Effect And Mechanism Of Honeysuckle Petroleum Ether On Pulmonary Fibrosis

Objective: The pharmacodynamic activity and mechanism of petroleum ether part of traditional Chinese medicine honeysuckle on idiopathic pulmonary fibrosis were investigated,and on this basis,preliminary prediction on its possible effective components was carried out.Efficacy research and mechanism analysis were conducted on petroleum ether part of honeysuckle through the establishment of cell model in vitro,and on the basis of it,the conclusions of animal models were drawn and verified by using in vivo cell model to further confirm the efficacy.At the same time,GC-MS and LC-MS techniques were applied to analyze the chemical composition of petroleum ether of honeysuckle and to conduct the preliminary prediction of its possible effective components.Methods: 1.In vitro experiment: Based on the in vitro TGF-?1 induced A549 epithelial-to-mesenchymal transition cell model that has been confirmed and used in the laboratory,RT-PCR was applied to detect the expression of epithelial-to-mesenchymal transition(EMT)related genes,E-cadherin gene,Vimentin gene and ?-SMA gene;ELISA was used to detect the content changes of HYP in the cell supernatant,and in vitro efficacy and mechanism of petroleum ether part of honeysuckle on idiopathic pulmonary fibrosis were preliminarily studied.2.In vivo experiment: Based on the IPF model of bleomycin(BLM)induced C57BL/6 mice,lungs pathology was observed by HE and Masson staining through paraffin sections of lung tissue of mice;the expression of HYP in lung tissue of mice was detected by RT-PCR;the expression of Col I and marker genes in EMT,E-cadherin,vimentin and ?-SMA was detected by RT-PCR.Then,in vivo validation was carried out on the in vitro cell test results.3.GC-MS and LC-MS analysis on petroleum ether part of honeysuckle were conducted,and its possible effective components were preliminary predicted.Results: 1.In vitro experiment: The morphological observation showed that petroleum ether part of honeysuckle can effectively inhibit epithelial-mesenchymal transition of TGF-?1-induced A549 cells,so that fibrocyte cells would be transformed into normal epithelial cells;RT-PCR results showed that compared with TGF-?1-induced group,the expression of E-cadherin mRNA in the group of honeysuckle petroleum ether with drug was up-regulated,and the expression of Col I,?-SMA,Vimentin mRNA and protein was down-regulated;the ELISA results indicated that compared with the group with drug,the content of hydroxyproline in the model group was decreased.2.In vito experiment: The results of HE and Masson staining showed that in the sham-operation group,the structure of lung tissue was clear,and there was no bleeding and small collagen deposition.In the model group,the lung wall was obviously thicken,the lungs septum was significantly smaller;substantial lesion showed piece-shaped,and there was obvious collagen deposition;in the honeysuckle petroleum ether group,the alveolar wall was slightly thickened,alveolar septum was clear;substantial lesion was reduced,and there was no obvious collagen deposition,improved compared with the model group.The ELISA results showed that in the group of honeysuckle petroleum ether with drug,the content of hydroxyproline in lung tissue homogenate was significantly lower than that of the model group,and the RT-PCR results showed that the E-cadherin m RNA expression was up-regulated,and Col I,?-SMA and Vimentin mRNA was down-regulated.3.By GC-MS technology and database retrieval,22 compounds were identified,including fatty acids,lipids,sterols and aldehydes.4.The UPLC-Q-Extractive/MS technology was used to qualitatively analyze the effective parts of honeysuckle,and a total of 14 chemical constituents were identified,such as chlorogenic acid,chlorogenic acid,caffeic acid and other phenolic acids.Conclusions: Honeysuckle petroleum ether can effectively inhibit the gene expression of important protein of extracellular matrix of TGF-?1-induced A549 cell,Col I,reduce thesecretion of the main amino acid component of this collagen,hydroxyproline.It is proved that in the in vitro experiment,honeysuckle petroleum ether has therapeutic effect on idiopathic pulmonary fibrosis.On the basis,RT-PCR technique was applied to investigate the level changes of marker proteins?-SMA,Vimentin and E-cadherin during EMT.The results indicated that honeysuckle petroleum ether can inhibit the EMT process,proving that the mechanism of the therapeutic effect of honeysuckle on idiopathic pulmonary fibrosis in the in vitro experiment may be related to its inhibition on EMT.On this basis,its therapeutic efficacy was further validated through the bleomycin-induced in vivo fibrosis animal model,and it can be seen from HE,MASSON and ELISA results that honeysuckle petroleum ether has therapeutic effect on bleomycin-induced pulmonary fibrosis mice.RT-PCR results also showed that its action mechanism may be achieved through the inhibition of EMT process of epithelial cells.the main components of the petroleum ether of honeysuckle found by GC-MS and LC-MS techniques are fatty acids,lipids,sterols,aldehydes,and phenolic acids,suggesting that such components and their combinations may be potential active substances for anti idiopathic pulmonary fibrosis.In conclusion,honeysuckle petroleum ether is a potential therapeutic drug for idiopathic pulmonary fibrosis,and its mechanism may be achieved through the regulation on marker genes of EMT,vimentin,?-SMA and E-cadherin.This study provides an important experimental basis for the discovery of new drugs for the treatment of pulmonary fibrosis.