Effects of diacetylmorphine on caspase-8 gene in apoptosis of cerebellar granule neurons

doi:10.3969/j.issn.2095-4344.2015.02.010

Abstract

Abstract:

BACKGROUND: Caspase-8 plays an important role in starting the process of cell apoptosis, and diacetylmorphine can induce neuronal apoptosis. The relationship between the apoptosis of cerebellar granule neurons cells induced by diacetylmorphine and caspase-8 has not been reported.
OBJECTIVE: To investigate the effect of caspase-8 on diacetylmorphine-induced neuronal apoptosis.
METHODS: Cerebellar granule neurons from Sprague-Dawley rats aged 7 days were cultured in vitro. At 7 days, the cells were cultured with different dosage of diacetylmorphine (10, 40, 80, 100, 120 mg/L) and SP600125 for 24 hours, and were divided into control group, 80 mg/L diacetylmorphine group, diacetylmorphine+SP600125 group. Cell morphology was observed by Hoechst 33258 fluorescent staining, and cell apoptosis rate was measured by flow cytometry. Immunofluorescence staining, RT-PCR and western blot assay were used to detect caspase-8 mRNA and protein expression.
RESULTS AND CONCLUSION: (1) After different dosage of diacetylmorphine was used to induce neuronal apoptosis, dark blue translucent apoptotic bodies were found in apoptotic cells, appearing with nucleus condensation, cohesion and fracture, and the apoptosis rate presented an increasing trend with increasing of drug dosage (P < 0.05). (2) Compared with the control group, caspase-8 mRNA and protein expression was remarkable under the intervention of 80 mg/L diacetylmorphine (P < 0.05); caspase-8 mRNA expression exhibited an increasing trend with increasing dosage of diacetylmorphine (P < 0.05), while caspase-8 mRNA and protein expression in the diacetylmorphine+SP600125 group was significantly lower than that in the 80 mg/L diacetylmorphine group (P < 0.05). These findings indicate that caspase-8 is involved in the process of diacetylmorphine-induced neuronal apoptosis, and meanwhile, it is also an important candidate of pro-apoptotic factors in the c-jun N-terminal kinase signaling pathway.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: Heroin, Neurons, Apoptosis, Caspase 8

CLC Number:

R318

Su Li-ping1, Liu Xiao-shan2, Wang Xue-mei, Chen Xiao, Zhang Li-ping, Pu Hong-wei, Wang Zhi-guo, Wang Hua, Li Kai-chao. Effects of diacetylmorphine on caspase-8 gene in apoptosis of cerebellar granule neurons[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(2): 213-219.

Figures/Tables 3

随二乙酰吗啡药物浓度的越高，细胞凋亡数量增多、凋亡率越高(表1)。流式细胞能够精确表达细胞凋亡率，进一步证实了二乙酰吗啡致神经元细胞凋亡。 2.3 Caspase-8 蛋白免疫荧光检测结果与对照组相比，Caspase-8蛋白在80 mg/L的二乙酰吗啡处理下，胞质中的Caspase-8被大量激活，呈高表达状态，进一步级联激活下级促凋亡因子而产生凋亡(图3A)，Caspase-8蛋白相对表达量较对照组明显升高，差异有显著性意义(P < 0.05，图3B)。 2.4 检测caspase-8 mRNA和蛋白表达 Caspase-8是凋亡过程中重要调节因子，其改变是否参与了二乙酰吗啡诱导神经元凋亡? C-Jun氨基末端激酶通路已证实参与细胞凋亡过程[4-5]，那么Caspase-8是否也参与二乙酰吗啡诱导凋亡过程的C-Jun氨基末端激酶途径呢。为了探索这个机制，首先观察caspase-8 mRNA在不同质量浓度的二乙酰吗啡作用下，其mRNA表达水平情况。半定量PCR所示(图4A)，在以GAPDH为内参基因基础上，发现在不同质量浓度二乙酰吗啡影响下，caspase-8 mRNA 出现显著上调，并呈现相应的升高趋势；通过反转录聚合酶链反应在 80 mg/L二乙酰吗啡时，与对照组相比Caspase-8 mRNA水平明显高表达，在加入C-Jun氨基末端激酶通路特异性抑制剂SP600125后，二乙酰吗啡+SP600125组中caspase-8 mRNA表达与单因素二乙酰吗啡(80 mg/L)作用小脑颗粒细胞表达水平比较，有明显下降趋势(图4B)。同时，在以β-Tubulin为内参的基础上，与对照组相比，Caspase-8蛋白量分别在10，40 mg/L开始上调80，100，和120 mg/L 出现高表达(图4C)。同理，加入SP600125抑制剂后，二乙酰吗啡+ SP600125组中caspase-8 蛋白表达水平与80 mg/L二乙酰吗啡组作用小脑颗粒细胞表达水平比较，有明显下降趋势(图4D)。这表明SP 600125在二乙酰吗啡致神经元凋亡过程中具保护作用。在二乙酰吗啡及SP 600125作用影响下，caspase-8 mRNA和蛋白水平改变，提示caspase-8参与二乙酰吗啡诱导凋亡过程并被C-Jun氨基末端激酶通路相应调节。"

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