Effects Of Chronic Cold Exposure On Proteomics Of Lung Tissue In Mice

ObjectiveA large number of clinical epidemiological studies have shown that chronic cold exposure is one of the independent risk factors for adverse respiratory events.Long term exposure to cold weather can increase the morbality and mortality of respiratory diseases,leading to an increase in the number of emergency inpatients in the respiratory department.Continuous inhalation of a large amount of dry cold air not only directly damages the respiratory tract,but also increases the risk of respiratory infection,induces asthma attacks,and aggravates lung injury caused by PM2.5 particles,endotoxin and other factors.Studies have shown that the lung injury caused by chronic cold exposure may be directly related to the local inflammatory reaction caused by cold stress,but the exact mechanism is still unclear.Although many previous studies have revealed that cold exposure can cause a series of pathophysiological changes in the respiratory system,including lung injury,and elaborated the possible molecular pathways related to the injury event and the corresponding potential treatment options,so far there is still a lack of effective clinical treatment.This is mainly because most of the previous studies only focus on limited signal pathways.At the same time,it is also related to the lack of experimental animal models of chronic cold exposure lung injury used to simulate long-term exposure to extremely cold environment.Proteomics is a large-scale study of the structure and function of proteins at the overall level.It is a technology that reveals biological behavior and gene expression regulation mechanism through quantitative detection of gene expression at the protein level.So far,a variety of proteomic analysis meth ods have been widely used in the research of respiratory diseases.Proteomics based on liquid chromatography-tandem mass spectrometry(LC-MS/MS)is a specific and sensitive tool for determining lung protein abundance,predicting biochemical reactions and clarifying specific activated proteins.Based on this,this study intends to first establish an experimental animal model of chronic cold exposure lung injury in mice that is used to simulate long-term exposure to extreme cold,and analyze the total protein in the lung tissue of mice after cold exposure through the proteomics method of LC-MS/MS,to explore the possible mechanism of lung inflammation reaction under low temperature exposure from the overall level,in order to further screen efficient diagnostic markers and explore potential therapeutic targets,And then provide the basis for protecting patients from the inflammatory damage induced by cold stress.MethodsForty healthy male C57BL/6 mice,weighing 18-22 g,aged 8 weeks,were randomly divided into a control group and a cold exposure group,with 20 mice in each group.All mice were fed adaptively for one week before conducting the experiment.The cold exposure group mice were used to establish a chronic cold exposure model using our self-developed method.Namely,the mice were placed in a low temperature artificial climate box at-20 ℃,without wind,and exposed for 4 hours per day for 2 weeks.The control group mice were exposed in a no wind chamber at 20-24 ℃.Two weeks later,the mice were euthanized and evaluated for lung tissue damage using HE staining.The inflammatory mediators interleukin-6(IL-6)and tumor necrosis factor(TNF-α)in bronchoalveolar lavage fluid and serum were analyzed using ELISA.The total proteome in mouse lung tissue was quantitatively analyzed using LC-MS/MS proteomics method.After bioinformatics analysis,differentially expressed proteins were screened and 20 of them were selected for PRM validation.ResultsHE staining showed that the lung tissue structure of the control group mice was normal,the alveolar septa were intact,and no significant pathological changes were observed.The lung tissue structure of mice in the cold exposure group was severely damaged,with alveolar septal rupture,edema thickening,bleeding,and infiltration of inflammatory cells.The lung injury score,IL-6 and TNF-α levels in alveolar lavage fluid and serum were significantly higher than those in the control group(P <0.05).The above results indicate that the model was successfully established.A total of 708483 secondary spectra were obtained by mass spectrometry analysis.After searching the database of protein theoretical data,a total of 201422 available effective spectra were obtained.After analysis,117361 peptides were identified,including 112583 specific peptides.The number of identified proteins was 6498,of which 5520 were quantifiable.The change threshold of the differential expression level>1.2 is significantly up-regulated,and<1/1.2 is significantly down-regulated.A total of 151 up-regulated proteins and 95 down-regulated proteins were found.In the enrichment analysis,we found a significant enrichment trend in GO functional types.In biological process,it includes 216 subclasses,the first three are thioester biosynthesis process and acetyl-coenzyme A biosynthesis process,Polysaccharide biosynthesis process,a total of 216 subclasses.In terms of cell composition,the first three are pigment complex,respiratory chain complex,mitochondrial respiratory chain and other related functions,totaling 72 subclasses.In terms of molecular function,the first three are pyruvate dehydrogenase activity,pyruvate dehydrogenase NADP+ activity and pyruvate dehydrogenase NAD+ activity,a total of 83 subclasses.A total of 26 distinct enrichment pathways were obtained for KEGG,the first three were dicarboxylic acid metabolism(mmu010630),citric acid cycle(TCA cycle)(mmu00020)and 2-oxycarboxylic acid metabolism(mmu01210),and immune-related complement pathways also changed.GABAA receptor α 2 subunits(gabra 2 gene coding),Pkal(Kl Kb1 gene coding)and complement related proteins were significantly up-regulated.In the PRM validation analysis,the results showed that most of the selected proteins were consistent with the results of proteomics,which proved the reliability of the results of proteomics.ConclusionsIn this study,we independently established an experimental animal model of chronic cold exposure induced lung injury in mice,and applied the proteomics technology based on LC-MS/MS to analyze the total proteomics of lung tissue.It has been confirmed that the pathological process of chronic cold exposure lung injury may be involved in multiple factors,including glycolysis,gabra2,KKS,and complement pathways.The above pathways involve inflammation,stress,energy metabolism,and other aspects,indicating that chronic cold exposure induced lung injury in mice is a complex multifactorial injury.The results of this study help to reveal the possible mechanisms of lung injury under cold exposure and provide new experimental data for potential biomarkers and therapeutic targets.However,more research is needed to further evaluate and confirm.