| Objectives The gene expression profile of A549 cells treated with nano-silica dioxide（SiO2）is analyzed by bioinformatics.At the same time,the in vitro model of alveolar typeⅡepithelial cells exposed to nano-SiO2 is established by A549 cells,and the toxic effect of nano-SiO2 on alveolar epithelial cells are observed.The changes of pulmonary surfactant protein-A（SP-A）and pulmonary surfactant protein-B（SP-B）influenced by nano-SiO2 in A549 cells and its regulatory mechanism are discussed from JNK signaling pathway.It is expected to provide a new research direction for the lung injury and toxic mechanism of nano-SiO2.Methods 1 Firstly,the gene chip data was searched according to the keywords"nano-SiO2,A549 and 24 h".Then,the differential genes were screened by online analysis platform,and the biological process and regulatory pathway of genes were also enriched and analyzed.Secondly,A549 cells were routinely cultured and divided into control group（0μg/m L）and nano-SiO2 group（25,50,75,100μg/m L）for 24 h.The cells morphology of each group was observed,and the survival rate,apoptosis rate and reactive oxygen species（ROS）of each group were also detected.The CD63 was observed by immunofluorescence,and the expression of SP-A and SP-B m RNA in each group were detected by RT-q PCR,the protein expression of SP-A and SP-B were detected by western blot.Finally,the optimal concentration of nano-SiO2 on A549 cells was eventually screened after compared the differences among the groups.2 Cells were set for different groups:control group,nano-SiO2 group,N-acetyl-L-cysteine（NAC）group and nano-SiO2+NAC group.After treated with optimal concentration of nano-SiO2 for 1,3,6,12 and 24 h respectively,fluorescence labeled probe method was used to detect the expression of ROS levels in different groups,while SP-A,SP-B,JNK,c-jun and c-fos protein in each group were detected by western blot,and univariate analysis of variance and repeated measurement analysis were used to compare the differences among the groups to screen the appropriate time of nano-SiO2.Then,after pretreated with ROS inhibitor NAC and JNK inhibitor SP600125,the cells were set to different groups including control group,nano-SiO2 group,inhibitor group and nano-SiO2+inhibitor group.After cells were treated with the optimal concentration of nano-SiO2for a certain time,the expression of JNK,c-jun,SP-A and SP-B protein in each group were detected.Finally,after A549 cells transfected with c-jun-siRNA,the cells were set for control group,nano-SiO2 group,c-jun-siRNA group and nano-SiO2+c-jun-siRNA group.Treating cells with the same method,the expressions of c-jun,SP-A and SP-B protein in each group were detected and compared.Results 1 The gene chip data GSE63806 screened according to the keywords including160 differential genes with 84 up-regulated genes and 76 down-regulated genes.In addition,the gene expression of SFTPA,SFTPB,SFTPC and SFTPD in nano-SiO2 group were all down-regulated,but there had no different compared to the control group（P>0.05）.Biological process enrichment were mainly concentrated in cell surface receptor signal pathway,regulation of signal transduction,regulation of response to stimulus and so on,while pathway enrichment were mainly concentrated in pathway in cancer,MAPK signal pathway and so on.2 Compared with the control group,A549 cells gradually lost their normal morphology with the increase of the concentration of nano-SiO2,while the apoptosis rate increased and the survival rate decreased（P<0.01）.In addition,the expression of ROS increased gradually and reached the maximum at 75μg/m L nano-SiO2 treated group（P<0.05）.Compared with the control group,the expression of CD63 and SP-A,SP-B m RNA and protein decreased gradually with the increase of nano-SiO2（P<0.01）.Combined with the level of ROS and the changes of SP-A and SP-B,the concentration of nano-SiO2 of 75μg/m L was selected for the follow-up experiments.3 Compared with the control group（0μg/m L）,when A549 cells were treated with 75μg/m L nano-SiO2 for 1,3,6,12,24 h,the expression of ROS increased at first and then decreased,reaching the maximum at 6 h,and the inhibitor NAC could successfully inhibit the expression of ROS（P<0.05）.In addition,the expression of SP-A protein were significantly inhibited from 1 h,and the inhibitory effect was strong at 6 h（P<0.05）,but there were no significant changes at 12～24 h.The expression of SP-B protein decreased significantly from 1 h and reached the lowest at 24 h（P<0.05）.The JNK and c-jun protein in nano-SiO2 group were significantly activated and reached the maximum at 6 h（P<0.05）,while the c-fos protein was not activated and there were no significant difference between the nano-SiO2 group and control group（P>0.05）.Finally,6 h was selected as the best treatment time for the follow-up experiments.4 Compared with nano-SiO2 group,it was found that JNK and c-jun protein were significantly inhibited at 6 h after pretreatment with inhibitor NAC,while SP-A and SP-B protein expression were increased in nano-SiO2+NAC inhibitor group（P<0.05）.After pretreatment with inhibitor SP600125,it was found that compared with nano-SiO2 group,JNK and c-jun protein were significantly inhibited in nano-SiO2+SP600125 inhibitor group,while SP-A and SP-B protein expression were increased（P<0.05）.After transfected with c-jun-siRNA,it was found that the level of c-jun protein was successfully inhibited.The c-jun protein decreased in nano-SiO2+c-jun-siRNA group compared with nano-SiO2 group,while the expression of SP-A and SP-B protein increased（P<0.05）.Conclusions 1 The differential gene expression profile suggest that nano-SiO2 may down-regulate the expression of SFTPA and SFTPB genes in A549 cells and could activate biological processes such as oxidative stress and signal transduction pathways such as MAPK in A549 cells.2 Nano-SiO2 could reduce the survival rate of A549 cells,promote apoptosis,activate oxidative stress,and could also decrease the number of lamellar bodies and inhibit the expression of SP-A and SP-B protein.3 Nano-SiO2 could induce oxidative stress in A549 cells,and the produced ROS could activate the JNK/c-jun signal pathway which eventually leads the SP-A and SP-B protein decreased.The mechanism would be that the formated c-jun homodimers interfere the transcription of SP-A and SP-B genes.Figure 18;Table 12;Reference 117... |
