Role Of Changes In Intestinal Microbial Composition And Structure In Epilepsy

Object:To explore the effect of fecal microbiota transplantation（FMT）on gut microbiota compositional structure,cognitive function and seizure threshold in young rats.To investigate the changes of lipopolysaccharide（LPS）and toll like receptor 4（TLR4）in FMT mice.Methods:Part I: healthy male young C57 BL / 6 mice aged 3-5 months,weighing 25-35 g,were randomly divided into fecal bacteria transplantation group（FMT）and blank control group（control）,10 mice per group,the feces of old C57 BL / 6 mice were collected in advance to make fecal bacteria suspension,and the young mice in FMT group were gavaged.The control group was given an equivalent volume of normal saline.Before and after modeling,the cognitive functions of mice were assessed using the Morris water maze and eight arm maze,and the escape latency,target phase time,distance traveled in the target phase,and number of platform crossings were analyzed;Latency of the eight arm maze,number of reference memory errors,working memory.C57 mice of the same standard were selected and randomly divided into fecal microbiota transplantation group（FMT）and blank control group（control）with 20 mice each,before and after fecal microbiota transplantation using 6 Hz seizure threshold test,and the current intensity eliciting 50% of the mice’s seizures（cc50 current value）was chosen as the seizure threshold reflecting seizure susceptibility.T-tests were used to compare the differences in cognitive function and seizure threshold between the FMT and control groups;Paired ttests were used to compare differences in cognitive function and seizure threshold between pre-and post implantation mice.Part II: 18 C57 mice were selected according to the same criteria and randomly divided into three groups: the FMT group,the fecal transplant group injected with the TLR4 antagonist tak-242（FMT + tak-242）intraperitoneally（i.p.）at the same time as fecal transplantation,and the control group administered an equal dose of saline,six mice per group.After 7 days,colonic feces and plasma were collected for LPS content detection using the Limulus amoebocyte lysate（LAL）method,and hippocampal and cortical tissues were stripped and subjected to enzyme-linked immunosorbent assay（ELISA）for LPS detection.Protein immunoblotting（Western blot,WB）and quantitative reverse transcription polymerase chain reaction（QRT PCR）methods were used to detect TLR4 of cortex and hippocampus tissues.Proinflammatory factor IL-1 in plasma and brain tissue β、 IL-6、TNF-α、 COX-2,CCL2 and the anti-inflammatory factor TGF-β 1、TGF-β 2、TGF-β 3.For IGF-1,G-CSF,and GM-CSF,ELISA was used,and RT-PCR was used to determine the m RNA of the above inflammatory factors.One way ANOVA was used to compare the levels of LPS,TLR4,and various inflammatory factor proteins among the above 3 groups.Results Part I:（1）In the Morris water maze test,after FMT in young rats,the escape latency was longer,the number of crossing the platform was reduced,the percentage of target quadrant stay time and the percentage of target quadrant walking distance were decreased compared with the pre FMT and control groups;In the eight arm maze test,the FMT group showed longer latency,increased numbers of working memory and reference memory errors,and impaired spatial cognition,learning and memory in mice.（2）Mice after FMT had a significantly higher seizure threshold than pre FMT and controls and increased seizure susceptibility.Part II:（1）No differences were observed in the results of HE staining between hippocampal and cortical tissues.（2）Mouse pro-inflammatory factor IL-1 in the FMT group β、 IL-6、TNF-α、 COX-2,CCL2 showed an upregulation trend;The antiinflammatory factor TGF-β 1、TGF-β 2、TGF-β 3.IGF-1,G-CSF,GM-CSF showed a trend of downregulation.（3）The expression of gta8,PHF-1,p-tau,and t-tau was significantly increased in mice of the FMT group.（4）The amount of LPS in plasma increased in FMT group.Conclusions: We found that the gut microecology of healthy young mice underwent FMT perturbed,the mice showed decreased cognitive function,impaired spatial memory ability,decreased seizure threshold,and increased seizure susceptibility.And it was found that the disordered alteration of gut flora was through the activation of TLR4 by LPS to exert Pro epileptogenic effects.