| Backgrounds:In recent years,the development of traditional Chinese medicine has become a national strategy.As a traditional Chinese medicine,Semen Strychni is widely used clinically,but it has central nervous system toxicity,and reactions such as shivering will occur in maximum dose.The toxic dose can lead to central inhibition and death.In addition,overdose of drugs with neurotoxicity is one of the causes of cognitive impairment.Therefore,it is of great significance to study the mechanism of its neurotoxicity to make its application safe and effective.The extracellular release of high mobility group protein B1(HMGB1)induces an inflammatory response,it has been used to study various injury mechanisms and to predict the early progression of neurological diseases such as cognitive impairment.Glycogen synthase kinase-3β(GSK-3β)is abundant in the central nervous system,intervention of GSK-3β activity is considered to be one of the important prevention and treatment strategies for neurodegenerative diseases.Our previous experiments found that HMGB1 was released in a large amount in the cytoplasm of the hippocampus after Semen Strychni poisoning,so we hypothesized that Semen Strychni may impair cognitive function and induce cognitive impairment through HMGB1-mediated.Therefore,this project took HMGB1 as the entry point to evaluate the impairment of cognitive function caused by Semen Strychni,and to explore whether its mechanism is related to the increased release of HMGB1 and the activation of GSK-3β.Objectives:To evaluate the degree of cognitive impairment caused by Semen Strychni and explore the possible molecular mechanism.Methods:1.To evaluated the degree of damage to brain tissue and cognitive abilities caused by Semen Strychni central neurotoxicity through animal experiments.72 SD rats were randomly divided into 4 groups: blank control group,Semen Strychni group,GSK-3β inhibitor + Semen Strychni group,GSK-3β inhibitor control group,with 18 rats in each group.The Semen Strychni extract was administered for 7 consecutive days to induce acute poisoning in rats.According to behavioral,biochemical and pathological analysis,the ability of learning and memory,cholinergic function,oxidative stress,neuron structure,neurotoxic protein accumulation,glial cell status,and synaptic plasticity were evaluated in rats.The mRNA and protein expression of GSK-3β,p-GSK-3β(ser9),Nrf2,HO-1,HMGB1 were detected by Western Blot and Real-time PCR,the nucleocytoplasmic distribution of HMGB1 was observed by immunofluorescence.2.The network pharmacology was used to predict the common core target proteins,related biological functions,and signaling pathways of Semen Strychni and cognitive impairment.To further verify the involvement of GSK-3β in the mechanism of central neurotoxicity of Semen Strychni.Results:1.The results of Morris Water Maze and Novel Object Recognition showed that the ability of learning and memory of the rats in Semen Strychni group was decreased slightly.Hippocampal cells were damaged,neurons were degenerated and necrotic,synaptic ultrastructure was abnormal,the expression of synaptic functional protein PSD-95 was significantly reduced.APP mRNA level,Tau protein expression and mRNA level increased.The levels of MDA increased.An increase in the protein expression of microglia and astrocytes markers Iba-1 and GFAP.However,the levels of ACh and Ch AT increased at the same time.In addition,the expression of p-GSK-3β(Ser9)protein was significantly decreased and the mRNA level of GSK-3β increased,the total protein and nuclear protein expressions of Nrf2 decreased,and Nrf2 mainly distributed in the cytoplasm,the expression of HO-1 protein reduced,and then the secretion of HMGB1 increased,which was released from the nucleus to the cytoplasm.However,the levels of these injury indicators significantly decreased in GSK-3β inhibitor + Semen Strychni group,and there was no significant difference compared with the Control group.GSK-3β inhibitor treatment can reduce GSK-3β mRNA expression,increase protein expression of total Nrf2 and HO-1,and decrease total protein content of HMGB1.2.Network pharmacology results showed that GSK-3β is the common target protein of Semen Strychni and cognitive impairment,it involved in secretion of neurotransmitters,synthesis of synaptic and neuronal structures related to cognitive function.GSK-3β can affect synaptic plasticity in dopaminergic synapse pathways.Conclusions:The cognitive function of rats poisoned with Semen Strychni was impaired.GSK-3β inhibitor can activate Nrf2/HO-1 pathway and inhibit HMGB1 secretion and nuclear cytoplasmic transfer,thereby alleviating central neurotoxicity of Semen Strychni and improving cognitive function impairment in rats.GSK-3β is a key target of Semen Strychni causing cognitive impairment. |
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