The Role And Mechanism Of LXRα And Its Upstream And Downstream Molecules In Experimental Silicosis Model

ObjectiveSilicosis is a disease mainly caused by lung tissue fibrosis caused by long-term inhalation of dust with excessive free Si O₂.It is known that myofibroblasts are the main terminal effector cells that cause widespread fibrosis of lung tissue.A number of studies have shown that when transcription factors（TFs）are activated or inhibited,a significant phenotypic transition can occur between fibroblasts and myofibroblasts.In the early stage,our research group has screened out 7 core TFs closely related to silicosis through high-throughput transcriptome sequencing technology,namely EGR2,BHLHE40,TBX2,LXRα,NR2F1,PPARγ,EPAS1,among which the liver X receptor（LXRα）is at a low expression level in the silicosis model.As a ligand-activated transcription factor,LXRαcan participate in the regulation of tumors and fibrosis and other diseases.However,its regulatory role and mechanism in the occurrence and development of silicosis have not been reported in detail at present.Therefore,this research group intends to explore the role of LXRαand its upstream and downstream molecules in silicosis by constructing experimental mouse silicosis models and in vitro models of silica-induced fibroblast transdifferentiation,in order to provide certain theoretical basis and methods for monitoring and intervention in silicosis.Methods1.The expression of related molecules upstream and downstream of LXRαin the silicosis model1.1 Construction and identification of mouse silicosis model100 male C57BL6/N mice were randomly divided into experimental groups at different time points（including 1 day,3 days,7 days,14 days,28 days,42 days and56 days）and the corresponding normal saline control group.Instillation of 50μL of Si O₂dust suspension（50 mg/m L）or equal volume of normal saline by tracheal instillation was used to establish a mouse silicosis model.The lung tissues of mice on the 1,7,28,and 56 days after dust staining were taken,and the lesions were observed by HE and Masson staining.1.2 Observation of LXRαupstream and downstream gene expression in lung tissue of silicosis modelThe lung tissues of mice on day 1,3,7,14,28,42 and 56 days after dust were collected,and the change trend of LXRαexpression level before and after treatment was detected by RT-q PCR.Based on the change trend of LXRαlevel,select the appropriate dust time point to observe the changes in the expression levels of upstream molecules PPARγand downstream molecules ABCA1,PLTP,SREBF1 and BHLHE40.2.Experimental observation on the intervention effect of PPARγand LXRαin transdifferentiation model induced by Si O₂2.1 Construction and observation of Si O₂induced fibroblast transdifferentiation model in vitroMouse lung fibroblasts（NIH/3T3 cells）and mouse macrophages（RAW264.7cells）are routinely cultured.Si O₂dust（100μg/ml）stimulated macrophages and collected their supernatants,and constructed the co-culture model with lung fibroblasts,and then detected the changes in the expression of transdifferentiation-related indicators before and after the dust exposure.2.2 Experimental observation on the intervention effect of PPARγin the transdifferentiation model induced by Si O₂PPARγagonist（RSG）was added to the Si O₂-induced cell model,and Western Blot was used to detect the changes in the expression levels of PPARγ,LXRαand their downstream related molecules and transdifferentiation indicators COL1A1 and FN1 before and after lung fibroblasts transdifferentiation.2.3 Experimental observation of the intervention effect of LXRαin the transdifferentiation model induced by Si O₂After overexpressing or silencing the expression of LXRα,RT-q PCR and Western Blot were used to detect the expression of COL1A1 andα-SMA before and after Si O₂stimulation.At the same time,the expression levels of LXRαand its downstream molecules ABCA1,SREBF1 and PLTP were detected after in vitro intervention.4.Statistical analysisSPSS 27.0 was used for experimental data processing.Under the normal distribution of the data,the result was represented by the mean±standard deviation（±s）.The Student’s t test was used to compare the means between two independent samples,and the one-way analysis of variance was used to detect the difference in means between multiple groups.The inspection levelα=0.05.Results1.Observation of LXRαupstream and downstream gene levels in silicosis mouse model1.1 Identification of Si O₂induced mouse silicosis modelHE and Masson staining were performed on pathological sections of mouse lung tissues on day 1,3,7,28,and 56 after treatment.The results showed the degree of inflammatory infiltration of mouse lung tissue from day 1 to day 56 after dusting and the accumulation of collagen gradually increased.RT-q PCR detected that the expression level ofα-SMA in the lung tissues of mice increased significantly on the42th and 56th days after the dust treatment（P<0.05）,and the expression level of COL1A1 had increased significantly since the 28th day after the treatment.（P<0.05）,and its changing trend continued until the 56th day.1.2 Expression levels of LXRαupstream and downstream genes in lung tissues of mouse silicosis modelThe lung tissues of mice at different time points（including 1 day,3 days,7 days,14 days,28 days,42 days,and 56 days）after being infected with dust were collected,and then the LXRαexpression levels at different time points were detected by RT-q PCR technology.The results showed During the 14th to 56th days after dust exposure,the downward trend was relatively stable（P<0.05）.Therefore,the lung tissues were selected for detection of LXRα-related genes on the 28th to 56th days after dust exposure.RT-q PCR results showed that the expression level of PPARγshowed a significant downward trend on the 28th,42th and 56th days（P<0.05）;the expression of BHLHE40 increased significantly on the 42nd and 56th days after dust exposure（P<0.05）;ABCA1 decreased on the 42th and 56th days after dust exposure（P<0.05）;SREBF1 was significantly down-regulated on the 42th and 56th days after dust exposure（P<0.05）;PLTP was not significantly down-regulated until the 56th day after dust exposure（P<0.05）.2.Experimental observation on the intervention effect of LXRαupstream and downstream molecules in the transdifferentiation model in vitro2.1 Identification and observation of Si O₂induced transdifferentiation model of pulmonary fibroblasts in VitroWestern blot results showed that after Si O₂induced NIH/3T3 lung fibroblasts for48 h,the expression of COL1A1,FN1,andα-SMA were significantly increased（P<0.05）.RT-q PCR results showed that FN1 and COLIA1 showed a significant increase after Si O₂stimulation（P<0.05）.2.2 PPARγand LXRαhave an upstream and downstream targeted regulation relationshipAfter comparing the binding site of PPARγwith the selected LXRαpromoter sequence,it was concluded that PPARγmay regulate the transcriptional activity of LXRαgene through this binding site,and Western blot detected that after RSG induces NIH/3T3,PPARγand LXRαincreased expression level.2.3 Experimental observation on the transdifferentiation process of lung fibroblasts after intervention with LXRαAfter silencing LXRα,RT-q PCR results showed that compared with the si R-1+Si O₂group,SREBF1,PLTP,and ABCA1 showed a significant downward trend（P<0.05）.At the same time,the levels of COL1A1 and FN1 were significantly increased（P<0.05）;Western blot results showed that the expression levels of SREBF1 and PLTP were significantly down-regulated in the si R-1+Si O₂group compared with the si R-NC+Si O₂group（P<0.05）.After overexpression of LXRα,RT-q PCR results showed that:p EGFP-N1-LXRα+Si O₂compared with p EGFP-N1-vector+Si O₂group,PLTP and ABCA1 showed an up-regulation trend（P<0.05）,while the expression level of COL1A1 and FN1 were decreased（P<0.05）;Western blot results showed:p EGFP-N1-LXRα+Si O₂compared with p EGFP-N1-vector+Si O₂group,the expression levels of SREBF1 and ABCA1 were significantly increased（P<0.05）,while COL1A1 showed an obvious downward trend（P<0.05）.ConclusionsOverexpression of PPARγand LXRαcan slow down the transdifferentiation of lung fibroblasts induced by Si O₂,and in this process,LXRαcan play a significant regulatory role on lipid metabolism-related genes PLTP,SREBF1 and ABCA1.