Inhibitory Effect of CCK-8 on Methamphetamine-Induced Apoptosis

doi:10.12116/j.issn.1004-5619.2021.310206

Abstract

Abstract: 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 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.

Conclusion

CCK-8 can bind to CCK2R and exert an inhibitory effect on METH-induced apoptosis by activating the β-arrestin 2 signal.

Key words: forensic toxicology, apoptosis, methamphetamine, cholecystokinin octapeptide, neuron, receptor, β-arrestin

CLC Number:

DF795.1

Wu-hua ZHANG, Ming-long ZHANG, Wei-wei JING, Bing XIE, Hai-tao BI, Feng YU, Bin CONG, Chun-ling MA, Di WEN. Inhibitory Effect of CCK-8 on Methamphetamine-Induced Apoptosis[J]. Journal of Forensic Medicine, 2021, 37(6): 796-805.

Figures/Tables 19

Fig. 1 Apoptosis of SH-SY5Y cells detected by Annexin Ⅴ-FITC/PI staining and flow cytometry

Tab. 1 The apoptosis rate of SH-SY5Y cellstreated with different concentrations ofMETH detected by flow cytometry

组别	细胞凋亡率
对照	8.48±0.46
1 mmol/L METH	15.05±0.92^1）
2 mmol/L METH	17.06±1.73^1）

Fig. 2 Morphological changes of apoptotic nucleidetected by Hoechst 33258 staining

Fig. 3 Expression of apoptosis-related proteins induced byMETH in SH-SY5Y cells detected by Western blotting

Tab. 2 Expression of apoptosis-related proteinsinduced by METH in SH-SY5Y cells

组别	Bax	Bcl-2	cleaved caspase-3	Bax/Bcl-2
0 h	1.00±0.00	1.00±0.00	1.00±0.00	1.00±0.00
6 h	1.26±0.14	1.08±0.16	1.10±0.05	1.26±0.31
12 h	1.38±0.09^1）	0.99±0.14	1.41±0.08^1）	1.47±0.27
18 h	1.56±0.05^1）	0.79±0.09	1.46±0.06^1）	2.04±0.26^1）
24 h	1.54±0.16^1）	0.75±0.03	1.26±0.01^1）	2.04±0.17^1）

Fig. 4 Apoptosis of SH-SY5Y cells detected by Annexin Ⅴ-FITC/PI staining and flow cytometry

Tab. 3 Effects of CCK-8 on the apoptosis rateinduced by METH in SH-SY5Y cellsdetected by flow cytometry

组别	细胞凋亡率
对照	8.13±0.70
METH	14.18±0.40
0.01 μmol/L CCK-8+METH	12.20±1.24
0.1 μmol/L CCK-8+METH	10.56±0.15^1）
1 μmol/L CCK-8+METH	10.77±0.29^1）
0.01 μmol/L CCK-8	5.76±0.73
0.1 μmol/L CCK-8	5.53±0.89
1 μmol/L CCK-8	5.95±0.84

Fig. 5 Morphological changes of apoptotic nucleidetected by Hoechst 33258 staining

Tab. 4 Effects of CCK-8 on the expression of apoptosis-related proteins induced by METH in SH-SY5Y cells

组别	Bax	Bcl-2	cleaved caspase-3	Bax/Bcl-2
对照	1.00±0.00	1.00±0.00	1.00±0.00	1.00±0.00
METH	1.65±0.15	0.90±0.04	1.66±0.09	1.83±0.10
0.01 μmol/L CCK-8+METH	1.46±0.08	1.16±0.13	1.33±0.10^1）	1.31±0.22^1）
0.1 μmol/L CCK-8+METH	1.33±0.11^1）	1.16±0.12	1.23±0.02^1）	1.18±0.19^1）
1 μmol/L CCK-8+METH	1.10±0.08^1）	1.29±0.13^1）	1.07±0.04^1）	0.86±0.08^1）

Fig. 6 Effects of CCK-8 on the expression of apoptosis-related proteins induced by METH in SH-SY5Y cellsdetected by Western blotting

Fig. 7 Morphology of HEK293 cells transfected with lentivirus vectors under phase and fluorescence （×200）

Fig. 8 The confocal images of lentivirus-transfected HEK293 cells

Fig. 9 Effects of CCK-8 on the expression of apoptosis-related proteins induced by METHin HEK293-CCK1R and HEK293-CCK2R detected by Western blotting

Tab. 5 Effects of CCK-8 on the expression of apoptosis-related proteinsinduced by METH in HEK293-CCK1R and HEK293-CCK2R

蛋白	HEK293-CCK1R				HEK293-CCK2R
蛋白	对照组	METH	CCK-8+METH	CCK-8	对照组	METH	CCK-8+METH	CCK-8
Bax	1.00±0.00	1.65±0.08	1.59±0.07	0.94±0.02	1.00±0.00	1.67±0.13	1.18±0.04^1）	1.11±0.13
Bcl-2	1.00±0.00	0.88±0.07	0.91±0.09	0.97±0.09	1.00±0.00	0.83±0.05	1.00±0.15	0.96±0.08
cleaved caspase-3	1.00±0.00	1.85±0.14	1.87±0.17	0.99±0.17	1.00±0.00	1.76±0.06	1.25±0.06^1）	1.02±0.10
Bax/Bcl-2	1.00±0.00	1.92±0.23	1.80±0.24	0.99±0.11	1.00±0.00	2.01±0.15	1.23±0.15^1）	1.15±0.04

Fig. 10 Morphology of SH-SY5Y cells transfected withsiRNA under phase and fluorescence （×200）

Fig. 11 Expression of β-arrestin 2 in SH-SY5Y cellstransfected with different siRNA sequencesdetected by Western blotting

Tab. 6 Effects of siRNA on the expressionof β-arrestin 2 in SH-SY5Y cells

组别	相对表达量
对照	1.00±0.00
阴性对照	1.09±0.06
β-arrestin 2 siRNA-1	0.96±0.06
β-arrestin 2 siRNA-2	0.61±0.04^1）
β-arrestin 2 siRNA-3	0.28±0.04^1）2）

Fig. 12 Effects of CCK-8 on the expression ofapoptosis-related proteins induced by METHin β-arrestin 2 knockdown SH-SY5Ydetected by Western blotting

Tab. 7 Effects of CCK-8 on the expression of apoptosis-related proteinsinduced by METH in β-arrestin 2 knockdown SH-SY5Y

组别	Bax	Bcl-2	cleaved caspase-3	Bax/Bcl-2
对照	1.00±0.00	1.00±0.00	1.00±0.00	1.00±0.00
METH	1.81±0.04^1）	0.92±0.04	1.80±0.07^1）	1.98±0.13^1）
CCK-8+METH	1.79±0.09^1）	0.89±0.04	1.70±0.08^1）	2.02±0.02^1）
CCK-8	1.07±0.02	1.07±0.11	0.91±0.05	1.02±0.09

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