Study On PD-1/IL-17A/TGF-β1 Molecular Immune Mechanism Of Gancao Ganjiang Decoction In The Treatment Of Idiopathic Pulmonary Fibrosis

Objective:Gancao Ganjiang decoction(GGD)is a prescription of traditional Chinese medicine for the treatment of“atrophic lung disease”.The purpose of this study was to explore the mechanism of GGD in the treatment of bleomycin(BLM)-induced idiopathic pulmonary fibrosis(IPF)in C57BL/6J mice and to explore its immune mechanism in the treatment of IPF.This will provide a basis for the research and development of new traditional Chinese medicine about GGD.Methods:1.To study the mechanism of GGD in the treatment of IPF based on network pharmacology.GGD compound targets and IPF targets were searched in databases.Compound target network,compound-disease target network and target interaction network were constructed by Cytoscape 3.7.2 software,and the key targets were analyzed.Cytoscape 3.7.2 software plug-in Clue Go was used for GO and KEGG analysis of key targets,and the potential mechanism of GGD in the treatment of IPF was obtained.2.Preparation of GGD and HPLC analysis of its compounds.According to the Jingui Yaolue,the proportion of Glycyrrhiza uralensis Fisch.and Zingiber officinale Roscoe.is2:1.It was soaked in 10 times the volume of distilled water for 0.5 h,then heated for 0.5 h,filtered and collected,repeated twice.The extract was concentrated under reduced pressure and freeze-dried.According to the records of Pharmacopoeia of the People’s Republic of China(2020 edition)and the results of network pharmacology,the contents of liquiritin,glycyrrhizic acid and 6-gingerol in GGD freeze-dried powder were detected by HPLC.3.Replication of IPF murine model and treatment of GGD.The mice were randomly divided into control group,BLM group,positive control group(pirfenidone,0.3 g/kg),high dose GGD group(4.8 g/kg),middle dose GGD group(2.4 g/kg)and low dose GGD group(1.2 g/kg).The model of IPF in C57BL/6J mice was established by intratracheal instillation of BLM(3.5 mg/kg).The mice were treated with GGD for 14 days and 28 days.The weight changes of mice were recorded.After anesthesia,blood was collected from the inferior vena cava,the lung tissue was collected and the pulmonary index was calculated.4.Improvement of GGD on inflammation and extracellular matrix(ECM)deposition in IPF mice.The lung tissue structure,inflammation and collagen deposition were observed by HE and Masson staining.According to the reported evaluation methods,the inflammation and fibrosis of lung tissue in mice were scored.The content of hydroxyproline,a marker of collagen deposition in ECM,was detected by ELISA.5.Immunomodulatory effect of GGD on IPF mice.The proportion of PD-1~+CD4~+T cells in peripheral blood of mice was detected by flow cytometry.The contents of PD-1,p-STAT3 and IL-17A in lung tissue of mice were detected by immunohistochemistry.6.Inhibition of GGD on epithelial-mesenchymal transformation(EMT)in IPF mice.The content of TGF-β1 in lung tissue was detected by ELISA.According to the level ofβ-actin normalization,the gene expression and protein content of EMT markers E-cadherin,vimentin andα-SMA in lung tissue were detected by q RT-PCR and western blot.Results:1.34 compounds were selected from GGD.Network topology analysis screened out 88 key targets.The enrichment analysis results of KEGG include 145significant related pathways including HIF-1 signaling pathway,PI3K-Akt signaling pathway,Fox O signaling pathway,TNF signaling pathway,IL-17 signaling pathway,etc(P<0.01).It involves 966 significantly related biological processes such as regulating cell proliferation,migration,adhesion,hematopoietic or lymphoid organ development and leukocyte differentiation,and regulating response to external stimuli(P<0.01).2.The HPLC results showed that the retention times of liquiritin,glycyrrhizic acid and 6-gingerol in GGD freeze-dried powder were 17.75min,39.91min and 41.34min,respectively.The contents of liquiritin,glycyrrhizic acid and 6-gingerol are 30.06 mg/g,71.21 mg/g and 7.08 mg/g(n=3).3.The body weight of mice induced by BLM decreased significantly with time(P<0.01).GGD could significantly increase the body weight of mice,especially in the high dose group.On the other hand,the pulmonary index of the BLM group was higher than that of the control group(P<0.01).The pulmonary index decreased after treatment with high-dose GGD(P<0.05).4.HE staining showed that the inflammatory infiltration and inflammatory score of lung tissue in GGD group were lower than those in BLM group.The results of Masson staining showed that the lung tissue structure of mice in GGD group tended to be regular,the aggregation of collagen fibers decreased,and the score of fibrosis in GGD group was significantly lower than that in BLM group(P<0.05).The results of ELISA showed that the content of hydroxyproline in lung tissue of GGD group was significantly lower than that of BLM group(P<0.01,P<0.05).5.After GGD treatment,compared with BLM group,the proportion of PD-1~+CD4~+T in peripheral blood(P<0.01)and the content of PD-1 in lung tissue decreased(P<0.01,P<0.05).The results of immunohistochemical showed that the contents of p-STAT3 and IL-17A in lung tissue of mice decreased(P<0.01,P<0.05).6.After GGD treatment,compared with BLM group,the content of TGF-β1 in lung tissue of mice decreased(P<0.05),the expression of E-cadherin increased(P<0.01,P<0.05),and the expression of vimentin(P<0.01,P<0.05)andα-SMA decreased.Conclusion:The main mechanism of GGD in the treatment of IPF is to inhibit inflammation and immune regulation,and then affect the EMT and ECM deposition in lung tissue.These effects were associated with the decrease of the expression of PD-1 in lung tissue and the decrease of the proportion of PD-1~+CD4~+T cells in peripheral blood,which negatively affected the production of IL-17A and TGF-β1,and interfered with the levels of E-cadherin,vimentin,α-SMA and hydroxyproline.These results suggested that GGD can be used as a potential drug for the treatment of IPF,which provides a basis for related research and clinical drug use.