| Objective:Pulmonary fibrosis,also known as pulmonary interstitial fibrosis(PF),is a diffuse inflammatory disease of the lungs caused by a variety of causes.The lesions mainly involve the lung interstitial and alveolar epithelial cells and pulmonary blood vessels.The main pathological changes are alveolar structural disorders,diffuse alveolitis and pulmonary fibrosis.The clinical manifestations are progressively increased dyspnea and irritating dry cough.The pathological characteristics include restricted ventilation dysfunction,hypoxemia,and chronic,Progressive,diffuse fibrosis of the pulmonary interstitial.Clinical morbidity and mortality are high,which highly affect people’s life,health and safety.At present,researchers have not developed very targeted therapeutic drugs.The whole-genome high-throughput sequencing analysis of pulmonary fibrosis rats revealed that Notch1 / Jagged1 signaling pathway is widely present in pulmonary fibrosis.Studies have found that some traditional Chinese medicine compounds or single drugs can regulate the occurrence and development of pulmonary fibrosis by activating the Notch1/Jagged1 pathway.This experiment is based on the research results of the National Natural Science Foundation of China “Study on the Mechanism and Material basis of Buyang Huanwu Decoction for pulmonary fibrosis in rats by the microvascular angiogenesis based on miRNA”(No.81874442).The human embryo lung fibroblasts(MRC-5)were stimulated with TGF-β before and after the model was given to the treatment of Jiawei Buyang Huanwu decoction,and the Notch/Jagged1 pathway was intervened by si-RNA technology for exploring the specific mechanism and characteristics of Jiawei Buyang huanwu decoction against pulmonary fibrosis.Method:1.High-throughput sequencing analysis of the whole transcriptome of pulmonary fibrosis rats.Twelve 7-week SD rats were randomly divided into two groups,the control group and the experimental group each had 6 rats.The model group was subjected to fibrosis modeling.The blank group was not treated.After 28 days of treatment,the rats were treated with broken vertebrae and lung tissues were taken.They were stored in liquid nitrogen and 4% paraformaldehyde fixatives for subsequent RNA sequencing and pathological observation.Section HE staining confirmed the success of lung fibrosis modeling.Sequencing and analysis of experimental group and control group by high-throughput sequencing,analysis of expression patterns of lnc RNAs,mRNAs,circ RNAs,and miRNAs,respectively,and screening of ce RAN from the difference table,and GO and KEGG enrichment Analyze the pathways and genes related to fibrosis.2.Experimental Observation of Jiawei Buyang Huanwu Decoction on Pathological Morphology of Lung Tissue in Rats with Pulmonary Fibrosis.Divided into blank group,model group,Chinese medicine(Jiawei Buyang huanwu Decoction)group,prednisone group.Bleomycin was used to replicate a rat model of pulmonary fibrosis.On the 14th and 28th days,the general conditions of rats in each group were observed,the general morphological changes of lung tissue,HE staining,masson staining,and Westen blot and RT-PCR were used to detect Notch1,Jagged1 and Hes-1Protein and mRNA expression.3.Jiawei Buyang Huanwu Decoction Intervenes Notch Signaling Pathway to Regulate MRC-5 Cells.RNA interference technology(siRNA)was used to screen out the target sites with the best interference effect,which were divided into normal cell group,siRNA-silent Notch1 group,TGF-β stimulation group alone,siRNA-silencing TGF-β stimulation group,and Jiawei Buyang Huanwu Decoction was given to the TGF-β-treated group after 1 hour of intervention,and 48 hours after the Notch1 siRNA transfection was added to the TGF-β-stimulated group.Cells biochemically detected the expression of Jagged1 and Hes-1 proteins after siRNA silencing Notch;Western blot and RT-PCR were used to detect the expression levels of Notch1 protein and mRNA in each group of cells.Result:1.High-throughput sequencing analysis of the whole transcriptome of pulmonary fibrosis rats.In this study,the experimental group and control group were sequenced and analyzed by high-throughput sequencing,and the expression patterns of lnc RNAs,mRNAs,circ RNAs,and miRNAs were sequenced,and differentially expressed non-coding RNAs were screened,and Perform GO and KEGG enrichment analysis to mine related signal pathways and genes.Full transcriptome sequencing analysis found that Notch signaling pathway was significantly enriched in pulmonary fibrosis rats(Figure 2-6B),and the expression of related genes had changed significantly.It is speculated that in the occurrence of pulmonary fibrosis,The Notch signaling pathway has important regulatory effects,and the role of anti-fibrotic drugs may be closely related to intervention in this pathway.The specific regulatory mechanism needs to be further verified,providing favorable evidence for subsequent experiments.2.Experimental Observation of Jiawei Buyang Huanwu Decoction on Pathological Morphology of Lung Tissue in Rats with Pulmonary Fibrosis.2.1 HE staining state: compared with the blank group,the lung tissue remained relatively intact at the 14th day of each administration group,showing a slight edema inside the tissue,the infiltration of related inflammatory cells was obvious,and the disease state was modified by Buyang Huanwu Decoction The stronger Songsong group was lighter;on the 28th day,the relevant cells showed hyperplasia,collagen deposition occurred in the pleura and other places,and a clear trend of pulmonary fibrosis could be observed.The degree was stronger in other groups.2.2 Masson staining state: Compared with the blank group,the thickness of the collagen layer in the bronchus of the experimental rats is very small,and a certain color of fiber distribution can be observed.For the model group,the width of different internal alveoli becomes larger on the 14th day,and the appearance of fibroblasts can be observed in the lung interstitial and other locations,and there is a certain degree of blue form of deposition;on the 28th day The structure of the alveoli showed a high degree of destruction,and the bundled or other forms of fibrous deposition were observed in the interstitial lung,which developed into a representative fibrotic state.The degree of fibrosis in the lung tissue of the Jiawei Buyang Huanwu decoction group was significantly lower than that of the model group,and also significantly improved compared with the prednisone group.2.3 The results of Westen blot showed that compared with the blank group,the Notch1,Jagged1,and Hes-1 proteins in the lung tissue of the model group were significantly increased(P <0.01),and the expression of the 28th day protein was the most significant.The expression levels of Notch1,Jagged1,and Hes-1 protein at the respective time points of the Huanwutang group and the prednisone group were significantly lower than those of the model group.Among them,the Jiaweibuyanghuanwutang group had the most significant decrease on the 28th day,which was similar to the prednisone group The ratio was statistically significant(P<0.05).The protein expression at each time point in the drug group was lower than that in the model group.2.4 The results of RT-PCR method can be seen,compared with the normal group,the levels of Notch1,Jagged1,Hes-1 mRNA in the model group,prednisone group,and flavored Jaiwei Buyang Huanwu decoction group were significantly increased,and the differences between the two groups were different.Compared with the model group,the relative expression levels of Notch1,Jagged1,Hes-1 mRNA were decreased after intervention with prednisone and Jiawei Buyang Huanwu decoction,and the modified expression was reduced with modified flavor.The 28-day intervention in the Jiawei Buyang Huanwu decoction group had the most significant decrease,and the pairwise comparison within the group was significantly different(P<0.05),which had statistical significance.The prednisone group was statistically significant compared with the Jiawei Buyang Huanwu doction group in the same period.3.Modified Buyang Huanwu Decoction Intervenes Notch Signaling Pathway to Regulate MRC-5 Cells.3.1 Cell biochemical detection of Jagged1 and Hes-1 protein expression after siRNA silencing Notch,the results suggest that compared with the normal cell group,the expression of Jagged1 and Hes-1 in the Notch1 siRNA silencing group was significantly reduced.After β-modeling,the expression of Jagged1 and Hes-1 in cells increased significantly.Compared with the model group,the expression of Jagged1 and Hes-1 in cells decreased after Notch silencing and TGF-β stimulation.But higher than the normal group.In comparison,compared to the modified flavor of Jiawei Buyang Huanwu decoction group,the content of siRNA silencing was significantly increased with or without siRNA,compared with the blank group,but the value of siRNA silencing group was significantly lower.3.2 Western blot and RT-PCR showed that compared with the normal cell group,the Notch1 expression in the siRNA-silent Notch1 group and the TGF-β stimulation group after siRNA silencing were significantly reduced.Compared with the TGF-βstimulated group,the Notch1 group in siRNA silencing and the TGF-β stimulated group in siRNA silencing reduced the expression of Notch1 in the cells more significantly.After the intervention of Jiawei Buyang Huanwu Decoction for 1 hour,the group was treated with TGF-β,and Notch-siRNA was transfected 48 hours later.Significantly lower than the TGF-β-stimulated group.Conclusion:Jiawei buyang Huanwu Decoction can inhibit the Notch/Jagged1 signal pathway,down-regulate the expression level of Notch1 to play a protective role,and then improve the fibrosis level of MRC-5 cells.It is speculated that Modified Buyang Huanwu Decoction may interfere with Notch/Jagged1 signal transduction Pathway to play an anti-fibrotic effect,and has a certain pre-protection effect. |
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