The Study On Anti-pulmonary Fibrosis Effect And Anti-inflammatory Mechanism Of Dry Extract Of Compound Mango Leaves

Pulmonary fibrosis is a disease caused by many factors with diffuse alveolar inflammation and alveolar structure disorder eventually leading to the pulmonary interstitial fibrosis.The main pathological characteristics of the disease are early acute inflammatory response of the lower respiratory tract following pathological hyperplasia and transformation of a large number of fibroblasts,accompanying to some sedimentation of extracellular matrix in alveoli and interstitial tissue and the disorder of lung tissue structure,alveolar damage and collagen deposition as a result of excessive fibrous tissue repair,eventually leading to the formation of pulmonary fibrosis.Along with the development of the industrialization,there is an upward trend in incidence of pulmonary fibrosis,because of its lack of effective treatment,poor prognosis,high mortality,which cause serious damage to human health and life.The pathogenesis of pulmonary fibrosis is not clear.Inflammation,immunoregulation and collagen deposition are the important reasons to the pathogenesis of pulmonary fibrosis.At present,glucocorticoid and immunosuppressants are the main drugs in the clinic.It can alleviate some symptoms only to a certain extent,but cannot fundamentally reverse and defense the disease with varying degrees of side effects.For pulmonary fibrosis,the Chinese medicine with the characteristic of rich clinical experience and multiple-component,multiple-target,multiple-pathway treatment had received the scholars' attention at home and aboard in recent years.Dry extract of CML is a compound prescription set up according to clinical experience for many years by Jia Gang Deng,who is the guangxi tenured professor and director doctor of traditional Chinese medicine.The research taked dry extract of CML as target to investigate the effects or mechanism on the relevent pathology,cytokines,different proteins of the pulmonary fibrosis model caused by BLM.Then,it screened the effective anti-inflammatory compouds by inflammation cell model and investigate the effects of related inflammatory pathways on pulmonary fibrosis.The main research contents and results are as follows:1 ? Study of the treatment of dry extract of CML on pulmonary fibrosis: The anti-inflammatory,antioxidant and anti-pulmonary fibrosis effects of dry extract of CML were evaluated in terms of the observation of representation and pathology,levels of HYP,inflammation cytokines and oxidative stress cytokines by establishing the model of pulmonary fibrosis tracheal intubation with BLM.The results showed that general condition of the mice in high dose presented smooth breathing,lively action,clean hair and normal diet and the general condition of the mice in middle or low dose were also improved.The lung index of all administration groups were significantly lower than the model group(P<0.001).It suggested that dry extract of CML could protect the status and lung tissue of pulmonary fibrosis mice.Pathology showed that there was a wide distribution structural damage and a large number of inflammatory exudate in part of the bronchial and alveolar cavity leading to moderate to severe alveolar inflammation with fibrous tissue hyperplasia and fibrosis of lung consolidation by HE staining.The lung structures had normal morphology with less pulmonary alveolitis,fibrosis and thick alveolar interval after administration.The lung tissue with less fibrosis after administration by Masson staining.The degree of alveolitis inflammation of all administration groups were significantly lower than the model group by Szapiel score(P<0.001).The degree of Fibrosis were significantly lower in the high and middle dose groups than the model group by Ashcroft score(P<0.001).The levels of HYP of all administration groups were significantly lower than the model group(P<0.001).It suggested that dry extract of CML could protect the lung tissue by reducing alveolitis,collagen deposition and fibrosis in lung.Study of anti-inflammatory and antioxidant showed that the levels of IL-1?,IL-6 and TNF-? were significantly lower in the high dose group than the model group(P<0.001 or P<0.01).The levels of IL-6 and TNF-? were significantly lower in the middle dose group than the model group(P<0.001 or P<0.05).The level of MDA and the levels of SOD and T-AOC were significantly lower and higher after administration,respectively(P<0.001 or P<0.01 or P<0.05).The level of GSH was significantly higher in the high and middle dose groups than the model group(P<0.001).It suggested that dry extract of CML had anti-inflammatory and antioxidant effects on pulmonary fibrosis.2?Study of the mechanism of resistance to pulmonary fibrosis of dry extract of CML: The mechanism of resistance to pulmonary fibrosis of dry extract of CML was analyzed by i TRAQ to quantitative analysis of different proteins in the lung tissue of mice and by functional annotation to analyze the enrichment of GO?COG?Pathway.The results showed that there were about 1700 different proteins which participated in biological process of defense response ? inflammatory response ? immune response ?regulation of blood coagulation etc.with molecular function such as peptidase regulator activity?enzyme inhibitor activity?structural molecule activity?calcium ion binding etc.in some places including extracellular region?extracellular space?extracellular matrix?cell surface?nucleosome etc.in the dry extract of CML treatment.The mechanism of dry extract of CML treatment of pulmonary fibrosis was that 73 main different proteins such as Itgb1 and FGF1 worked on 26 pathways such as PI3K-Akt signaling pathway and Focal adhesion closely related to inflammation,immunoregulation,ECM deposition,muscle contraction and Oxidative stress.It suggested that the pathogenesis of pulmonary fibrosis is a complex biological processe which is built of factors in multiple target proteins,signal transduction pathways and biological functions.The dry extract of CML treatment of pulmonary fibrosis could work by the biological functions including inhibition of inflammation,regulation of immunity,reduction of extracellular matrix formation,ease of muscle contraction and antioxidation.3?Study of chemomics of dry extract of CML: The chemomics of dry extract of CML was studied according to determination of the molecular ion peak in the primary mass spectrogram and the analysis of pieces information in the secondary mass spectrogram to presume compound Structure by the UPLC-Q-TOF/MS analysis technology.The study characterized and analyzed 48 ingredients including 26 saponins,10 benzophenones,3 flavonoids,5 phenolic acids,3 anthraquinones and 1 coumarin.4?Study of anti-inflammatory compound screening of dry extract of CML and mechanism prediction: The anti-inflammatory compound screening of dry extract of CML was studied in TNF-? stimulated bronchial epithelial cell(BEAS-2B)by UPLC-Q-TOF/MS combined with NF-?B dual-luciferase reporter assay systems.The anti-inflammatory ingredients were studied in network pharmacology to predicting their target proteins and related pathways by Pharm Mapper and KEGG bioinformatics softwares.The anti-inflammatory verification and synergistic effect of antiinflammatory ingredients Mangiferin,Psoralen,Ginsenoside Rg1 were analysised by dual-luciferase reporter assay systems and ELISA.The results showed: it is 16 anti-inflammatory ingredients including Iriflophenone-3-C-?-D-glucoside,Methyl gallate,mangiferin,Iriflophenone-3-C-(2-O-galloyl)-?-D-glucoside,2,4',6-trihydroxy-4-methoxybenzophenone-3-C-?-D-glucopyranoside,Notoginsenoside R1,Ginseno side Rg1,Ginsenoside Re,Psoralen,physcion,Notoginsenoside R2,Ginsenoside Rg2,Ginsenoside Rh1,Ginsenoside Rc,Rubiadin-1-methyl ether,Notoginsenoside K in dry extract of dry extract of CML.16 anti-inflammatory components work on 11 pathways such as MAPK and Focal adhesion by acting on 40 target proteins such as HRAS and MAP2K1.There is a high correlation between the pathways such as MAPK,Focal adhesion,VEGF and the anti-inflammatory components.The about suggested dry extract of CML played a role of anti-inflammatory by multiple components,targets and pathways.The network pharmacology about MAPK pathway showed Mangiferin worked on HRAS and FGFR1,Psoralen worked on MAP2K1 and FGFR1,Ginsenoside Rg1 worked on MAP2K1 and BRAF in MAPK pathway.The verify study of anti-inflammatory components showed Mangiferin?Psoralen?Ginseno side Rg1 decreased the levels of NF-?B,IL-6 and IL-8 in the cells(P<0.001 or P<0.01 or P<0.05).It suggested that Mangiferin?Psoralen?Ginsenoside Rg1 can play a role of Inflammation inhibiting respectively.The collaborative study of anti-inflamma tory components showed the level of IL-6 was obviously lower by the cooperation of Mangiferin and Psoralen than that Mangiferin or Psoralen worked alone(P<0.01).The levels of NF-?B,IL-6 and IL-8 were obviously lower by the cooperation of Psoralen and Ginsenoside Rg1 than that Psoralen or Ginsenoside Rg1 worked alone(P<0.001 or P<0.01 or P<0.05).It suggested that Mangiferin?Psoralen?Ginseno side Rg1 can effectively suppress inflammation synergistically.In summary,firstly,these studies demonstrated that dry extract of CML played a role in protecting lung tissue by resistancing to lung tissue damage and pulmonary fibrosis on the animal level.Inhibition of collagen deposition and effects of anti-inflammatory and antioxidant may be the mechanisms.Secondly,it certificated that dry extract of dry extract of CML played a biological mechanism in multipletargets,multiple-pathways and multiple-factors treatment of pulmonary fibrosis on the protein level.The anti-inflammatory signaling pathways are related to the treatment of pulmonary fibrosis mostly.Thirdly,it analyzed the chemical characterization of dry extract of CML by UPLC-Q-TOF/MS.Then,it illuminated the anti-inflammatory material basis on a cellular level and manifested the components cooperate mutually in the process of resistancing to inflammation.Finally,the relevance of “ingredientstargets-pathways” of dry extract of CML pointed out the mechanism for the treatment of pulmonary fibrosis was a complex biological processes.So,the study has the important significance and application prospect and it will be required to refine research plan further according to the requirements of new drug to research and development to be the sixth class national new drugs.