| Purpose : This study was conducted to investigate the in vitro molecular biological mechanism of the efficacy of the liver and spleen formula in the treatment of paediatric bronchial asthma,based on the clinical and pre-application experimental study of the major project of "micro RNA155-based regulation of Th17/Treg cells to investigate the effect and intervention mechanism of the liver and spleen formula on paediatric bronchial asthma".The study was carried out to investigate the in vitro molecular biological mechanism of the efficacy of the liver and spleen formula in the treatment of paediatric bronchial asthma.The blank group,the induction group,the dexamethasone containing serum group,the liver and spleen formula containing serum low dose group,the liver and spleen formula containing serum medium dose group and the liver and spleen formula containing serum high dose group were used to induce the differentiation of Th17/Treg cells in vitro and to investigate whether the liver and spleen formula containing serum could interfere with paediatric bronchial asthma through the JAK2/STAT3 signalling pathway from an epigenetic perspective.This study was conducted to investigate whether the liver and spleen formula containing serum could interfere with pediatric bronchial asthma through the JAK2/STAT3 signaling pathway and whether it could influence the expression of endogenous mi R-155 in Th17/Treg cells in vitro.Materials and methods:The expression of Foxp3,RORγt and mi R-155 m RNA in Th17/Treg cells was detected by Real-time PCR and the expression of ρ-JAK2,ρ-STAT3 and SOCS1 protein in Th17/Treg cells was detected by Western Blot.Results:1.Real-time PCR results of Foxp3 gene content:Th17 cell differentiation: when compared with the standard control group,the level of Foxp3 expression in Th17 cell differentiation was clearly lower than that in the model group(P<0.01);when compared with the level of Foxp3 gene expression in the group of model group,the level of Foxp3 expression showed an increasing trend in the group of low,medium and high doses of Liver and Spleen Concentrate Serum and the group of Dexamethasone Concentrate Serum,especially the level of Foxp3 expression in the group of Liver and Spleen Concentrate Serum was the best(P<0.05).The expression of Foxp3 in the rest of the groups was similar;in Treg cell differentiation:when compared with the natural control group,the gene expression of Foxp3 was dramatically increased in the model group(P<0.0001);comparing with the model group: the expression of Foxp3 gene were all increased significantly in the low,medium and high doses of the liver and spleen formula containing serum and the dexamethasone containing serum(P<0.05).In addition,the expression of Foxp3 in the high-dose group of the liver and spleen formula-containing serum was significantly increased compared with the dexamethasone containing serum group(P<0.05),while the expression of Foxp3 in the rest of the groups was similar.2.Real-time PCR results for RORγt gene content:Th17 cell differentiation: when compared with the normal group,the level of RORγt expression in the model group was dramatically increased(P<0.0001);comparing with the RORγt in the group of model,the level of RORγt expression in all dose groups(low,medium and high dose groups)and the dexamethasone containing serum group of Liver and Spleen Tongzhi Formula had different degrees of decrease.The expression of RORγt was significantly lower in the medium dose group(P<0.001)and dramatically decreased in the high dose group of Liver and Spleen Tongzhi Formula(P<0.0001);Treg cell differentiation:when compared with the standard group,the level of RORγt expression was obvioursly lower in the model group(P<0.05);when compared with the model group,the level of RORγt expression were lower than those of the model group in different dose groups(low,medium and high dose groups)of Liver and Spleen Tongzhi Formula.On the contrary,the level of RORγt expression of dexamethasone containing serum group was stronger than that of the model group.3.Real-time PCR results for mi R-155 gene content:Th17 cell differentiation: The level of mi R-155 expression in this model group was more than that in the standard control group(P<0.0001);in this study,the expressed amount of mi R-155 was remarkably lower than that in the model group(P<0.05)in different doses of the liver and spleen formula-containing serum group and the dexamethasone containing serum group.Treg cell differentiation: comparing with the standard group,the level of mi R-155 expression in the model group was decreased,however,the level of mi R-155 expression was not significant in the two groups,because the level of mi R-155 expression was similar in the two groups.The lever of mi R-155 expression was similar in all groups and there was no clear difference between the different groups.4.Western Blot assay of ρ-JAK2 protein content:Th17 cell differentiation: ρ-JAK2 is significantly higher in the model group than the standard group(P<0.0001);when compared with the group of the model,ρ-JAK2 has been reduced in each dosage group of the liver and spleen formula containing serum and the dexamethasone containing serum group(P<0.0001),among which ρ-JAK2 expression is the lowest in the high dosage group of the liver and spleen formula containing serum,but still higher than the normal group;Treg cell differentiation:the level of ρ-JAK2 expression was dramatically improved in the group of the model when compared with the standard group(P<0.0001);comparing with the model group,ρ-JAK2 expression has been significantly reduced in the mid-and high-dose groups of the liver and spleen formula-containing serum and the dexamethasone containing serum group(P<0.0001),but still remains higher than the standard group,but we could not fond significant difference in the low-dose group of the liver and spleen formula-containing serum(P>0.05).5.Western Blot assay of ρ-STAT3 protein content:Th17 cell differentiation:When compared with the standard group,the level of ρ-STAT3 expression was obviously higher in the group of model(P<0.0001);comparing with the model group,the expression of ρ-STAT3 was greatly reduced in all the groups of the liver and spleen formula containing serum and dexamethasone containing serum(P<0.0001),and was the lowest in the group of the high-dose liver and spleen formula containing serum,although it was still more than the normal group;Treg cell differentiation: the level of ρ-STAT3 expression in the model group was greatly higher than that in the standard group(P<0.0001),and the lever of ρ-STAT3 expression in the group with high-dose liver and spleen formula containing serum was clearly lower than that in the group with model(P<0.001),however,there was no apparent difference with other therapeutic groups(P>0.05).6.Western Blot assay of SOCS1 protein content:Th17 cell differentiation: comparing with the standard group,the lever of SOCS1 expression in the group of model was greatly reduced(P<0.0001);when compared with the model group,the expression of SOCS1 in the groups of each dose of the liver and spleen formula containing serum and the group of dexamethasone containing serum were all seriously improved(P<0.0001),while all of them were lower than the standard group;Treg cell differentiation: the expression of SOCS1 was dramatically decreased in the model group comparing with the standard group(P<0.0001);it was markedly decreased in the medium-dose group of the liver and spleen formula containing serum and the dexamethasone containing serum group compared with the model group(P<0.0001),and the the expression of this protein was also clearly decreased in the high-dose group of the liver and spleen formula containing serum,and showed a statistically considerable difference(P<0.01).However,the expression of SOCS1 was not dramatically different from that of the liver and spleen formula-containing serum low-dose group(P>0.05).Conclusions:1.By activating the expression of Foxp3 and inhibiting the expression of RORγt and mi R-155,Ganpi Tongzhi Formula may affect the differentiation of Th17 and Treg cells,thus intervening in the development of paediatric bronchial asthma.2.Ganpi Tongzhi Formula inhibits the expression of ρ-JAK2 and ρ-STAT3 proteins by elevating the expression of SOCS1 protein,which may be the result of Ganpi Tongzhi Formula targeting SOCS1 and inhibiting the activation of JAK2/STAT3 signaling pathway and participating in the pathogenesis of paediatric bronchial asthma.3.Based on the physiological characteristics of the liver and the spleen and the theory of the five viscera in children,the application of liver and spleen treatment can significantly reduce the chronic inflammation in the airways of paediatric bronchial asthma,which may be related to the JAK2/STAT3 signalling pathway and the regulation of Foxp3,RORγt and mi R-155 expression levels,thus improving the Th17/Treg immune balance. |
PDF Full Text Request