| Idiopathic pulmonary fibrosis(IPF)is a chronic progressive fibrotic interstitial pneumonia with unknown etiology,mainly manifested as follows: dry cough,progressive dyspnea,restrictive ventilation dysfunction and gas exchange disorder,usually accompanied with lung transplantation and gas exchange impairment,which would result inhyoxemia and even failure of respiration.There is no effective treatment to cure such a disease except the lung transplantor and long-term oxygen therapy.Therefore this is really an urgent and difficult topic and it's necessary to explore some new and effective methods to treat IPF.At present,it's found that traditional Chinese medicine has some advantages in the clinical treatment of idiopathic pulmonary fibrosis.There's a report about some scholars researching on the treatment of pulmonary fibrosis by Radix Astragali and Angelica Sinensis,but still left some doubtful points unquestioned.For instance,from what signaling pathways do the cell factors effected by the Qi Gui recipe play an anti-fibrotic role since the cytokine network is huge and complex with many signal pathways? Does Qi Gui recipe regulate other cytokines against pulmonary fibrosis except the cytokines detected in this report? How much quantities could make the best anti-pulmonary fibrosis effects by the Qi Gui recipe? What is the effect of clinical treatment? What are the results of different treatments with diverse principles of Chinese medicine? There are still no relevant papers about that reported both china and abroad.In order to solve these problems,the experiment was designed and a comparative study was carried out meanwhile on Maimen Dong Tang,Qi Gui recipe,and Polygonum cuspidatum,which are the representative of the main treatment methods,namely,Qingre Yangyin Zhike,Yiqi Huoxue Tongluo,clearing heat and detoxification and activating blood circulation,altogether to observe the effect and mechanism of Qi Gui recipe on idiopathic pulmonary fibrosis,to analyze the basic rule of clinical treatment group,and to further explore the antagonistic effect of Qi Gui recipe on idiopathic pulmonary fibrosis in rats.Part one: clinical observation and studyObjective: To observe the efficacy and safety of Qigui recipe in the treatment of IPF,and explore its possible mechanism.Methods:IPF patients with deficiency of lung qi and internal obstruction of blood stasis were randomly divided into observation group and control group in TCM and respiratory out-patient department of Zhongnan Hospital of Wuhan University.The control group was treated with routine therapy and the observation group was treated with Qigui recipe on the basis of the control group.The two groups were treated continuously for 12 weeks to evaluate the clinical efficacy and the influence on the side effects of prednisone,and observe the TCM syndrome integral of the two groups.6-minute walking test(6MWT),quality of life score,pulmonary function(DLco%,VC%)changes,arterial blood gas analysis(Pa O2?Sa O2),The serum levels of tumor necrosis factor-?(TNF-?)and transforming growth factor-?1(TGF-? 1)were measured by enzyme-linked immunosorbent assay(Elisa).The changes of indexes before and after treatment were monitored,and the clinical curative effect and mechanism were discussed.Results:(1)the total effective rate was 84% in the observation group and 76% in the control group,which was significantly higher in the observation group than in the control group(P<0.05),and the side effects of prednisone such as acne and insomnia were significantly higher in the observation group.There was significant improvement in the observation group of tachycardia(P<0.01).(2)the scores of TCM syndrome and quality of life in the two groups decreased after treatment,the difference was statistically significant(P<0.01).The 6-minute walking distance(6MWD)in the observation group was significantly higher than that in the control group(P < 0.05).(4)comparison of pulmonary function and arterial blood gas analysis: there was no difference in VC% between the two groups after treatment,but DLco%in observation group was improved compared with that before treatment(P<0.05).(5)comparison of inflammatory factors: the levels of TNF-? and TGF-?1 in the observation group were significantly lower than those in the control group before and after treatment,and the observation group was better than the control group(P < 0.05 or P < 0.01).Conclusion: Qi Gui recipe can effectively improve the clinical symptoms of patients with idiopathic pulmonary fibrosis,improve exercise endurance and quality of life,reduce the level of inflammatory factors in patients,and the curative effect is better than that of prednisone alone.There was no significant improvement in blood gas index and pulmonary function,but it had a positive effect on delaying the progress of the disease.Part two: Explore the intervention effect of Qi Gui recipe on idiopathic pulmonary fibrosis in rats by TGF-?/smad/miRNA-targeted signaling pathway.Objective:Through in vivo animal experiments,To explore the pharmacodynamic mechanism of Qigui recipe in the treatment of idiopathic pulmonary fibrosis based on the mi RNA targeting regulation of TGF-?1 /smad signaling pathway.methods:Rats with idiopathic pulmonary fibrosis were induced by a one-time intratracheal injection of bleomycin?The model of idiopathic pulmonary fibrosis was interacted with Qigui recipe,normal saline and prednisone at low,medium and high doses,and healthy rats were set up as blank control group?The survival status,body weight,lung morphology and lung coefficient of the rats were observed after7 d and 28 d treatment.Hematoxylin-eosin staining(HE)was used to observe the grade of pulmonary alveolar inflammation.masson dyeing observation lung organization fibrosis degree,The levels of serum CTGF,TGF-?1 and NF-k B were detected by ELISA method examination.The expression of Smad3,p-smad2 and p-smad3 proteins was analyzed by immunohistochemis?The content of Smad7 in rat lung tissue was detected by Wester blot?The expressions of mi R-21,mi R-326 and mi R-29 b in lung tissues were detected by real-time PCR.Results:(1)General condition observation,The rats in the blank group were strong and fat,with shiny fur,in good mental state and no death?All the rats showed cough,wheezing,dullness,reduced diet,arched back and dark fur.A week later,the results were slightly better,but worse than the blank group?(2)Pathological findings:The appearance of lung tissues in the blank group was always pink and red,with uniform color,clear lung profile,good luster and elasticity,and no bleeding spots or nodules?Bright red edematous changes and focal ecchymosis were observed in both lung tissues of the model group at day 7?At day28,the surface of the lung tissue was pale,uneven,hard and markedly less elastic?Compared with the model group,the color,hardness,elasticity and volume of the bilateral lungs were improved in all groups?A few spots of bleeding or gray-white plaques can be seen in some of the lobes,and the nodular changes and bleeding spots can be alleviated?Among them,the improvement was most prominent in the medium and high dose group?By HE coloring,Rats in the blank group had normal lung tissue structure,uniform alveolar spacing,no obvious exudation,no congestion,edema and acute or chronic inflammation?No significant pathological changes were observed at all time points?Changes of alveolar inflammation were observed on day 7 in the model group,with significant infiltration of inflammatory cells and increased secretion in alveolar cavity?On day 28,the alveolar structure was destroyed,the alveolar cavity was reduced,and there were obvious collagen deposition and fibrosis areas?Similar pathological changes were observed in both the traditional Chinese medicine group and prednisone group,but to a lesser extent?Masson staining results:In the blank group,the collagenous layer in the bronchial wall of the rats was thin,and a small amount of pale blue collagenous fibers could be seen in the alveoli?In the model group,the alveolar septum was widened at day 7,and fibroblast hyperplasia was observed in the interstitial and alveolar septum?On day 28,the alveolar structure was significantly destroyed,and there were bundles or large sheets of collagen fibers in the interstitium?Compared with the model group,there were fewer alveolar septal fibrous hyperplasia lesions,smaller lesion range and less pulmonary parenchyma structural damage in Qigui recipe group.(3)the comparison of expressions of CTGF,TGF-?1 and NF-k B?The contents of CTGF,TGF-?1 and NF-k B in serum at different periods in the model group were increased compared with those in the blank group?Compared with the model group,the Qigui recipe dose group can improve the secretion of inflammatory factors in rats with pulmonary fibrosis.The contents of CTGF,TGF-?1 and NF-k B in serum of rats were all decreased?Compared to the prednisone group,At all time points,moderate and high doses of Qigui recipe could improve the contents of NF-k B,TGF-?1 and CTGF in serum of IPF rats?(4)Expression of Smad protein,Compared with the blank group,the protein expressions of Smad3,p-smad2 and p-smad3 in the model group were significantly increased,and the content of Smad7 was decreased?Compared with the model group,immunohistochemistry indicated that the expression of p-smad2 and p-smad3 in lung tissues of Qigui recipe group decreased at both time points(P < 0.05 or P < 0.01)?The results of western-blot showed that:Qigui recipe can improve the expression level of Smad7 in lung tissues?(5)Comparison of the expression of mi R-29 b,mi R-326 and mi R-21: In the lung tissues of rats in the model group,the levels of mi R-29 b and mi R-326 were decreased,and the levels of mi R-21 were increased?Qigui recipe can inhibit the expression of mi R-21,enhance the activity of mi R-29 b and mi R-326,and reduce pulmonary fibrosis?(6):Compared with the results on day 7 and 28,there were statistically significant differences between the prednisone group and the traditional Chinese medicine group(P<0.05)?Qigui recipe has the time dependence,the treatment time is long,the effect is more obvious;Medium and high dose Qigui recipe has obvious effects?However,there was no significant difference in efficacy between the medium and high dose groups?The above results suggested that Qigui recipe's improvement of pulmonary fibrosis was dose-related,but too high dose could not enhance its efficacy?Therefore,the clinical efficacy is more ideal with medium dose.conclusion :The anti-pulmonary fibrosis effect of Qigui recipe may be related to inhibiting mi R-21,improving the expression of mi R-326 and mi R-29 b,thus interfering with TGF-?1 /Smad signaling pathway and reducing extracellular matrix deposition ? It can effectively inhibit alveolar inflammation and fibrosis.Part three: study on the intervention effect of traditional Chinese Medicine on Idiopathic Pulmonary Fibrosis in RatsObjective: To observe the effects of Qi Gui recipe(QG group),maimendong decoction(M group)and Polygonum cuspidatum(H group)on IPF model rats with idiopathic pulmonary fibrosis,and to evaluate the effects of traditional Chinese medicine under the guidance of different treatment principles on idiopathic pulmonary fibrosis rats.Methods: The rat model of idiopathic pulmonary fibrosis was induced by intratracheal injection of bleomycin.The rats were randomly divided into groups: Qi Gui recipe group,maimendong decoction group,Polygonum cuspidatum group and model group,and normal rats were injected with normal saline.After 7days and 28 days,the changes of body weight,lung coefficient and alveolar inflammation were observed by HE staining,and pulmonary fibrosis by Masson staining.Serum levels of CTGF,TGF-?1 and NF-?B were measured by Elisa.Results: the body weight of the normal group increased gradually every week,no animal died,the model group and the Polygonum cuspidatum group had animal death,all the treatment groups reduced the mortality of the model animal to some extent,and antagonized the weight loss of the model animal.Compared with the model group,the levels of serum CTGF,TGF-?1 and NF-?B in each treatment group were decreased(P<0.05).Conclusion:traditional Chinese medicine have different protective effects on IPF model animals,but Qi Gui recipe has more obvious effect. |
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