Effect Of Tongqiaoyizhi Granule On Synaptic Plasticity Of Hippocampal Neurons In Vascular Dementia Rats Based On CAMP/PKA-CREB Signaling Pathway

Objective This study aims at exploring the effect of Tongqiaoyizhi granule on vascular dementia by detecting the behavior,neuronal apoptosis in hippocampal CA1 region of vascular dementia rats,the key factors of the cAMP/PKA-CREB signaling pathway and the postsynaptic NMDA receptor subunit GluN1,GluN2 B and AMPA receptor subunit GluA2 as well,thus illuminating the potential mechanism of Xuanfu method in the treatment of vascular dementia rats.Methods 1.Modeling and grouping: Morris water maze was used to screen clean SD rats with similar learning and memory ability.Then rats were randomly divided into sham group and operation group.The VaD rats was established by permanent ligation of bilateral common carotid arteries(also known as 2-VO method).30 days after the operation,Morris water maze test was performed to qualify whether the model rats were eligible.They were randomly divided into model group,nimodipine group and Tongqiao yizhi granule high,middle and low dose groups(hereinafter referred to as traditional Chinese medicine(TCM)group)with 10 rats each.2.Mode of aministration: according to the conversion of equivalent dose between human and animal,nimodipine group was given nimodipine suspension 6.25mg/kg.d intragastrically and the high,middle and low dose groups of traditional Chinese medicine were given Tongqiaoyizhi granule concentrated solution 24.8g/kg.d,12.4g/kg.d and 6.2g/kg.d respectively for 30 days.The model group and sham group were given the same amount of distilled water(3ml)at the same time.3.Specimen collecting: after Morris water maze test was performed to detect the behavior of rats,5 rats were randomly selected to cardiac perfuse with normal saline and fresh hippocampal tissue was quickly collected on ice for ELISA and Western Blot experiments.The other 5 rats were perfused with 4% paraformaldehyde phosphate buffer and brain tissue was taken out to be fixed in paraffin embedded sections for TUNEL and immunohistochemistry experiments.4.Detecting methods: Morris water maze was performed to detect the results of orientation navigation and spatial exploration to evaluate the ability of learning and memory.The apoptosis rate of neurons in hippocampal CA1 region is detected by TUNEL.ELISA is used to measure the cAMP level in hippocampus and the hippocaml BDNF expression is detected by immunohistochemical method.Western Blot was used to detect the protein level of PKA C-?,pCREB,GluN1,GluN2 B and GluA2.Results 1.Behavior: compared with the sham group,the EL of the model group was prolonged significantly,with the frequency of crossing the virtual platform and the duration in the third quadrant being significantly decreased(P<0.05).The EL of nimodipine group and high,middle and low dose groups were distinctly shortened,and the frequency of crossing the platform and the duration in the third quadrant were increased in varying degrees compared with the model group(P<0.05),especially in the high dose group(P<0.05).2.Apoptosis rate of hippocampal CA1 neurons: the apoptosis rate of neurons in the model group was significantly higher than that in the sham group(P<0 05),and was decreased to some extent after treated with nimodipine or Tongqiaoyizhi granule of different doses compared with the model group(P<0 05).The high dose group had a lower rate than the middle,low dose group and nimodipine group(P<0.05).3.BDNF: BDNF positive cells showed brownish yellow precipitation in the cytoplasm of neurons.The expression of hippocampal CA1 BDNF in model group was significantly lower than that in sham group(P<0.05),and was up-regulated by giving nimodipine or Tongqiaoyizhi granule(p<0.05),especially in high dose group(P<0.05).4.cAMP: compared with sham group,the content of cAMP in hippocampal neurons of model group was significantly decreased(P<0.05).The expression of cAMP in nimodipine group and each dose group of Tongqiaoyizhi granule was significantly increased(P<0.05),especially in the high dose group(P<0.05).5.PKA and pCREB protein level: the expression of PKA and pCREB in hippocampal neurons of the model group was significantly lower than thoset of the sham group(P<0.05).After oral administration,the contents of PKA and pCREB in Tongqiaoyizhi group and nimodipine group were distinctly increased(P<0.05),with the effect of high dose group was the most significant(P<0.05).6.GluN1,GluN2 B and GluA2 protein level: compared with sham group,the expression of postsynaptic GluN1,GluN2 B and GluA2 protein in hippocampal neurons of model group was significantly reduced.(P<0.05).After 30 days of treatment with nimodipine and different dose of Tongqiaoyizhi granule,GluN1,GluN2 B and GluA2 were significantly up-regulated(P<0.05),while the effect in the high dose group was the most distinct among them(P<0.05).Conclusion 1.Tongqiaoyizhi granule can significantly improve the cognitive ability of vascular dementia rats.2.Tongqiaoyizhi granule can significantly inhibit the apoptosis of hippocampal CA1 neurons,increase the expression of cAMP,PKA and the phosphorylation of CREB,thus mediating the BDNF downstream and playing a neuroprotective role.3.Tongqiaoyizhi granule can up-regulate the expression of postsynaptic GluN1,GluN2 B and GluA2 in VaD rats,thus improving learning and memory of vascular dementia rats by recovering the synaptic structure and improving the transmission efficiency after cerebral hypoperfusion.All the effects above showed a dose-dependent relation.