Study On The Mechanism Of Rheumatoid Arthritis Combined With Interstitial Lung Disease Based On Wnt/β-catenin Signaling Pathway And The Intervention Effect Of Guiqi Bufei Decoctio

Objective:1.To sort out and refine the origin,connotation,treatment ideas,and medication rules of traditional Chinese medicine（TCM）for Feibi in ancient literature2.To establish a rat model of rheumatoid arthritis associated interstitial lung disease（RA-ILD）that mimics the pathological features of human RA with interstitial pneumonia.3.To investigate the effect of Guiqi Bufei Decoction on Epithelial-mesenchymal transition（EMT）through regulating Wnt/β-catenin signaling pathway in RA-ILD.Methods:1.Searching the ancient medical literature on Feibi and establishing a database of ancient literature on Feibi.Using descriptive statistics to analyze descriptive sentences,sort out the origin and treatment ideas of Feibi.Analyzing and summarizing the characteristics,taste,meridian tropism,frequency,and medication rules of the prescription through the TCM inheritance assistance platform software（V3.0）.2.Eight of 24 SD rats were randomly selected as contol group,and then the collagen induced arthritis（CIA）modeling model was established.Eight of them were randomly selected as the RA group,and the rest were treated with bleomycin（BLM）intratracheal instillation for pulmonary fibrosis model as the RA-ILD group.The body weight and arthritis index of rats were observed.Serum rheumatoid factor（RF）,anti-cyclic citrullinate peptide antibody（Anti-CCP-Ab）,C-reactive protein（CRP）,matrix metalloproteinase 7（MMP-7）,transforming growth factorβ1（TGFβ-1）and Tumor necrosis factor-α（TNF-α）levels were measured by ELISA;The histopathological changes of synovium and lung were observed by HE and Masson staining;The incidence of RA-ILD in rats was evaluated according to the results of HE and Masson staining;The expression levels of MMP-7 and IL-22 in lung tissue were measured by immunohistochemistry and WB.The histopathological changes of synovium and lung were observed by HE and Masson staining;To evaluate the incidence of RA-ILD in rats;The expression levels of MMP-7 and interleukin-22（IL-22）in lung tissues were detected by immunohistochemistry and western blot.3.The effect of Guiqi Bufei Decoction on Wnt/β-catenin signaling pathway in rheumatoid arthritis associated interstitial lung disease:Sixty SD rats were randomly divided into normal group（10 rats）and modeling group（50 rats）.The model groups after being constructed with RA-ILD induced by CIA combined with BLM were randomly divided into model groups;methylprednisolone group;Guiqi Bufei Decoction low-concentration group(0.85g﹒ml^-1);Guiqi Bufei Decoction medium concentration group(1.7g﹒ml^-1);Guiqi Bufei decoction high concentration group(2.55g﹒ml^-1),respectively 10 cases in each group.HE and Masson staining were performed to observe the pathological changes of lung tissues and to evaluate the degree of pulmonary fibrosis and alveolitis in each groups.The contents of RF,TNF-α,Anti-CCP-Ab,CRP,TGFβ-1and MMP-7 in serum of rats were detected by ELISA.Immunohistochemistry and Western-blot were applied to detected the protein expression of Wnt5a,β-catenin,GSk3β,p-GSK-3β,MMP-7,E-cadherin andα-SMA in lung tissues.RT-PCR was applied to detect the expression of Wnt5a,β-catenin,GSk3β,MMP-7,E-cadherin andα-SMA genes in lung tissues.Results:1.Feibi originated from the Warring States to the Qin and Han Dynasties,improved during the Sui,Tang,and Song dynasties,and flourished during the Ming and Qing dynasties.The etiology of Feibi is related to the five zang organs,with weakness of lung qi as the main factor.The theory of"seven emotions"as the etiology of Feibi has also emerged.There are 52 prescriptions and 111 drugs included,with the main meridians of the drugs being the lung,spleen,and stomach meridians.The efficacy is mainly in the categories of resolving phlegm,relieving cough,relieving asthma,and tonifying deficiency.The top ten drug frequencies are almond,platycodon grandiflorum,licorice,ginseng,pinellia ternata,tulip,coix seed,aster,poria cocos,and loquat leaf in order.The drug combination mainly includes three drugs:almond,platycodon grandiflorum,and tulip,13 drug association rules,two groups of core drugs to relieve cough,dissipate phlegm,relieve asthma,replenish deficiency and strengthen health.Cluster analysis based on K-means predicts three new clusters.As a result,it has obtained three new prescriptions based on k-means clustering analysis.2.（1）Compared with the blank group,there was no significant difference in body weight between RA group and RA-ILD group at day 0,day 7,day 14,day 21,day 28,while the body weight of RA group and RA-ILD group at day 35 was lower than that of the blank group（P<0.05）;（2）Compared with the blank group,the arthritis scores of RA group and RA-ILD group at day 7,day 14,day 21,day 28,day 35 were increased（P<0.05）.（3）ELISA results showed that the serum levels of RF,Anti-CCP-Ab,TNF-α,MMP-7,TGFβ-1 and CRP in RA and RA-ILD groups were increased compared with those in the blank group（P<0.05）,while the serum levels of Anti-CCP-Ab and TNF-αin RA-ILD group were increased than those in RA group（P<0.05）,at day 37 after the construction of RA-ILD for 4 weeks.（4）HE staining showed that the synovium of RA group and RA-ILD group showed obvious synovial hyperplasia and inflammatory infiltration at day 37 after the construction of RA-ILD for 4 weeks.The lungs of RA group and RA-ILD group showed obvious alveolar structural disorder,widened alveolar septum,thickened alveolar wall,and massive inflammatory cell exudation;On day 37 after 4weeks of RA-ILD construction,Masson staining observed obvious blue collagen fiber deposition and fiber foci.（5）The incidence of RA-ILD in RA-ILD group was higher than that in RA group at day 37 after the construction of RA-ILD for 4 weeks（P<0.05）.（6）Immunohistochemistry and WB results showed that the expressions of MMP-7 and IL-22 in lung tissue of RA group and RA-ILD group were significantly up-regulated compared with the blank group at day 37 after the construction of RA-ILD for 4 weeks（P<0.05）.3.（1）Compared with normal group,the degree of alveolar inflammation and fibrosis degree,the contents of RF,TNF-α,Anti-CCP-Ab,CRP,TGFβ-1and MMP-7 in serum,the levels of Wnt5a,β-catenin,GSk3β,p-GSK-3β,MMP-7,E-cadherin andα-SMA protein expression,Wnt5a,β-catenin,GSk3β,MMP-7,E-cadherin andα-SMA gene expression were increasedin lung tissues（P<0.05）.（2）Compared with model group,the degree of alveolar inflammation and fibrosis degree,the contents of RF,TNF-α,Anti-CCP-Ab,CRP,TGFβ-1and MMP-7 in serum,the levels of Wnt5a,β-catenin,GSk3β,p-GSK-3β,MMP-7,E-cadherin andα-SMA protein expression,Wnt5a,β-catenin,GSk3β,MMP-7,E-cadherin andα-SMA gene expression were decreased in Guiqi Bufei Decoction groups（P<0.05）,and the effect was more obvious in Guiqi Bufei Decoction medium concentration group and high-concentration group（P<0.05）.Conclusion:1.The etiology of Feibi is related to the five zang organs,and the main treatment is the weakness of lung qi.The main idea for the treatment is to strengthen the spleen and benefit the lung,and reduce qi and dissipate phlegm.2.The RA-ILD model of SD rats induced by CIA combined with BLM can be used as an experimental animal model to study the pathogenesis of RA-ILD.3.Guiqi Bufei Decoction may be delay the development of RA-ILD pulmonary fibrosis through inhibiting the activation of Wnt/β-catenin signaling pathway and regulating EMT.