银杏酮酯对抑郁症大鼠的治疗作用及其对海马CA1区组织5-HT1AR/cAMP/PKA通路的调控机制

Effect of Ginkgo biloba ester on depressive rats and regulation of 5-HT1AR/cAMP/PKA pathway in hippocampal CA1 region

【目的】观察银杏酮酯对抑郁症大鼠的作用,并探讨其对海马CA1区组织5-羟色胺受体(5-hydroxytryptamine receptor,5-HT1AR)/环腺苷酸(cyclic adenosine mono-phosphate,c AMP)/蛋白激酶A(protein kinase A,PKA)通路的调控作用。【方法】取50只SD大鼠随机分为A组、B组、C组、P组和M组,各10只;另取10只正常SD大鼠记为N组。A、B、C、P和M组建模,同时A、B和C组分别给予35、70、140 mg/kg银杏酮酯灌服,P组给予10 mg/kg氟西汀灌服,N组和M组均给予等体积蒸馏水灌服,各组均1次/d,持续3周。对比各组体质量变化;糖水偏爱实验和强迫游泳实验评价干预前后抑郁样行为学改变;ELISA检测干预前后血清5-羟色胺(5-hydroxytryptamine,5-HT)含量,竞争法检测干预后大脑皮质组织5-HT含量;q RT-PCR检测海马CA1区组织5-HT1AR、c AMP反应元件结合蛋白(c AMP responsive element binding,CREB)、PKA m RNA表达;免疫组化法检测海马CA1区组织5-HT1AR、CREB、PKA蛋白表达并对比其光密度值(D),125碘(125I)标记放免法检测海马CA1区组织c AMP的含量。【结果】体质量、糖水偏爱实验、血清5-HT干预前后差值对比,M组均高于N组(P<0.05),P组和银杏酮酯3剂量组均低于M组(P<0.05),P组、B组和C组均低于A组(P<0.05),C组均低于P组、B组(P<0.05);强迫游泳不动时间干预前后差值对比,M组远长于N组(P<0.05),P组和银杏酮酯3剂量组均短于M组(P<0.05),P组、B组和C组均短于A组(P<0.05),C组短于P组、B组(P<0.05);干预后大脑皮质5-HT含量对比,M组低于N组(P<0.05),P组和银杏酮酯3剂量组均高于M组(P<0.05),P组、B组和C组均高于A组(P<0.05),C组高于P组、B组(P<0.05);各组海马CA1区组织5-HT1AR、CREB、PKA m RNA及蛋白表达、c AMP含量对比,M组上述指标均显著低于N组(P<0.05),A、B、C组和P组均显著高于M组(P<0.05),且银杏酮酯的作用均呈剂量依赖性。【结论】银杏酮酯可控制抑郁症大鼠的体质量下降,减轻抑郁样行为,改善血清和大脑皮质5-HT含量,推测该药物与氟西汀均可能通过调整5-HT1AR/c AMP/PKA通路,上调海马CA1区组织5-HT1AR、CREB、PKA m RNA及蛋白表达、增加c AMP含量实现抗抑郁作用,且银杏酮酯的抗抑郁作用呈剂量依赖性。

【Objective】To observe the effect of Ginkgo biloba ester on depressive rats, and explore its regulation of 5-HT1 AR/c AMP/PKA pathway in hippocampal CA1 region.【Methods】Fifty SD rats were randomly divided into group A, group B, group C, group P and group M, 10 rats per group. Ten normal SD rats were noted as group N. The rat models were established in group A, B, C, P and M. The rats in group A, B and C were fed with 35, 70, 140 mg/kg ginkgo ketone ester, the rats in group P with 10 mg/kg fluoxetine, and those ingroup N and M with distilled water of equal volume once a day for 3 weeks. The changes of body mass were compared among each group.Sugar water preference test and forced swimming test were used to evaluate depression-like behavioral changes before and after intervention. Enzyme-linked immunosorbent assay(ELISA) was usedto detect serum 5-hydroxytryptamine(5-HT) content before and after intervention, and competitive method was used to detect 5-HT content in cerebral cortex after intervention. The expression of 5-HT1 AR, c AMP response element binding protein(CREB) and PKA in hippocampal CA1 region was detected by real-time fluorescence quantitative polymerase chain reaction(RT-q PCR).Immunohistochemistry was used to detect the expression of 5-HT1 AR, CREB and PKA in hippocampal CA1 region and compare their integrated optic density(D). And 125-iodine(125 I) labeled radioimmunoassay was used to detect the content of c AMP in hippocampal CA1 region.【Results】The difference of body mass, sugar water preference test and serum 5-HT before and after intervention in group M was higher than that in group N(P<0.05). It was lower in group P and ginkgolide 3 dose group than that in group M(P<0.05), and it was lower in group P, group B and group C than that in group A(P<0.05). It was lower in group C than that in group P and group B(P<0.05). The difference before and after the intervention of forced swimming immobility time was significantly longer in group M than that in group N(P<0.05), shorter in group P and ginkgolide group 3 than that in group M(P<0.05), shorter in group P, group B and group C than that in group A(P<0.05), shorter in group C than in group P and group B(P<0.05). After intervention, the 5-HT content in cerebral cortex in group M was lower than that in group N(P<0.05), higher in group P and ginkgolide group 3 than that in group M(P<0.05),higher in group P, group B and group C than that in group A(P<0.05), higher in group C than that in group P and group B(P<0.05).According to the m RNA and protein expression of 5-HT1 AR, CREB, PKA and c AMP content in hippocampal CA1 region in each group, the above indexes in group M were significantly lower than those in group N(P<0.05), while those in group A, B, C and P were significantly higher than those in group M(P<0.05), and the effect of Ginkgolides was dose-dependent.【Conclusion】Ginkgo biloba ester can control the decline of body mass and alleviate depression-like behavior in depressive rats and improve the content of 5-HT in serum and cerebral cortex of depressive rats. It is speculated that both the drug and fluoxetine may achieve antidepressant effect by regulating the 5-HT1 AR/c AMP/PKA pathway, up-regulating the expression of 5-HT1 AR, CREB and PKA in hippocampal CA1 region,and increasing the content of c AMP. The effect of anti-depression is dose-dependent.