Toxoplasma Chinese 1 Strains Of WH6 Causes Mice Abnormal Cognitive Behavior Through Affecting Hippocampal Neurons

Toxoplasma gondii(T.gondii)is an obligate intracellular parasite that can infect almost all warm-blooded animals,including humans.Many studies have confirmed that Toxoplasma is a neurotropic parasite.In the acute phase,T.gondii tachyzoites proliferate and enter various tissue sites,forming tissue cysts,especially the central nervous system(CNS).When Toxoplasma reaches the central nervous system,it invades all nucleated cells,activates microglia and astrocytes in the brain,and secretes a large number of inflammatory cytokines.There is evidence that Toxoplasma gondii can directly regulate the NF-?B pathway and that the toxoplasma effector molecule GRA15 II plays a key role in this regulation.In recent years,the main genotype of Toxoplasma gondii in China has been found to be Chinese 1(Toxo DB # 9),which has GRA15 II and ROP16 I / III effector molecules.The WH6 strain is one of the Chinese1 genotypes of Toxoplasma gondii,which is called the Wh6 strain of T.gondii Type Chinese 1,Tg Ctwh6.Parlog et al.Further found that cysts of Toxoplasma gondii are mainly distributed in the somatosensory cortex,motor cortex and hippocampus of the brain,and chronic infection of Toxoplasma gondii gradually affects the function of neurons.This suggests a possible connection between Toxoplasma gondii and host nerve and behavioral disorders.In-depth research shows that on the seventh day after Toxoplasma infection,it can cross the blood-brain barrier through the "Trojan horse mechanism",reach the central nervous system and erode neurons,triggering a series Neuropsychiatric disorders,such as schizophrenia,epilepsy,depression,and Alzheimer's disease.Objective: The purpose of the present study aims to investigate the effect of Tg Ctwh6 infection on cognitive behavior in mice,and to study the cellular and molecular mechanisms of this effect from the perspective of hippocampal neuron apoptosis and A? deposition.Materials: The male C57BL/6 mice were divided at random into two groups: the control group(Saline group)and the infected group.After 8 weeks,the body surface morphology and brain tissue compression microscopy of Toxoplasma gondii cysts were observed in mice to observe changes in body posture behavior and determine whether the modeling was successful.Water maze test and Open field experiments were performed to detect the motor exploration ability,cognition and spatial memory ability of mice infected with Tg Ctwh6.HE,Nissl staining and Western blotting were used to detect the pathological changes of mouse hippocampal tissues,the changes of the number of neurons in hippocampal tissues,and the expression of synaptotagmin1 in hippocampal neurons respectively.IHC was carried out to detect the expression of A? and BACE1 protein in mouse hippocampal tissue.At the same time,A? expression was also detected by Thioflavin S staining.The detection of BACE1,A?,and GAPDH protein and their m RNA expression in their hippocampal tissue was conducted by Western blotting and q RT-PCR,respectively.In order to study the mechanism of Tg Ctwh6 infection leading to a decrease in the number of neurons,morphological changes,and A? deposition in the hippocampus of mice,we performed further in vitro studies using cell experiments.Tg Ctwh6 tachyzoites were used to directly infect hippocampal neuron cell line HT22.The expression of apoptosis-related proteins Bax,Caspase-3 and Bcl-XL and the expression of Synaptotagmin1 in HT22 were detected by WB to determine the apoptosis and synaptic;Fluorescence staining was used to detect the expression of BACE1,APP and A? in protein and gene levels in HT22.In order to understand the molecular mechanism of A? deposition in cells induced by Tg Ctwh6 tachyzoite infection with HT22,we used WB and immunofluorescence staining to detect the cells of HT22 after infection with or without the pretreatment of PDTC,a specific inhibitor of NF-?B signaling pathway.Expression of NF-?B p65,p-NF-?Bp65,BACE1,APP and A? protein.In order to find out whether Tg Ctwh6 infection can indirectly affect hippocampal neurons through microglial activation,causing the latter to apoptosis and produce A? deposition,we used the Transwell system to co-culture microglial cell lines BV2 and HT22.experiment.Flow cytometry was used to detect whether infected BV2 was activated as M1,and then WB was used to detect the expression of Hes1 and Notch,two key signaling proteins in the Notch signaling pathway of BV2 cells.After confirming that BV2 was activated by Tg Ctwh6 tachyzoite infection,we used q RT-PCR to detect the expression of IL-6,TNF-?,i NOS and TGF-?1 inflammatory factors at the gene level in BV2 in the upper layer of the Transwell system.Then we took HT22 in the lower layer of the Transwell system,used WB to detect the expression of apoptosis-related proteins Bax,Caspase-3,and Bcl-XL in HT22,and used flow cytometry to detect the apoptosis rate of HT22.At the same time,we also used WB to detect The expression level of APP protein in HT22 was investigated to understand whether Tg Ctwh6 tachyzoite infection with BV2 could indirectly affect HT22 apoptosis and A? production.Results: In vivo experiments :(1)Tg Ctwh6 infection led to impairment of cognitive behavior in mice;(2)Tg Ctwh6 infection caused a small number of neurons in the hippocampus of the mice,a small volume,and disordered alignment accompanied by a decrease in neuronal synapses.(3)Tg Ctwh6 infection resulted in A? deposition in the hippocampus of mice.In vitro experiments;(1)Tg Ctwh6 tachyzoite infection led to apoptosis directly after HT22 infection;(2)Tg Ctwh6 tachyzoite infected with HT22 through NF-?B signal pathway directly caused excessive secretion of A?;(3)Tg Ctwh6 tachyzoites induced BV2 to polarize to M1 through Notch signal pathway after infection.The proinflammatory factors secreted by BV2 led to HT22 apoptosis and excessive secretion of A?.Besides,Tg Ctwh6 tachyzoites acted on HT22 indirectly to make it apoptotic and secrete A?.Conclusions: The results of this study showed that Tg Ctwh6 infection could lead to changes in posture and cognitive behavior in mice.Tg Ctwh6 tachyzoites directly promoted HT22 apoptosis and directly induced HT22 to produce a large number of A? through the NF-?B signaling pathway.In addition,Tg Ctwh6 tachyzoites activated BV2 through the Notch signaling pathway to indirectly promote HT22 apoptosis and produce A?.It suggested that the cognitive and behavioral changes caused by Tg Ctwh6 infection in mice may be the direct and indirect effects of Tg Ctwh6 on mouse hippocampal neurons.This study initially revealed the mechanism of Tg Ctwh6-mediated cognitive and behavioral impairment,promoted our further understanding of the relationship between Toxoplasma gondii and cognitive impairment.Also,it provided new clues for the prevention and treatment of cognitive and behavioral disorders,including Alzheimer's disease.