基于cAMP/PKA/CREB信号通路探讨醒脑益髓汤对血管性痴呆大鼠海马神经元的影响

Effect of decoction for restoring consciousness and replenishing marrow on hippocampal neurons in rats with vascular dementia based on cAMP/PKA/CREB signaling pathway

目的探讨醒脑益髓汤对血管性痴呆大鼠海马神经元凋亡及环磷酸腺苷(cAMP)/蛋白激酶A(PKA)/环磷酸腺苷反应元件结合蛋白(CREB)信号通路的影响。方法随机选取10只SD大鼠作为假手术组,取50只SD大鼠采用双侧颈总动脉永久结扎法建立血管性痴呆模型。将造模成功大鼠随机分为模型组、多奈哌齐组及醒脑益髓汤低、中、高剂量组,每组10只。假手术组和模型组给予生理盐水灌胃,多奈哌齐组给予0.45 mg/mL的多奈哌齐溶液灌胃,醒脑益髓汤低、中、高剂量组分别给予0.98 g/mL、1.96 g/mL、3.92 g/mL的醒脑益髓汤灌胃,均1次/d,连续30 d。定位航行实验和空间探索实验观察大鼠行为学改变,TUNEL法检测海马CA1神经元凋亡情况,ELISA法检测海马组织中cAMP及脑源性神经营养因子(BDNF)含量,Western blot法检测海马组织中PKA和CREB表达情况。结果与假手术组比较,模型组大鼠穿越平台所在象限次数和穿过第Ⅲ象限次数均明显减少(P均<0.05),发现平台并爬上平台时间明显延长(P<0.05),第Ⅲ象限游泳时间明显缩短(P<0.05),海马CA1神经元凋亡细胞明显增加,海马组织中cAMP、BDNF含量和PKA、CREB蛋白表达量均明显降低(P均<0.05);与模型组比较,多奈哌齐组及醒脑益髓汤各组大鼠穿越平台所在象限次数和穿过第Ⅲ象限次数均明显增加(P均<0.05),发现平台并爬上平台时间明显缩短(P均<0.05),第Ⅲ象限游泳时间明显延长(P均<0.05),海马CA1神经元凋亡细胞明显减少,海马组织中cAMP、BDNF含量和PKA、CREB蛋白表达量均明显升高(P均<0.05);醒脑益髓汤中剂量组各指标改善更明显,与醒脑益髓汤高、低剂量组比较差异均有统计学意义(P均<0.05)。结论醒脑益髓汤可以改善血管性痴呆大鼠的学习记忆能力,其机制可能与调控cAMP/PKA/CREB信号通路关键因子的表达,减少神经元凋亡有关。

Objective It is to investigate the effects of decoction for restoring consciousness and replenishing marrow on apoptosis of hippocampal neurons and cyclic adenosine monophosphate(cAMP)/protein kinase A(PKA)/cAMP response element binding-protein(CREB)signaling pathway in rats with vascular dementia.Methods Ten SD rats were randomly selected as the sham operation group,and another 50 SD rats were taken to establish vascular dementia models by permanent ligation of bilateral common carotid arteries.The successful modeled rats were randomly divided into model group,donepezil group,and low,medium,and high dose groups of decoction for restoring consciousness and replenishing marrow,with 10 rats in each group.The sham operation group and the model group were given normal saline by gavage,the donepezil group was given 0.45 mg/mL of donepezil solution by gavage,and the low,medium and high dose groups of decoction for restoring consciousness and replenishing marrow were given 0.98 g/mL,1.96 g/mL and 3.92 g/mL of decoction for restoring consciousness and replenishing marrow by gavage respectively,all once per day,continuously treated for 30 days.The behavioral changes of the rats were observed by the localization navigation test and spatial exploration test,the apoptosis of hippocampal CA1 neurons was detected by TUNEL method,the contents of cAMP and brain-derived neurotrophic factor(BDNF)in hippocampal tissues were detected by ELISA,and the expression of PKA and CREB in hippocampal tissues were detected by Western blot.Results Compared with the sham operation group,the times of the rats crossed the quadrant where the platform was located and the times they crossed the third quadrant were significantly reduced in the model group(all P<0.05),the time to find and climb the platform was significantly prolonged(P<0.05),the swimming time in the third quadrant was significantly shortened(P<0.05),the apoptotic cells of CA1 neurons in the hippocampus were significantly increased,the contents of cAMP and BDNF and the expression of PKA and CREB in the hippocampal tissue were significantly decreased(all P<0.05);Compared with the model group,the times of the rats crossed the quadrant where the platform was located and the times they crossed the third quadrant in the donepezil group and each dose group of decoction for restoring consciousness and replenishing marrow were significantly increased(all P<0.05),the time to find and climb the platform was significantly shortened(P<0.05),the swimming time in the third quadrant was significantly prolonged(P<0.05),and the apoptotic cells of hippocampal CA1 neuronal were significantly reduced,the contents of cAMP,BDNF and the expression of PKA and CREB in hippocampal tissues were significantly increased(all P<0.05);The improvement of each index was more obvious in the medium-dose group of decoction for restoring consciousness and replenishing marrow,and the difference was statistically significant compared with the high and low dose groups of decoction for restoring consciousness and replenishing marrow(all P<0.05).Conclusion Decoction for restoring consciousness and replenishing marrow can improve the learning and memory ability of rats with vascular dementia,and the mechanism may be related to regulate the expression of key factors of cAMP/PKA/CREB signaling pathway and reduce neuronal apoptosis.