Observation Of TCM Syndromes Of Idiopathic Pulmonary Fibrosis And Experimental Study On Anti-pulmonary Fibrosis With Nourishing Yin Yiqi Mixture

Idiopathic pulmonary fibrosis(IPF)is a special type of interstitial pneumonia characterized by chronic inflammatory injury and excessive interstitial fibrosis.It occurs mainly in the elderly,and lesions are confined to the lungs.Its histopathology or/ and HRCT manifestations are UIP.The disease has a poor prognosis and a high mortality rate.As the progress of IPF,it may eventually develop into respiratory failure and lead to death.At present,there is no specific treatment method for IPF.The treatment efficiency of drugs used by clinical is very limited,and Chinese medicine has shown significant advantages in treatment of IPF,and has gradually become a research hotspot.This study had three parts: The first part is literature research,which mainly describes the general situation of IPF in Chinese and Western medicine research and the role of TGF-?/Smad pathway and its mediated Epithelial-mesenchymal transition(EMT)in IPF.The second part was clinical observation,for this part,we discussed the distribution of TCM syndromes of IPF and its relationship with HRCT,and the clinical adaptability of Yangyin Yiqi mixture was verified.The third part is animal experiment research,from the angle of TGF-?/Smad pathway and TGF-? mediated EMT,to investigate the effect of Yangyin Yiqi Mixture on bleomycininduced pulmonary fibrosis in rats.ObjectiveTo investigate the distribution characteristics of TCM syndromes of IPFand its relationship with HRCT through collecting clinical data of patients with IPF.To verify the clinical applicability of Yangyin Yiqi Mixture in the treatment of IPF.To investigate the protective effect of Yangyin Yiqi Mixture on Bleomycin-induced pulmonary fibrosis in rats based on TGF-?/ Smad signal pathway and TGF-? mediated epithelial to mesenchymal transition(EMT)by animal experiments.Methods1.Clinical TCM syndrome characteristic of IPFClinical questionnaire for patients with IPF was developed according to the diagnostic criteria of Chinese and Western medicine.The clinical data of 120 patients with IPF were collected.The characteristics of TCM syndromes in patients with IPF were analyzed by factor analysis method.Then discuss the relationship between TCM syndromes and HRCT.To verify the clinical applicability of Yangyin Yiqi Mixture in the treatment of IPF.2.Animal experiments:120 Wistar rats were randomly divided into six groups: control group,BLM group,BLM + Pred group,BLM+YYYQ-L group,BLM+YYYQ-M group,and BLM+YYYQ-H group.Rats were given an intratracheal instillation of 3 mg/kg BLM to establish the pulmonary fibrosis model,and followed by different dosages of YYYQ(11,22,44g/ kg,via intragastric gavage)or prednisone soluble(4.2mg/kg,via intragastric gavage)or water.After 14 days and 28 days,tissue sections were stained with hematoxylin-eosin and Masson's trichrome to observe histopathological changes.Protein level of TGF-?1,CTGF,Interleukin-18 and hydroxyproline were detected by ELISA method,and mRNA expression of TGF-?1,T?RI,T?RII,Smad3,Smad7,?-SMA,E-cadherin,laminin and collagen I were detected by RT-PCR.Results1.TCM syndromes characteristics of IPF1.1 General information: In 120 patients with IPF,HRCT scores were significantly positively correlated with mMRC dyspnea index and symptom scores(p <0.05).1.2 TCM Syndrome of IPF: In this study,we obtained 6 TCM syndromes by method of factor analysis from 120 patients with IPF.Among them,the syndrome of deficiency of qi accounted for the largest proportion,about 42.5%,followed by deficiency of yin syndrome,then blood stasis syndrome > phlegm-dampness syndrome > phlegm-heat syndrome > deficiency of yang.The essential pathogenesis of IPF is the standard is incidental excess and fundamental deficiency.The manifestation of fundamental deficiency include qi deficiency,yin deficiency,and yang deficiency,and the manifestation of incidental excess include blood stasis,phlegm-dampness and phlegm-heat,of which qi deficiency and yin deficiency are the main sydromes,,and different pathological factors often appear together.The disease position of IPF is mainly in lung,relating to spleen,kidney,and heart.There was no significant correlation between TCM syndrome distribution and HRCT.The HRCT score of yang deficiencysydrome was significantly higher than the sydromes of deficiency of qi and deficiency of yin in IPF patients(p <0.05).2.Research on the effect of Yangyin Yiqi Mixture on Pulmonary Fibrosis2.1 Pathological changes in lung tissue: H&E staining was used to observe the changes of alveolar inflammation in lung tissues.Under the microscope,samples in control group indicated that the alveolar wall was intact,the alveolar cavity was clear,no bleeding or exudate was found in the alveolar and bronchial cavity,and no obvious inflammatory cells infiltration was observed on day 14 and day 28.On day 14,samples from BLM group showed that alveolar cavity and part of bronchi structure had collapsed and destructed,alveolar septal became thick and swollen,and large amount of inflammatory cells infiltration were observed in pulmonary interstitial tissues and alveolar tissues.On day 28,pulmonary interstitial inflammation was reduced,while the alveolar structure was replaced by collagen fibers because of the fibroblast proliferation.In summary,the histopathological results showed that lesions of lung tissues from the rats in BLM group and all the treatment groups were more severe compared to that of rats in control group.However,thickness of alveolar septa and infiltration of inflammatory cells among the rats in BLM + Pred group and different dosages of YYYQ groups were significantly reduced when compared to that of rats in BLM group.HE staining showed that inflammatory cell infiltration was significantly relieved on day 28 compared with day 14.Masson's staining was used to observe the degree of pulmonary fibrosis in lung tissues.Control group showed that normal lung tissue structure and slight collagen deposition in the alveolar septa.On the other hand,BLM group indicated severe collagen deposition,obliteration of interalveolar septum,and damaged of lung structure,whereby the alveolar space was replaced by collagen fibrosis and normal alveoli was further deteriorated on day 28.The results also indicated that the level of destruction of pulmonary interstitium and severity of collagen fibrosis in the alveolar septa were reduced in BLM + Pred group and different dosage of YYYQ groups.Meanwhile,BLM+YYYQ-M and BLM+YYYQ-H groups shown better treatment effect than other groups.2.2 YYYQ decreased the protein expression of TGF-?1,CTGF,IL-18 and HYP in sera.On day 14 and 28,the levels of protein concentration of TGF-?1,CTGF IL-18 and hydroxyproline in BLM group were significantly higher when compared to that of control group(p < 0.01),whereas the levels of protein concentration of CTGF and hydroxyproline in BLM+ Pred group were significantly higher than that of control group(p <0.05).On day 28,the levels of protein concentration of TGF-?1 and hydroxyproline in YYYQ-L group were significantly higher than that in the control group(p <0.05),but were lower than that in BLM group(p <0.05).On day 28,the levels of protein concentration of TGF-?1,CTGF and hydroxyproline in the sera samples of BLM+ Pred and BLM+YYYQ groups were significantly decreased compared to that of BLM group,especially in YYYQ-M and YYYQ-H groups(p < 0.01 or p < 0.05).The levels of protein concentration of interleukin-18 in other treatment groups were decreased when compared to that of BLM group,but were not statistically significant(p > 0.05).2.3 YYYQ suppressed BLM-activated TGF-?/ Smad signaling pathway in ratsThe mRNA expression of TGF-?1,T?RI and T?RII in BLM,BLM+ Pred and BLM+YYYQ-L group were significantly higher than that of control group on both day 14 and day 28(p <0.01 or p <0.05).On day 14,BLM+YYYQ-M and BLM+YYYQ-H group had down-regulated mRNA expression of TGF-?1,T?RI and T?RII(p <0.05).Meanwhile,mRNA expression of T?RII was significantly down-regulated in YYYQ-M group than that of BLM + Pred group(p <0.05).On day 28,the mRNA expression of TGF-?1,T?RI and T?RII in all treatment groups was significantly down-regulated when compared to that in BLM group(p <0.01).The overall results indicated that the BLM+YYYQ-H group had significant advantage over the BLM+ Pred and BLM+YYYQ-L group(p <0.05).On day 14 and 28,the mRNA expression of Smad3 in BLM and BLM+ Pred group significantly up-regulated(p <0.05 or p <0.01),whereas mRNA level of Smad7 was significantly down-regulated in BLM group when compared to that of control group(p <0.01).BLM+YYYQ group could significantly down-regulated the mRNA expression of Smad 3 than BLM group(p <0.05 or <0.01).Moreover,the mRNA level of Smad 3 in BLM + YYYQ-M group and BLM + YYYQ-H group were lower than BLM + Pred group(p < 0.05).Apart from that,the results also showed that mRNA expression of Smad 7 was significantly up-regulated in all treatment groups,especially in BLM + YYYQ-M group(p < 0.05).2.4 YYYQ inhibited BLM-induced EMT in ratsOn day 14,compared with control group,the mRNA expression of ?-SMA in BLM group was significantly higher(p < 0.01),while the mRNA expression of E-cadherin was significantly lower(p < 0.05).On day 28,the mRNA expression of ?-SMA in BLM group and BLM + Pred group were significantly up-regulated(p < 0.01),whereas mRNA expression of E-cadherin was significantly down-regulated(p < 0.01).Treatment by prednisone and different dosage of YYYQ could reduce the mRNA expression of ?-SMA compared with BLM only(p <0.05 or p <0.01).Meanwhile,mRNA expression level of E-cadherin in BLM+ YYYQ-H group was significantly up-regulated than BLM group(p < 0.05),and it revealed more remarkable improvement than BLM+ Pred group(p < 0.05).On both day 14 and day 28,mRNA expression of laminin and collagen I in BLM group,BLM + Pred group and BLM + YYYQ-L group were significantly up-regulated when compared to that of control group(p < 0.05 or p < 0.01).In summary,mRNA expression of laminin in all the treatment groups were significantly down-regulated than BLM group on both day 14 and day 28(p < 0.01),especially on day 28,mRNA level of laminin in different dosage of YYYQ group were significantly decreased compared with BLM + Pred group.The mRNA expression of collagen I in all the treatment groups was significantly down-regulated when compared to that of BLM group(p < 0.05 or p < 0.01).ConclusionIn 120 patients with IPF,The HRCT scores of patients with IPF were significantly positively correlated with the mMRC dyspnea index and the symptom scores.TCM syndrome distribution: qi deficiency and blood stasis syndrome > deficiency of yin syndrome > deficiency of qi and yin syndrome > deficiency of qi and phlegm-dampness syndrome > deficiency of qi syndrome > deficiency of yang and blood stasis syndrome = deficiency of qi and phlegm-heat syndrome > blood stasis syndrome.The common pathological factors include qi deficiency,blood stasis and yin deficiency,followed by yang deficiency,phlegm dampness and phlegm heat,and various pathological factors often appear together.This is in line with the efficacy of YYYQ,which is promoting blood circulation and removing blood stasis.The HRCT manifestation of deficiency yang sydrome is the most serious.The result of our animal study concluded that YYYQ has the potential of ameliorating progression of pulmonary fibrosis,and the mechanism may be related to suppressing TGF-?/ Smad signal pathway and TGF-? mediated EMT in BLM induced pulmonary fibrosis of rats,but the effect of YYYQ on interleukin-18 was not obvious.