| Objectives To observe the influence of particulate matter(PM2.5)exposure on the cognitive function and the composition of intestinal flora in mice,and to explore the role of intestinal flora and microbe-gut-brain axis in the cognitive impairment of mice induced by PM2.5 exposure.The purpose of this study is to provide a new idea for the prevention and treatment of neurocognitive dysfunction caused by PM2.5exposure in the future.Methods After adaptive feeding for one week,a total of 48 healthy adult male C57BL/6J mice were randomly divided into control group（NS group）,Lactobacillus rhamnosus GG group（LGG group）,PM2.5-exposed group（PM group）and PM2.5-exposed+Lactobacillus rhamnosus GG group（PML group）,with 12 mice in each group.NS group and PM group were given normal saline daily according to their body weight,while LGG group and PML group were given Lactobacillus rhamnosus GG daily（0.1 ml per 10 g body weight）,the concentration of bacterial suspension is 1×109 CFU/mL.After intragastric administration,mice in PM group and PML group were exposed to PM2.5 by PM2.5 on-line enrichment system and whole body dynamic exposure system.The concentration was 8 times higher than outdoor concentration,6 hours a day,6 days a week for 7 weeks.After exposure,open field test,the elevated plus maze test and new object recognition test were used to detection the cognitive function of mice.The diversity,composition and abundance of intestinal microflora in mice feces were detected by 16s r RNA high-throughput sequencing.The structural changes of hippocampus and colon were observed under light and electron microscope.The proteins expression of ZO-1,Occludin and Claudin 1 in mice colon and hippocampus was detected by Western-blot.The levels of 5-HT and GABA and the contents of inflammation-related factors LPS and TNF-αin serum and hippocampus of mice were measured by ELISA,and the content of short-chain fatty acids in feces of mice was quantitatively analyzed by gas chromatography-mass spectrometry.Finally,the relationship between intestinal flora and neurobehavioral related indexes was analyzed by spearman correlation.Results 1 During exposure,the PM2.5 concentrations in clean room,outdoor and exposure cabinet were 11.5（6.0,15.8）,50.2（36.0,66.1）and 407.8（294.9,545.4）μg.m-3,respectively.2 In the open field test,compared with NS group,the total exercise distance of mice both in PM and PML group decreased（P<0.05）,the stay time in central area in PM group also reduced（P<0.05）.In the elevated plus maze test,compared with NS group,the percentage of entries in the open arms in PM group was reduced（P<0.05）.Compared with PM group,the percentage of entries in the open arms in PML group was increased（P<0.05）.In the new object recognition test,compared with NS group,the recognition index of PM and PML groups were decreased,while compared with PM group,the recognition index of mice in PML group increased（P<0.05）.3 Compared with NS group,intestinal floraαdiversity index simpson and shannon index increased andβdiversity index decreased in PM group.While compared with PM group,simpson and shannon index decreased andβdiversity index increased in PML group.The analysis of differential species showed that compared with NS group,the abundance of Lactobacillus decreased,and the abundance of Dubosiella and Faecalibaculum increased in PM group.Compared with PM group,the abundance of Odoribacter decreased in PML group,while the abundance of Porphyromonas and Neisseria increased.The LEf Se analysis showed that the dominant species in PM group were Dubosiella and Erysipelotrichaceae.4 Under light microscope,the colonic tissue structure of NS group and LGG group was intact and the mucosal surface was smooth.In PM group,the mucosal structure of colon tissue was incomplete,glandular structural destruction,the number of both columnar and goblet cells decreased,at the same time,there was a large number of inflammatory cells infiltrated.Inflammatory cell was observed in PML group.The pathological sections of CA1 area of mice hippocampal tissue under light microscope showed that the cells of NS group and LGG group were closely ordered and morphologically regular.The cells of PM group were disordered and some cell nucleus condensation.The cell arrangement in PML group was significantly improved than that in PM group.Under ultrastructure,NS and LGG groups hippocampal neuron structure was basically normal,and the number of organelles was abundant.Mitochondria in PM group were swollen and vacuolated.Compared with PM group,the ultrastructure of PML group was significantly improved.5 Compared with NS group,the expression levels of tight junction protein Occludin and ZO-1 in colon and hippocampus of PM group were decreased（P<0.05）.The protein expression levels of Occludin and ZO-1 in PML group were higher than those in PM group（P<0.05）.6 Compared with NS group,the level of 5-HT in hippocampus of PM group was decreased,and the level of GABA in serum of LGG group and PML group was decreased（P<0.05）;compared with PM group,the level of GABA in serum of PML group was decreased（P<0.05）.7 Compared with NS group,LPS and TNF-αlevels in serum and hippocampus of PM group were increased,while TNF-αlevels in PML group were increased（P<0.05）.Compared with PM group,LPS levels in hippocampus and TNF-αlevels in serum and hippocampus were decreased in PML group（P<0.05）.8 Compared with NS group,the levels of caproic acid in PM group and PML group were significantly reduced.9 At the genus level,the dominant bacterium Faecalibaculum in PM group was negatively correlated with recognition index,the percentage of entries in the open arms and 5-HT level,but positively correlated with LPS level in mice.Dubosiella was negatively correlated with recognition index,the percentage of entries in the open arms and GABA level in mice.Conclusions 1 PM2.5 exposure can cause cognitive dysfunction and intestinal flora imbalance in mice.2 PM2.5 exposure damaged the structure of colon and hippocampal tissue and endothelial tight junctions in mice.3 PM2.5 exposure resulted in imbalance of 5-HT,a cognitive function-related neurotransmitter,increased levels of endotoxin and inflammatory factors,and decreased levels of caproic acid,a metabolite of intestinal flora.4 Dubosiella and Faecalibaculum increased after PM2.5exposure,which was negatively correlated with the cognitive function of mice.After adding lactobacillus rhamnosus GG to mice exposed to PM2.5,cognitive function,histopathological damage,intestinal epithelial barrier,blood-brain barrier and related inflammatory indicators could be improved.Therefore,the research results suggest that the influence of PM2.5on the central nervous system is partly due to its intestinal flora imbalance,neurochemical transmitter imbalance and inflammation,and the microbial-gut-brain axis plays a powerful role in this process.Figure 19;Table 13;Reference 114... |
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