MWYF Decoction Exerts Anti-pulmonary Fibrosis Effect By Regulating PINK1-mediated Mitophagy In AECⅡ

Objective:Pulmonary fibrosis is an irreversible interstitial disease in the respiratory system.At present,the drugs and methods for the treatment of pulmonary fibrosis are relatively single,with great differences in clinical efficacy and obvious side effects.On this basis,we use the method of integrated traditional chinese and western medicine,with modern biotechnology as a tool,to explore the molecular mechanism of traditional chinese medicine compound Maiwei Yangfei Decoction(MWYF)in the treatment of pulmonary fibrosis,and give full play to the characteristics of traditional Chinese medicine.Methods:1 Composition analysis of MWYFFirstly,the MWYF Decoction was prepared,the standard and test samples were treated well,and the chromatographic and mass spectrometric conditions were set.Finally,the MWYF Decoction was tested on the machine and the results were compared with the standard.2 Effect of MWYF on Pulmonary Fibrosis in MiceC57BL/6J mice were randomly divided into 6 groups,Control,Model,low-dose group(20 g/kg/d),medium-dose group(40 g/kg/d),high-dose group(60 g/kg/d)and pirfenidone group,with 10 mice in each group.After a week of adaptive feeding,the groups were given bleomycin intratracheal atomization,except for control group.The mice were administrated by by gavage on the second day after modeling.The Control group and the pirfenidone group were administrated of normal saline and pirfenidone corresponding to body surface area,respectively,while the other groups were administrated of MWYF.At the same time,the body weight changes of mice were detected every day.CT of the lungs was taken after the last administration at 21d,and lung function was detected.Lung tissues of mice that died suddenly after cervical spine amputation were taken and stored at-80℃ for later use.Paraffin section of lung tissue was performed,HE and Masson staining were performed,and hydroxyproline content in lung tissue of mice was detected.3 To explore the mechanism of MWYFDecoction against pulmonary fibrosis based on network pharmacologyThe active components of MWYF were obtained by TCMSP,TCMID database and HPLC.The obtained compounds were imported into TCMSP,SEA,PharmMapper and SwissTargetPrediction databases to obtain the potential targets of all compounds.The potential targets of MWYF can be obtained by integrating all the targets and eliminating the duplication.All the target information related to disease was obtained through OMIM,CTD,TTD and GeneCards suite database with the term "Pulmonary fibrosis".Mapping drug-related targets to disease-related targets to obtain the public target which is the potential target of MWYF in the treatment of pulmonary fibrosis.We obtain the core targets among the potential targets through the study of the relationship between potential targets and topological analysis.KEGG and go enrichment analysis of the core target was performed by DAVID database.4 MWYF Decoction exerts anti-pulmonary fibrosis effect by regulating PINK1-mediated mitophagy in AECⅡReal time qPCR was used to detect the differential expression of core targets screened by network pharmacology in lung tissue.The content of mitochondrial DNA(mtDNA)in lung tissue of mice was detected.The expression of TOM20 protein in lung tissue was observed by immunohistochemistry.ATP synthase and SP-C protein were co stained,and the location of mitochondrial aggregation was observed by immunofluorescence.The morphology of mitochondria was observed by transmission electron microscope.The expressions of PINK1,p62 and LC3 in lung tissues were detected by Western Blot.CCK8 was used to determine the concentration of intervention cells in drug-containing serum.MLE-12 and fibroblasts were co-cultured in vitro and divided into Control group,TGF-β group,20 g/kg/d group,40 g/kg/d group,and 60 g/kg/d group.The Control group was treated with drug-containing serum of control,the TGF-β group was treated with 10ng/mL TGF-β intervention(drug-containing serum in the Control group),and the 20 g/kg/d,40 g/kg/d and 60 g/kg/d drug-containing serum intervention in groups A,B and C(all containing TGF-β),respectively.JC-1 fluorescent probe was used to detect the changes of mitochondrial membrane potential of MLE-12 cells in the co-culture model.ROS content of MLE-12 in the co-culture model was detected by DCFH-DA.The changes of mitophagy in MLE-12 cells in co-culture model were observed by immunofluorescence.MLE-12 cells were cultured in vitro and transfected with PINK 1-shRNA plasmid and Con-shRNA empty plasmid,respectively,and cell co-culture model was established with mouse lung fibroblasts,respectively.The co-culture models were divided into Control group,TGF-β group,PINK1-shRNA group,Con-shRNA group,MWYF-DS+TGF-β group,and MWYF-DS+TGF-β+PINK1-shRNA group.In the Control group,non-transfected MLE-12 and fibroblasts were co-cultured with drug-containing serum of the control group,and in the TGF-β group,non-transfected MLE-12 and fibroblasts were interfered with with drug-containing serum of the Control group containing 10 ng/mL TGF-β.In PINK1-shRNA group,MLE-12 transfected by PINK1-shRNA plasmid and fibroblasts were interacted with drug-containing serum in the control group.In Con-shRNA group,we used drug-containing serum of the control group to interfere with MLE-12 transfected with Con-shRNA empty plasmid and fibroblasts.The MWYF-DS+TGF-β group was treated with group C drug-containing serum(10 ng/mL TGF-β)to intervene fibroblasts and MLE-12 transfected with Con-shRNA plasmid.The MWYF-DS+TGF-β group was treated with group C drug-containing serum(10 ng/mL TGF-β)to intervene fibroblasts and MLE-12 transfected with PINK1-shRNA plasmid.Twenty-four hours later,the protein expression and gene transcription ofα-SMA and Collagen I in lung fibroblasts were detected by Western Blot and real-time qPCR.The content of hydroxyproline in the medium of cell co-culture model was detected by ELISA.The changes of mitophagy in MLE-12 cells in co-culture model were observed by immunofluorescence.Results:1 Composition analysis of MWYFAfter comparing with the standard,9 compounds were found,which were ephedrine,Liquiritin,ferulic acid,hesperidin,lobetyolin,rosmarinic acid,baicalin,glycyrrhizic acid and schisandrol A.2 Effect of MWYF on Pulmonary Fibrosis in MiceMaiwei Yangyang Fei Decoction can significantly alleviate the rate of weight loss and the changes of lung imaging,improve the lung function,and enhance the compliance of the lung tissue of mice.HE and Masson staining showed that the formula could reduce inflammation,collagen deposition and hydroxyproline content in lung tissue.3 To explore the mechanism of MWYFDecoction against pulmonary fibrosis based on network pharmacologyA total of 179 active components and 260 potential targets of MWYF were obtained.There are 1657 disease-related targets and 124 public targets of drugs and diseases.19 core targets were screened by topology analysis,and the most different signal pathway was mitochondrial autophagy.4 MWYF Decoction exerts anti-pulmonary fibrosis effect by regulating PINK1-mediated mitophagy in AEC ⅡThe detection of mtDNA in lung tissue showed that the number of mitochondria in the model group was significantly increased,and mtDNA showed an obvious downward trend after drug intervention,especially in the high-dose group;The immunohistochemical results of TOM20 were similar to the trend of mtDNA,and immunofluorescence of ATP synthase showed that the accumulated mitochondria were mainly concentrated in epithelial cells;Transmission electron microscopy of lung tissue showed that there were a large number of abnormal mitochondria in the epithelial cells of the model group,and the number of abnormal mitochondria decreased significantly with the increase of MWYF concentration;The analysis of the mitochondria’s morphology showed that in the lung tissue treated by MWYF tended to be normal gradually;CCK8 assay showed that the optimal drug-containing serum concentration of the intervention cells was group C;Through the detection of MLE-12 cells in the cell co-culture model,it was shown that MWYF could improve the mitochondrial membrane potential in MLE-12 and reduce the intracellular ROS content;Mitochondrial autophagy related proteins PINK1,p62 and LC3 in lung tissues were detected by Western Blot,which showed that MWYF could reduce the contents of p62 and LC3Ⅱ/LC3Ⅰ in lung tissues treated with bleomycin(n=3),and increase the expression of PINK1 protein in lung tissues(n=3);Immunofluorescence detection of lysosomal and PINK1 in MLE-12 cells showed that MWYF could promote mitochondrial autophagy;MLE-12 transfected with PINK1-shRNA plasmid were co-cultured with fibroblasts.Immunofluorescence showed that the effect of drug-containing serum of MWYF decreased significantly after low expression of PINK1(n=3);Western Blot and real-time qPCR showed that the regulation of α-SMA and Collagen Ⅰ proteins and genes in the co-cultured fibroblasts was dependent on the expression of PINK1;The content of hydroxyproline in cell supernatant was detected by ELISA;After PINK1 expression was low,the drug-containing serum lost its regulation effect on hydroxyproline.Conclusions:1 MWYF can alleviate bleomycin-induced pulmonary fibrosis in mice.2 MWYF can reduce the number of mitochondria and improve the function of mitochondria in AECⅡ.3 MWYF plays an anti-pulmonary fibrosis role by regulating PINK 1-mediated mitochondrial autophagy in AEC Ⅱ.