| Objective:To test the hypothesis "lung and large intestine are interior exteriorly related,through investgating into the effect of Dachengqi decotion,as our experimental prescription,on mice of allergic asthma.In our research,we decide to treat allergic asthmatic mice with Dachengqi dection.Then,the differentiation of immune cells in lung and intestinaltissues as well as the changes of intestinal microbiota were observed to explore the cellular and molecular mechanism of "treating intestine for lung diseases" on mice with allergic asthma.Finally,we expect to find the associated substances between the lung and gut.Methods:1 Establishment of mice with allergic asthma and grouping:In this study,6-week-old female C57BL/6 mice were divided into four groups:normal group,model group,Dachengqi Decoction group and positive control group.The four groups of mice were treated as follows:(1)the normal group received sterile saline intraperitoneally and saline intranasally;(2)the model group received 1%OVA in PBS intranasally after intraperitoneal injection of 20μgOVA plus alum;(3)the gut-treatment group received 1%OVA in PBS intranasally after intraperitoneal injection of 20μgOVA plus alum,and were fed 19g/kg/day of Dachengqi decotion from day21 to day27;(4)The positive control group received 1%OVA in PBS intranasally after intraperitoneal injection of 20μgOVA plus alum,and were fed smg/kg/day of Dexamethasone from day21 to day27.Mice were sensitizedby intraperitoneal injection of 20μg of OVA(Sigma-Aldrich)emulsified in 2 mg of aluminum hydroxide(Pierce)or saline on day 0 and day 14.On days 21 to 27,mice were challenged with aerosolized OVA(l%w/v in saline for 30 minutes)or saline alone delivered via an ultrasonic nebulizer(PARI TurboBOYN).2 The lung and instestine histopathological assessment of mice with allergic athmaOn days 7,21,26 and 28,the lung and intestine tissues were removed separately,which were fixed by 10%formaldehyde and embedded in paraffin.Then,these tissues were cut into 4mm slices,and stained with HE.The airway inflammation and inflalmatory cell inifltration in the intestine tissue were detected by hematoxylin and eosin staining with a magnification ratio of 1:200.3 The pathological changes of other organs include heart,liver and kidney of allergic asthma miceThe histopathological assessment of heart,liver and kidney followed the same procedure as lung tissue.The serum were collected after centrifugation from blood of mice.The levels of ALT and BUN in serum were determined by the Automatic biochemical analyzer.4 Evaluation on gut-treatment of allergic asthma miceAfter sacrificing the mice,their lung and intestine tissues were removed and fixed.Morphological histology of all mice was observed.What’s more,the index of lung and the length of intestines were compared among all groups.Inflammatory cell infiltration of lung and intestinal tissues was assessed by stanining with HE.The secretion of mucus in the lung tissues were detected by staining with periodic acid-Schiff(PAS).5 Analysis of the ratioes of Th17 to Treg cell of lung and intestine by flow cytometrySingle cell suspensions from lung and intestinetissues were prepared for further flow cytometryassay.Cells were stimulatedfor 5 hours with phorbol 12-myristate 13-acetate and ionomycin and Golgistop in the presence of complete medium.After stimulation,cells were stained with FITC-labeled anti-mouse CD4antibody and Percp-cy5.5-labeled anti-mouse CD25antibody for 30min at 4℃.Then these cells were incubated with the fix/perm solution for 20-50min,and were stained with PE-labeled anti-mouse IL-17A and APC-labeled anti-mouse Foxp3 antibodies for 40min.After washing,labelled single cell suspensionswere analyzed by flow cytometry.6 Effect on gut microbiota of mice with allergic asthma after gut-treatmentThe diversification of gut microbiotawas assayed by PCR-DGGE technique after the end of the experiments.At the same time,the amounts of five specific bacteria in per gram of stool were detected by colony counting method.These five bacteria included Escherichia coli,Enterococcus,Bifidobacterium,Lactobacillus and Bacteroides.Results:1 Comparison of the common reaction between normal and model group:In the aspect of body weight,there was no significant difference between the normal group and model group.The weight of all mice increased stablely.However,the average numbers of feces as well as fecal water content of the model group decreased(p<0.05),compared with those of the normal group.In addition,its wet weight deereased significantly(p<0.01).It’s worth noting that mice from model group behaved extremely active,with the symptoms of scratching nose,shortness of breath,wheezing and sneezing etc.2 Specific injury in lung and colon from mice with allergic asthmaOnly on days 26 and 28,inflammatory cells infiltration was significantly detected in the lung tissue as well as lamina pripria of colon in mice with allergic asthma.In contrast,nonmucosal heart,liver and kidney tissues premained normal after sentization and OVA-challenged,which was also supported by ALT and BUN analysis.3 Amelioratioin of pulmonary inflammation of allergic asthma mice after gut-treatmentMice from model group showed abnormal morphology in lung tissue,include congestion and edema.Furthemore,lung index increased significantly(P<0.01).The results of H&E staining revealed that there were plenty of mucus secreted inside the airway and a large number of inflammatory cells infiltrating around the airway.The results of PAS staining fromlung tissue showed inceased goblet cell metaplasia.In "gut-treatment"group,lung index decreased(p<0.05)compared with model group.What’s more,its inflammatory cells infiltration and mucus secretion around and inside the airway was attenuated.As to positive control group,its lung index decreased dramatically(p<0.01),with the inhibition of lung inflammation and goblet cell metaplasia.4 Improvement in intestine inflammation of allergic asthmatic mice after "gut-treatment"Colon length increased notablely in model group(p<0.01),compared with normal group.Additionally,there were a plenty of lymphocytes in the mucosal lamina propria.Afler gut-treatment,the colonic injury was reduced.Among the three groups,there were no significant changes in the length of small intestines as well as their morphological histology.(p>0.05).5 Comparison of the numbers of Thl7 and Treg cells from lung and colon among groups.The experimental asthmatic mice had higher numbers of lymphocytes and IL-17A secreting CD4+cells while the numbers of CD4+CD25+Foxp3+cells were decreased in lung,compared with normal mice ep<0.01).Though the numbers of lymphocytes and IL-17A secretingCD4+cellsin colon from model groupalso increased(P<0.01),the numbers of CD4+CD25+Foxp3+cellsp did not decreased.After gut-treatment,the numbers of IL-17A secreting CD4+cells from lung and colon in mice with allergic asthma were obviously reduced.what’s more,the numbers of CD4+CD25+Foxp3+cells in lung were prominently raised(p<0.05).However,there seemed no obvious changes in CD4+CD25+Foxp3+cell populations from the colon in gut-treatment group,compared with model group.6 The effect of gut-treatment on gut microbiota in mice with allergic asthma.The results of sequencing indicated that total number of bacteria from colons in model group had an obvious decreased(p<0.01).Among them,the number of Enterobacteriaceae and Enterococcaceae increased(p<0.05),the number of Lactobacillaceae decreased significantly(p<0.05),no significant changes in Bacteroidaceae,Bifidobacteriaceae and Clostridiaceae.And Segmented filamentous bacteria were seldomly found in mice with allergic asthma.After treatment,there were no significant changes in bacteria,Lactobacillaceae and Enterococcaceae,while the number of Enterobacteriaceae increased(p<0.05).After selectively culturing in medium,the amounts of Escherichia coli and Enterococcus in the model group were significantly increased(p<0.01).Its content of Bifidobacterium and Bacteroides was not significantly different(p>0.05)compared with normal group.In gut-treatment group,the numbers of Escherichia coli and Enterococceis decreased(p<0.01)while those of Lactobacillus increased(p<0.05).The numbers of Bifidobacterium and Bacteroides were not significantly different among these three groups(p>0.05).Conclusion:1 Pulmonary and colonic immune injury were detected in mice with allergic asthma.2 "Gut-treatment" could relieve pulmonary inflammationin mice with allelgic asthmain in some degree,which might be attributed to the rebalance of gut microbiota.The later possibly affected the differentiation of Thl7and Treg cells in lung and colon through the interactions of the mucosal immune system between gut and lung. |
PDF Full Text Request