表达。采用Annexin Ⅴ-FITC/PI染色和流式细胞术检测细胞的凋亡率，Western印迹法检测凋亡相关蛋白的表达。结果: 1 mmol/L、2 mmol/L METH可诱导SH-SY5Y细胞凋亡，核碎裂、固缩的细胞数量显著增加，凋亡相关蛋白Bax和活化型胱天蛋白酶3（cleaved caspase-3）表达升高。0.1 mmol/L和1 mmol/L CCK-8预处理均可逆转METH诱导的SH-SY5Y细胞凋亡，并抑制METH引起的核碎裂、固缩的细胞数量增多以及凋亡相关蛋白的改变。慢病毒转染构建HEK293-CCK1R和HEK293-CCK2R细胞，发现CCK-8对METH诱导HEK293-CCK1R细胞凋亡相关蛋白的变化无明显影响，但可抑制METH诱导的HEK293-CCK2R细胞凋亡相关蛋白表达水平的升高。敲减SH-SY5Y细胞的β-arrestin 2表达后可阻断CCK-8对METH诱导细胞凋亡的抑制作用。结论: CCK-8可与CCK2R结合，并通过激活β-arrestin 2信号抑制METH诱导的细胞凋亡。.
Keywords: apoptosis; cholecystokinin octapeptide; forensic toxicology; methamphetamine; neuron; receptor; β-arrestin.