Study On The Mechanism Of Jitian Jiannao Decoction Regulating Synaptic Plasticity Of Rat Hippocampal Neurons Under Low Glucose And Hypoxia

Background:Vascular dementia(VD)is a kind of cognitive disorder caused by cerebrovascular disease and ischemic or hemorrhagic brain injury.Under the influence of cerebrovascular disease,the synaptic plasticity of neurons changes or even dies during cerebral ischemia and hypoxia.The pathogenesis of VD is complex.Current studies have shown that the related pathogenesis of VD includes the impairment of cholinergic pathway,changes in synaptic plasticity,oxidative stress and inflammatory response,and genetic mechanism.The main clinical symptoms of VD are the damage of neurons caused by the lack of oxygen in the brain.Currently,there is still a lack of fundamental methods for the prevention and treatment of vascular dementia.In addition to the effective control of risk factors for vascular dementia,the treatment methods are mainly to improve patients’ cerebrovascular metabolism such as Nimodipine or improve patients’ cognitive function such as cholinesterase inhibitors.Clinical observation has shown certain efficacy,but large-scale clinical verification is still needed.TCM treatment of VD focuses on the concept of overall regulation and syndrome differentiation,and has achieved certain results.VD belongs to the category of "dementia" and "idiotic disease" in traditional Chinese medicine.The main pathogenesis of VD is deficiency of kidney essence and insufficient cerebral pulse.Traditional Chinese medicine treatment of VD treatment is mainly to "tonify",the brain for the house of mental activity,the sea of marrow,brain loss of nourishment causes dementia,so the treatment of dementia should tonify the innate essence,raise the acquired gas.Ji Tian Jian Nao Decoction tonifies kidney expelling dampness,quieting spirit and stabilizing mind,and has a definite effect on the treatment of mild VD.It can significantly improve the cognitive ability and the ability of daily living activities.The newly discovered micro RNA-132(miR-132)can be as one of the important factors in regulating synaptic plasticity,can interact with a variety of target genes and related proteins,forming network signal,regulate synaptic morphology change and plasticity procession,this study aims to apply low sugar low oxygen conditions induced rat hippocampal neurons injury of brain neurons,simulating the pathological feature of VD patients to observe the protective effect and mechanism of Ji Tian Jian Nao formula and miR-132 on rat hippocampal neuron cell injury model.Objective:To analyze the intervention effect and target mechanism of Ji Tian Jian Nao prescription and miR-132 mediated signal network on regulating synaptic plasticity of hippocampal neurons induced by hypoglycemia and hypoxia in rats.Methods:SD rats were divided into five groups,which were given high,medium and low doses of Ji Tian Jian Nao prescription,nimodipine solution and normal saline,respectively.and prepared drug-containing serum after 7 days of intragastric administration.The hippocampal neurons were cultured in vitro,and the neurons were collected in 4 × 105 cells per well and inoculated in 24-well plates.The cells were cultured in complete medium(high glucose).The experiment was divided into 10 groups:blank group,model group,positive group,Ji Tian Jian Nao prescription highdose,medium-dose and low-dose groups,miR-132 enhancement group,miR-132 inhibition group,miR-132 positive control group and miR-132 negative control group.The transfection group transfected corresponding RNA fragments with liposomes.Microtubule-associated protein 2(MAP-2)was used for cell identification when the cells were cultured to 70%-80%.The blank group was cultured with blank drugcontaining medium(high glucose);the positive group,Ji Tian Jian Nao prescription high-dose,medium-dose and low-dose groups were cultured with their own drugcontaining medium(low glucose);the remaining model group and the miR-132 transfection group were cultured with blank drug-containing medium(low glucose).Cultured for 6 hours under hypoxic condition,removed and recovered for 20 hours under normal oxygen condition.The expression of postsynaptic dense substance 95(PSD-95),methylCpG binding protein 2(MeCP2),and guanosine triphosphate activated protein(p250GAP)protein were detected by immunohistochemistry.The synaptic specific indicators,synaptic growth associated protein 43(GAP-43)and synaptophysin(Syn)protein sites were observed by immunofluorescence method.The levels of cyclic adenosine phosphate response element binding protein(CREB)and brain-derived neurotrophic factor(BDNF)were determined by enzym-linked immunosorbent assay(ELISA).Results:Compared with blank group,the expressions of PSD-95,MeCP2,GAP43 and Syn in hippocampal neurons of model group were significantly decreased,the expression of p250GAP was significantly increased,and the contents of CREB and BDNF were significantly decreased(P<0.05 or P<0.01).Compared with model group,the expressions of PSD-95,MeCP2,GAP-43 and Syn in positive group and Ji Tian Jian Nao prescription high-dose,medium-dose and low-dose groups were significantly increased,the expression of p250GAP was significantly decreased,and the contents of CREB and BDNF were significantly increased(P<0.05 or P<0.01).Compared with the model group,the expression of GAP-43 and Syn in the miR-132 positive control group was significantly increased,and the content of CREB was significantly increased(P<0.05 or P<0.01);the expression of GAP-43 and Syn in the miR-132 enhanced group was significantly increased(P<0.05 or P<0.01).The expression of GAP-43 in miR-132 inhibited group was significantly decreased(P<0.05).Conclusions:Ji Tian Jian Nao prescription may mediate the expression of miR132 signal network by regulating MeCP2,p250GAP and other proteins,and increase the content of CREB and BDNF,thereby enhancing the expression of PSD-95,GAP43 and Syn,promoting synaptic remolding and protecting hippocampal neurons in rats.