摘要
目的研究八肽胆囊收缩素(cholecystokinin octapeptide,CCK-8)与胆囊收缩素2受体(cholecysto⁃kinin 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AnnexinⅤ-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1mmol/L、2mmol/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。0.1mmol/L和1mmol/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。
Objective 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 ofβ-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 ofβ-arrestin 2.AnnexinⅤ-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 ofβ-arrestin 2 expression in SH-SY5Y cells.Con⁃clusion CCK-8 can bind to CCK2R and exert an inhibitory effect on METH-induced apoptosis by activating theβ-arrestin 2 signal.
作者
张武华
张明龙
景伟伟
谢冰
毕海涛
于峰
丛斌
马春玲
文迪
ZHANG Wu-hua;ZHANG Ming-long;JING Wei-wei;XIE Bing;BI Hai-tao;YU Feng;CONG Bin;MA Chun-ling;WEN Di(Collaborative Innovation Center of Forensic Medical Molecular Identification,Hebei Key Laboratory of Fo-rensic Medicine,College of Forensic Medicine,Hebei Medical University,Shijiazhuang 050017,China)
出处
《法医学杂志》
CAS
CSCD
2021年第6期796-805,共10页
Journal of Forensic Medicine
基金
国家自然科学基金面上项目(81772019)。
