| Objective:To investigate the effect of Ganoderma triterpenoids combined with exogenous monosialotetrahexosylganglioside(GM1)on learning and memory ability of epilepsy rats induced by pentylenetetrazol(PTZ),and the mechanism of intervention in the reconstruction of synaptic structure and functional plasticity of hippocampus.Methods:(1)A total of 50 healthy male adult SD rats were randomly divided into blank control group,epilepsy model group,Ganoderma triterpenoids group,GM1 group and Ganoderma triterpenoids combined with GM1 group,with 10 rats in each group.All the groups,except the blank control group,were intraperitoneally injected with pentylenetetrazol(PTZ)sub convulsant dose(35 mg/kg)once a day for 28 days to replicate the models of chronic epilepsy.Medication groups were given corresponding administration based on daily intraperitoneal injection of PTZ.The Morris water maze experiment was performed 28 days after the administration,and samples were taken after the experiment.(2)HE staining was used to observe the pathological changes of hippocampal neurons;transmission electron microscope was used to observe the ultrastructure of neurons in hippocampal CA1 area of epilepsy rats.(3)The expressions of actin-binding protein(Cofilin)and its phosphorylated p-Cofilin,synaptophysin(SYN)and nerve growth associated protein 43(GAP-43)in the hippocampal CA1 region were observed byimmunohistochemistry staining.(4)Western blot and were used to detect the protein expression of Cofilin,p-Cofilin,SYN,GAP-43 in hippocampus of rats,and real time PCR was used to detect the m RNA expression level of related genes.Results:(1)Compared with the epilepsy model group,the duration of seizure was shortened(P<0.05),and latency was prolonged in the combined group(P<0.05).Compared with Ganoderma triterpenoids group and GM1 group,the duration of attack in the combination group were shortened,and the latency of attack was prolonged.(2)Morris water maze showed that the escape latency of rats in each group was shortened with the increase of experimental training time.Compared with the blank control group,the escape lantency of epilepsy model group was prolonged(P<0.05),the time of staying in the target quadrant was significantly shortened(P<0.01),and the times of crossing the platform was significantly reduced(P<0.01);compared with the epilepsy model group,the escape latency of each medication group was shortened,and there was a significant difference in some time points(P<0.05),the time of staying in the target quadrant was significantly prolonged(P<0.01),and the times of crossing the platform were significantly increased(P<0.01).The escape latency of the combination group was significantly shorter than that of the Ganoderma triterpenoids group on the first day(P< 0.05),and the escape latency of the combination group was shorter than that of the Ganoderma triterpenoids group and GM1 group on the other days,the time of staying in the target quadrant was prolonged,and the times of crossing the platform were increased.(3)HE staining showed that there were few neurons in the hippocampus of epilepsy model group,which were arranged loosely and disorganized,some cells were swollen and deformed,and the nucleus was dissolved and fragmented.Compared with the model group,the number of hippocampal neurons in each group increased,the morphology of neurons tended to be normal,the arrangement was relatively neat,the gap was obviously reduced,and the nucleus fragmentation and shrinkage were reduced.(4)The results of transmission electron microscopy showed that in the epilepsy model group,the shape of neuron cells was irregular,the number of organelles such as mitochondrion was small,the crista of mitochondrion was broken,some crista disappeared,the number of synapses decreased,the number of synaptic vesicles decreased,and the structure of presynaptic and postsynaptic membrane was unclear.In each treatment group,the nucleus shape of neurons was more regular,the number of mitochondria and other organelles increased,the structure improved,the number of synapses increased,the synaptic structure was more complete,the number of synaptic vesicles increased,and the space between presynaptic and postsynaptic membrane was clear.(5)The results of immunohistochemistry showed that: compared with the blank control group,the expression of Cofilin protein in epilepsy model group increased(P<0.01),and the expression of p-Cofilin,SYN and GAP-43 decreased(P<0.05);compared with the epilepsy model group,the expression of Cofilin decreased in each treatment group(P<0.05),and the expression of GAP-43 increased(P<0.05);the expression of p-Cofilin increased(P<0.05)in combination group;the expression of SYN in Ganoderma triterpene group and the combination group increased(P<0.05).Compared with GM1 group and Ganoderma triterpene group,the expression of p-Cofilin in the combination group increased(P<0.05).(6)Protein expression in hippocampus: compared with the blank control group,the expression of Cofilin protein in hippocampus of epilepsy model group increased(P<0.05),the expression of p-Cofilin,SYN and GAP-43 decreased significantly(P<0.05);compared with the model group,the expression of Cofilin protein in hippocampus decreased(P<0.05),and the expression of GAP-43 increased in each treatment group(P<0.05),and the expressions of p-Cofilin and SYN protein increased in the combination group(P<0.05).(7)Gene m RNA expression in hippocampus: compared with the blank control group,the expression of Cofilin m RNA increased(P<0.05),and the expression of SYN m RNA and GAP-43 m RNA decreased(P<0.05)in the epilepsy model group;compared with the epilepsy model group,the expression of Cofilin m RNA decreased(P<0.05),and the expression of SYN m RNA increased(P<0.05)in all the treatment groups,while the expression of GAP-43 m RNA increased(P<0.05)only in the combination group.Conclusiong:(1)The damage of learning and memory ability in epileptic rats may be related to the abnormal expression of Cofilin,p-Cofilin,SYN,GAP-43 protein and gene,the loss and rearrangement of synapse,and the influence of synaptic remodeling.(2)Ganoderma triterpenoids,exogenous GM1 and Ganoderma triterpenoids combined with exogenous GM1 can improve the cognition,learning and memory ability and the morphological structure of hippocampal neurons in PTZ induced epileptic rats.In particular,Ganoderma triterpenoids combined with exogenous GM1 can better improve the cognition,learning and memory ability and the morphological structure of hippocampal neurons in PTZ induced epileptic rats.In particular,Ganoderma triterpenoids combined with exogenous GM1 can better improve the drug safety to a certain extent,improve the cognition,learning and memory ability and the morphological structure of hippocampal neurons in rats,and the mechanism of action may be possible related to the increase of SYN,GAP-43,p-Cofilin protein and gene expression,the down-regulation of Cofilin protein and gene expression,the promotion of synaptic reconstruction and nerve growth,and the intervention of hippocampal synaptic plasticity in order to protect brain neurons. |
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