| Purpose:Bronchial asthma is a common chronic respiratory disease of the respiratory system,the pathogenesis of which is abnormal immune response,causing airway hyperresponsiveness,reversible respiratory dyspnea,increased mucus secretion,eosinophilia,airway remodeling and other clinical manifestations.The etiology of asthma is complicated,not fully understood still,closely related to heredity and environment.It is well known that dust,pollen,mites and other allergens in the air could lead to asthma attacks.The current studies show that exposure to environmental microorganisms in early life was negatively correlated with the incidence of allergic diseases such as asthma and allergic rhinitis.Investigations show that children who are exposed to environmental microorganisms earlier and more through contact with pets in their early lives are less likely to have asthma attack than those who are not;Children born and grown on the farm,having access to abundant environmental microorganisms in their early lives,have a lower incidence of asthma than those who are raised in an over-clean environment in the city.Residents in Western developed countries with an over-clean environment have a higher incidence of asthma than those in Eastern Europe and Asia,and the incidence rate has increased over the past two decades.All the evidences above indicate that environmental factors have an effect on the incidence of allergic diseases such as asthma.But the specific approaches and mechanisms of the effects of environmental microorganisms on body are still remained unclear.Preliminary consideration is that intestinal microorganisms are the mediums between the environment and body's immune system.Intestinal microbial community,also known as intestinal flora,is avariety of microorganisms collective existed in the intestinal tract of the body.There are more than 500 kinds of microorganisms known in the human intestinal tract,and the total weight reach up to 1.5 kg approximately,which is mutually beneficial and symbiotic with the host,forming a dynamic and stable micro-ecological structure with intestinal mucosa and digestive enzymes together.Intestinal micro-organisms in the body are gradually colonized from the mother,the environment and the diet in the early life,participating the development of the intestinal mucosal immune and systemic immune system.Environmental microorganisms can not only affect the order and species of intestinal microbes in the early life,but also affect the intestinal microbial community structure of adult organisms.The microbial community structure of migratory population also changes.This study was to investigate the changes of intestinal microbial community structure and the expression of related allergenic immune factors in sensitized mice after cultured in different environments,and to explore its underlying mechanism.Methods:1.Thirty 4-week-old BALB / C mice were randomly divided into three groups: asthma group(group A),intervention group(group B),control group(group C),n=10 for each group.The asthma group and the intervention group were injected intraperitoneally with 200?l of normal saline solution containing40?g OVA and 4mg aluminum hydroxide on the 1st,14 th and 14 th day,and then treated with OVA saline solution by aerosol inhalation(Volume fraction5%,1 day / time,30 min / time)on the 22 nd to 28 th day.The rats were injected or inhale with normal saline intraperitoneally in the control group.The behavioral changes of mice were observed during aerosol inhalation.2.Soil rich in rot was collected in the same place at the same time,mixed with sterile granular padding by a certain proportion,for preparing the padding rich in environmental microorganisms for the intervention group.On the 28 th day after the aerosol inhalation treatment,the intervention group was transferred to the common animal room,the environment with abundant environmental microorganisms and the control group,the asthma group wasstill kept in the SPF animal room.Feces were collected after 2 weeks and stored in-80? refrigerator.Then the mice were separately treated by aerosol inhalation again according to the method as mentioned above.3.Blood samples and bronchoalveolar lavage fluid were collected within24 hours after the last aerosol inhalation,and the serum samples were prepared.The expression of OVA-IgE,IL-4 and IFN-? of serum samples were determined by ELISA.Bronchial alveolar lavage fluid was prepared for cell smear after low-speed centrifugation,and Wright's staining and microscopic classification count was administered later.4.The DNA in the feces were extracted and the concentration and OD of the total DNA were measured.The DNA samples were sequenced for 16 s DNA.The biological information was analyzed by QIIME software under the Linux system.Results:1.Behavioral changes of mice during aerosol inhalation:After OVA sensitization,behavioral changes such as anxiety,nausea,wheezing,hair messy,incontinence were observed.The mental state,the degree of wheezing,the level of anxiety of mice in intervention group during the last aerosol inhalation were improved after cultured in the environment rich in environmental microorganism,and was better than asthma group.Control group shows no abnormal change.2.Differences in flora of each group:(1)The PCA coordinate shows that the position of each group were close to each other,and the coordinate positions of the three groups were separated from each other.(2)Phylogenetic tree showed that the differences between the three groups of mice were small and the differences between the groups were large.(3)The number of OTU in intestinal microbes in the intervention group was significantly higher than that in asthma group and control group(p<0.05).There was no significant difference between asthma group and control group(p>0.05).(4)The proportion of phylum firmicutes in the intervention group was significantly higher than that in the asthmatic group and the control group(p<0.05).There was no significant difference between asthma group and control group(p>0.05).(5)In the intervention group,the increase of phylum firmicutes was mainly lactobacillus.The proportion of lactobacilli was significantly higher than the other two groups(p<0.05).There was no significant difference between asthma group and control group(p>0.05).3.The levels of OVA-IgE in the serum of the intervention group,asthma group and control group were 281.86 ± 37.75(ng / ml),972.44 ± 120.53(ng / ml),230.42 ± 46.53(ng / ml).There were significant differences in IgE data between the asthma group and the control group,the intervention group and the control group,the intervention group and the asthma group,the difference was statistically significant(p <0.05).The content of OVA-IgE in the serum of the intervention group was significantly lower than that of the asthma group,but the content of IgE in the serum group was significantly higher than that in the control group.4.The levels of IL-4 in the serum of the intervention group,asthma group and control group were 224.61 ± 28.73(pg / ml),327.98 ± 35.48(pg / ml),160.41 ± 27.67(pg / ml).There were significant differences in serum IL-4levels between the asthma group and the control group,the intervention group and the control group,the intervention group and the asthma group,the difference was statistically significant(p <0.05).The levels of IL-4 in the serum of the intervention group were significantly lower than those in the control group.and the levels of IL-4 in the asthma group was significantly higher than the control group,the intervention group was significantly higher than the control group.5.The results showed that the levels of IFN-? in the serum of the intervention group and the asthma group were 784.45.64 ± 22.47(pg / ml),363.59 ± 28.14(pg / ml)and 877.64 ± 36.33(pg / ml)respectively(p<0.05).The level of IFN-? in the serum of the intervention group wassignificantly lower than that in the asthmatic group.The expression of IFN-?in the serum of the asthma group was significantly higher than that in the control group The expression of IFN-? was significantly higher than that of the control group.6.The total number of white blood cells in the BALF group was 54.24± 5.05(×104ml-1),121.23 ± 6.04(×104ml-1),30.41 ± 5.33(×104ml-1),the eosinophilic The total number of granulocytes was 1.75 ± 0.55(×104ml-1),13.02 ± 3.51(×104ml-1),0.21 ± 0.07(×104ml-1),the difference was statistically significant(p <0.05).The number of white blood cells and eosinophils in BALF of mice were significantly higher than those in the control group and the intervention group.The number of white blood cells and eosinophils in BALF of the intervention group were significantly higher than those in the control group.Conclusion:1.Increasing the diversity and abundance of environmental microbes can change the community structure of intestinal microbes,and the proportion of bacteria will change,which can improve intestinal microbial diversity and abundance2.Intestinal microbial community structure changed by rich environmental microbial can affect the immune system,inhibit the expression of inflammatory factors in the serum,increase serum IFN-? content,reduce the number of inflammatory cells in the lungs secretions,relieve asthma attack symptoms or help to control the number of asthma attacks. |
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