The Relationship Between Gut Microecology And Allergic Diseases In Children , And The Influence Of ShenQiJianPi Decoction On Restoring Gut Mucosa Barrier Function And Modulating Immune Function

Many studies have confirmed increases in the incidence and prevalence of allergic diseases over the past 2 decades in the worldwide, but much remains unknown. These observations support the hygiene hypothesis, i.e. the lack of microbial exposure during infancy or early childhood leading to allergic diseases. Moreover, the lack of microbial stimulation also results in the imbalance of Th1/Th2 and hypofunction of regulatory T cells. There is growing evidence that early-life allergy is significantly related to the development of asthma and other allergic diseases. Although genetic predisposition is clearly evident, gene-by-environment interaction probably explains much of the international variation in prevalence rates for asthma and other allergic diseases. Environmental factors such as infections and exposure to endotoxins,some prenatal risk factors, including maternal smoking, diet and nutrition, stress, use of antibiotics and mode of delivery may also affect the early development of asthma and other allergic diseases. It is likely that detailed studies of gene-by-environment interactions, molecular biology and epigenetics will eventually untangle the inconsistencies among the many putative exposures and outcomes. Much of the epidemiology, many of environment risk factors and pathophysiological mechanisms in asthma and Henoch-Schonlein purpura remain to be adequately explained. A better understanding of these mechanisms may reduce the risk factors for asthma and find effective drugs to regulate gut microecology and immune function, which will eventually lead to opportunities for primary prevention of allergic diseases. Part one: Alteration of gut microecology and regulation of the CD4+T cell subsets and their cytokines and master transcription factors in immune networks in bronchial asthma pathogenesisObjective: To study alteration of gut microecology and mutual regulation of the CD4+ T cell subsets and their cytokines and master transcription factors in immune networks in bronchial asthma pathogenesis.Methods: Forty-seven children with asthma and thirty healthy children as control group were enrolled into this study. Flow cytometrie (FCM) analysis was performed to detect the percentage of CD3+CD8-INF-γ+ IL-4-(Th1),CD3+CD8-INF-γ-IL-4+(Th2),CD4+CD25+T cell(Treg)and CD3+CD8-IL-17+(Th17)cell; Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of INF-γ,IL -4,TGF-β1 and IL- 17 in plasma ; The specific master transcription factors for Th1, Th2, Treg and Th17 cells, T-bet, GATA3, Foxp3 and ROR-γt, are respectively involved in their differentiation. Reverse transcription-polymerase chain reaction(RT-PCR) was used to analyze the mRNA expression of T-bet, GATA3, Foxp3 and ROR-γt in peripheral blood mononuclear cell (PBMC).16SrDNA fluorescent quantitative PCR was applied in determining the content of bifidobacterium, lactobacillus,escherichia coli and enterococcus in feces.Results: The amount of anaerobes represented by the bifidobacterium and lactobacillus declined. Howere, the amount of aerobes represented by escherichia coli and enterococcus did not change obviously, Bifidobacteria / Escherichia coli (B/E) which indicated a balance of intestinal flora declined. The results showed the intestinal dysbacteriosis occured in children with acute stage of asthma. With treatment and the improvement of symptoms, the amount of bifidobacteria increased faster than other bacteria, but the overall flora structure did not restore to its normal levels in recovery stage. Based on the immunological indexes in children with acute stage of asthma, the percentage of Th2 and Th17 cells and the concentrations of their related cytokines in plasma increased, and the percentage of Th1 and Treg cells and the concentrations of their related cytokines decreased as compared to that in the control group, indicating that there were immune imbalances of Treg/Th17 and Th1/Th2. Though the percentage of Th2 and Th17 cell and the concentrations of cytokines in plasma decreased in remission of asthma, they were still higher than that in healthy control group, and there was still the imbalance of Treg/Th17 and Th1/Th2. Analysis of the correlation between the level of cytokines in blood plasma and the expression of specific master transcription factors in PBMC showed that positive correlations between INF-γand T-bet, IL-4 and GATA3, TGF-β1 and Foxp3, and IL-17 and ROR-γt in children with bronchial asthma, indicating that the balances of Th1/Th2 and Treg/Th17 was mainly regulated by their master transcription factors. Th1/Th2 ratio and Tregs/Th17 ratio had positive correlation with B / E ratio.Conclusions: The imbalance of Th1/Th2 cells is critical in the pathogenetic mechanisms of bronchial asthma. Besides its importance for Th2 cell development, Treg cell and Th17 cell play significant roles in the process. Thus, besides the imbalance of Th1/Th2 cells, the imbalance of Treg/Th17 cells also involved in the pathogenetic mechanisms of bronchial asthma. Commensal intestinal bacteria are crucial in providing immune stimulation for normal immune system development, and can correct the imbalance of Th1/Th2 by downregulating Th2 responses. The integrity of intestinal flora structure and their function are important to maintain the function of CD4+CD25+ Treg cell. Intestinal dysbacteriosis in children with bronchial asthma compromises the ability of CD4+CD25+Treg cell to suppress responses, the process leading to the disturbance of immune tolerance which is important in asthma pathogenesis. Our study have provided an important theoretical basis for future development and application of medicines, and prevention and treatment of bronchial asthma through timely restoring the balance of gut microecology and abrogating any component element of abnormal immune responses at early stages. Part two: Alteration of gut microecology and regulation of the CD4+ T cell subsets and their cytokines and master transcription factors in immune networks in pathogenesis of Henoch-Schonle in purpura in childrenObjective: To study alteration of gut microecology and mutual regulation of the CD4+ T cell subsets and their cytokines and master transcription factors in immune networks in pathogenesis of Henoch-Schonlein purpura in children.Methods: Forty-one children with Henoch-Schonlein purpura and thirty healthy children as control were enrolled into this study. Flow cytometrie (FCM) analysis was performed to detect the percentage of CD3+CD8-INF-γ+ IL-4-(Th1), CD3+CD8-INF-γ- IL-4+(Th2), CD4+CD25+T cell(Treg)and CD3+CD8-IL-17+(Th17)cell; enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of INF-γ, IL -4,TGF-β1, IL-9 and IL- 17 in plasma ;the master transcription factors for Th1, Th2, Treg and Th17 cells, T-bet, GATA3, Foxp3 and RORγt, are respectively involved in their differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze the mRNA expression of T-bet, GATA3, Foxp3 and ROR-γt in peripheral blood mononuclear cell (PBMC). 16S rDNA fluorescent quantitative PCR was applied in determining the bacterial content of bifidobacterium, lactobacillus, escherichia coli and enterococcus in feces. Urine samples were collected before and after the test solution administrated ( lactulose, L and mannitol, M) and analyzed by high performance liquid chromatography (HPLC) and the intestinal permeability can be evaluated by L/M ratio.Results: The amount of the bifidobacterium and lactobacillus declined, especially lactobacillus. However, the amount of escherichia coli and enterococcus did not change obviously. The results showed that intestinal dysbacteriosis occured in children with HSP during acute stage. With clinical treatment and the improvement of symptoms, the amount of bifidobacterium and lactobacillus gradually increased, and the amount of bifidobacterium increased faster than that of lactobacillus, but there was still intestinal dysbacteriosis during the recovery stage of HSP. Moreover, the amount of lactobacillus had negative correlation with B/E and L/M ratio. Based on the other indexes during the acute stage of HSP, the percentage of Th2 and Th17 cells in PBMC, the levels of IL-4 and IL-17 in plasma, the expression of GATA3 and ROR-γt in PBMC increased when compared with that in the control group. The percentage of Th1 and Treg cells, the levels of INF-γand TGF-β1, the expression of T-bet and Foxp3 decreased as compared to that in the control group. The results indicated that there were imbalances of Treg/Th17 and Th1/Th2 during the acute stage of HSP. Though the percentage of Th2 and Th17 cells, the levels of their cytokines in plasma and the expression of their master transcription factors increased during the recovery stage of HSP, there were still the imbalance of Treg/Th17 and Th1/Th2. Analysis of the correlation between the expression of master transcription factors in PBMC, the percentage of cells in PBMC and the levels of cytokines in plasma in children with HSP showed that Th1 and T-bet had positive correlations with INF-γ; Th2 and GATA3 had positive correlations with IL-4; Treg and Foxp3 had positive correlations with TGF-β1; Th17 and ROR-γt had positive correlations with IL-17. Similar to B/E ratio, the amount of the lactobacillus also had positive correlations with Th1/Th2 ratio and Tregs/Th17 ratio and negative correlation with L/M ratio.Conclusion: B/E ratio decreased, especially for the amount of lactobacillus, intestinal permeability increased, and intestinal barrier function was damaged, indicating that the intestinal dysbacteriosis occurred in children with HSP in acute stage. The decrease of the percentage of CD4+CD25+T cell, the level of TGF-β1 in plasma and the expression of Foxp3 in PBMC showed that immune tolerance induced by commensal intestinal bacteria was reversed. The finding was a supplement to Th1/Th2 paradigm .In our study, besides Th1/Th2 paradigm, the imbalance of Treg/Th17cells was involved in the pathogenesis of HSP. Bifidobacterium and lactobacillus in gut played significant roles in restoration of the immune homeostasis and tolerance induction. From the ecological and immunological perspective, our study clarified pathogenesis of allergic purpura and investigated the regulation of commensal intestinal bacteria, immune cells, cytokines and transcription factors in the development of the disease, which provided theoretical basis for future development of medicines and clinical treatment of Henoch-Schonlein purpura not only through regulating the host immune function, but also through improving gut microecology.Part three : The mechanisms of ShenQiJianPi decoction in restoring gut mucosa barrier function and modulating immune function in SD rats with intestinal dysbacteriosis.Object: Based on the studies above, we demonstrated that intestinal dysbacteriosis and immune function disorders were involved in pathogenesis of allergic diseases such as bronchial asthma and allergic purpura in children. Therefore, it is very important to develop new traditional medicines which can improve gut microecology, regulate immune function and break vicious cycle at early stage of allergic diseases. This study investigated the mechanisms of ShenQiJianPi decoction in restoring the gut mucosa barrier function, inhibiting bacterial translocation phenomenon and modulating of immune function in SD rats with intestinal dysbacteriosis, which provided theoretical basis for future development of traditional chinese medicines to treat allergic diseases.Methods: The dynamic changes of intestinal flora and the amount of translocated bacteria in mesenteric lymph nodes, liver, spleen and colon in SD rats were observed by bacterial culture after using antibiotic; bowel tissue electronmicroscopic and opticalmicroscopic changes in SD rats were performed; the reverse transcription polymerase chain reaction (RT-PCR)was used for determining Toll-like receptor mRNA expression of intestinal tissues, mesenteric lymph nodes, liver and spleen from SD rats.Results: The amount of the bifidobacterium, lactobacillus and escherichia coli decreased as compared to that in the control group, indicating that the intestinal dysbacteriosis occured in SD rats with intestinal dysbacteriosis. Concerning indexes of measuring intestinal barrier function, intestinal epithelial cell, the intestinal villus and intestinal tight junction were damaged, suggesting that intestinal barrier integrity loss occured. The expression of Toll-like receptor mRNA was inhabited in the early of intestinal dysbacteriosis, which leading to bacterial translocation from gastrointestinal tract to mesenteric lymph nodes, liver and spleen. Escherichia coli pathogenic attack may increase the degree of intestinal dysbacteriosis and the damage gut barrier function, while the upregulation of Toll-like receptor mRNA expression may increase the degree of bacterial translocation. ShenQiJianPi decoction and lactobacillus intervention could be applied to reduce the degree of bacterial translocation, lessen the inhibition of Toll-like receptor of mRNA expression, restore the structure of intestinal flora and lessen the damage gut barrier function.Conclusion: The correlations among the structure of intestinal flora, gut mucosa barrier function, bacterial translocation phenomenon and immune function have been clarified. Intestinal dysbacteriosis can induce the alteration of gut microecology, damage of intestinal epithelial cells, bacterial translocation phenomenon and inhabition the expression of Toll-like receptor mRNA. Early intervention of ShenQiJianPi decoction provided the protective effect on maintaining the balance in microecosystem, restoring gut barrier function, inhibiting bacterial translocation phenomenon and modulating the immune function , especially on modulating host pattern recognition receptor in innate immunity, which are critical in the prevention of allergic disease.