基于BDNF/TrkB/CREB通路研究六味地黄丸对丙戊酸钠诱导的孤独症谱系障碍模型仔鼠的作用机制

Mechanism of action of Liuwei Dihuang Pill on sodium valproate-induced neonatal rat model of autism spectrum disorder based on BDNF/TrkB/CREB pathway

目的基于脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)/酪氨酸激酶受体B(tyrosine kinase receptor B,TrkB)/cAMP反应元件结合蛋白(cAMP response element binding protein,CREB)通路,探讨六味地黄丸对丙戊酸钠(sodium valproate,VPA)诱导的孤独症谱系障碍(autism spectrum disorder,ASD)仔鼠的作用机制。方法将13只SD孕鼠随机分为两组,其中10只孕鼠在第12.5天时腹腔注射VPA溶液(600 mg·kg^(-1))为VPA组,另外3只孕鼠注射等体积生理盐水为对照组。第21天对两组雄性仔鼠开展行为学检测,筛选出符合ASD疾病模型的仔鼠30只,随机分为模型组(等体积生理盐水),维生素D组(1480 IU·kg^(-1)),六味地黄丸高(3 g·kg^(-1))、中(1.5 g·kg^(-1))、低(0.75 g·kg^(-1))剂量组,每组6只。正常雄性仔鼠6只,设为空白组(等体积生理盐水)。各组仔鼠连续灌胃14 d,1次/d,给药后再次开展行为学检测。尼氏染色观察各组仔鼠海马组织神经元形态学变化,比色法检测各组仔鼠海马组织中谷氨酸(glutamic acid,GLU)、γ-氨基丁酸(gamma-aminobutyric acid,GABA)含量;qRT-PCR检测各组仔鼠海马组织中BDNF、TrkB、CREB mRNA相对表达。结果与对照组比较,VPA组仔鼠体质量、身长、尾长更小(P<0.05)。与空白组比较,模型组社交障碍症状明显(P<0.01),焦虑障碍症状明显(P<0.01),重复刻板行为增多(P<0.05或P<0.01),海马神经元结构损伤,GLU升高(P<0.01)、GABA下降(P<0.01),BDNF、TrkB、CREB mRNA表达降低(P<0.05或P<0.01);与模型组比较,维生素D组及六味地黄丸中、低剂量组仔鼠社交能力增强(P<0.05或P<0.01),焦虑障碍减轻(P<0.05或P<0.01),重复刻板行为减少(P<0.01或P<0.05),海马神经元结构明显复原,GLU下降(P<0.01),BDNF、TrkB、CREB mRNA表达增加(P<0.05或P<0.01),六味地黄丸中、低剂量组GABA上升(P<0.05或P<0.01)。结论六味地黄丸能显著改善VPA诱导的ASD仔鼠行为表现,增强海马组织神经元的再生与修复,其机制可能与平衡GLU、GABA水平,上调仔鼠海马组织中BDNF/TrkB/CREB的表达有关。

Objective To explore the mechanism of action of Liuwei Dihuang Pill(LWDHP)on the neonatal rats with autism spectrum disorder(ASD)induced by sodium valproate(VPA)based on the brain-derived neurotrophic factor(BDNF)/tyrosine kinase receptor B(TrkB)/cAMP response element binding protein(CREB)pathway.Methods Thirteen SD pregnant rats were randomly divided into two groups,with 10 pregnant rats receiving intraperitoneal injection of VPA solution(600 mg·kg^(-1))on the day 12.5th as the VPA group,and the other 3 pregnant rats receiving injection of equal volume of normal saline as the control group.Behavioral tests were carried out on the male neonatal rats of two groups on the 21th day,then 30 male neonatal rats conforming to the ASD disease model were selected and randomized into model group(equal volume of normal saline),vitamin D group(1480 IU·kg^(-1)),as well as high-(3 g·kg^(-1)),medium-(1.5 g·kg^(-1)),and low-dose(0.75 g·kg^(-1))LWDHP groups,with six rats in each group.And six normal male neonatal rats were set as blank group(equal volume of normal saline).The neonatal rats in each group were given the corresponding medicine by gavage once a day,for continual 14 days,and behavioral tests were carried out again after administration.The morphological changes of hippocampal neurons in neonatal rats of each group were observed using Nissl staining;the content of glutamic acid(GLU)and gamma-aminobutyric acid(GABA)in the hippocampal tissue was measured by colorimetry;the mRNA relative expressions of BDNF,TrkB,and CREB in the hippocampal tissue were determined by qRT-PCR.Results Compared with the control group,the VPA group had smaller body weight,body length,and tail length(P<0.05).Compared with the blank group,the model group showed significant symptoms of social disorders(P<0.01),anxiety disorders(P<0.01),and increased repetitive stereotyped behaviors(P<0.05 or P<0.01),structural damage of hippocampal neurons,increased GLU content(P<0.01),decreased GABA content(P<0.01),as well as reduced mRNA expressions of BDNF,TrkB,and CREB(P<0.05 or P<0.01).Compared with model group,the social ability of neonatal rats in vitamin D group and medium-and low-dose LWDHP groups was enhanced(P<0.05 or P<0.01),anxiety disorder was alleviated(P<0.05 or P<0.01),repetitive and stereotyped behaviors were reduced(P<0.01 or P<0.05),the structures of hippocampal neurons were significantly restored,GLU content was decreased(P<0.01),and the mRNA expressions of BDNF,TrkB,and CREB were higher(P<0.05 or P<0.01).Additionally,GABA content was elevated in medium-and low-dose LWDHP groups(P<0.05 or P<0.01).Conclusion LWDHP can significantly improve the behavioral outcome of VPA-induced ASD neonatal rats,and enhance the regeneration and repair of hippocampal neurons.The mechanism may be related to the balance of GLU and GABA levels as well as up-regulation of BDNF/TrkB/CREB expression in the hippocampal tissue.