Effect Of Invitro Hypomethylation Of CD4~+T Cells Promoter Of Perforin On Apoptosis Of Alveolar Septal Cells In Autoimmune Emphysema Rats

Objective:One relatived artical has suggested that hypomethylation of CD4~+T cells promoter of perforin and apoptosis of alveolar septal cells were found in autoimmune emphysema rats.It is speculated that hypomethylation of the promoter of perforin may induce the apoptosis of alveolar septum cells in autoimmune emphysema rats.In order to explore the relationship between the two,we injected intraperitoneally in vitro hypomethylated CD4~+T lymphocytes into normal rats,and observed the apoptosis of alveolar septum cells and pathological changes of lung tissue,providing ideas for further research on the pathogenesis of emphysema and finding new treatment methods.Methods:Thirty rats were randomly divided into three groups:model group(n=10),normal control group(n=10)and sham operation group(n=10).Model group:CD4~+T lymphocytes in spleens of normal SD rats were isolated by magnetic beads,and the cells were treated with 5-azacytidine(5-Aza)(the concentration of 5-Aza was 10μmol/l)and incubated for 72 hours.1×10~7 cells were used to centrifuge for removing supernatant,and mixed with 1ml complete Freund’s adjuvant and 1ml Phosphate Buffered Saline(PBS)then injected intraperitoneally other normal SD rats which marked as Model group.The left 1×10~7 cells that were not injected into the rats were used to centrifuge for removing supernatant and detected methylation level of perforin promoter.We marked the cells as Cell Group 1.Normal control group:CD4~+T lymphocytes in spleens of normal SD rats were isolated by magnetic beads,and the cells were incubated for 72 hours without the intervention of 5-Aza.Then we dealed with the cells as the above,respectively marked the rats as Control group and the cells as Cell Group 2.Sham operation group:Healthy SD rats were injected intraperitoneally 1ml complete Freund’s adjuvant and 1ml PBS.Three groups of rats were raised in the same environment.After 21 days,the right lower lung tissue of rats were used to make paraffin sections and stained with Hematoxylin and Eosin(HE),we then observed its pathological changes,and measured the Mean Linear Intercept(MLI)and Mean Alveolar Numbers(MAN).Serum and left lung Broncho Alveolar Lavage Fluid(BALF)samples were prepared,and the content of Antiendothelial Cell Antibodies(AECA)was detected by Enzyme-linked Immunosorbent Assay(ELISA).The expression of Vascular Endothelial Growth Factor(VEGF)in right lower lung tissue was measured by immunohistochemistry.We used Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick end Labeling(TUNEL)technique to detect the apoptosis of alveolar septum cells,then we calculated Apoptosis Index(AI).We also extracted the DNA of rat spleens CD4~+T lymphocytes and investigated the methylation level of promoter of perforin using Bisulfite Amplicon Sequencing(BSAS).Results:(1)the MAN in the model group was lower than that in the control group and sham operation group,the MLI in the model group was higher than that in the control group and sham operation group,the above P<0.05.(2)Compared with the control group and the sham operation group,the concentration of AECA was higher in the model group both in serum and BALF,and the same with VEGF and AI,the above P<0.05.(3)The promoter of perforin methylation level of cells interfered by 5-Aza(Group 1)was lower than that cells without 5-Aza’s intervention(Group 2)(P<0.05),the methylation level of spleens CD4~+T lymphocytes perforin promoter in model group was lower than that in control group and sham operation group(P<0.05).(4)There was no significant difference between the control group and the sham operation group(P>0.05).(5)The methylation level of perforin promoter was positively correlated with MAN(r=0.747,P<0.05)and negatively correlated with AI,AECA(serum and BALF),MLI,VEGF(r was respectively:-0.789、-0.746、-0.661,-0.743,-0.660,P<0.05).Conclusion:(1)5-Aza can reduce the methylation level of perforin promoter in rats spleens CD4~+T lymphocytes.(2)CD4~+T lymphocytes of rats spleens treated with 5-Aza may induce apoptosis of rat alveolar septum cells,increase expression of AECA and VEGF,and lead to autoimmune emphysema by reducing methylation level of perforin promoter.