Theoretical And Experimental Research On The Treatment Of Allergic Asthma From The "hepatic And Pulmonary Treatment"

Objective: Based on efficacies of Modified Guo Min Jian(MGMJ), its TCM mechanisms of treating allergic asthma can be preliminarily deduced from the principle of “treating the liver and lungs together” . Moreover, efficacies of MGMJ on allergic asthma can be observed with mice experimental models of allergic asthma induced by OVA, so as to discuss the immune molecule mechanisms and therapeutic targets of MGMJ on anti-allergy, anti-inflammation and anti-asthma. Therefore, studies mentioned above will not only laid a theoretical and experimental foundation for developing a new drug with both safety and efficacy for asthma, but also provide scientific basis for the clinical treatment in allergic diseases with the same method.Methods: 1. On traditional Chinese medicine (TCM) mechanisms of MGMJ in the treatment of allergic asthma.1.1 The study of modern literatures on allergic asthma of TCM: With the SPSS17.0 statistical software, TCM patterns, Zang-Fu organs, pathological factors and therapies involved in those relevant literatures were standardized.1.2 The theoretical study on allergic asthma with the principle of "treating the liver and lungs together": A series of methods, such as the literature management, comparison, analysis, generalization and conclusion, were applied in the study.2. The pharmacodynamic study on allergic asthma with MGMJ: 60 female BALB/c SPF class mice, four to five weeks old, were randomly divided into 6 groups,namely blank control group, the model control group, the dexamethasone positive control group (Dex, 1. 25mg ·kg-1), the high-dose MGMJ group (crude drug, 2. 72g/ml),the middle-dose MGMJ group (crude drug, 1. 36g/ml), the low-dose MGMJ group (crude drug, 0. 68g/ml), with 10 mice respectively. The OVA were used to induce mice models of allergic asthma. Furthermore, performances of mice, peripheral WBC differential counts in cell smears, inflammatory cell infiltrations of pathological slices in lung tissues with HE staining and the content of OVA-sIgE in serum with ELISA test were observed.3. The study of immunological molecule mechanisms on allergic asthma with MGMJ: With the same method of grouping and molding above, the percentage of CD4~+IFN-?~+ cells, CD4~+IL-4~+cells, CD4~+IL-17~+ cells and CD4~+Foxp3~+cells in CD4~+T cells were detected by FACS. In addition, RT-PCR was used to test the mRNA expression of Thl-type cytokines IFN-? as well as Th2-type cytokines IL-4 and IL-5 in lung tissues. With the Real-time FQ-PCR and WB test, the mRNA and protein expression of transcription factors including T-bet, GATA-3 and Foxp3 in lung tissues were determined as well.Results: 1. On TCM mechanisms of MGMJ in the treatment of allergic asthma:1.1 The study of modern literatures on allergic asthma in TCM : According to the frequency for their correlations with allergic asthma, the sequences of TCM patterns, Zang-Fu organs, pathological factors and therapies were demonstrated as following: A total of 12 excessive patterns in allergic asthma are cold, heat,phlegm-heat accumulation in lungs, liver fire attacking lungs, cold-phlegm obstructing lungs, wind-phlegm obstruction, turbid phlegm obstructing lungs,wind invasion, phlegm-stasis obstructing lungs, liver qi invading lungs,wind-cold invading lungs and wind-heat invading lungs, respectively; there are 6 deficient patterns in allergic asthma, including lung-kidney deficiency, lung qi deficiency, yin deficiency of lung, liver and kidney, lung-spleen deficiency,spleen qi deficiency as well as kidney yang deficiency; Moreover, there are 6 syndromes of intermingled deficiency and excess, namely pulmonary cold and kidney deficiency, lung heat and kidney deficiency, Qi deficiency and blood stasis, deficiency of liver blood, endogenous liver wind, lung heat and Yin deficiency, kidney deficiency and phlegm stagnancy; the lung, spleen, kidney,liver, stomach, heart as well as small intestine relating to allergic asthma,respectively; a total of 10 pathological factors include phlegm, deficiency,wind, qi stagnation, cold, stasis, heat(fire), dampness, dryness as well as summer heat; the sequences of 12 therapies widely used in allergic asthma, namely the therapy of resolving phlegm, clearing heat, ascending and descending lung qi to relieve asthma, regulating the liver (including nourishing liver yin,promoting liver qi and clearing liver heat), eliminating wind, warming lungs to dispel cold, activating blood circulation and removing blood stasis,replenishing lungs and the kidney, reinforcing lungs and the spleen,strengthening the spleen and tonifying qi, strengthening body resistance and dispelling pathogens as well as resolving phlegm to promote qi, respectively.1.2 The theoretical study on allergic asthma with the principle of "treating the liver and lungs together": With complex pathogenic mechanisms, the asthma could be induced by disorders of the liver and lungs, including wind, qi, phlegm,stasis and deficiency. However, the allergic asthma, as a seasonal disease, often occurred in spring due to wind pathogen first, which was related to the liver and lungs according to TCM. The specific mechanism of allergic asthma could be generalized as “Based on the qi-yin deficiency in the liver and lungs, the disease occurs with the manifestation of wind invading lungs. Moreover,excessive liver yang caused by liver yin and blood deficiency produces interior wind. As a result, integrating the interior and exterior wind together, the phlegm, qi and stasis all obstructed in the air way, which finally led to the disease." Following the principle of "treating the liver and lungs together",the MGMJ, as a representative formula for allergic asthma, focused on the primary therapy of "regulating the liver and lungs, nourishing yin and dispelling wind as well as resolving phlegm and dredging collaterals".2. The pharmacodynamic study on allergic asthma with MGMJ: Compared with the model group, both manifestations of asthmatic mice in each MGMJ group,including sneezing, bucking, polypnea, restlessness, contraction of abdominal muscles and cyanosis, and the contain of OVA-sIgE in serum were reduced (P<0. 01?0.05, respectively) . It was observed that not only the ratio between total number of peripheral WBC and EOS decreased, but inflammatory infiltrations with EOS in lung tissues were improved in MGMJ groups with a high or middle dose (P<0. 01?0.05,respectively).3. On immunological molecule mechanisms of MGMJ in the treatment of allergic asthma:3.1 The effect of MGMJ on differentiations and functions of CD4~+ T cell subsets and cytokines: As the FCM test showed, comparing with the model group, the percentage of CD4~+IL-4~+cells and CD4~+IL-17~+ cells in CD4~+ cells in spleen tissues decreased in the high-dose MGMJ group (P<0.05), while there was no statistical significance though a descending tendency was showed in MGMJ groups with a middle or low dose (P>0. 05). Moreover, in the high-dose MGMJ group, the percentage of CD4~+IFN-?~+ cells in CD4~+ cells increased (P<0.05), and there was no statistical significance in MGMJ groups with a middle or low dose though the increasing tendency for the percentage of CD4~+IFN-?~+cells was showed (P >0. 05). It was also found the increasing tendency for the percentage of CD4~+Foxp3~+ cells in CD4~+cells in MGMJ groups with a high or middle dose, but there was no statistical significance (P >0.05).3.2 The effect of MGMJ on the expression level of Thl/Th2-type cytokines in serum: With the ELISA test, comparing with the model group, it was found that the content of serum IL-4 decreased in the high-dose MGMJ group (P<0. 01), and there was no statistical significance in MGMJ groups with a middle or low dose though the decreasing tendency of the content of serum IL-4 was showed (P>0. 05).The content of serum IL-5 decreased in MGMJ groups with a high or middle dose(P<0.05),whereas there was no statistical significance though it showed a decreasing tendency for serum IL-5 in the low-dose MGMJ group (P>0. 05). The content of serum IFN-? increased in MGMJ groups with a high or middle dose(P<0. 01-0. 05),but there was no statistical significance though it showed an increasing tendency for serum IFN-? in the low-dose MGMJ group (P>0. 05).3.3 The effect of MGMJ on the mRNA expression of Thl/Th2-type cytokines in serum: According to the result of RT-PCR, the mRNA expression level of IL-4 and IL-5 in lung tissues was reduced in each MGMJ group (P<0. 01?0. 05), compared to the model group. It was observed that mRNA expression levels of IFN-?increased in both dexamethasone group and high-dose MGMJ group (P<0. 05), and there was no significant manifestations found in other groups (P>0. 05)except the increasing tendency.3.4 The effect of MGMJ on the mRNA and protein expression of transcription factors in lung tissues: By the comparison with the model group, the RT-PCR test showed that the mRNA expression level of Th2-type transcription factor GATA -3 in lung tissues decreased in MGMJ groups with a high or middle dose (P<0. 01 ?0.05), and the high-dose MGMJ groups was more effective (P<0.01). The mRNA expression level of both Th1-type transcription factor T-bet and Treg cell-specific transcription factor Foxp3 were enhanced in the high-dose MGMJ group (P<0.01?0. 05),and only the mRNA expression level of Foxp3 was enhanced in MGMJ groups with a low or middle dose (P<0. 01?0. 05). There was an increasing tendency for the expression level of T-bet in MGMJ groups with a low or middle dose, and a decreasing tendency for the mRNA expression level of GATA - 3 in the low- dose MGMJ group (P>0. 05, respectively). Moreover, resulting from the WB test, the protein expression of GATA - 3 was inhibited in MGMJ groups with a high or middle dose (P <0. 01-0. 05), and there was no statistical significance though the protein expression of T-bet and Foxp3 increased in different degrees (P>0.05), by comparing the model group.Conclusion: 1. According to document statistics, a close relationship was found to exist between the liver and lung and allergic asthma. Although asthma is not related to these two organs only, such organs are the most critical ones triggering asthmatic attack (acute state) ; the type of syndrome, generation of pathological factors, treatment methods and other aspects of asthma correlate closely with physiological and pathological changes of the liver and the lung,and type of syndrome, pathological factors, treatment methods that are related to the liver and the lung are very important in terms of asthma.2. Based on the principle of “treating the liver and lungs together",the MGMJ is considered as the representative formula for treating allergic asthma,with functions of "regulating both liver and lung qi,nourishing yin and dispelling wind as well as resolving phlegm and dredging collaterals". Most ingredients in MGMJ have sound effects on liver and lung meridians by treating exterior, interior, yin and yang together, so as to eliminate pathogenic factors,including wind, phlegm and stasis, as well as strengthen body resistance.3. In mice models, it shows that the MGMJ can relieve asthma decrease the ratio between total number of WBC and EOS, inhibit inflammatory infiltrations with EOS in lung tissues and reduce the content of OVA-sIgE, which all demonstrates sound effects of MGMJ on anti-allergy, anti-inflammation and anti-asthma.4. The immunological mechanism of MGMJ on anti-asthma may be as follows:firstly, it can regulate the imbalance of differentiations between Thl and Th2 cells, by increasing the ratio of Thl-type cell subsets in CD4~+cells while decreasing that of Th2-type cell subsets in CD4~+cells in spleen tissues. In addition, it can promote the expression of Thl-type cytokines IFN-?,and has a reverse effect on Th2-type cytokines IL-4 as well as IL-5. It also has an inhibitory effect on the gene and protein expression level of Th2-type transcription factor GATA ? 3,and, on the contrary, has a reinforcing effect on the gene expression level of Thl-type transcription factor T-bet in lung tissues. Secondly, it can regulate the imbalance of Treg/Th17 cell differentiations via reducing the ratio of Th17-type cell subsets in CD4~+cells and up-regulating the gene expression of Treg cell-specific transcription factor Foxp3.