The Preliminary Study On The Biological Responses Of Pulmonary Fibrosis On Silicon Stress

Pneumoconiosis is a common occupational disease worldwide,especially in developing countries.The patient’s long-term exposure to a high concentration of dust particles causes the dust particles to continuously accumulate in the lungs,futher inducing chronic lung inflammation and irreversible fibrosis,which eventually leads to respiratory failure and death.The pathogenesis of pneumoconiosis is extremely complex.Researchers have proposed a variety of theories to explain the pathogenesis of pneumoconiosis,but most of them have not been validated by effective experiments.And there are no effective measures to treat fibrosis so far.Alternative splicing is a major contributor of proteome diversity and the regulation of gene expression,and has important significance in the process of biological growth and cell differentiation.When an organism or cell is strongly stimulated by the outside stress,it will produce a stress response and changes at the transcription level.At present,it has been proved that alternative splicing is closely related to the occurrence of various diseases.Therefore,alternative splicing is considered to be the basis for identifying new diagnostic biomarkers or new treatment methods for disease occurrence.The formation of pneumoconiosis requires four stages:injury,inflammation,fibroblast proliferation and migration,and fibrosis.Under the continuous stimulation of silica,macrophages,as an important defense line of the immune system,will secrete a large amount of cytokines and chemokines,such as IL6,IL1-β,TNF-α,TGF-βand so on.TGF-βis of great significance in the process of inflammatory response,and has the effect of stimulating the proliferation and activation of fibroblasts and promoting the development of fibrosis.The cell signaling pathway related to TGF-βis involved in various metabolic pathways in the human body,and the TGF-β—Smad3 signaling pathway has been shown to be closely related to the development of pulmonary fibrosis.In this paper,from the two perspectives of TGF-β—Smad3 signaling pathway and alternative splicing,the relationship between pneumoconiosis inflammation and fibrosis stage was studied,and the expression changes of gene alternative splicing events during fibrosis explored.Firstly,the occurrence of alternative splicing events in pneumoconiosis animal models was examined,and secondly we established a cell model from a single cell perspective to explore the biological responses of macrophages and fibroblasts in the pathogenesis of pneumoconiosis and the cascade between them.1.Verification of alternative splicing events in an animal model of pneumoconiosis:Based on the results of high-throughput sequencing analysis of fibrotic lung tissue in pneumoconiosis rats under SiO₂ stress,gene alternative splicing events with significant differences were selected for experimental verification analysis.Six genes with obvious differential alternative splicing events were identified by using semi-quantitative PCR.Compared with the control group,the inclusion rate of exon 4 in Dgkg and exon 6 in Tle4 under silica stress did not change,the inclusion rate of exon 23 in Ect2l,exon 4 in Mocs2,exon 27 in Rapgef6 are increased,while the rate of exon 2 inclusion of Phtf1 gene is decreased.2.Biological response of macrophages under silica stress:Macrophages were inoculated at a concentration of 3×10⁵ Cells/mL,and after overnight culture,macrophages were simultaneously stressed with silica at 100 ng/mL LPS for 48 h.The expression levels of IL1-βand TNF-αgenes in macrophages were significantly increased after stress.3.Cascade response of macrophages and fibroblasts under silicon dioxide stress:macrophages were seeded at a concentration of 3×10⁵ Cells/mL,and after overnight culture,macrophages were simultaneously stressed with 100 ng/mL LPS and 200μg/mL SiO₂ cell 48h.Fibroblasts were seeded in the transwell cell chamber at 5×10⁵ Cells/mL,and they were cultured for 24 h after adherence.The adherent fibroblasts for 24 hours were co-cultured with macrophages for 24 hours after stress.After co-cultivation,the macrophages showed an increase in volume,cell fragmentation,and long antennae.The number of fibroblasts increased,and the cells showed an irregular shape of slenderness.After 24 hours of co-culture,the expression of fibroblasts collagen genes is increased.4.Biological effect of fibroblasts stimulated by TGF-βfactor:Fibroblasts are seeded in a six-well plate at a concentration of 5×10⁵ Cells/mL,after adherence,the culture medium is replaced with a medium containing 0.5%FBS,and the culture continues 24 h.Add 10 ng/mL TGF-βfactor to stimulate fibroblasts for 12 h.After TGF-βstimulation,the expression of collagen andα-SMA genes in fibroblasts increased.This work verifies several differentially alternative splicing events in animal models during the fibrosis stage,and at the same time,a cell model under silica stress has been established.It may be helpful in understanding pathogenesis of pneumoconiosis and identifying new therapeutic targets for the pneumoconiosis.