| Idiopathic pulmonary fibrosis(IPF)is a chronic,progressive,fibrotic interstitial pneumonia.The cause of IPF remains unknown,and the prognosis for this disease is often poor with no definitive effective treatment available so far.At present,conventional treatment for IPF mainly focuses on the applications of hormones,pirfenidone,and nintedanib,in which these treatment plans,which are related to certain adverse effects,cannot cure IPF and are reported to have relatively limited effects in controlling the progression of the disease.On the contrary,Traditional Chinese medicine offers numerous advantages in treating IPF.Feixian formula(FXF)is a prescription formed based on the clinical experience from Professor Liangduo Jiang in treating IPF.This study explores the clinical effect and mechanism of FXF in the treatment of IPF via clinical observation and experimental researchObjectiveIn order to explore the potential of FXF in treating IPF patients with the syndromes of deficiency of lungs and kidneys,complicated with retention of phlegm,this study compared the treatment effects among different treatment groups,namely FXF group,FXF combined with hormones group,and FXF combined with pirfenidone group via clinical observation.On the other hand,FXF was also used as an intervention in treating bleomycin-induced IPF rats during experimental research.The mechanisms of FXF in treating IPF were determined through observation of the pathological changes of lung tissues dissected from the bleomycin-induced rats after the intervention,as well as its effects in regulating Wnt/β-catenin signaling pathway and the process of epithelial-mesenchymal transitionMethodsClinical observation:A clinical observation table for IPF patients with syndromes of deficiency of lungs and kidneys,complicated with retention of phlegm was prepared by the researcher.A total number of forty-nine IPF patients who met the inclusion criteria were recruited into the study and these patients were divided into different treatment groups accordingly to their pre-existing treatment plans,via the method of convenient sampling,with 18 patients in FXF group,17 patients in FXF+hormone group,and 14 patients in FXF+pirfenidone group.Clinical data such as the clinical manifestations,results of lung function test,chest high resolution CT and modified mMRC dyspnea index score of the patients before and after the intervention were recordedExperimental research:A total number of 120 bleomycin-induced IPF male rats,weighed between 180-220g,were randomly divided into six different groups,namely control group,BLM group,BLM+Pred group,BLM+FXF groups,in which BLM+FXF groups can be subdivided into high-dose group(19.8 g/kg,BLM+FXF-H),moderate-dose group(9.9 g/kg,BLM+FXF-M),and low-dose group(4.95 g/kg,BLM+FXF-L),with 20 rats in each group.The rat models from each group were then equally divided into two different subgroups,in which data from half of the rat models will be collected on the 14th day of the experiment,whilst data from the other half of the rat models will be collected on the 28th day of the experiment.The general states of the rat models,which include the skin color,mental state,eating,breathing,etc are carefully observed and recorded.HE and Masson’s trichrome were used to observe the degree of alveolities and fibrosis of the lung samples which were collected,on 14th day and 28th day of the experiment respectively,from the rat models.Furthermore.western blot was used to detect the protein levels of wnt3a,β-catenin,wnt inhibitory factor,glycogen synthase kinase-3 beta,phosphorylated GSK-3p,a-SMA,E-cadherin and collagen I Also,RT-PCR was used to detect the mRNA expression of wnt3a,β-catenin,WIF1,LRP,oc-SMA and E-cadherinResults1 Clinical observation1.1 General informationA total number of 49 patients were initially recruited into three different groups,with three patients withdrawal from the study,a total number of 46 patients were included in the analysis.There were no significant statistical differences in gender,age,course of disease,and degree of HRCT among the three treatment groups(p>0.05)before the intervention1.2 Comprehensive effectThe results of Kruskal-Wallis H test among the three treatment groups were not significantly difference(p>0.05)from each other,suggesting that three treatment groups possessed similar curative effects in IPF patients,in which the comprehensive effect of FXF group,FXF+hormone group,and FXF+pirfenidone group were 68.75%,56.25%and 78.57%respectively.1.3 Main symptomsAll three groups can significantly alleviate the symptoms of wheezing,coughing and expectoration(p<0.05 or p<0.01).The effect of FXF+pirfenidone group in improving shortness of breath was significantly better(p<0.05)than that of the other groups.1.4 Main signsAll three groups can significantly alleviate the signs of Velcro and cynosis(p<0.05 or p<0.01),while the sign of clubbing finger among the three groups did not have significant improvement(p>0.05).1.5 Lung functionThe effect of FXF group in improving TLC%was significantly better(p<0.05)than that of the other groups.The effect of FXF+pirfenidone group in improving DLco%was significantly better(p<0.05)than that of the other group.All three groups did not exhibit significant effect in improving VC%(p>0.05).1.6 Chest HRCTAll three groups can significantly alleviate the intensity of ground-glass opacity(p<0.01).The effect of FXF+pirfenidone group in alleviating ramification was significantly better(p<0.05)than that of the other groups.All three groups showed no effect in improving the intensity of the sign of honeycomb of the lungs.1.7 mMRC dyspnea index scoreThe effect of FXF group and FXF+pirfenidone group in improving the mMRC dyspnea index scores were significantly better(p<0.05 or p<0.01)than that of the other group.FXF+hormone group did not exhibit significant effect in improving mMRC dyspnea index scores(p>0.05).2 Animal experiments2.1 Histological examination of lung tissuesHE staining:The scores of HE staining in BLM-induced IPF rats were significantly higher than that in the control group on the 14th and 28th days(p<0.01),and the sign of alveolitis was more severe on the 14th day.However,the severity of alveolitis on the 14th and 28th day were significantly improved among all treatment groups when compared to that of control group(p<0.05 orp<0.01)Masson’ s trichrome staining:The score of Masson’s trichrome staining in BLM-induced IPF rats were significantly higher than that in the control group on the 14th and 28th days(p<0.01),and the condition of pulmonary fibrosis was more severe on the 28th day.The intensity of pulmonary fibrosis among all treatment groups were significantly improved on the 14th and 28th days,in which the BLM+FXF-M group showed the most significant improvement when compared to that of BLM group(p<0.05 orp<0.01)2.2 Effects of FXF on Wnt/β-catenin signaling pathway in BLM-induced IPF ratsThe protein expression:The protein expressions of wnt3a,β-catenin and p-GSK3β in BLM group were significantly up-regulated when compared to that of control group on the 14th and 28th days(p<0.05 or p<0.01).The protein expression of GSK-3β in BLM group was significantly down-regulated when compared to that of control group on the 14th and 28th days(p<0.05).Meanwhile,the protein expressions of wnt3a and β-catenin were significantly down-regulated,whilst the protein expressions of WIF1 and GSK-3β were significantly up-regulated in all treatment groups,in which BLM+FXF-M group exhibited the most significant impact when compared to that of BLM group(p<0.05 orp<0.01).The mRNA expression:The mRNA expressions of wnt3a,β-catenin and LRP in BLM group were significantly up-regulated when compared to that of control group on the 14th and 28th days(p<0.05 or p<0.01).The mRNA expression of WIF1 in BLM group was significantly down-regulated when compared to that of control group on the 14th and 28th days(p<0.05 or p<0.01).The BLM+FXF-H group and BLM+FXF-M group significantly down-regulated the mRNA expression of wnt3a,β-catenin,and LRP and up-regulated the mRNA expression of WIF1 when compared to that of BLM group(p<0.05 orp<0.01)2.3 Effects of FXF on EMT in BLM-induced IPF ratsThe protein expression:The protein expression of a-SMA and Collagen Ⅰ in BLM group was significantly up-regulated when compared to that of control group on the 14th and 28th days(p<0.01).The protein expression of E-cadherin in BLM group was significantly down-regulated when compared to that of control group on the 14th and 28th days(p<0.05 or p<0.01).The BLM+FXF-H group showed the most significant effect in down-regulating the expressions of α-SMA and Collagen Ⅰ when compared to that of BLM group(p<0.05 or p<0.01).The BLM+FXF-M exhibited the most significant effect in up-regulating the expression of E-cadherin(p<0.05).The mRNA expression:The mRNA expression of α-SMA in BLM group was significantly up-regulated when compared to that of control group on the 14th and 28th days(p<0.01).The mRNA expression of E-cadherin in BLM group was significantly down-regulated when compared to that of control group on the 28th day(p<0.05).The expression of α-SMA was significantly downregulated whilst expression of E-cadherin was significantly up-regulated in the BLM+FXF-H group on the 14th and 28th days when compared to that of BLM group(p<0.05 or p<0.01).Conclusion1 Clinical observation1.1 FXF group,FXF+hormone group and FXF+pirfenidone group can improve the overall efficacy of IPF patients with the syndromes of deficiency of lungs and kidneys,complicated with retention of phlegm.Although the results from the present study suggested that the effect of FXF+pirfenidone group in treating IPF was the most significant than that of the other groups,it has also proven that FXF is an effective prescription for IPF patients with the syndromes of deficiency of lungs and kidneys,complicated with retention of phlegm.1.2 In terms of improving main symptoms and signs,all three groups have similar effects.In terms of improving lung function,the effect of FXF+pirfenidone group was better compared to that of the other groups.In terms of improving chest HRCT，the effect of FXF+hormone group and FXF+pirfenidone group were better compared to that of the other groups.In terms of improving the mMRC dyspnea index score,the effect of FXF group and FXF+pirfenidone group were better compared to that of the other groups2 Animal experiments2.1 FXF can reduce the intensity of alveolitis and pulmonary fibrosis in rats with pulmonary fibrosis,especially in the high and middle dose groups of FXF,despite the difference between the two groups was not significant.In other words,excessive dose of FXF did not significantly enhance the effect of the prescription,hence,the result of the present study suggested that the middle dose of FXF could be the most ideal dosage.2.2 FXF improved the condition of pulmonary fibrosis by inhibiting the expression of proteins and mRNA expressions of the related molecules in the Wnt/β-catenin signaling pathway and EMT.FXF exerted anti-inflammatory and anti-fibrotic effects on BLM-induced IPF rats probably by interfering with the Wnt/β-catenin signaling pathway and EMT. |
