Establishment Of A Rat Model Of Pulmonary Fibrosis Induced By Silica And The Analysis Of Transcriptome Sequencing Data

At present,pneumoconiosis is one of the most serious occupational diseases in China.With the continuous development of social economy and industrialization,especially in developing countries,the problem of dust pollution has become increasingly serious.Long-term work in high-concentration dust environments without protective measures can seriously threaten workers'health.According to the data released by the National Health and Family Planning Commission for the National Occupational Disease Report,the number of Chinese pneumoconiosis patients is extremely large and has been ranked first in the world for many years.Since the reform and opening up,China has reported more than830,000 occupational diseases,and the number of new cases is still rising year by year.The pathogenesis of pneumoconiosis is extremely complicated,and there are many related theories,including the body's immune response,tissue damage repair,accumulation of myofibroblasts and extracellular matrix.However,there is no complete and reliable theory at present to explain this pathological mechanism,and related research is still concerned by many scholars.Biological tissues are prone to pathological changes under external stimulation or stress.At the same time,RNA changes extensively at the level of the whole transcriptome,regulating the expression of genes,which is an important mechanism of biological stress response.At the same time,external stimuli,growth factors or ligands affect the intracellular kinase and other factors through receptor transmembrane conduction,further affecting the splicing factor activity and regulating the alternative splicing of many pre-mRNA.Related studies have found that spirulina has the effect of improving the body's immunity and resisting external stimuli to some extent.Dexamethasone is a common adjuvant drug for the treatment of fibrotic diseases,which can improve microcirculation in the body and relieve the body's inflammatory response.In this paper,based on the establishment of rat silicosis model under SiO₂ stress and high-throughput sequencing,the genes involved in the regulation of lung fibrosis expression and changes were deeply screened,and the changes of gene expression level and alternative splicing pattern were studied.At the same time,the effects of spirulina and dexamethasone on the fibrosis process of lung tissue were explored,which provided a reference for the follow-up study on the prevention and treatment of pneumoconiosis.The details are as follows:1.Establishment of a pulmonary fibrosis rat model:dust stress was applied to SD rats.Normal saline was infused with SiO₂ suspension of 45 mg/mL and 125 mg/mL by non-tracheal exposure method.On 14 days,28 days and 56 days,rats were sacrificed at daytime,and their lung tissues were taken for morphological observation,HE staining and Sirius red staining.The degree of pulmonary fibrosis was determined by detecting the content of hydroxyproline and cytokines in the lung tissue.The sectioning results showed that the degree of fibrosis of the lung tissue was aggravated with the increase of the concentration of SiO₂ treatment.The content of hydroxyproline and cytokines in lung tissue was positively correlated with the treatment concentration and time.The results are consistent with the pathological features of various stages of pneumoconiosis.The establishing of a rat silicosis model will lay the foundations for the subsequent study of the molecular biological mechanism of pneumoconiosis.2.High-throughput sequencing of gene changes in lung tissue fibrosis:high-throughput sequencing was used to detect gene changes in the 45 mg/mL SiO₂ treatment group and the saline control group at 56 days,and to screen for differentially significant expression genes or differentially alternative splicing pattern.It was found that differential genes were mainly enriched in metabolic pathways such as immune response and signal transduction.Molecular biological experiments were carried out by means of real-time fluorescent quantitative PCR and semi-quantitative PCR to investigate the molecular mechanism of pneumoconiosis.The results showed that the expression trends of each differential gene were consistent with the sequencing results,and the correlation was good.Partially variable alternative splicing genes did not change significantly.3.Effects of spirulina and dexamethasone on pulmonary fibrosis:based on the establishment of rat lung fibrosis model under SiO₂ stress,the effects of spirulina and dexamethasone on pulmonary fibrosis were investigated.A blank control group,a saline control group,a 125 mg/mL SiO₂ perfusion group,a spirulina group and a dexamethasone group were respectively set.Among them,the spirulina group and the dexamethasone group were also perfused with 125 mg/mL SiO₂ suspension.The rats were sacrificed at 14days,28 days,and 56 days.The effects of spirulina and dexamethasone on the fibrosis of lung tissue were analyzed by morphological observation,HE staining,Sirius red staining and Elisa of related cytokines.It has been found that spirulina and dexamethasone have a certain retardation effect on the fibrosis process of lung tissue,and dexamethasone is more effective in preventing pneumoconiosis.