| purpose:This study uses OVA to copy allergic asthma model rats to verify the anti-allergic effects of Masonite,and then explore Max Xifei Gan Tang to an allergic asthma model rat auxiliary T lymphocyte TH2,TH9,TH7 regulation and main regulation of cells;and use in vitro cultured peripheral blood lymphocytes to explore the role of allergic asthma;explore Mao Xikan Tang intervene in the main mechanism of allergic asthma cellular immunity,Provide experimental basis for explaining the apricotous almights and action mechanisms.Material and method:1.The anti-allergic effect of Maxinganshi Decoction on allergic asthma model rats.To observe the regulating effect of Maxinganshi Decoction on serum Ig E,lung tissue pathology(HE staining),and degranulation of abdominal mast cells in allergic model rats.The allergic asthma model rats were replicated by OVA sensitization and stimulation,and then given Maxinganshi Decoction by oral gavage intervention.The Ig E in the rat serum was detected by ELISA,and the lung tissue pathology was observed by HE staining.Granule experiment to observe the stabilizing effect of Maxinganshi Decoction on mast cell membrane.2.The regulating effect of Maxinganshi Decoction on Th2,Th9 and Th17 in rats with allergic asthmaObserve the effect of Maxinganshi Decoction on the serum IL-4,TGF-β,IL-9,IL-5,IL-10,IL-17 levels in allergic model rats.Observe the effect of Maxinganshi Decoction on the expression of IL-4,TGF-βand IL-9 in the lung tissue of rats with allergic asthma.To observe the effect of Maxinganshi Decoction on the ratio of Th2,Th9,Th17 of peripheral blood lymphocytes in rats with allergic asthma.The allergic asthma model rats were replicated by OVA sensitization and stimulation,and then the Maxinganshi Decoction was administered orally for intervention,and the serum IL-4,TGF-β,IL-9,and IL-5 of the rats were treated by ELISA.,IL-10,IL-17 content.WB and IHC were used to detect the expression of IL-4,TGF-βand IL-9 in rat lung tissue.The FC method was used to detect the ratio of Th2,Th9,Th17 in peripheral blood lymphocytes of rats.3.Using the peripheral blood lymphocytes of rats in vitroUsing cytokines and blood-free intervention CD4~+T cell development,Macao TH2,TH9,TH17 positive rate and cells were observed in PBMC using immunofluorescence and ELISA.Factor content,the antibody mechanism of Maxinganshi Decoction regulates TH2,TH9,TH17.Results:1.Animals in vivo experiments1.1 After intraperitoneal injection of OVA and then nebulized inhalation of OVA,the rat model of allergic asthma can be successfully induced.1.2 Maxinganshi Decoction can significantly improve the general state of the modeled rats.In the Maxingshigan group,the lung tissue of the rats in the Maxingshigan group is stained with HE staining of tracheal mucosa intact,not broken,chronic inflammation of bronchial mucosa is mild,goblet cells,mucosal proliferation and protrusions It is mild,emphysema and pulmonary interstitial inflammation are mild,and there are a few mast cells,eosinophils and other inflammatory cell infiltrations around it.It shows that Maxing Ganshi Decoction can improve the degree of lung tissue lesions in rats with allergic asthma as a whole.1.3 In the detection of mast cell degranulation,the mast cell count,degranulation mast cell count,and degranulation rate of rats in the western medicine control group and the Maxing Ganshi group were significantly reduced(p<0.05);compared with the western medicine control group,the anesthesia There was no significant difference in mast cell count,degranulation mast cell count,and degranulation rate in the apricot ganshi group(p>0.05).It is suggested that the Maxingshigan group can reduce the recruitment of mast cells in lung tissue,reduce mast cell degranulation,and relieve airway inflammation.And the effect is equivalent to western medicine.1.4 The content of Ig E in the serum of OVA model rats was significantly increased,and there was no significant change in serum Ig E of rats in the blank group,which was consistent with the results reported in the literature.Compared with the model control group after drug intervention in the other two groups,the serum Ig E levels of rats in the Maxing Ganshi group and the western medicine control group were significantly down-regulated(p<0.05).Although the difference between the groups was not statistically significant(p>0.05),compared with the data,the Maxingshigan group has a slightly better Ig E regulation effect than the western medicine control group.It is speculated that the traditional Chinese medicine compound granules may inhibit Ig E and enhance the anti-allergic effect,thereby reducing airway inflammation.1.5 In this experiment,helper T lymphocytes and cytokines were determined by flow cytometry and ELISA.Compared with the blank control group,IL-4,TGF-β,IL-9,and IL-5in the lung tissue of the model control group The expression of IL-10,IL-17 was significantly up-regulated(p<0.05),and the proportion of Th2 cells,Th9 cells,and Th17 cells increased.It shows that Th2,Th9,Th17 and their main cytokines play an important role in the occurrence and development of bronchial asthma,which is consistent with reports in the literature.The experiment also found that Th2 cytokines(IL-4,IL-5)had a significant up-regulation trend and the positive rate of Th2 cells was the highest,confirming that Th2 cells play a dominant role in the development of asthma.After drug treatment,compared with the model control group,the expressions of IL-4,TGF-β,IL-9,IL-5,IL-10,and IL-17 in the lung tissues of the Maxingshigan group and the western medicine control group were all There are different degrees of down-regulation(p<0.05).The indexes of Maxingshigan group are better than those of western medicine control group,and the expressions of IL-4,TGF-βand IL-9 in Maxingshigan group are down-regulated significantly(p<0.05)Flow cytometry proved that the positive rate of Th9 cells in the Maxingshigan group was roughly the same as that of the blank control group(p>0.05)and lower than that of the western medicine control group(p<0.05),which proved that Maxinganshi Decoction could regulate Th2,Th9,Th17 has good effects,among which Maxing Ganshi Decoction may mainly interfere with allergic asthma by regulating Th9 cells.1.6 The results of immunohistochemistry suggest that IL-4,TGF-β,and IL-9 can be detected in epithelial cells,lymphocytes,eosinophils,smooth muscle cells,goblet cells,and mast cells.Western blot experiments proved that the Maxingshigan group can down-regulate the expression of IL-4,TGF-β,and IL-9(p<0.05).Lateral observation of the values ??of each group measured by immunohistochemistry,western blot and ELISA,Maxing Shigan Decoction significantly down-regulated the expression of IL-9(p<0.05),while inhibiting the expression of IL-4 and TGF-β(p<0.05),indicating that Maxing Ganshi Decoction may simultaneously inhibit the differentiation of CD4~+T cells into Th9 cells and the transformation of Th2 cells into Th9 cells.It is speculated that Maxing Shigan Decoction may interfere with the development and differentiation of Th9 cells by regulating cytokines to achieve the purpose of treating allergic asthma.2.Animals experiments in vitro part2.1 Using Ficoll-paque to successfully extract PBMC。2.2 It is proved by immunofluorescence that the differentiation of Th9 cells is inseparable from the synergistic effect of IL-4 and TGF-β.The medicated serum of Maxing Ganshi Decoction can effectively regulate the proliferation of CD4~+T cells.2.3 Maxing Ganshi Decoction effectively regulates the expression of L-4,TGF-β,IL-9,IL-5,IL-10,and IL-17 in PBMCIn the detection of IL-4,the IL-4 content in the cytokine control group and the drug-containing serum group was significantly higher than that in the blank control group(p<0.05),and the IL-4 group increased the most significantly(p<0.05).The serogroup increased the least(p<0.05).It indicates that under the condition of high IL-4 expression,the original CD4~+T cells may differentiate into Th2 cells.When TGF-βexpression is also present,CD4~+T cells may differentiate into non-Th2 cells,and Maxing Ganshi Decoction may inhibit Th2.The role of cell differentiation.When detecting TGF-β,the content of TGF-βin the IL-4group was the lowest in the cytokine group(p<0.05).The content of TGF-βin the drug-containing serum group was lower than that in the cytokine group(p<0.05),which proved that Maxing Ganshi Decoction may have the effect of regulating the expression of TGF-β.In the detection of IL-5 content,there was no statistical difference between the IL-4group and the IL-4+TGF-βgroup and the TGF-βgroup content was very small(p<0.05),which proved that IL-5 was mainly secreted by Th2 subgroup.The IL-5 content of the drug-containing serum group is basically the same as that of the TGF-βgroup(p>0.05),which proves that Maxing Ganshi Decoction may have the effect of inhibiting the expression of IL-5.When detecting IL-10,the IL-10 content in the IL-4+TGF-βgroup was significantly higher than that in the remaining cytokine group(p<0.05),and there was no significant difference in the IL-10 content between the IL-4 group and the TGF-βgroup(p>0.05),the above results are consistent with the literature.The level of IL-10 in the drug-containing serum group was significantly reduced,which proved that Maxing Ganshi Decoction may have the effect of interfering with the expression of IL-10 in Th9 cells.In order to further verify the ability of Maxing Ganshi Decoction to interfere with Th9 cells,we tested the content of IL-9,the main Th9 cytokine,in each group.The results showed that the IL-9 content of the IL-4+TGF-βgroup was a cytokine The highest in the group(p<0.05),and the IL-9 content in the drug-containing serum group was significantly decreased(p<0.05),which further proves that Maxing Ganshi Decoction can regulate Th9 cells by interfering with the cytokine IL-9.Finally,when IL-17 was detected,IL-17 was highly expressed in the TGF-βgroup and IL-4+TGF-βgroup(p<0.05),proving that TGF-βis an essential cytokine for Th17 cell differentiation.The drug-containing serum group The expression of IL-17 decreased(p<0.05),indicating that Maxing Ganshi Decoction may have the effect of inhibiting the expression of IL-17.Conclusion:1.Through the animal in vivo,Maxing Ganshi Decoction can reduce the daemon of asthma rat fertilizer,so that the content of Ig E in rat serum is reduced,and the role of treating airway inflammation;Th2,TH9,TH17 cells were regulated by intervention IL-4,TGF-10,IL-9,IL-5,IL-10,IL-17,and the like to achieve the purpose of controlling asthma.At the same time,Maxing Ganshi Decoction can significantly reduce the expression of IL-9,IL-4 and TGF-β,and it is possible to achieve the role of anti-asthma by intervention of Th9 cells.2.Through the animal in vitro experiment,IL-4 and TGF-βhave decisive effects on TH9 cell differentiation;Maxing Ganshi Decoction can effectively inhibit the proliferation capabilities of TH2,TH9,TH17 in CD4~+T cells,where the TH9 cells are inhibited The proliferation capacity is the strongest;further proves that Maxing Ganshi Decoction can effectively interfere with the differentiation of TH9 cells and the expression of corresponding cytokines to play an anti-asthma. |
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