Based On The Theory Of Lung Heat, Collateral Blood Stasis And Pathogenesis, The Mechanism Of Chinese Herbal Compound Intervention On AE-IPF

Purpose:Acute exacerbation of idiopathic pulmonary fibrosis(AE-IPF)which has high incidence and death rate,is a special and critical stage in the development of idiopathic pulmonary fibrosis(IPF),and it is progressing rapidly and being the most common cause of death during the process of IPF.At present,the research foundation of the disease is relatively weak,and there is no consensus on the pathogenesis and treatment.Clinically,although the effectiveness remains controversial,supportive care and glucocorticoids are the main treatment methods for alleviating symptoms,correcting hypoxemia.In addition,there is no unified animal model establishment,which greatly limits the depth and breadth of experimental research.Therefore,based on this research status,collaterals theory and clinical experience of my doctoral supervisor,this paper presents the chinese medicine pathology of AE-IPF which is summarized as ’lung heat and collaterals stasis’.And we have developed a traditional chinese medicine compound prescription called ‘Qing Luo Yin’,which has the efficacy of ‘ clearing heat,detoxicating,nourishing yin and dredging collaterals’.Then,we have tried to build an animal model of AE-IPF which has characteristics of mobility,repeatability and economy.And based on the model,we have uesd multiple methods to explore the curative effect of‘Qing Luo Yin’ in correcting the imbalance of Th1/Th2 and MMPs/TIMPs at all stages of the experiment to reveal the mechanism of delaying the development of AE-IPF.On the whole,this study aims to lay the ground for the disease animal experiment research,reveal the unique advantages of Chinese medicine in the treatment of the AEIPF,and to provide first-hand research data for future drug discovery.Material and method:1 Establishing a rat model of AE-IPF1.1 The intratracheal injection of bleomycin(BLM)was performed twice in the model experiment.1.2 A total of 120 SPF male Wistar rats were randomly divided into three groups using a random number table method.The three groups are named AE-IPF group,IPF group and Control group.After 3 days of adaptive feeding,the first intratracheal injection of BLM was performed,and on the 28 th day after the first time,the second intratracheal injection of BLM was performed.According to the dynamic pathological evolution rule of IPF,the point-in-time of sampling and index observation were set up at 5time points of 7d,14 d,31d,35 d and 42 d.1.3 Observating the indexes such as survival condition,W/D ratio,lung coefficient,gross appearance of lung tissue,HE-staining and Masson of lung tissue,the pathologic score of alveolar inflammation and the pathologic score of fibrosis of each group at five different times.1.4 Measuring the arterial partial pressure of oxygen of each group at 31 d,35d and 42 d.1.5 Assaying the hydroxyproline content in the rat lung of each group at five different times.1.6 Measuring the protein expression level of alpha-smooth muscle actin by the method of immunohistochemistry of each group at five different times.2 To explore the effect and mechanism of Chinese herbal compound ‘Qing Luo Yin’in proventing IPF processing which induced by BLM at different stage of the disease.2.1 Re-establishing the rat model of AE-IPF by the way of twice intratracheal injection of BLM.2.2 A total of 240 SPF male Wistar rats were randomly divided into six groups using a random number table method.The six groups are named Control group,AE-IPF group,Low-dose group,Middle-dose group,High-dose group,Glucocorticoids(GCS)group.The point-in-time of sampling and index observation were set up at 4 time points of 7d,14 d,28d and 31 d.2.3 Observating the indexes such as survival condition,W/D ratio,lung coefficient,gross appearance of lung tissue,HE-staining and Masson of lung tissue,the pathologic score of alveolar inflammation and the pathologic score of fibrosis of each group at four different times.2.4 Measuring the arterial partial pressure of oxygen of each group at 28 d and 31 d,and calculate the average descent rate of arterial partial pressure of oxygen from 28dto 31 d.2.5 Assaying the hydroxyproline content in the rat lung of each group at four different times.2.6 Observating the effects of various experimental drugs in changing the expression level of alpha-smooth muscle actin by the experimental methods such as western-blot,immunohistochemistry and quantitative real-time polymerase chain reaction.2.7 Observating the imbalance of Th1 and Th2 cytokines of each group at four times by various methods,such as enzyme-linked immuno sorbent assay,western-blot,immunohistochemistry and quantitative real-time polymerase.The contents,protein expression levels and m RNA expression levels were used to detect the levels of interleukin-12(IL-12),interleukin-4(IL-4),and Interferon-gama(IFN-γ)in each experimental group.And then we calculated the correlation between IL-12 levels and IL-4/ IFN-γ levels,the results show that IL-12 could regulate the balance of Th1 and Th2 cytokines by adjust the IL-4/ IFN-γ levels.Based on this,the mechanism of ‘Qing Luo Yin’ in preventing IPF was clarified,which can be described as clarifying the imbalance of Th1/Th2 cytokines.2.8 Observating the imbalance of MMP-9 and TIMP-1 of each group at four times by various methods,such as enzyme-linked immuno sorbent assay,western-blot,immunohistochemistry and quantitative real-time polymerase.The expression of mmp-9,timp-1 protein and m RNA in rats were measured at different levels.By this means we discovered that ‘Qing Luo Yin’ could restore balance between MMP-9 and TIMP-1,and accelerate degradation speed of extracellular matrix.Results:1 Establishing a rat model of AE-IPF1.1 Survival condition:There was no difference between IPF group and AE-IPF group,befor the second intratracheal injection of BLM,but they are worse than the Control group at the same period.After the second building operation,compared with the IPF group and Control group,the rats in the AE-IPF group showed symptoms such as shortness of breathing,dyspnea,nosegrab,cyanosis,loss of weight and Increasing mortality.1.2 W/D ratio and lung coefficient:After the second building operation,compared with the IPF group and Control group,the W/D ratio and lung coefficient of AE-IPF group was significantly higher,and appeared obvious manifestation of acute pulmonary edema.1.3 Lung tissue morphology:Compared with Control group and IPF group,in the AEIPF group,alveolar catarrh changes were present mainly at 7d,fibroblast proliferation were mainly at 14 d,and diffuse alveolar damage,lots of inflammatory infiltration,pulmonary hyaline membrane formation,lots of collagenous deposition and fibroblast foci activation were all appeared after 31 d.1.4 Arterial partial pressure of oxygen:Compared with the other groups,the rats of AEIPF group showed the lowest arterial partial pressure of oxygen at 31 d,and the level of it were gradually rised at 35 d and 42 d.Finally at 42 d,the arterial partial pressure of oxygen between AE-IPF group and IPF group have no significant differences.1.5 Hydroxyproline content of lung tissue:The hydroxyproline content of lung tissue in AE-IPF group were progressively increased during the whole experimental stages,and the level of it was significally higher than that of IPF after the second building operation.1.6 Immunohistochemistry of alpha-smooth muscle actin:In the Control group,the alphasmooth muscle actin were showed weak positive expression only in the smooth muscle cells of the larger vessels and bronchial walls during the whole experimental stages.In the IPF group,the protein expression level of alpha-smooth muscle actin were increased slowly after 7d,and there was no significant difference of positive expression intensity between the IPF group and AE-IPF group before the second building operation.But after the second operation,the protein expression level of alpha-smooth muscle actin were continuously increased,and the positive expression intensity was significantly higher than that of IPF group.2 To explore the effect and mechanism of Chinese herbal compound ‘Qing Luo Yin’in proventing IPF processing which induced by BLM at different stage of the disease.2.1 Successfully re-establishing the rat model of AE-IPF by the way of twice intratracheal injection of BLM.2.2 Survival condition,W/D ratio and lung coefficient:The Chinese herbal compound‘Qing Luo Yin’could obviously improve the survival condition,promote weight gain,reduce mortality,alleviate acute pulmonary edema,and it had effect on promoting general functional status recovery.Compared with other doses,the middle dose had the best effect.And compared with GCS group,it had better effect on improving some indicators.2.3 Lung tissue morphology:Chinese herbal compound ‘Qing Luo Yin’could effectively reduce the acute diffuse alveolar and extracellular matrix deposition,inhibit active fibroblast foci formation.Compared with other doses,the middle dose had the best effect,and it showed better effect on reducing pulmonary fibrosis.2.4 Arterial partial pressure of oxygen:Chinese herbal compound ‘Qing Luo Yin’could increase arterial oxygen partial pressure of organism and improve the hypoxia state in rats with acute exacerbation.The average descent rate of arterial partial pressure of oxygen from 28 d to 31 d in the Middle-dose group is the lowest compared with other groups.2.5 Hydroxyproline content of lung tissue:Chinese herbal compound ‘Qing Luo Yin’could reduce the hydroxyproline content in various stages of IPF,especially AE-IPF stage.Thus it could reduce collagen synthesis and deposition.Among them,the middledose showed the best effect.2.6 Protein and m RNA expression of alpha-smooth muscle actin:Chinese herbal compound ‘Qing Luo Yin’could decrease the protein and m RNA expression levels of alphasmooth muscle actin,inhibit the transformation of Fb to MFb,block the formation of fibroblastic foci and delay IPF pathological process.The middle-dose showed the best effect in this mechanism.2.7 Th1 and Th2 cytokines:Compared with the Control group,AE-IPF showed significant Th2 dominance.The expression level of IL-4 was higer and level of IL-12 and IFN-γ were lower in AE-IPF group than that in Control group.‘Qing Luo Yin’could reduce IL-4 content and increase IFN-γ and IL-12 contents in rat serum,reduce protein and m RNA expression levels of IL-4 and increase IFN-γ and IL-12 expression levels in lung tissues.It could delay the pathological process of pulmonary fibrosis by the mechanism of restoring Th1/Th2 cytokine balance.The middle-dose showed the best effect in this mechanism.2.8 MMP-9/TIMP-1:Compared with the Control group,the ratio of MMP-9m RNA/TIMP-1m RNA showed tendency as ‘increasing at early phase,reducing at middle and advanced phase,re-increasing at acute exacerbation phase’ in the AE-IPF group.As a whole,the protein and m RNA expression levels of MMP-9 and TIMP-1 were increased in the AE-IPF group.But the increasing tendency between them are different,the expression of TIMP-1 showed continuously increasing during the experimental process,however the expression of MMP-9 showed increasing at 7d,briefly reducing during 14 d to 28 d and re-increasing after 28 d.The experimental results displayed that‘Qing Luo Yin’could reduce the protein and m RNA expression of MMP-9 and TIMP-1,alleviate injury of alveolar basement membrane,accelerate degradation of collagen and then prevent the process of IPF.By the way,the middle-dose showed the best effect in this mechanism and it had a better effect on reducing the protein expression level of TIMP-1 during acute exacerbation period.Conclusion:1.We have successfully built the rat model of AE-IPF by the method of twice intratracheal injection of bleomycin(0d,5mg/kg & 28 d,7mg/kg).At 31 d,the clinical manifestations and pathological changes of rats were the closest to human beings,so we set 31 d as the key observation points of AE-IPF models.2.Chinese herbal compound ‘Qing Luo Yin’could alleviate the symptoms such as wheeze,dyspnea,cough,loss of weight of AE-IPF.Then it could treat AE-IPF by the way of relieving pneumatorexis,reducing alveolar inflammation,collagen synthe-sis and extracellular matrix deposition.3.‘Qing Luo Yin’could inhibit expression of IL-4,promote expression of IL-12 and IFN-γ,and then rebuild the balance of Th1/Th2 cytokines.4.IL-12 plays an important role in maintaining Th1/Th2 cytokine balance.5.‘Qing Luo Yin’could inhibit expression level of MMP-9 and TIMP-1,and rebuild the balance of MMP-9/TIMP-1.6.Theoretical research indicates that the basic pathogenesis of AE-IPF is ’lung heat and collaterals stasis’.The concept of ‘lung heat’ includes phlegm-heat,toxic-heat and heat due to yin deficiency,at the same time the cause of ‘collaterals stasis’ includes qi stagnation,qi deficiency,phlegm congealing,collaterals injury due to toxic and bloodheat accumulation.