The Possible Mechanism Of PAD Enzyme In Rheumatoid Arthritis Induced By Lung Injury In CIA Mice And The Effect Of Sinomenine

BACKGROUND:Rheumatoid arthritis(RA)is an autoimmune disease.Chronic synovitis is the main pathological change.It can be divided into two categories:ACPA+ and ACPA-.ACPA+ RA patients are associated with specific HLA ? alleles.Gene mutation can only explain the RA susceptibility of less than 20%of ACPA+ and lower contribution to ACPA-RA.Environment is an important inducing factor.The immune response of anti-citrullinated protein antibodies(ACPA)is earlier than the symptoms of RA arthritis.The citrulline reaction catalyzed by peptidyl arginine deiminase(PAD)provides an antigen for ACPA production.This reaction may occur in the lung,gingiva and intestine.The role of lung is particularly important.OBJECTIVE:To find the theoretical basis for treatment of RA of TCM and modern medical basis from the lung through theoretical research;through clinical research to verify lung injury and possible role in the pathogenesis of RA;through animal experiments to further verify whether lung injury through the activation of the PAD enzyme can promote the citrullinated protein production induced by RA,and whether sinomenine can inhibit PAD the enzyme activity inhibition of citrullinated protein produced and inhibit or slow down the development of RA.METHODS:The first part of the study using literature review method,acquisition of Chinese traditional literature and modern medical research on the theory and practice basis for treatment of RA were reviewed and analyzed from the lung;the second part is clinical research using retrospective study method,selecting 46 cases objects between January 2014-2017 year in January to who accept the inspection with respiratory abnormalities can not explain,and in accordance with the terms of the ACPA positive individuals,24 patients with RA onset time of less than 1 years according to RA classification criteria,and 32 cases of ACPA negative controls.Evaluation of chest CT of 3 groups of individual imaging were performed.The third part of animal experiments,30 male DBA/1 mice were randomly divided into 2 groups,one group 5 as blank control group,the other group 25 models,the mice were immunized with type II collagen at the d 0 and d 21 in tail roots,respectively,and induced CIA mice model.15 successful established models were randomly divided into 3 groups on d 24:model group(n=5),BB-CI group(n=5),Chinese medicine group(n=5)and blank control group(n=5),all were 4 groups.d 28 begins to give medicine.The mice were killed 14 days after the drug was given.The arthritis index,synovial pathological changes,lung pathological changes,lung PAD-2,PAD-4 and citrullinated protein immunohistochemistry and were assessed by western blotting.PAD-2,PAD-4 mRNA expression were observed,too.At the same time,the changes of Thl related cytokines TNF-a,IL-6 and Th2 related cytokines IL-4 and IL-10 were also observed.RESULTS:The first part of the theoretical research results,to fully understand the relationship between the lung and the joint in traditional Chinese medicine,through the theories about the lung is in charge of the fur,Qi and breathing,these knowledge will be linked to lung and arthralgia.Modern medical research shows that lung as a potential site of rheumatoid arthritis occured,through a variety of trigger factors(especially smoking)in genetically susceptible individuals with immune tolerance is broken,PAD enzyme and its product citrullinated protein is important trigger.The second part of clinical study results:both parenchymal and airway HRCT abnormalities were more frequent among ACPA-positive cases than among ACPA-negative controls(52%versus 19%with parenchymal changes and 52%versus 19%with airway changes;each P=0.003).Fibrosis(6[13%]of 46)was detected solely in ACPA-positive cases.Parenchymal abnormalities were identified in both ACPA-positive cases and early RA patients(52%versus 38%;P=0.243).And no differences in the distribution of airway abnormalities were observed between ACPA-positive cases and early RA patients(52%versus 67%;P=0.245).Mediastinal lymph node enlargement were found in ACPA-positive cases,compared with ACPA-negative controls(19%versus 0,P=0.033).The results of the third part of the animal experiment result:d 24 to d 42,the AI score of the model group was significantly higher than that in the blank group,and the difference was statistically significant(P<0.01).After initial immunization,d 24 to d 42.The AI score of Chinese medicine group showed a downward trend compared with the model group,but there was no significant difference.d 39 to d 39,BB-C1 group AI score was significantly lower than that of the model group,the difference was statistically significant(d 36 P<0.05,d 39 P<0.01).The control group synovial cells of about 1-2,the distribution of neat,no significant inflammatory cell infiltration;model group synovial cells in 5-10 layers,showing a large number of neutrophils and mononuclear lymphocyte infiltration;BB-C1 group and Chinese medicine group improved the proliferation of synovial cells,infiltration of inflammatory cells.The model group serum TNF-a alpha levels were elevated significantly compared with control group,the difference was statistically significant(P<0.01);compared with the model group,the serum TNF-alevel.The traditional Chinese medicine group decreased significantly,the difference was statistically significant(P<0.01);the level of serum IL-6 in model group was significantly higher than the control group,the difference was statistically significant(P<0.01);compared with the model group,BB-C1 group and Chinese medicine group serum IL-6 level were significantly decreased,the difference was statistically significant(P<0.01).The model group serum IL-4 levels were elevated significantly compared with control group,the difference was statistically significant(P<0.01);compared with the model group,the serum level of IL-4 of Chinese medicine group was significantly increased,the difference was statistically significant(P<0.01);the level of serum IL-10 in model group was significantly higher than the control group,the difference was statistically significant(P<0.01);compared with the model group,BB-C1 group and Chinese medicine group serum IL-10 levels were significantly increased,the difference was statistically significant(P<0.05).The alveolar septum in the normal lung tissue is homogeneous,with about 2 red cell thickness,clear and uniform structure and no exudation in the alveoli.Occasionally lymphocytes and macrophages infiltrate in the bronchi and vessels and alveolar septum.The model group mice lung had inflammation,alveolar serous effusion,interstitial inflammation,alveolar exudation of alveolar septum thickening,the structure of fuzzy,infiltration around the bronchi and blood vessels,lymphatic macrophages.In the treatment group the pneumonia was relieved,the alveolar septum was slightly widened,and the lymphocyte and macrophage were small.Compared with the control group,immunohistochemical staining in model group of PAD-2,PAD-4 and peptidyl-citrulline was significantly enhanced,the gray value was significantly increased,the difference was statistically significant(PAD-4,P<0.05,PAD-2 and peptidyl-citrulline P<0.01);compared with the model group,the expression of BB-C1 group and Chinese medicine group PAD-2 and peptidyl-citrulline decreased obviously,gray value is significantly reduced,there is significant differences(traditional Chinese medicine group PAD-2 P<0.05,others P<0.01);compared with the model group,BB-C1 group and Chinese medicine group PAD-4 decreased,but the difference was not statistically significant.Compared with the control group,the expression of PAD-2 in the model group,PAD-4 and peptidyl-citrulline protein was increased,the relative gray value was significantly increased,the difference was statistically significant(P<0.01);compared with the model group,BB-C1 group and Chinese medicine group PAD-2,PAD-4 and peptidyl-citrulline protein expression was significantly decreased,the relative gray value was significantly decreased,the difference was statistically significant(P<0.01).Compared with the control group,the expression of PAD-2 and PAD-4 mRNA in the model group increased significantly,the difference was statistically significant(P<0.01).Compared with the model group,the expression of PAD-2 and PAD-4 mRNA in the BB-CI group and the Chinese medicine group decreased significantly,the difference was statistically significant(P<0.01).The PCR amplification plot and the melt curve showed that the amplification efficiency of the target gene was consistent,the amplification product was single,and the experimental results were reliable.CONCLUSION:The theoretical results show that Bi has a close relationship with lung;lung is a potentially important starting site for RA,RA is associated with increased local PAD enzyme and citrullinated protein products induced by lung.Clinical research results show that lung parenchymal lesions and airway abnormalities are an inflammatory state.The proportion of lung abnormal is higher in ACPA positive people induced by citrullinated protein than that of ACPA negtive people,suggesting that lung inflammation may be a possible inducement factor of RA.The animal experiment showed that the collagen induced arthritis in mice and the presence of pneumonia and arthritis,can reduce pneumonia and arthritis symptoms by suppressing generation of local PAD-2 and PAD-4,while reducing the secretion of Thl related inflammatory cytokines,increased secretion of Th2 related cytokines.Sinomenine can inhibit the production of local citrullinated protein in pneumonia by inhibiting PAD-2 and PAD-4 enzymes,thereby alleviating arthritis in mice and playing a therapeutic role.