| Asthma is harmful to our people's health.As allergic asthma is the most common subtype of asthma,we pay more attention to it.Kinds of cataplasms of Chinese Traditional medicine for treating asthma can not only play a good role,but also have the advantages of safety and convenience,which makes up for the disadvantages of side effects of Western medicine.Now,these cataplasms are recognized by more and more people in clinic practice.In this study,Majie cataplasm is a typical kind of cataplasm consisting of Ephedra Herba(Mahuang),Semen Armeniacae Amarum(Kuxingren),Semen Sinapis(Baijiezi),and Rhizoma Corydalis(Yanhusuo)and ginger(Shengjiang).It has good clinical effect for asthmatic patients.In previous experiments,we have found that Majie cataplasm can effectively alleviate airway hyperresponsiveness(AHR)in allergic asthma model animals.As Th2 type inflammation in the respiratory tract of allergic asthma is the main cause of AHR,it can be seen that Majie cataplasm can reduce AHR by regulating Th2 type inflammation.Therefore,the main task of this study is to explore the mechanism of relieving Th2 inflammation of allergic asthma and reducing AHR.Objectives1.To verify the relief of Majie cataplasm for asthmatic inflammation2.To investigate whether Majie cataplasm can regulate Th1/Th2 directly.3.To investigate whether Majie cataplasm can regulate Th1/Th2 indirectly by affecting ILCs.4.To explore whether Majie cataplasm can regulate Thl/Th2 indirectly by affecting neuropeptides associated with allergic asthma.5.To explore whether Majie cataplasm can relieve the AHR by inhibiting the mucus secretion induced by IL-13.6.To explore whether Majie cataplasm can regulate M1/M2 to affect Th1/Th2.Methods1.Establishing the OVA induced asthmatic model of mice,after the intervention of dexamethasone/Majie cataplasm,H&E staining of lung tissue was used for routine histological examination.Contents of serum IgE and HIS were tested by ELISA.2.Establishing the OVA induced asthmatic model of mice,after the intervention of dexamethasone/Majie cataplasm.The numbers of IL-4+T cells,IFN-?+T cells,Tregs and B10s from spleen tissues were tested by FCM,expressions of GATA-3 mRNA,STAT6 mRNA and T-bet mRNA in spleen tissues were detected by qPCR and expressions of JAK2,STAT3 and pSTAT3 in lung tissues were detected by WB.3.Establishing the OVA induced asthmatic model of mice,after the intervention of dexamethasone/Majie cataplasm,the numbers of ILC1s,ILC2s,ILC3s and NKs in lung tissue were detected by FCM.The 40 inflammatory factors in lung tissue were tested by Mouse Inflammation Array Kit.4.Establishing the OVA induced asthmatic model of mice,after the intervention of dexamethasone/Majie cataplasm,the content of CGRP and NKA in serum was detected by ELISA,the expressions of CGRP and CD3 together with CGRP and Neutrophil in lung tissue were detected by IF,and expressions of CGRP mRNA,SP mRNA,IL-5 mRNA and IL-17 mRNA in lung tissue were detected by qPCR.5.Establishing the OVA induced asthmatic model of mice,after the intervention of dexamethasone/Majie cataplasm,the airway resistance was detected.And the expressions of IL-13 mRNA,CLCA3 mRNA and MUC5AC mRNA of lung tissue were detected by qPCR.The expressions of PI3K and AKT in lung tissue were detected by WB,and the secretions of mucus and MUC5AC in lung tissue were detected by PAS and IHC respectively.6.Preparing the water extract of Majie cataplasm,CCK-8 kits were used to detect the cytotoxicity of Majie cataplasm on M?,and the differentiation models of Ml and M2 were established in vitro.FCM was used to detect the effect of Majie cataplasm on the differentiation of M1/M2.ELISA was used to detect the contents of IL-1? and IL-12p70 in the supernatant after Majie cataplasm acted on M2 differentiation.Results1.Compared with the control group,the results showed that the content of IgE and HIS in the asthmatic model group increased significantly(P<0.001,P<0.0001).Both dexamethasone and Majie cataplasm significantly reduced the content of IgE(P<0.0001,P<0.001)and HIS content(P<0.001,P<0.01).The result of H&E staining showed that there were some substantiation,congestion and edema in the blood vessels,and a large number of inflammatory cells in the alveoli and bronchioles in the asthmatic model group comparing to the control group.In the dexamethasone group,the alveolar wall was slightly thickened,and a small number of inflammatory cells were found in interstitial lung.After treatment of Majie cataplasm,the pulmonary inflammation improved,but the effect was not as obvious as that of dexamethasone.2.The results of FCM experiments showed that the expressions of IFN-?+T cells and IL-4+T cells from spleen tissue in the asthmatic model group were significantly higher than those of the control group(P<0.0001,P<0.01).After treatment of dexamethasone and Majie cataplasm,the levels of IL-4+cells(P<0.0001,P<0.0001)and IFN-?+cells(P<0.01,P<0.05)were significantly decreased.For Tregs and B 10s,compared with the control group,they boomed in the asthmatic model group(P<0.0001,P<0.001).After the treatment of dexamethasone,Tregs decreased(P<0.0001),but B10s only had the downward trend.The.contents of Tregs and B10s decreased significantly in the Majie cataplasm group(P<0.0001,P<0.01).The results of qPCR experiments showed that expressions of STAT6 mRNA and GATA-3 mRNA from spleen tissue in the asthmatic model group were higher than that of the control group(P<0.0001,P<0.001),and dexamethasone and Mamie cataplasm could inhibit the expression of STAT6 mRNA(P<0.0001,P<0.0001)and GATA-3 mRNA(P<0.05,P<0.01).For T-bet mRNA,there was no obvious change between the asthmatic model group and the control group.It decreased(P<0.01)in the dexamethasone group,while increased in the Majie cataplasm group(P<0.0001).The results of WB experiments showed that the expression of JAK2 in the asthmatic model group was higher than that in the control group(P<0.01).Both and dexamethasone and Majie cataplasm could inhibit the expression of JAK2(P<0.05,P<0.001).For STAT3,it had not changed much in experimental groups.For pSTAT3,the expression of pSTAT3 in the asthmatic model group was higher than that in the control group,while it was lowered in the dexamethasone group and the Majie cataplasm group.The ratio of pSTAT3/STAT3 was higher in the asthmatic model group comparing with the control group(P<0.05),while dexamethasone and Majie cataplasm reduced the ratio of pSTAT3/STAT3(P=0.05,P<0.05).3.The results of FCM experiments showed that for ILCs in lung tissue,the number of ILCls,ILC2s ILC3s in the asthmatic model group remained unchanged compared with the control group,while the numbers of ILCls,ILC2s ILC3s increased after the intervention of dexamethasone(P<0.0015,P<0.001,P<0.01)and Majie cataplasm(P<0.01,P<0.01,P<0.05),respectively.For NKs,the number of NKs in the control group,the asthmatic model group and the dexamethasone group were kept at a low level and changed little.However,the number of NKs in the Majie cataplasm group shot up(P<0.0001).The result of the inflammatory cytokines in lung tissue experiment showed that there were significant differences in the expressions of TNF-?,CXCL-9,CCL-12,GM-CSF,CCL-9,CCL-2,IL-1?,CCL-5 and TNFR2 between the asthmatic model group and the control group.The levels of TNF-?,TNFR2,CXCL-9,CCL-12,CCL-9,CCL-2 and CCL-5 increased in the asthmatic group,while the levels of GM-CSF and IL-1? decreased.Compared with the asthmatic model group,the levels of G-CSF,CCL-9,CCL-5 and TNFR2 decreased after the intervention of dexamethasone.The levels of TNF-?,TNFR2 and CCL-9 in the Majie cataplasm were lower than those in the asthmatic model group,while the levels of IFN-?,IL-1?,ICAM-1 and IL-4 increased.4.The results of ELISA experiments showed that the contents of CGRP and NKA increased significantly in the asthmatic model group(P<0.01,P<0.01).Both dexamethasone and Majie cataplasm could reduce the content of CGRP(P<0.05,P<0.0001)and NKA(P<0.01,P<0.0001).The results of IF experiments showed that compared with the control group,the expression of CGRP around the trachea increased with a large number of CD3+T cells gathered in the asthmatic model group.Some of these CD3+T cells also expressed CGRP.There was no obvious infiltration of CD3+T cells in the dexamethasone group and the Majie cataplasm group,and the secretion of CGRP around the trachea decreased compared with the asthmatic model group.For the double staining of CGRP and neutrophil,CGRP was highly expressed in the cells around the trachea with intensive neutrophil in the asthmatic model group.Some neutrophils also expressed CGRP.In the dexamethasone group,the secretion of CGRP and the infiltration of inflammatory cell were decreased,while cells expressed both neutrophil and CGRP were still obvious.In the Majie cataplasm group,there was less CGRP secretion and inflammatory cells secreting CGRP were scattered with lower cells expressed CGRP and neutrophil than those in the group of dexamethasone.The results of qPCR experiments showed that CGRP mRNA,SP mRNA,IL-5 mRNA and IL-17 mRNA were all increased in the asthmatic model group(P<0.001,P<0.01,P<0.0001,P<0.001).After the treatment of dexamethasone and Majie cataplasm,the expressions of CGRP mRNA(P<0.001,P<0.01),SP mRNA(P<0.0001,P<0.0001),IL-5 mRNA(P<0.0001,P<0.0001)and IL-17 mRNA(P<0.01,P<0.001)were inhibited.5.The results showed that both of them could alleviate the increased airway hyperresponsiveness.Comparing with the control group,the levels of MUC5AC mRNA,CLCA3 mRNA and IL-13 mRNA in the asthmatic model group were significantly higher than those in the control group(P<0.001,P<0.001,P<0.0001),and they could be reduced after the intervention of dexamethasone(P<0.001,P<0.01,P<0.001)and Majie cataplasm(P<0.001,P<0.01,P<0.001).The results of WB experiments showed that the expression of PI3K changed little among the control group,the asthmatic model group and the dexamethasone group,it could be reduced with the intervention of Majie cataplasm(P<0.05).The expression of AKT in the asthmatic model group was higher than that in the control group(P<0.01),and it was inhibited by the dexamethasone and Majie cataplasm(P<0.05,P<0.05).The results of qPCR experiments showed that contents of lung mucus and MUC5AC in the asthmatic model group were significantly higher than that of the control group,and they also could be reduced after the treatment of dexamethasone and Majie cataplasm.6.The water extract of Majie cataplasm affected the differentiation of M1/M2 in vitro.When the concentration is less than 10 mg/mL,there is no obvious cytotoxic effect on M?.Considering that water the extract of traditional Chinese medicine may affect the cell morphology,we chose 1 mg/mL for subsequent experiments.After LPS stimulation,Mcp differentiated into M1(P<0.001),which could not be reversed by the intervention of water extract of Majie cataplasm.IL-4 stimulated the differentiation of M? into M2(P<0.0001),while the intervention of water extract of Majie cataplasm could promote the increase number of M1(P<0.0001).The contents of IL-1? and IL-12p70 increased with the influence of water extract of Majie cataplasm after IL-4 stimulation(P<0.01,P<0.01).Conclusions1.Majie cataplasm can relieve the Th2 inflammation of allergic asthma.2.Majie cataplasm can regulate Th1/Th2 directly.3.Majie cataplasm can regulate Th1/Th2 indirectly by IFN-?+NKs.4.Majie cataplasm can regulate Th1/Th2 indirectly by inhibiting neuropeptides related to allergic asthma.5.Majie cataplasm can inhibite the mucus secretion induced by IL-13 to relieve the AHR.6.Majie cataplasm can promote the M1 differentiation in M2 induced model and have the potential to boost Th1 differentiation. |
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