| Purposes:Alzheimer′s disease(AD)is one of the most common diseases that afflict the elderly,with complex and irreversible causes,and has been ranked as a global public health priority.The pathogenesis of AD is still unclear,but in recent years,it has been shown that the intestinal flora and the central nervous system have a bidirectional dialogue through the gut-brain axis.Dysbiosis of the intestinal flora,intestinal inflammation,and increased intestinal permeability are strongly associated with the worsening of AD.Many studies have shown that exercise can improve the structure of intestinal flora as well as promote the repair of intestinal mucosal system,and there are relatively few studies on exercise modulation of intestinal flora to interfere with AD.Therefore,this study was conducted in APP/PS1 double transgenic mice to investigate the effect of exercise modulation of intestinal flora on the changes of proteins related to intestinal TLR4/NF-κB signaling pathway and further on the cognitive behavior of APP/PS1 mice.PS1 mice,to provide new ideas for the treatment and intervention of AD.Method:In this study,24 APP/PS1 double transgenic mice were randomly divided into two groups of 12 males each,as APP/PS1 locomotion group(APP-Y)and APP/PS1 control group(APP-D),and 24 littermate C57 mice were randomly divided into two groups of 12 males each,as C57 locomotion group(WT-Y)and C57 control group(WT-D).The mice were housed as usual,free range and subjected to a ten-week aerobic exercise intervention for the exercise group,with training days from Monday to Saturday and rest on Sunday.At the end of the tenth week,all mice were subjected to Morris water maze experiment for 6 days to test the cognitive-behavioral ability of the mice.Protein blotting experiments were performed,and then feces were taken for high-throughput sequencing of intestinal flora.Results:1.localization navigation experiment: the differences were significant on the third,fourth and fifth days,and the mean evasion latency of mice in the APP-D group was significantly longer compared to the WT-D group(P<0.01);compared to the APP-D group,the evasion latency of the APP-Y group was slightly reduced on the second and third days,but there was no significant difference,and on the fourth day,the evasion latency of the APP-Y group was shortened(P<0.05),and on the On the fifth day,the escape latency of the APP-Y group was significantly shortened(P<0.01).2.Spatial exploration experiment: the WT-Y group stayed in the fourth quadrant for the longest time and shuttled the most times,followed by the WT-D group.Compared with the WT-D group,the exploration time of the original platform quadrant in the APP-D group decreased significantly(P < 0.01),and the dwell time and the number of crossing the platform in the target quadrant in the APP-Y group increased significantly compared with the APP-D group(P < 0.05).3.Comparing with WT-D group,it was found that the diversity of intestinal microflora in APP-D group mice were significantly different,and the dominant flora in the intestine was changed,the abundance of Trichoderma spp.NK4A136 group,Bifidobacterium spp.and Lactobacillus spp.in WT-D group was significantly higher than that in APP-D group,after exercise intervention in APP-Y group Trichoderma spp.NK4A136 group,Bifidobacterium spp.The abundance of Trichoderma spp.,Bifidobacterium spp.and Lactobacillus spp.increased after the exercise intervention,and the distribution of flora was significantly better,and the dominant flora returned.Compared with the WT-D group,the abundance of Escherichia coli was higher in the APP-D group and decreased in the APP-Y group after the exercise intervention.4.HE staining experiment: From the HE staining results of mouse colon,it could be observed that the intestinal mucosal epithelial cells of mice in the WT-D and WT-Y groups were continuously The intestinal mucosa of the mice in the APP-D group showed residual damage,rupture,disorderly arrangement of glands,reduction or even disappearance of cup cells,accompanied by inflammatory cell infiltration,dilated capillaries,damage to the integrity and edema,while the colonic mucosa of the mice in the APP-Y group was repaired to some extent,with less residual damage to the intestinal mucosa and less glandular arrangement than in the APP-Y group.The neuronal cells in the CA1 area of the hippocampus of mice in the WT-D group were neatly arranged and had a higher number of cells,and the size and morphology of the cells did not differ greatly.Compared with the WT-D group,the neuronal cells in the APP-D group were more sparsely arranged,cells existed in the state of extinction and lysis,a large number of cells appeared dead,the size of cell gaps was inconsistent,and the cell structure was incomplete,while the cell morphology and structure of the WT-Y group were more compact and complete.Compared with APP-D group,the cells in APP-Y group mice were relatively neatly arranged,the cell gap gradually became smaller,and the number of cell death decreased.5.The results of thioflavin staining: from the cortical area of mice,it can be seen that the difference between WT-D group and APP-D group was very obvious,no Aβ1-40 deposition was seen dispersed in the cortical area in WT-D group,while Aβ1-40 expression was more in APP-D group and aggregated into plaques.6.ELISA experiment: the expression of IL-1β in mouse serum was significantly increased in APP-D group compared to WT-D group(P<0.01);significantly decreased in APP-Y group compared to APP-D group(P<0.01).the expression of LPS in mouse serum was significantly increased in APP-D group compared to WT-D group(P<0.01);significantly decreased in APP-Y group compared to The expression of LPS in mouse serum was significantly increased in the APP-D group compared with the APP-D group(P<0.01)and significantly decreased in the APP-Y group compared with the APP-D group(P<0.01).7.Western Blot experiment: TLR4 protein: compared with WT-D group,APP-D group mice colonic tissue expression increased(P<0.05);compared with APP-D group,APP-Y group mice colonic tissue expression significantly decreased(P<0.01).NF-κB protein:compared with the WT-D group,the expression in the colonic tissues of mice in the APP-D group was significantly increased(P<0.05),while that in the WT-Y group was significantly decreased(P<0.05);compared with the APP-D group,the expression in the colonic tissues of mice in the APP-Y group was significantly decreased(P < 0.01).IL-1β protein: compared with the WT-D group,the expression in the hippocampus of mice in the APP-D group was significantly increased(P<0.01).TNF-α protein:compared with the WT-D group,the expression in the hippocampal tissues of mice in the APP-D group was increased(P<0.05);compared with the APP-D group,the expression in the hippocampal tissues of mice in the APP-Y group was significantly reduced(P<0.01).8.Immunohistochemical experiments: TLR4 protein: compared with the WT-D group,the expression in the colon tissue of mice in the APP-D group increased significantly(P<0.01),and the expression in the colon tissue of mice in the WT-Y group decreased significantly(P<0.01);compared with the APP-D group,the expression in the colon tissue of mice in the APP-Y group also had a tendency to decrease significantly(P<0.01).NFKB protein: compared with the WT-D group,the expression in the colonic tissues of mice in the APP-D group increased significantly(P<0.01),and the expression in the colonic tissues of mice in the WT-Y group decreased significantly(P<0.01);compared with the APP-D group,the expression in the colonic tissues of mice in the APP-Y group also had a tendency to decrease significantly(P< 0.01).Conclusions:1.Aerobic exercise can reduce the abundance of Escherichia coli spp.and increase the abundance of Trichosporon spp.NK4A136 group,Bifidobacterium spp.and Lactobacillus spp.in APP/PS1 mice to further repair the damaged intestinal mucosal barrier.2.Aerobic exercise can significantly reduce TLR4 and NF-κB protein expression and inhibit serum IL-1β and LPS production in APP/PS1 mice colonic tissues.3.Aerobic exercise can improve the inflammatory lesions in the colon tissue of APP/PS1 mice,possessing a certain degree of anti-inflammatory,mucosal protection and improvement of edema and congestion.4.Aerobic exercise can inhibit the deposition of Aβ in the brain of APP/PS1 mice.5.Aerobic exercise can effectively improve the general state of mice,learning cognitive memory function.6.Aerobic exercise inhibits TLR4 activity by blocking its downstream activation of NF-κB,impeding the signaling of the signaling pathway it mediates,leading to reduced production or release of pro-inflammatory factors,thus exerting a therapeutic effect on intestinal flora inflammation,which may be one of the mechanisms by which aerobic exercise intervenes in intestinal flora dysbiosis. |
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