Research Of Probiotics For Hypoxia-induced Cognitive Impairment Intervention And Its Mechanisms

Objective: The plateau area occupies about 26% of China's land area,which is of great strategic importance.Its low-pressure and low-oxygen environmental factors will significantly affect the body's cognitive function and gut microbiota.The gut microbiota is a general term for the microbial population in the intestine.They co-exist,interact,co-evolve with the host and related to human health closely.The imbalance of the gut microbiota can cause various diseases.With the development of bioinformatics and genetic engineering technology,the relations between gut microbiota and cognitive functions have been discovered.Probiotics,as a living microorganism that able to colony in the intestine,can be beneficial to health if supplemented properly.Studies have found that probiotic interventions can prevent cognitive decline in certain neurological diseases by regulating the gut microbiota.What is the relationship between the impairment of cognitive function caused by hypoxic exposure and changes in gut microbiota? Can probiotics be a new intervention to prevent hypoxic-induced cognitive impairment? What is the mechanism behind this? The solution to these problems will provide a strong support for the mechanism of dietary nutrition regulating the microbiota-gut-brain axis to prevent the special environment of the plateau induced cognitive damage.It will also provide scientific evidence for development of nutraceutical and functional foods that enhance the cognitive function of people working on the plateau.Therefore,firstly,this study explored the relationship between cognitive impairment and gut microbiota changes in young soldiers under hypoxic conditions,and then discusssed the protection effect of Lactobacillus LP45 and Bifidobacterium bifidum TMC3115 on cognitive impairment induced by hypoxia in mice,and explored the possible mechanism.Methods:1.Field study of plateau and plain populations: The research subjects were officers and soldiers from three different altitude areas in Tibet and Beijing,and the basic cognitive ability test(BCAT)software was used to test their five cognitive domains including perception of speed,mental arithmetic efficiency,spatial representation efficiency,Working memory and memory recognition.Representative fecal samples from each group of military personnel were collected and 16 S r RNA gene sequencing technology was used to analyze the structural characteristics of intestinal microbiota of officers and soldiers stationed at different altitudes on the plain and plateau.2.Simulated plateau hypoxia exposure mice intervention experiment: 60 6-weekold C57/6 male mice were randomly divided into five groups: control group(Control),hypoxia model group(Hypoxia),hypoxia lactobacillus intervention group(HL),hypoxic bifidobacterium intervention group(HT)and hypoxic combined intervention group(HLT).There were ten animals in each group,which were gavaged saline,saline,lactobacillus plantarum LP45,bifidobacterium bifidum TMC3115 and the mix of two microbiota.The last four groups of mice were placed in a hypobaric oxygen chamber daily to simulate hypobaric hypoxia exposure at an altitude of 6000 meters.After 30 days,conducted behavioral tests on the mice,including open field test,new object recognition test,and step-down test;the mice were then sacrificed.ELISA measures the levels of gamma aminobutyric acid(GABA),serotonin(5-HT),tryptophan(Trp),and endotoxin(ET)in serum.Luminex detects interleukin-1?(IL-1?),contents of cytokines such as interleukin-6(IL-6),interleukin-10(IL-10),tumor necrosis factor(TNF-?).16 Sr RNA gene sequencing technology was used to analyze the intestinal microbiota structure,and western blotting,the expression levels of brain-derived neurotrophic factor(BDNF),tight junction protein ZO-1,occludin and hypoxia-inducible factor-1?(HIF-1?)in hippocampus,cortex and intestinal tissues.3.Fecal bacteria transplantation experiment: 36 6-week-old C57 / 6 male mice were randomly divided into CFMT,HFMT,and PFMT groups,and then treated with mixed antibiotics for 3 weeks.Each group was transplanted with feces from the Control group,Hypoxia group,and HLT group.After 4 weeks of fecal transplantation,open field test,new object recognition test,and step-down test were conducted on each group of animals immediately.After the behavioral experiments were completed,serum,hippocampus,prefrontal cortex,and intestinal tissues were collected.Western blot was used to detect the expression levels of BDNF,ZO-1,Occludin,CLDN1 and other genes.Results:1.Field test of plateau and plain populations: In the BCAT test,the scores of speed of perception,working memory,memory recognition,and total BCAT test of soldiers at different altitudes in the plain and plateau decreased with altitude increasing,and there were significant differences between groups(p<0.05);16Sr RNA gene sequencing results showed that as the altitude increased,there was no significant difference in the alpha diversity of the gut microbiota of each group of military personnel(p>0.05),and the relative abundance of Firmicutes significantly decreased(p<0.05).Bacteroides became the dominant microbiota,and the proportion of beneficial bacteria Lactobacillus and Bifidobacteria decreased(p<0.05).2.Simulated plateau hypoxic exposure mice intervention experiment:(1)Hypoxia exposed mice showed reduced spontaneous vitality in open field test(p<0.05),platform jumping experiments showed learning and memory impairment(p<0.05),and new object recognition experiments showed new object exploration ability decreased(p<0.05);hippocampus and intestinal tissue structure were damaged;intestinal microbiota diversity decreased(p<0.05),structure changed significantly;serum IL-1?,IL-6 and ET contents increased,The levels of IL-10,GABA,5-HT,and Trp were decreased;the expressions of BDNF,ZO-1,and Occludin in hippocampus,cortex,and intestinal tissues were down-regulated,and the expression of HIF-1? protein was up-regulated.(2)Compared to Hypoxia group mice,three probiotics treated groups showed improved cognitive ability;relieved tissue damage;decreased relative abundance of Parabacteroides,and increased abundance of Lactobacillus and Bifidobacterium of gut microbiota;serum IL-10 and 5-HT levels were increased The levels of IL-1 ? and ET were decreased;the expressions of ZO-1 and Occludin in the cortex were up-regulated;the expressions of BDNF,ZO-1 and HIF-1? in the hippocampus were up-regulated;the expressions of BDNF and ZO-1 in the intestine were up-regulated.3.Fecal bacteria transplantation experiment:(1)The HFMT mice showed decreased moving distance and crossings(p<0.05)in the open field test,prolonged latency(p<0.05),and increased number of errors(p<0.05)in step-down test,decreased number of new object visits in the ORT test(p<0.05);BDNF,ZO-1,Occludin,and CLDN1 protein expressions were down-regulated in the hippocampus and the cortex,and ZO-1,Occludin,and CLDN1 protein expressions were down-regulated in the intestinal tissue.(2)The PFMT mice showed increased moving distance and crossings(p<0.05)in the open field test,decreased latency(p<0.05)and number of errors(p<0.05)in step-down test,increased number of new object visits in the ORT test(p<0.05);The expressions of BDNF,ZO-1,and Occludin in the hippocampus and cortex were up-regulated,and ZO-1,Occludin protein expression in intestine,is up-regulated.Conclusion:1.High altitude hypoxic environment leads to a decline in soldiers' cognitive ability such as attention and memory;at the same time,it significantly affects the structure of the human gut microbiota,changes the diversity and richness of the microbiota,increases the proportion of harmful bacteria,reduces beneficial bacteria,and The effect becomes more pronounced as the altitude rises.2.Hypobaric hypoxia exposure can cause changes in the structure of the hippocampus and intestinal tissue of mice,reduce the diversity of gut microbiota,and disrupt the balance composition,which in turn leads to cognition impairments such as decreased spontaneous vitality,ability of exploring new objects,learning and memory in mice;Intervention of Lactobacillus and Bifidobacterium can reverse hypoxia-induced changes in mouse microbiota and improve hypoxia-induced cognitive impairment;combined intervention of Lactobacillus and Bifidobacterium might protect cognitive impairment in hypoxic exposed mice through the microbiota-gut-brain axis pathway.The specific mechanism involves improving the gut microbiota disorder,reducing the secretion of serum pro-inflammatory factors,increasing the level of certain neurotransmitters,and enhancing the expression of cognitive-related neurochemicals in the brain.