The Mechanism Of SelM Knockout In Nickel Induced Pulmonary Fibrosis In Mice

Rock weathering,nickel-containing metal smelting,fertilization of farmland,decay of animal and plant debris and other reasons will lead to soil nickel pollution,which in turn leads to excessive nickel in feed,posing a serious threat to animal health.Animal toxicological studies and human epidemiological studies have shown that nickel inhalation can lead to pulmonary fibrosis,emphysema,alveolar protein deposition,and even lung cancer.However,the mechanism of pulmonary toxicity caused by dietary nickel intake is not clear.Selenium,as a part of the enzyme that catalyzes redox reactions in organisms,can protect cells from the harmful effects of free radicals and play an important antioxidant role.Selenium also helps prevent or treat lung diseases such as silicosis,asbestosis and coal workers’pneumoconiosis.Previous studies have found that selenoprotein M（SelM）is highly expressed in the lungs.However,the mechanism of lung injury caused by SelM deficie ncy and its protective mechanism against lung injury caused by nickel exposure have not been fully elucidated.Therefore,in order to explore the molecular mechanism of lung injury induced by SelM knockout and nickel exposure,and to study the role of SelM in lung injury of nickel-exposed mice,C57BL/6N male mice with wild-type and SelM knockout were selected as research objects,and the wild-type control group was given normal saline in the morning and evening.The wild type nickel exposure group was given nickel chloride（10 mg/kg）in the morning and normal saline in the evening.SelM^-/-control group was given normal saline in morning and evening;SelM^-/-nickel exposure group was given nickel chloride（10 mg/kg）in the morning and normal saline in the evening.After 14 days of continuous gavage,mice were sacrificed,and lung tissues were taken for H&E staining,Masson staining and electron microscope examination.q RT-PCR and Western blot were used to detect the expression of pulmonary fibrosis markers,TGF-β1/Smad signaling pathway and JAK2/STAT3 signaling pathway related genes,in order to better understand the mechanism of SelM in nickel-induced pulmonary fibrosis.Specific research results are as follows:1.The mental state of wild type control mice w as stable.In the wild type nickel exposure group,the mice were slightly excited in the early stage,fluffy and unglossy in the middle stage,reduced food intake and water intake,low response to external stimuli,and wheezing in the late stage.SelM^-/-mice in the control group suffered from mental depression and mild wheezing.SelM^-/-nickel exposure group was characterized by depression,shortness of breath,lethargy,loss of appetite,emaciation,unkempt,dry and dull coat,and some mice died later.2.The H&E staining results showed that the lung tissues of the wild-type control group showed normal morphology.In the wild-type nickel exposure group,the alveolar structure was destroyed,the alveolar wall was thickened,the alveolar cavity became smal ler,and a large number of inflammatory cells were infiltrated and fibrotic nodules were observed.In SelM^-/-control group,the alveolar wall was thickened,the lung tissue structure was damaged,and the alveolar cavity collapsed.The SelM^-/-nickel exposed group had the thickest alveolar walls with surrounding inflammatory cell infiltration and dense fibrosis.Compared with SelM^-/-control group,wild-type nickel exposure group showed increased alveolar wall thickness and more inflammatory cell aggregatio n.Masson staining showed that the alveolar structure of wild type control group was clear.In the wild-type nickel exposed group,the number of collagen fibers increased,there was extensive blue collagen deposition,and the collagen fibers expanded.In SelM^-/-control group,the number of collagen fibers increased significantly and there was obvious blue staining.In the SelM^-/-nickel exposed group,the matrix was significantly increased with diffuse distribution and collagen fibers fused into clusters.Compared with SelM^-/-control group,the area of blue staining increased in the wild-type nickel exposure group.The results of electron microscopy showed that some elastic fibers and collagen fibers were deposited in the lung interstitial of the wild-type nickel exposure group,and the basement membrane was obviously thickened.In SelM^-/-control group,collagen fibers appeared in lung interstitium,and the cell structure was incomplete.Compared with the wild-type nickel exposure group,the SelM^-/-nickel exposure group had a large amount of collagen fiber proliferation and deposition in the lung interstitium,and collagen fibers appeared in bundles.3.The determination results of malondialdehyde（MDA）,superoxide dismutase（SOD）,total antioxidant capacity（T-AOC）and glutathione peroxidase（GSH-Px）related indexes of lung oxidative stress in mice showed that SelM knockout alone or nickel treatment alone could cause lung oxidative stress in mice（P<0.05）.Nickel exposure resulted in more severe oxidative stress than the SelM^-/-control group.SelM knockdown increased nickel-induced oxidative stress in lung tissue（P<0.05）.4.The mRNA and protein expression levels of SelM in lung were significantly decreased by nickel exposure in wild-type control group and wild-type nickel exposure group（P<0.05）.Compared with wild type control group and wild type nickel exposure group,SelM mRNA and protein were not expressed in SelM^-/-control group and SelM^-/-nickel exposure group（P<0.05）,indicating that SelM knockout model was successfully established.SelM knockout alone or nickel treatment alone could significantly up-regulate the protein and gene expression levels ofα-smooth muscle actin（α-SMA）and type I collagen（COL-I）（P<0.05）,and the protein and gene expression levels of TGF-β1/Smad signaling pathway marker were significantly up-regulated（P<0.05）.The ratio of P-SMad2/Smad2 and P-SMad3/Smad3 increased（P<0.05）.Nickel exposure can lead to more obvious up-regulation of fibrosis marker protein and gene expression levels,and SelM knockout aggrava ted the up-regulation ofα-SMA,COL-I,TGF-β1 mRNA and protein levels in nickel-induced lung tissue（P<0.05）.The ratio of P-SMad2/Smad2 and P-SMad3/Smad3 was increased（P<0.05）.These results suggest that TGF-β1/Smad signaling pathway is involved in SelM knockout and dietary nickel-induced pulmonary fibrosis in mice,and plays an important role in exacerbating nickel-induced pulmonary fibrosis by SelM knockout.5.SelM elimination alone or nickel treatment alone can up-regulate the gene expression levels of JAK2 and STAT3（P<0.05）,and increase the ratio of P-JAK2/JAK2 and P-STAT3/STAT3（P<0.05）.Nickel exposure can lead to the upregulation of protein and gene expression levels of more obvious fibrosis markers.SelM knockout increased the upregulation o f JAK2and STAT3 mRNA levels in nickel-induced lung tissues（P<0.05）,and increased the ratio of P-JAK2/JAK2 and P-STAT3/STAT3（P<0.05）.These results suggest that the JAK2/STAT3signaling pathway is involved in SelM knockout and dietary nickel-induced pulmonary fibrosis in mice,and plays an important regulatory role in the intensification of nickel-induced pulmonary fibrosis by SelM knockout.Combined with these findings,dietary nickel intake and SelM knockout both contribute to the development of pulmonary fibrosis through activation of oxidative stress,TGF-β1/Smad signaling pathways and JAK2/STAT3 pathways.SelM knockout exacerbates nickel-induced pulmonary fibrosis through activation of oxidative stress,TGF-β1/Smad signaling pathways,and JAK2/STAT3 pathways.It lays a foundation for further research on the mechanism of lung injury induced by nickel exposure and the protective effect of SelM,and provides certain insights.