Therapeutic Effect Of Gene Silencing Peptidyl Arginine Deaminase 4 On Pulmonary Interstitial Lesions Induced By CIA Mouse

Objective To study the therapeutic effects and possible mechanisms of Peptidyl arginine deaminase 4(PAD4)expression on pulmonary interstitial lesions induced by collagen-induced arthritis(CIA)mouse.Method CIA mouse models were established in 6-8 weeks old male DBA/1 mouse.The experiment was divided into blank group,model group,negative control group,PAD4-siRNA1 group,PAD4-siRNA2 group and PAD4-siRNA3 group,a total of 6 groups,4 Only / group.The blank group did not undergo any treatment,and the other groups used Immunization Grade Bovine Type II Collagen(CII)to establish CIA model.Then,CIA mouse were injected into the tail vein,the model group was not treated,the negative control group was injected with adenovirus liquid prepared by empty vector,the PAD4-siRNA1 group was injected with PAD4-siRNA1 vector,and the PAD4-siRNA2 group was injected with PAD4.Adenovirus liquid prepared by injection of the PAD4-siRNA3 vector into the adenovirus liquid prepared by the siRNA2 vector and the PAD4-siRNA3 group.Inject once a week for 8 weeks.At the end of the experiment,the mice were sacrificed.Real-time Quantitative polymerase chain reaction(q RT-PCR)and fluorescence immunohistochemistry were used to detect the expression of PAD4 m RNA in the lung.The spleen and thymus tissues were taken for cell culture,and Tfh cells were detected by flow cytometry.Changes in the ratio of cells to Tfr cells;serum levels of TNF-α,IL-6 and IL-9 were measured by Enzyme-linked immuno sorbent assay(ELISA)after cardiac blood sampling;lung staining was performed by HE staining in lungs.Results(1)Compared with the blank group,the expression of PAD4 m RNA was increased in the model group(P<0.05).The expression of PAD4 m RNA in the lungs of CIA mouse after PAD4-siRNA treatment was higher than that in the model group and the negative control group.The group was significantly reduced,the difference was statistically significant(P<0.05);(2)the red fluorescence in the lung tissue of the blank group was less,but the inflammatory cell infiltration area and the trachea around the lung tissue of the model group and the negative control group were visible.More red fluorescence,red fluorescence decreased significantly in the three groups after PAD4-siRNA treatment;(3)Compared with the blank group,the proportion of Tfh cells in the model group increased,the difference was statistically significant(P<0.05),PAD4 The proportion of Tfh cells in spleen cells of CIA mouse was significantly lower than that of the model group and the negative control group after siRNA treatment,and the difference was statistically significant(P<0.05).Compared with the blank group,the proportion of Tfr cells in the spleen cells of the model group was slightly lower.However,the difference was not statistically significant.The proportion of Tfr cells in the spleen cells of mice increased after PAD4-siRNA treatment,but the difference was statistically significant only in the PAD4-siRNA2 group compared with the model group and the negative control group(P<0.05).(4)The proportion of Tfh cells and Tfr cells in the thymocytes of the mouse group was not significantly different from that in the blank group.The difference was not statistically significant.The proportion of Tfh cells in the thymocytes of the three groups decreased after PAD4-siRNA treatment,and the model group and the negative control group.The difference was statistically significant(P<0.05),and the proportion of Tfr cells was increased,but only PAD4-siRNA1 group and PAD4-siRNA3 group were statistically significant(P<0.05).(5)The proportion of Tfh/Tfr in the spleen cells of the model group increased,compared with the blank group,the difference was statistically significant(P<0.05),but the proportion of Tfh/Tfr in thymocytes did not change significantly.After PAD4-siRNA treatment The proportion of Tfh/Tfr in the three groups decreased,whether it was spleen cells or thymocytes,and it was statistically significant(P<0.05).(6)Compared with the blank group,the alveolar wall of the lung tissue of the model group was thickened.The inflammatory cell infiltration increased,and the lung tissue destruction and inflammatory infiltration of CIA mouse decreased after PAD4-siRNA treatment,and the degree of fibrosis was reduced.Conclusions Gene silencing of PAD4 expression can reduce the proportion of Tfh cells,increase the proportion of Tfr cells,reverse the proportion of Tfh/Tfr,and reduce the degree of interstitial lesions and the degree of inflammatory infiltration in lung tissue.