激活Gs蛋白对胆红素诱导的大鼠小脑颗粒神经元凋亡影响的研究

Activation of Gs protein affects apoptosis of rat cerebellar granule neurons induced by bilirubin

目的 研究激活Gs蛋白对胆红素诱导的小脑颗粒神经元凋亡的影响及作用机制。方法 采用原代培养的新生大鼠小脑颗粒神经元 ,用霍乱毒素 (CT)激活Gs蛋白 ,用二乙酸荧光素(FDA)染色及四甲基偶氮唑蓝 (MTT)法测定胆红素诱导的小脑神经元存活率 ,12 5I放射免疫法测定细胞内环磷酸腺苷 (cAMP) ,Fura 2 /AM荧光比值成像技术测定细胞内钙浓度 ([Ca2 +]i)。结果 CT诱导细胞内cAMP升高并呈剂量依赖性地保护胆红素诱导的小脑颗粒神经元凋亡。当加入腺苷环化酶(AC)刺激剂 (Forskolin)及cAMP类似物 (8 溴 环磷酸腺苷 )后 ,虽导致细胞内cAMP升高 ,但不能阻断或减弱胆红素诱导的神经元凋亡。另发现胆红素浓度依赖性触发细胞内钙升高 ,而CT可降低胆红素导致的细胞内钙的升高。结论 Gs蛋白可能参与了小脑颗粒神经元凋亡的调控 。

Objective To investigate the effect and the mechanism of activation of Gs protein on bilirubin induced apoptosis of rat cerebellar granule neurons. Methods Primary cultured rat cerebellar granule neurons were exposed to Cholera toxin (CT). The effect of activation of Gs protein by CT on bilirubin induced apoptosis was observed. The survival rate of neurons was detected by fluorescein diacetate (FDA) staining and MTT assay. The intracellular [Ca 2+ ]i was measured by the ratio of fura 2/AM with the microspectrofluorometry ratio imaging system. Cytosolic cyclic AMP(cAMP) content was quantitated by using 125 I radioimmunoassay. Results CT could induce the elevation of cAMP content and protect cerebellar granule neurons from the bilirubin induced apoptosis in a dose dependent pattern. However, the exposure of cultured cerebellar granule neurons to Forskolin (which stimulates adenylate cyclase directly and enhances Gs cyclase coupling) and 8 bromo cAMP (a membrane permeable analogue of cAMP) did not block or attenuate apoptosis of cerebellar granule neurons induced by bilirubin. On the other hand, bilirubin elevated [Ca 2+ ]i in a concentration dependent pattern. CT attenuated the rise of [Ca 2+ ]i induced by bilirubin. Conclusion Gs protein might play a role in the regulation of apoptosis of cerebellar granule neurons. The mechanism of protection against bilirubin induced apoptosis of cerebellar granule neurons by activation of Gs protein was associated with decreasing [Ca 2+ ]i in a cAMP independent way.