| Objective:Based on the theory of "Treating pulmonary diseases from intestine",allergic asthma mouse model was established to determine the effect of Da Cheng Qi decoction(DCQD)on the changes of intestinal microbiota and Type 2 innate lymphoid cell(ILC2).Thus,exploring the experimental basis of this treatment rules in allergic asthma and finally enriching scientific connotation for the theory of "Lung being relative to large intestine".Methods:1 Experiment one:The effect of DCQD on intestinal microbiota in allergic asthma mice.Twenty female C57BL/6 mice aged 7 weeks were randomly divided into 4 groups(5 mice a group)as follow:Normal group(N),Allergic asthma model group(M),Da Cheng Qi decoction group(D),Positive control group(P).Mice in model group(M)and treatment group(D,P)were injected intraperitoneally with 0.2 ml of OVA and aluminum adjuvant on day 0 and day 14.And they were challenged with aerosolized OVA for 30 min/day from day 21 to day 27.Mice in group D and P were administered with Da Cheng Qi decoction and Dexamethasone respectively once per day via oral gavage at doses of 0.2ml half an hour before aerosolization.The normal group(N)received the same operation by phosphate buffer saline(PBS)at each time point as previously described.After all processes,mice received neck removal treatment.The contents of the jejunum,ileum and colon were aseptically taken out and collected in a 2 ml centrifuge tube,immediately frozen in liquid nitrogen then transferred to a-80 refrigerator.The detection of 16S rDNA was analyzed by Beijing Allwegene Technologies Co.,Ltd.Lung tissues were fixed and cut then applied Hematoxylin and eosin(H&E)stain and Periodic Acid-Schiff(PAS)stain.The tissues of jejunum,ileum and colon were fixed in the form of swiss-roll and applied HE stain.2 Experiment two:The effect of DCQD on intestinal ILC2 in allergic asthma mice.Fifteen female C57BL/6 mice aged 7 weeks were randomly divided into 3 groups(5 mice a group):Normal group(N),Allergic asthma model group(M),Da Cheng Qi decoction group(D).The method of giving rise to allergic asthma was the same as Experiment 1.After all sensitization,challenge,and drug administration,the lung and intestinal lamina propria tissue were digested by collagenase Ⅲ.Cells were separated by Percoll gradient centrifugation separating media and single cell suspension was analyzed for ILC2 by flow cytometric analysis.Meanwhile,spleen single cell suspension was obtained and used as unstained and compensation control.3 Experiment three:The effect of DCQD on ILC2 in allergic asthma mice via regulating intestinal microbiotaTen female C57BL/6 mice(age 7 weeks)were randomly divided into two groups(5 mice a group):allergic asthma model administrating DCQD group(MD),and allergic asthma model administrating DCQD after clearing intestinal microbiota group(QMD).Approaches of cleaning intestinal microbiota were described below:Dissolving in PBS,the mixture of antibiotic containing Vancomycin,Neomycin,Metronidazole and Amphotericin B was given through intragastric administration according mice weight.Moreover,Ampicillin was given in free drink from day 3 for 12 days.Amphotericin B(lmg/kg)was given orally once every 12 hours for 3 days at first,then Amphotericin B(1mg/kg),Vancomycin(5mg/kg),Neomycin(10mg/kg),Metronidazole(10mg/kg)were administrated every 12 hours for 12 days.Free drinking water containing Ampicillin were replaced every 6 days.Model establishing method and DCQD treatment for allergic asthma were the same as experiment 1.Pathological examination of lung and intestine tissue and flow cytometry analysis of ILC2 were performed after the establishment of allergic asthma model.4 Experiment Four:Preliminary study on the theory of "Treating pulmonary diseases from intestine" based on tissue immune cell trafficking.Congenic mice were used to track the migration of immune cells.Female donors(7 weeks age)expressing CD45.1 were equally divided into three groups:Normal group(N),Allergic asthma model group(M),Da Cheng Qi decoction Group(D).After establishing the model,single immune cell suspension was collected and transferred into the female recipients at the same age which expressed CD45.2.72 hours later detecting the transfer and trafficking results by flow cytometry.Results:1 Experiment 1DCQD could correct the disturbance of intestinal microbiota in allergic asthma mice and alleviate the inflammatory pathology of intestinal tissue and lung.1.1 DCQD improved pulmonary inflammation in allergic asthma miceCompared with normal group,the weight of model group mice decreased significantly(P<0.01)and the lung index increased(P<0.01).After treatment with Da Cheng Qi decoction,the weight recovered(P<0.05)and lung index was obviously decreased(P<0.01).In positive control group,changes of weight and lung index were almost the same as DCQD group(P<0.01).The alveolar structure was incomplete in allergic asthma mice accompanied by a large number of inflammatory cells infiltration around the bronchial vessels.And there was a large amount of secretion in bronchial lumen.These damages were recovered by DCQD treatment and positive control dexamethasone treatment.A great number of purple-red mucus could be observed in the airway cavity in allergic asthma model.After treatment with DCQD and dexamethasone,the purple-red substance around the bronchus was significantly reduced and the proliferation of airway goblet cells was obviously improved.1.2 DCQD alleviated the inflammatory infiltration of intestinal mucosal lamina propria in allergic asthma mice:(1)Jejunum:In normal group the structure remained unchanged.In allergic asthma model group,there were obvious inflammatory infiltration in jejunal mucosal lamina propria.After DCQD treatment,lymphocyte infiltration relieved clearly while this infiltration was not observed in positive control group.(2)Ileum:There was no obvious inflammatory cells infiltration in all of these four groups.(3)Colon:Compared with normal group,the colonic structure of model group had obvious inflammatory cells infiltration in mucosal lamina propria.And these pathological changes were significantly improved after treating with DCQD and positive control drug.1.3 DCQD improved the component and diversity disorder of intestinal microbiota in asthmatic mice(1)In terms of Alpha diversity:Compared with the allergic asthma model,the jejunum and ileum abundance decreased,the colony abundance increased,and the intestinal microbiota diversity increased.(2)In terms of species composition:The proportion of functional bacteria such as Firmicutes,Bacteroidetes,Lactobacillus and Lachnospiraceae increased in total microbiota.(3)In terms of principal component:In DCQD group,the principal component of microbiota in ileum and colon was far away from the model group and had a tendency to draw close to the normal group.2 Experiment 2:DCQD could inhibit the amount of ILC2 in intestinal mucosal lamina propria of allergic asthma miceThe proportion and number of ILC2 grew significantly in lung,small intestine and large intestine tissue of allergic asthma mice(P<0.01).Compared with model group,the proportion and number of ILC2 in these three tissues apparently decreased after DCQD treatment(P<0.05).3 Experiment 3:DCQD inhibited the differentiation of intestinal ILC2 by modulating intestinal microflora in allergic asthma mice.3.1 The effect of intestinal microbiota on correcting inflammatory pathology of lung tissue and inhibiting of lung ILC2 in allergic asthma mice.Broad-spectrum antibiotics were used to eliminate intestinal microbiota aiming to construct relative "germ-free" mice.After treatment with DCQD,the mucus secretion in airway cavity of model mice was significantly increased compared with wild type(WT)asthma mice,and the proportion and quantity of ILC2 differentiation in lung tissue were significantly increased.3.2 The effect of intestinal microbiota on correcting inflammatory pathology of intestine tissue and inhibiting of intestinal ILC2 in allergic asthma mice.Compared with allergic asthma mice,the proportion and number of small intestinal ILC2 were significantly increased(P<0.01)after DCQD treatment.And the colon showed obvious inflammatory cells infiltration.4 Experiment 4:Results of "Treating pulmonary diseases from intestine":4.1 The transfer result of lung-derived cellsAfter cells transferring from CD45.1 donor to CD45.2 recipient.Allergic asthma donor cells were observed in recipient in different tissues especially in the lung:lung(1.5%),small intestine(0.2%)and large intestine(0.3%).Moreover,the proportion of trafficking cells from DCQD-treated donor to recipient increased significantly compared to model donor:lung(2.7%)and small intestine(0.3%).4.2 The transfer result of intestine-derived cellsIntestinal immune cells from model donor can migrate to lung(0.2%),small intestine(0.3%)and large intestine(0.3%).After DCQD treatment,the proportion of intestine-derived immune cells in recipient obviously increased respectively in lung(1.7%),small intestine(0.4%)and large intestine(1.0%).Conclusion:Based on these results above.Da Cheng Qi decoction,the representative decoction of"Treating pulmonary diseases from intestine",could regulate the component of the microbiota by reducing the abundance of j ejunum and ileum and increasing the abundance of colonic flora.DCQD could restore the diversity of intestinal,thereby inhibiting the intestinal immune cell ILC2 and promoting the migration of immune cells from intestinal lamina propria to lung tissue.Finally,the pulmonary inflammation was improved. |
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