| Pulmonary fibrosis is the abbreviation of diffuse interstitial lung disease,in which the extracellular matrix is deposited progressively,the structure of lung tissue is destroyed,and pathologic processes of alveolate changes of pulmonary tissue and the gradual loss of lung function can be observed through imageology.The formation of myofibroblasts characterized by high expression of a-smooth muscle actin(a-SMA)is the core link of pulmonary fibrosis,and myofibroblasts are mainly derived from the activation and transformation of fibroblasts in situ or epithelial-mesenchymal transition two pathways,Thus,blocking the formation of myofibroblasts are expected to be a new strategies for the treatment of pulmonary fibrosis.Since there havn’t been fully effective western medicine to cure Pulmonary fibrosis,traditional anti-inflammatory,anti-oxidative and anti-fibrotic drugs can not bring substantial clinical efficacy.The research of targeted medicine is a novel direction of pulmonary fibrosis treatment currently.It highlights the importance of the exploration of mechanism of pulmonary fibrosis.Traditional Chinese medicine has unique features in the prevention and treatment of pulmonary fibrosis.The idea of prevention and treatment that combine the treatment upon syndrome differentiation and disease differentiation has received a well curative eflfects,such as the alleviation of the suffering caused by disease,improvement of the life quality of patients,and the prolongation of lifetime.The variety and complexity of Chinese medicine prescriptions have hindered the widespread application of Chinese medicine in the treatment of pulmonary fibrosis.Therefore,the selection and study in-depth of the traditional Chinese medicine compound with substantial clinical efficacy and reliable theoretical basis for the disease has important significance and practically clinical value.Yangfei Huoxue Decoction is a prescription has been applicated in clinic for many years,it has a good effect on the treatment of pulmonary fibers,and there have been many achievements in the study of its mechanism of treatment.Therefore,based on the research of the Doctoral Research Foundation of the Ministry of Education,this paper relies on the National Natural Science Foundation of China,focusing on the activation and transformation of lung fibroblasts-myofibroblasts and the role of traditional Chinese compound prescription Yangfei Huoxue Decoction in phenotypic transformation.Pulmonary fibrosis has a high incidence and mortality.Research on its pathogenesis and treatment methods has always been one of the hot spots in medical research.The study on pulmonary fibrosis has been widely reported by Chinese and Western medicine researcher.New progress and achievements are extremely prominent.Therefore,the first part of the dissertation focuses on the literature review,it summarizes the research progress of pulmonary fibrosis and phenotypic transformation of myofibroblasts in western medicine,describes the etiology and pathogenesis of Pulmonary fibrosis,and lays a foundation for discussion on the curative mechanism of traditional Chinese medicine.In addition,the research progress of traditional Chinese medicine on pulmonary fibrosis prescriptions is reviewed,and the theoretical basis for the treatment of pulmonary fibrosis by traditional Chinese medicine is clarified.The mechanism of effective prescription is the core part of the study of pulmonary fibrosis.Therefore,the second part of the thesis focuses on the intervention and regulatory mechanisrm of Yangfei Huoxue Decoction in the transformation of myofibroblasts.Methods:1.The whole genome methylation sequencing and functional analysis methods were used to observe the effects of the model group and the Yangfei Huoxue Decoction group.A rat model of pulmonary fibrosis was replicated by one-time administration of bleomycin(BLM).Normal rats were not modeled.Rats in the normal group and the model group were given intragastric administration for 14 days and the Yangfei Huoxue fang group was administered orally with Yangfei Huoxue Huoxue concentrated solution for 14 days.After 14 days,the rats were executed and the lung tissues were taken.HE staining,Masson staining,and electron microscopy experiments comprehensively reflect the success of rat pulmonary fibrosis model,and then whole genome methylation sulfite sequencing(WGBS)method were used for normal group,model group,Yangfei Huoxue Decoction medication group to completing Whole-genome methylation sequencing and functional analysis of lung tissue.2.The effect of Yangfei Huoxue Decoction on the function of myofibroblasts was observed on the basis of rat primary lung fiber model.Pulmonary fibroblasts were obtained by primary culture of rat lung tissue and purification.TGF-β1 was used to transform lung fibroblasts into myofibroblasts.At the same time,rabbit serum containing Yangfei Huoxue Decoction was prepared.We used the method of in vitro cell assay,such as immunofluorescence qualitative to detect the a-SMA fluorescence expression position and intensity,Western Blot quantitative to determinate the a-SMA protein expression,collagen contraction visually to reflect the clhanges in myofibroblast contractility.3.The effect of Yangfei Huoxue Decoction on myofibroblast transformation was studied from the Notch signaling pathway by in vitro cell assay.Western Blot was used to detect changes in Notch receptors,ligands,and downstream effectors,and the co-immunoprecipitation method was used to reflect the interaction of Notch intracellular domain(NICD1)with the nuclear effector protein CSL.Lentivirus was used to transfect Notch 1 gene into fibroblasts and the cells were also tested for the expression of intracellular COL-I after administration of different doses of Yangfei Huoxue Decoction.4.The main components of Yangfei Huoxue Decoction were detected by liquid chromatography and mass spectrometry(LC-MS).According to the compound dose of Yangfei Huoxue Decoction,the Chinese medicinal materials were weighed,the total composition of the compound was extracted with alcohol,and the composition of the Yangfei Huoxue Decoction was detected and analyzed by the LC-MS.Results:1.with the analysis of methylation levels of different chromosomes,different levels of chromosomal methylation we can be seen in different samples,CG regional methylation difference in blank,model and Yangfei Huoxue Decoction group is not significant,the main different genes was enriched in CHG,CHH regions.By clustering the methylation information of different functional regions of different sample genomes,the differences in the methylation status of the three groups,blank,model,and Yangfei Huoxue Decoction can slso be observed.At the same time,Yangfei Huoxue Decoction and blank group have more similarities,it indicate that Yangfei Huoxue Decoction has the effect of regulating DNA methylation.2.The treatment of fibroblasts with TGF-β1(5ng/mL)for 24 hours can induce the transformation of fibroblasts to myofibroblasts,The expression of a-SMA is induced with immunohistochemistry,and the fiberyarn in the TGF-β1 treated group are obvious.After treatment with Yangfeihuoxuefang drug-containing serum,fibroblast-muscular fibroblast transformation was significantly inhibited,expression of a-SMA,COL-I,and COL-III was decreased,and collagen contraction was inhibited.3.The serum containing Yangfei Huoxue Decoction in high,medium and low doses all can reduce the expression of Notch receptors Notch 1,Notch3,ligands Jagged 1,DLL4,and downstream effector CSL;Co-immunoprecipitation shows that drug-containing serum can reduce NICD1 the intracellular part of Notch 1 transformation to nucleus and interaction with CSL and then blocked the formatiuon of downstream products;the content of COL-1 in fibroblasts transfected with virus containing Notch 1 was increased,and the expression of COL-I in fibroblasts was significantly decreased after the treatment with Yangfei Huoxuefang-containing serum.4.The Chinese herbal compound contains the active ingredients of the single drug,seven standard products were referenced in this experiment and we totally detected catechins,ferulic acid,resveratrol glycosides,calycosin-7-O-glucoside,salvianolic acid B,Schizandrin B,isoimperatorin and other more ingredients.Conclusions:1.Yangfei Huoxue Decoction can regulate DNA methylation.2.Yangfei Huoxue Decoction drug-containing serum can inhibit the activation of myofibroblasts induced by TGF-β1;It also can inhibit the activation of Notch signaling pathway,and regulate fibroblast-myofibroblast phenotype Transformation.Main innovation:1.High-throughput whole-genome methylation sequencing and functional analysis methods were used to investigate the effect of Yangfei Huoxue Decoction on gene methylation of pulmonary fibrosis from the gene level,which laid a good foundation for further in-depth study.2.With the Notch signaling pathway as the focus,on the basis of the rat primary cell lung fibrosis model,from the perspective of affecting the phenotypic transformation of lung fibroblasts,the main mechanism of multi-target effect of Yangfei Huoxue Decoction was revealed..3,We use of liquid chromatography and mass spectrometry technology,for the first time to detect the composition analysis on Yangfei Huoxue Decoction,clarifying the main active ingredients and providing a reliable basis for traditional Chinese medicine on the clinical research of pulmonary fibrosis. |
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