Study On The Effect Of "Dumin Dingchuan Decoction Modified Recipe" In The Treatment Of Allergic Asthma In Mice And Its Mechanism Of Action On ILC2 Cells

Objective:To explore the asthma-curing efficacy and safety of the increasing or decreasing of"TuoMin DingChuan Decoction"(the TMDC Decoction)which was made up by the theory"Treatment on Constitutional Differentiation" by establishing the mice model of asthma,and to evaluate its influence on ILC2 cell,we'd like to find the pathogenesis of allergic asthma on mice.We expect to provide supporting for the theory "Treatment on Constitutional Differentiation" using on clinical application,and to enrich the scientific connotation of Traditional Chinese medicine.Methods:The project was divided into two parts:one is the study of the curing efficacy and the safety of the TMDC Decoction on the mice model of allergic asthma(including experiment 1 and experiment 2),the other one is its mechanism in curing the symptom(including experiment 3 and experiment 4).The experimental animal used in this experiment was a BALB/c mouse,which exhibited Thl/Th2 imbalance and was prone to a Th2-type reaction.Due to its own characteristics,BALB/c mice were susceptible to allergic diseases and were widely used in immunological experimental study.The first part was operated as the follows:The method of experiment 1 is as follows:48 female BALB/c mice were divided into 6 groups randomly,6 each group,namely normal group(N),model group(M),Dexamethasone group as positive control group(D),the TMDC Decoction high dose concentration group(Y20),the TMDC Decoction medium dose concentration group(Y10),the TMDC Decoction low dose concentration group(Y5),respectively.Set up the asthma mice model by using the ovalbumin(OVA)as aerosol,and to evaluate the model by observing and recording the symptom and weight of the mice,specifically,giving all the mice the mixture of OVA and Aluminum hydroxide gel by intraperitoneal injection in d0 and d13,and stimulate the mice by inhaling 10%OVA aerosol(30min/d,continuously)in PMMA cover at the day of 20,continued 7d.Synchronously with the model setting up,giving the mice the 3 groups of the TMDC Decoction by gavage 0.2mL per day started from d13,and continued for 14 days.Giving Dexamethasone to the mice 0.2mL per day for 7 days,and giving normal saline 0.2mL per day to the normal group 7 days started from d20 in the same way to group Y.After this,removal the eyeball of the mice to collect the blood,separate the serum via centrifugal after standing the blood 4 hours,to evaluate the concentration of IL-5 in the serum by the method of ELISA.Collecting the BALF of the mice,enriching the cells by centrifugal,and to calculate the inflammatory cells among which by using the method of Diff-quick staining.Then we dissect the mice to collect the lung fixation them by 10%formaldehyde,and dyeing by HE staining and PAS staining to observe after taking photos of them.The experiment 2 method is as follows:20 female BALB/c mice were divided into 4 groups randomly,5 each group,namely normal group(N),model group(M),the TMDC Decoction group(Y),Dexamethasone group as positive control group(D),respectively.To build up the model in the same way as experiment 1.Gavage from the day of 14 to the group Y for 14 days,0.2mL per day.Gavage from the day of 21 to the group D for 7 days,0.2 mL per day.The group N was treated in the same way as group D,giving normal saline rather than Dexamethasone.Dissect to get intestine,heart,liver,and kidney after model-building,fixation them by 10%formaldehyde,and dyeing by HE staining to observe after taking photos of them.Then measure the length of the large intestine and small intestine.The 2nd part was operated as the follows:The method of experiment 3 is as follows:20 female BALB/c mice were divided into 4 groups randomly,5 each group,namely normal group(N),model group(M),the TMDC Decoction group(Y),Dexamethasone group as positive control group(D),respectively.To build up the model in the same way as experiment 1,and gavage in the same way as experiment 2.After the model-building,we take mice eye balls and collect blood to kill and dissect to get the contents in cecum and colon,put which into liquid nitrogen.The specimen were all sent to Beijing allwegene Gene Technology Company to finish the test.The part 4 was operated as the follows:9 female C57BL/6 mice were divided into 3 groups equally,named normal group(N),model group(M),and TMDC Decoction group(Y),respectively.To build up the model and gavage in the same way as part one.Collecting the lung,spleen,and intestine organizations into 1640 cell culture medium,quantitative analysis the ILC2 cells among which by the method of Flow Cytometry(FC).Results:Part 1Experiment 1:1.The content of IL-5 in the mice serum decreased significantly in the D group and Y20 group compared to M group,and the decreasing in D group(p<0.01)is larger than in Y20 group.2.The index of lung and spleen from the mice decreased significantly in the D group and Y20 group compared to M group,and the decreasing in D group(p<0.01)is larger than in Y20 group.3.The score of the lung pathological staining by HE and PAS decreased significantly in the D group and Y20 group compared to M group,and the decreasing in D group(p<0.01)is larger than in Y20 group.Experiment 2:1.There is no significantly difference in heart,liver,and kidney of the mice among the different groups.2.The length of the large intestine and small intestine shortened significantly in the D group and Y20 group compared to M group,and the decreasing in D group(p<0.01)is larger than in Y20 group.Experiment 3:1.The index of Chaol of the mice cecum shows no significant difference,P>0.05,the index of Shannon indicated,compared to other groups,the group Y decreased a bit.The index of Chao1 of the mice colon shows,compared to group D,the group N and group Y increase significantly,when the difference among other groups show no statistical significance.The index of Shannon of mice colon indicated the 3 other groups show a significant decrease among the group D.2.The sequence of the amount of the caecum flora in the mice cecum was same among each groups,they are Thrombobacter,Bacteroidetes,and Proteobacteria.The sequence of the amount of the cecum flora in the mice cecum was same among each groups,they are Bacteroidetes,Thrombobacter and Proteobacteria.3.When compared to normal group,the first 5 bacteria in the 3 other groups mice cecum have changed.After the treatment of The TMDC Decoction or Dexamethasone,the sequence of the cecum bacteria has not changed compared to the normal group.4.When compared to the model group,The amount of Lactobacillus in cecum in the TMDC Decoction group and the Dexamethasone group show a significant increase,and the TMDC Decoction group increased more,P<0.01.When compared to the model group,after the treatment of the TMDC Decoction or the Dexamethasone,the amount of the Lactobacillus in the colon of the mice show a significant increase,P<0.01 in the D group.Experiment 4:1.Compared to the model group,the content of the ILC2 cells decreased significantly in the group D and group Ys.Conclusion:The TMDC Decoction,which embodies the concept of "Treatment onConstitutional Differentiation",may affect the composition of intestinal microflora,leading to the activation of ILC2 cells in the intestinal lamina propria.The ILC2 cells may migrate to the lung,thereby affecting the production of cytokines in the lung during the pathogenesis of allergic asthma.Ultimately,it affecting the eosinophilic chemotaxis toward the lung,and exerting its therapeutic effects on allergic asthma mice.