Effects of diacetylmorphine on caspase-8 gene in apoptosis of cerebellar granule neurons / 中国组织工程研究

ABSTRACT

Abstract BACKGROUND:Caspase-8 plays an important role in starting the process of cel apoptosis, and diacetylmorphine can induce neuronal apoptosis. The relationship between the apoptosis of cerebelar granule neurons cels induced by diacetylmorphine and caspase-8 has not been reported. OBJECTIVE:To investigate the effect of caspase-8 on diacetylmorphine-induced neuronal apoptosis. METHODS:Cerebelar granule neurons from Sprague-Dawley rats aged 7 days were culturedin vitro. At 7 days, the cels 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. Cel morphology was observed by Hoechst 33258 fluorescent staining, and cel 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 cels, 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.