Study On Molecular Mechanism Of DNA Damage And Mutations Induced By PM_2.5 In HBE Cells

Objective:PM2.5 can enter the bronchi and alveoli directly through the respiratory tract and deposit in the alveoli.When a certain amount is reached,it can cause inflammatory and oxidative stress reactions in upper respiratory cells and alveolar cells,and can regulate protein synthesis by regulating gene expression,thereby inducing cell apoptosis and DNA damage.The purpose of this study is to investigate the molecular mechanism of PM2.5 on Human bronchial epithelial（HBE）cells apoptosis,DNA damage and mutations,and to carry out a series of studies to provide a scientific basis for further research on PM2.5’s toxicity to human bronchus and to explore atmospheric fine particles The toxicological mechanism of PM2.5 carcinogenesis and mutagenesis is of great significance.Methods:1、HBE cells were poisoned with a low-dose 10μg/mL and a high-dose 50μg/mL PM2.5 aqueous solution,and untreated cells were used as blank control groups.At the same time,10μM Cr⁶⁺-infected HBE cells were established as positive controls,Real-time quantitative PCR（qPCR）was used to detect changes in mRNA expression levels of apoptotic genes Caspase-3,Caspase-8,Caspase-9,Bax,Bcl-2,Western blot detection of protein expression of apoptosis genes Caspase-3,Caspase-8,Caspase-9,Bax,Bcl-2.2、HBE cells were treated with p38MAPK inhibitor SB203580,ERK MAPK inhibitor PD98059 and PCK inhibitor CHE in advance for 30 min,and then HBE cells were infected with PM2.5 aqueous solution at a dose of 50μg/L for 24 h,at the same concentration and The PM2.5 aqueous solution was simply infected with HBE cells as the control group,and the untreated cells were used as the blank control group,and an inhibitor control group（ie,a simple inhibitor-treated group）was set.Detecting the changes of apoptotic genes Caspase-3,Caspase-8,Caspase-9,Bax,Bcl-2in gene and protein expression levels,Real-time quantitative PCR（Q-PCR）was used to detect changes in the expression level of apoptotic genes,and Western blot was used to detect changes in the expression of apoptotic genes.3、HBE cells were poisoned with PM2.5 aqueous solutions at a concentration of 10μg/mL and 50μg/mL,and untreated cells were used as blank control groups.At the same time,10μM Cr⁶⁺-infected HBE cells were established as a positive control group.10μM Cr⁶⁺was used as a positive control group;the changes in the expression levels of DNA damage repair genes hOGG1 and hMTH1 were detected;real-time fluorescent quantitative PCR（Q-PCR）was used to detect the changes in the expression levels of the above DNA damage genes;Western blot was used to detect the DNA Changes in protein expression of damage repair genes;HBE cells were exposed to PM2.5（0,8,20,50μg/mL）in gradient concentration,and single cell gel electrophoresis was used to detect cell DNA damage.4、Five kinds of histidine-deficient Salmonella typhimurium were used to detect the pollutant mutagenicity of the treated PM2.5 samples by the plate infiltration method,that is,the standard Ames test.The experiment set up four PM2.5 water soluble substance exposure dose groups:40,100,200 and 400μg/dish,Set up a negative control group and a positive control group at the same time,each bacteria is set up with and without S9 two test groups5、HBE cells were infected with PM2.5 water-soluble substance at a concentration of 50μg/mL for 24 h,and non-poisoned cells were used as a blank control group.RNA samples extracted from three parallel samples were set up to complete fluorescent labeling and purification and hybridize with the chip.The Rosetta Resolver?System（Rosetta Biosoftware）was used for data preprocessing and statistical calculation,and the differentially expressed genes were obtained by analysis.The data were analyzed by cluster profiler software for principal component and cluster analysis,Pathway and GO analysis,and the differential expression of genes in HBE cells before and after PM2.5 exposures was initially discussed;the protein-protein interaction relationship of differentially expressed genes was analyzed through the String protein interaction database The core protein was selected with the largest number of nodes.Finally,core genes related to PM2.5-induced HBE cell mutation,canceration,and DNA damage were selected,and gene verification was performed using q-PCR.Results1、The q-PCR results showed that compared with the control group,the relative expression levels of the pro-apoptotic genes Caspase-3,Caspase-8,Caspase-9,and Bax were increased,and the relative expression level of apoptotic suppressor gene Bcl-2 decreased significantly.The relative expression levels of pro-apoptotic genes Caspase-3,Caspase-8,Caspase-9,Bax and apoptotic suppressor gene Bcl-2 detected by Western blot were consistent with q-PCR results.2、Add P38 inhibitor（SB203580）,ERK inhibitor（PD98059）,and PKC inhibitor（CHE）before PM2.5 exposure,SB203580+50μg/mL PM2.5 group compared with the simple 50μg/mL PM2.5 group.The relative expression levels of the pro-apoptotic genes Caspase-3,Caspase-8,Caspase-9,and Bax all decreased,and the relative expression levels of the genes suppressing the apoptotic gene Bcl-2 significantly increased;The results of the relative expression levels of the caspase-3,Caspase-8,Caspase-9,Bax and Bcl-2 inhibitory genes were consistent with the q-PCR results.3、The q-PCR results showed that compared with the control group,the relative expression levels of the DNA damage repair genes hOGG1and hMTH1 were increased after HBE cells were exposed to 10μg/mL and 50μg/mL PM2.5 aqueous solution and the positive control Cr6+.Western blot results showed that compared with the control group,the relative expression levels of the DNA damage repair genes hOGG1 and hMTH1 were consistent with the relative expression levels of the genes.Single cell gel electrophoresis detected tail DNA content,tail length,tail distance,and olive tail distance of the PM2.5-stained groups at 8,20,and50μg/mL were significantly higher than those of the control group,and there was a dose-response relationship between the dose groups.4、Ames results showed that when the PM2.5 concentration was 40μg/dish and 200μg/dish,the difference between the colony return variables of the TA97 strain with and without S9 was statistically significant（p<0.01）;5 When the concentration is 400μg/dish,the difference between the colony return variables with S9 and without S9,TA98 and TA100 is statistically significant（p<0.05 or p<0.01）5、According to preset screening conditions,245 differentially expressed genes were selected from HBE cells exposed to PM2.5aqueous solution,of which 27 genes were up-regulated and 218 genes were down-regulated.After further analysis,the top ten core genes that were up-regulated and down-regulated were screened.The top 10differential gene GO（Gene Ontolog）functional annotation enrichment analysis results show that differentially expressed genes are mainly concentrated in molecular functions such as transmembrane receptor activity,receptor activity,cell signaling,and cell molecular conduction.At the same time,it is enriched in biological processes such as ion transmembrane transport,cell-to-polysaccharide inorganic ion transmembrane transport,and cell nutrient transport;Pathway and GO enrichment analysis results of the top 10 differentially expressed genes show that the differentially expressed genes are mainly concentrated in 7core pathways related to tumor cell migration,bile metabolism related,neural activity,genetic toxicity,etc.The protein interaction network map screened the five proteins with the most interactions,and finally selected WFDC1,SFRP1,and PHACTR1 as the core genes.The q-PCR verification results were consistent with the three core gene expression trends detected by the gene chip.Conclusion1.The results show that exposure to PM2.5 aqueous solution can cause apoptosis of human bronchial epithelial cells（HBE）.2.It is suggested that p38MAPK,ERK MAPK and PKC signaling pathways can effectively inhibit the apoptosis effect of PM2.5 exposure on HBE cells.3.The PM2.5 aqueous solution has an effect on the DNA damage of HBE cells.4.Ames experiment results The results show that PM2.5 has direct and indirect mutagenic effects.5、Gene chip results showed that PM2.5 caused HBE cells to differentially express their related genes through some pathways and biological functions,and the core genes screened from the differentially expressed gene profile.And the core genes selected from the differentially expressed gene profile can provide important clues for the further study of the molecular mechanism of PM2.5 on HBE cell apoptosis,DNA damage and its mutation.