| Objective:By observing the effect of Pingchuan Granules on the intestinal flora of asthmatic mice and the expression of IL33/ST2/ILC2s signaling pathway-related factors mediated by Pingchuan Granules,it is explored that Pingchuan Granules regulate IL33/ST2/ILC2s mediated by intestinal flora.The mechanism of the effect of signal axis on airway epithelial mucosal barrier and airway inflammation in asthmatic mice.Methods:1.Experiment 1:30 C57BL/6N mice were randomly divided into 3groups,each with 10 mice,namely the blank group,the model group,and the Pingchuan Granules group.Except for the blank group,the model group and Pingchuan Granules group all used OVA atomization and excitation methods to construct asthma mouse models.On the 21st day,mice in the Pingchuan Granules group were given Pingchuan Granules(0.1ml/10g)by gavage,and the blank group and model group were given normal saline(0.1ml/10g)by gavage for 4 weeks,once a day.Within 24 hours after the last challenge,the AHR of each group of mice was detected by EMKA plethysmograph;the pathological changes of lung tissue of each group of mice were observed by HE staining and PAS staining;the total cells in BALF of each group of mice were detected by microscope,EOS,Neu and Lym counts;through 16S r DNA gene sequencing to detect the alpha diversity analysis,Beta diversity analysis,species composition analysis,LEf Se analysis and KEGG function prediction in the fecal intestinal flora of each group of mice.2.Experiment 2:60 C57BL/6N mice were randomly divided into 6groups,each with 10 mice,namely blank group,sterile group,model group,model sterile group,model+Pingchuan Granules group,and model sterile+Pingchuan Granules.sterile group,model sterile group,model sterile+Pingchuan Granules group were given cefoperazone and sulbactam sodium-vancomycin hydrochloride physiological saline solution 20?L/only by gavage every morning before asthma model creation,blank group,model group,model+Pingchuan Granules group,20?L of normal saline was given to the stomach for 7 days.Model sterile group,model sterile+Pingchuan Granules group,model group,model+Pingchuan Granules group mouse asthma model method is the same as experiment one;the pathological examination of lung tissue and inflammatory cells in BALF of each group are the same as experiment One.ELISA method was used to detect the levels of IL-4,IL-5 and IL-13 in BALF of mice in each group.Experiment 3:40 C57BL/6N mice were randomly divided into 4 groups,each with 10 mice,namely the blank group,the model group,the Pingchuan Granules group,and the Lactobacillus rhamnosus group.Mice in the Lactobacillus rhamnosus group were given intragastrically with Lactobacillus rhamnosus at a dose of 5×10~5cfu/ml 200?L,4 times a week,and the course of treatment was 6 weeks.Model group,Pingchuan Granules group,Lactobacillus rhamnosus group mouse asthma model method is the same as experiment one;the pathological examination of lung tissue and inflammatory cells in BALF of each group are the same as experiment one;The detection of inflammatory factors in BALF of mice in each group is the same as experiment two;ELISA method was used to detect the level of IL-33 in the BALF of each group of mice;immunofluorescence,Western blot and q RT-PCR methods were used to detect the expression levels of ZO-1,Occludin protein and m RNA in the lung tissue of each group of mice;Immunohistochemistry,Western blot and q RT-PCR methods were used to detect the expression levels of IL-33,ST-2 protein and m RNA in the lung tissue of each group of mice;flow cytometry was used to detect positive ILC-2s in the lung tissue of each group of mice Cell expression rate.Results:experiment 1:1.Compared with the blank group,the airway responsiveness of mice in the model group was increased,and the airway responsiveness of the mice in the Pingchuan Granules group was improved compared with the model group,and the difference was statistically significant(P<0.05).2.HE staining:HE staining of lung tissue of the blank group showed no obvious asthma-related pathological changes;compared with the blank group,HE staining of lung tissue of the model group showed a large amount of asthma-related inflammatory cell infiltration;compared with the model group In the Pingchuan Granules group,pathological features such as asthma-related pathological changes and inflammatory cell infiltration were alleviated.3.PAS staining:PAS staining of lung tissue in the blank group has no obvious asthma-related pathological signs of airway epithelial goblet cell hyperplasia and increased airway mucus;compared with the blank group,PAS staining of lung tissue in the model group shows airway Mucus and goblet cells were significantly increased;compared with the model group,airway mucus and goblet cells and other asthma-related pathological signs of mice in the Pingchuan Granules group were significantly reduced.4.Compared with the model group,the counts of total cells,EOS,Neu and Lym in BALF of mice in the Pingchuan Granules group were significantly reduced(P<0.01).5.Alpha diversity analysis:Pingchuan Granules can significantly increase the Chao1 index,Shannon index and ACE index of asthmatic mice,and decrease the Simpson index,which affects the total number and diversity of intestinal flora species.6.Beta diversity analysis:PCA analysis,PCo A analysis and PLS-DA results show that the composition of the intestinal flora of mice in the blank group,model group and Pingchuan Granules group is significantly different.7.Species composition analysis:From the level of intestinal flora to the level of genus,the intestinal flora of mice in the blank group,model group and Pingchuan Granules group is significantly different,and the intervention of Pingchuan Granules can change the intestinal bacteria of asthmatic mice Group structure,among which Lactobacillus is the most obvious.8.LEf Se analysis:Pingchuan Granules can restore the abundance of Lactobacillus.9.KEGG function prediction:Pingchuan Granules group can affect the biosynthesis,transport and metabolism of polysaccharides,and regulate cell motility.Experiment 2:1.HE staining:HE staining of lung tissue of the blank group and sterile group has no obvious asthma-related pathological changes;compared with the blank group and sterile group,the HE staining of the lung tissue of the model group and the model sterile group shows asthma-related changes Inflammatory cell infiltration,airway wall thickening,and airway lumen narrowing to varying degrees;compared with model group and model sterile group,model sterile+Pingchuan Granules group,model+Pingchuan Granules group with the above pathological characteristics.The pathological changes in the model+Pingchuan Granules group were more significantly reduced than the model sterile+Pingchuan Granules group.2.PAS staining:PAS staining of lung tissue of mice in the blank group and sterile group has no associated pathological signs of asthma such as increased mucus;compared with the blank group and sterile group,PAS staining of the lung tissue of mice in the model group and model sterile group shows airways Mucus increased;compared with the model group and the model sterile group,the above symptoms of the model sterile+Pingchuan Granules group and the model+Pingchuan Granules group were significantly reduced,and the model+Pingchuan Granules group was more sterile than the model+Pingchuan Granules The pathological changes of the group were alleviated more obviously.3.The counts of total cells,EOS,Neu and Lym in BALF of the model group and model sterile group were significantly increased compared with the blank group and sterile group(P<0.01);model+Pingchuan Granules group and model sterile+Pingchuan Granules.The counts of total cells,EOS,Neu and Lym in BALF of mice in the Pingchuan Granules group were all lower than those in the model group and model sterile group(P<0.01);and the total cell,EOS,Neu and Lym counts in BALF of the model sterile+Pingchuan Granules group were higher than those of the model+Pingchuan Granules group(P<0.01).4.ELISA results showed that the levels of IL-4,IL-5 and IL-13 in BALF of the model group and model sterile group were significantly higher than those of the blank group and sterile group(P<0.01).The levels of IL-4,IL-5and IL-13 in BALF of the model+Pingchuan Granules group and model sterile+Pingchuan Granules group were significantly lower than those of the model group and model sterile group(P<0.01).The levels of IL-4,IL-5 and IL-13 in BALF of the model sterile+Pingchuan Granules group were higher than those of the model+Pingchuan Granules group(P<0.05).Experiment 3:1.HE staining:Compared with the model group,pathological features such as inflammatory cell infiltration and smooth muscle cell proliferation in the Pingchuan Granules group and Lactobacillus rhamnosus group were all reduced,while the asthma-related pathological changes in the Pingchuan Granules group were more significantly reduced.2.PAS staining:Compared with the model group,asthma-related pathological signs such as airway epithelial goblet cell hyperplasia in the Pingchuan Granules group and Lactobacillus rhamnosus group were significantly reduced,while the pathological changes in the Pingchuan Granules group were more significantly reduced.3.The counts of total cells,EOS,Neu,and Lym in BALF of mice in the Pingchuan Granules group and Lactobacillus rhamnosus group were significantly lower than those in the model group(P<0.01),while the Pingchuan Granules group decreased more obvious than that of the Lactobacillus rhamnosus group,the comparison is statistically different(P<0.01).4.ELISA results showed that the levels of IL-4,IL-5,IL-13 and IL-33 in BALF of mice in the Pingchuan Granules group and Lactobacillus rhamnosus group were significantly lower than those in the model group(P<0.01);The levels of the above-mentioned inflammatory factors in the Pingchuan Granules group were more significantly lower than those in the Lactobacillus rhamnosus group,with statistical differences(P<0.05).5.The results of immunofluorescence,Western blot and q RT-PCR showed that the expression levels of ZO-1,Occludin protein and m RNA in the lung tissue of the model group were significantly lower than those of the blank group(P<0.01);The expression levels of ZO-1,Occludin protein and m RNA in the lung tissues of the Pingchuan Granules group and Lactobacillus rhamnosus group were higher than those of the model group(P<0.01),but lower than those in the blank group(P<0.01);The expression levels of ZO-1,Occludin protein and m RNA in the lung tissue of the Pingchuan Granules group were higher than those of the Lactobacillus rhamnosus group(P<0.01).6.The results of immunohistochemistry,Western blot and q RT-PCR showed that the expression levels of IL-33,ST-2 protein and m RNA in the lung tissue of the model group were significantly higher than those in the blank group(P<0.01);The expression levels of IL-33,ST-2 protein and m RNA in the lung tissues of the Pingchuan Granules group and Lactobacillus rhamnosus group were lower than those of the model group(P<0.01),but higher than the blank group(P<0.01);The expression levels of IL-33,ST-2protein and m RNA in the lung tissue of the Pingchuan Granules group were lower than those of the Lactobacillus rhamnosus group(P<0.01).7.The results of flow cytometry showed that the rate of ILC-2s positive cells in the lung tissue of the model group was significantly higher than that in the blank group(P<0.01);The rate of ILC-2s positive cells in the lung tissues of the Pingchuan Granules group and Lactobacillus rhamnosus group was significantly lower than that of the model group(P<0.01),but still higher than that of the blank group(P<0.01).The rate of ILC-2s positive cells in the lung tissue of the Pingchuan Granules group was lower than that of the Lactobacillus rhamnosus group(P<0.01).Conclusions:1.Pingchuan Granules can alleviate the pathological changes of airway inflammation such as airway hyperresponsiveness and inflammatory cell infiltration in asthmatic mice,improve the structure of the intestinal flora,and protect the airway epithelial mucosal barrier function.2.Pingchuan Granules can reduce the levels of inflammatory cells and inflammatory factors in the BALF of asthmatic mice.3.Pingchuan Granules can reduce the expression level of IL-33,ST-2protein and m RNA in the lung tissue of asthmatic mice,increase the expression level of ZO-1,Occludin protein and m RNA in the lung tissue,and reduce the rate of ILC-2s positive cells in the lung tissue4.Pingchuan Granules improve the airway epithelial mucosal barrier through the IL33/ST2/ILC2s signaling pathway mediated by intestinal flora and inhibit airway inflammation in asthma. |
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