Mechanism Research Of Xinfeng Capsule On Inhibiting Platelet Activation To Improve Pulmonary Function In Rheumatoid Arthritis Based On FAK/Calpain Pathway

1.Objective:Using"treating illness from spleen"as the guiding principle of traditional Chinese medicine,it observed the effect of Xingfeng Capsule（XFC）on platelet activation and lung injury in RA based on FAK/Calpain signaling.In addition,it clarified the molecular mechanism of platelet activation and lung injury in RA and explore the molecular mechanism of the effect of XFC on platelet activation and lung injury in RA based on FAK/Calpain signaling.2.Methods:2.1 Animal experimental study Seventy-five rats were randomly divided into 5 groups including normal group（NC）,model group（MC）,sham-operated group（SHAM）,tripterygium wilfordii polyglucoside tablet group（TPT）and XFC group（XFC）,with 15 rats in each group.Except for the NC group,rats were injected with 0.1ml FCA into the metatarsal dermis to cause inflammation,0.05ml FCA was injected into the tail root to strengthen immunity at the 15th day,and given drugs on the 19th day.Rats were given0.034g/1ml/100g XFC in XFC group,0.57mg/1ml/100g TPT in TPT group,9mg/1ml/100g normal saline in NC and MC group,all the rats were given once a day and continue to 90 days,while there was nothing given to the SHAM group.Then the study observed the changes of E,AI,platelet parameters,pulmonary function parameters.Membrane and lung tissue morphology were observed by optical microscope,the expression of platelet activation markers,including GPIIb/IIIa,CD40L and CD147 were observed by flow cytometry,the levels of cytokins including IL-6,IL-8,IL-27,TGF-β1 and VEGF were detected by ELISA,and the FAK/Calpain signal and ColⅠα,ColⅢ,α-SMA were detected by RT-qPCR,immunohistochemical or western blot.2.2 Experimental study in vitro The study established ADP-induced platelet activation model,TGF-β1-induced lung fibrosis model,and further respectively culture platelet,lung fibroblast,and co-cultrue platelets and lung fibroblast in vitro.Platelets and lung fibroblast were respectively treated with XFC contains drug serum,triptolide,FAK inhibiter and Calpain inhibiter in vitro.Then,lung fibroblast proliferation rate was detected by CCK-8 kit,the levels of CD40L and GPⅡbⅢa were observed by flow cytometry,the level of IL-6 was detected by ELISA,ColⅠαmRNA and ColⅢmRNA were detected by RT-qPCR in lung fibroblast,the proteins expression of FAK,p-FAK,Calpain1,Calpain2 andα-SMA were detected by western blot and the expression of p-FAK andα-SMA were observed by immunofluorescencein lung fibroblast.3.Results 3.1 Results of animal experiment 3.1.1 Study of XFC efficacy 3.1.1.1 The effect of XFC on joint inflammation indexes in AA rats Compared with NC group,E and AI were significantly increased（P<0.01）.Compared with MC group,E and AI were significantly decreased in XFC and TPT group（P<0.01）.There was no statistical significance between the XFC treatment group and TPT group（P<0.01）.3.1.1.2 The effect of XFC on the changes of pathological morphological of joints and lung tissue in AA rats Compared with NC group,it can be found that synovial membrane hyperplasia,stratification and thickening,villous convex to articular lumen,a few neovascularization,and infiltration of a large number of inflammatory cells in the toe joint,and a large number of inflammatory cells mainly including neutrophile granulocyte,and some alveolar epithelial cells,lymphocytes,macrophages,as well as proliferating fibroblasts and deposition of large amounts of collagen fibers,moreover,it also can be observed that a large number of alveolar cavities is atrophy or disappear,large pulmonary vesicles were formed,alveolar structure becomes irregular,and some lung tissues showed substantial changes and the pulmonary septum become widened in lung tissue of AA rats in MC and SHAM group.After treatment,it can be observed that inflammatory cells significantly reduced in joint synovial membrane,some synovial tissue processes were embedded into the joint cavity,and synovial stratification was also reduced in the toe joint,and inflammatory cells,proliferating fibroblasts and collagen fibers were sinificantly reduced,additionally,alveolar structure was more regular,alveolar cavity was improved,and pulmonary septum also narrowed in lung tissue of AA rats in XFC and TPT group.Compared with TPT group,the synovial lining cells were less stratified,the articular surface was clear and synovial processes were improved,while inflammatory infiltration and neovascularization were not seen,and inflammatory cells were slightly reduced and alveolar septum was narrowed in lung tissue in XFC group.3.1.2 The effect of XFC on relevant indicators 3.1.2.1 The effect of XFC on lung function and ColⅠα,ColⅢandα-SMA in AA rats Compared with NC group,pulmonary function parameters including FVC,FEF₂₅,FEF₅₀,FEF₇₅ and PEF were significantly decreased,the expression of ColⅠα,ColⅢandα-SMA were significantly increased in MC and SHAM group（P<0.05,P<0.01）.Compared with MC group,FEF₅₀,FEF₇₅ and PEF were significantly increased,the expression of ColⅠα,ColⅢandα-SMA were significantly decreased in XFC and TPT group（P<0.05,P<0.01）.Compared with TPT group,FEF₅₀,FEF₇₅ were significantly increased,the expression of ColⅠα,ColⅢandα-SMA were significantly decreased in XFC group（P<0.05,P<0.01）.3.1.2.2 The effect of XFC on FAK/Calpain signaling in lung tissue of AA rats Compared with NC group,FAKmRNA,the protein of FAK and p-FAK were significantly increased,while Calpain1mRNA,Calpain2mRNA,Calpain1 and Calpain2 protein was significantly decreased in MC and SHAM group（P<0.05,P<0.01）.Compared with MC group,the expression of FAKmRNA,FAK and p-FAK protein were significantly decreased,while Calpain1mRNA,Calpain2mRNA,Calpain1 and Calpain2 protein was significantly increased in XFC and TPT group（P<0.05,P<0.01）.Compared with TPT group,the expression of FAKmRNA,FAK and p-FAK protein were significantly decreased,while Calpain2mRNA was significantly increased in XFC group（P<0.05,P<0.01）.3.1.2.3 The effect of XFC on platelet parameters and platelet activation markers in AA rats Compared with NC group,PLT,PCT,CD40L,CD147 and GPIIb/IIIa were significantly increased in MC and SHAM group（P<0.01）.Compared with MC group,PLT,CD40L,CD147 and GPIIb/IIIa were significantly decreased in XFC and TPT group（P<0.05）.Compared with TPT group,CD40L and GPIIb/IIIa were significantly decreased in XFC group（P<0.01）.3.1.2.4 The effect of XFC on FAK/Calpain signaling in platelets of AA rats Compared with NC group,the expression of FAK and p-FAK were significantly increased,while Calpain1 and Calpain2 was significantly decreased in MC and SHAM group（P<0.01）.Compared with MC group,the expression of FAK and p-FAK were significantly decreased,while Calpain1 and Calpain2 was significantly increased in XFC and TPT group（P<0.05）.Compared with TPT group,the expression of FAK and p-FAK were significantly decreased,while Calpain1 and Calpain2 was significantly increased in XFC group（P<0.01）.3.1.2.5 The effect of XFC on cytokines in AA rats Compared with NC group,the expression of IL-6,IL-8,TGF-β1 and VEGF were significantly increased,while IL-27 was significantly decreased in MC and SHAM group（P<0.01）.Compared with MC group,the expression of IL-8,TGF-β1 and VEGF were significantly decreased,while IL-27 was significantly increased in XFC group（P<0.05）,the expression of IL-6,IL-8,TGF-β1 and VEGF were significantly decreased,while IL-27 was significantly increased in TPT group（P<0.05）.Compared with TPT group,the expression of IL-8 and TGF-β1 were significantly decreased（P<0.01）.3.1.3 the correlation between the indicators 3.1.3.1The correlation between pulmonary function parameters,collagen fibrin and FAK/Calpain signaling,joint inflammation,cytokines,platelet related indexes The correlation analysis showed that FVC was negatively related to FAK and PLT（P<0.01）,FEF25 was negatively correlated with p-FAK,IL-6,TGF-β1 and PLT（P<0.05）,FEF50 was positively related to Calpain2,while negatively correlated with AI,E,IL-8 and PDW（P<0.05 or P<0.01）.FEF75 was positively related to Calpain2,while negatively correlated with E,TGF-β1,PLT,CD40L,CD147 and GPⅡbⅢa（P<0.05 or P<0.01）,PEF was positively related to Calpain2,while negatively correlated with E and IL-8（P<0.05 or P<0.01）,ColⅠαwas positively related to FAK,TGF-β1,VEGF,PLT,CD40L and GPⅡbⅢa（P<0.05 or P<0.01）,ColⅢwas negatively correlated with Calpain2,while positively related to AI,IL-8 and VEGF（P<0.05 or P<0.01）,α-SMA was positively related to FAK（P<0.05）.3.1.3.2 The correlation between platelets and FAK/Calpain pathway,joint inflammation index and cytokines The correlation analysis showed that PLT was positively correlated with E,IL-6,IL-8,FAK,p-FAK and Calpain1（P<0.05）.PCT was negatively correlated with Calpain2（P<0.05）.PDW was positively correlated with AI（P<0.05）.CD40Lwas positively correlated with E,IL-6,IL-8,FAK and p-FAK（P<0.05）.CD147 was positively correlated with IL-6,TGF-β1,FAK,p-FAK and Calpain1（P<0.05）.GPIIb/IIIa was positively correlated with E,IL-6,IL-8 and p-FAK（P<0.05）.3.2 Results of the vitro study 3.2.1 The effect of different concentrations of XFC on platelet activation in vitro Compared with control group,platelet activation markers including GPⅡb/Ⅲa and CD62p were significantly increased after treated with 20umol/L ADP（P<0.05）.Then platelets,which were treated with 20umol/L ADP,were respectively treated with high,middle,light dose of XFC.Compared with MC group,GPⅡb/Ⅲa was decreased after treated with different dose of XFC（P<0.05）,CD62p was decreased after treated with high,middle dose of XFC（P<0.05）,and the effect of XFC on platelet activation is at a dose-dependent.3.2.2XFC inhibits platelet activation by regulating FAK/Calpain signal pathway Compared with control group,the expression of GPⅡb/Ⅲa and CD62p were increased in MC group（P<0.05）.Compared with MC group,GPⅡb/Ⅲa and CD62p were decreased in XFC group（P<0.05）.Compared with XFC group,GPⅡb/Ⅲa and CD62p were decreased in the group treated with 20μM FAK inhibiter（P<0.05）,while GPⅡb/Ⅲa and CD62p were increased in the group treated with 50μM Calpeptin（P<0.05）.3.2.3 The effect of different drugs on proliferation rate of pulmonary fibroblasts The lung fibroblast,which were induced with 50ng/mLTGF-β1,were respectively treated with L-XFC,M-XFC,H-XFC,（5,10,15,20）nmol/L TPL,and cultured in incubator with 24h,48h and 72h.After that,detected the proliferation rate of lung fibroblast.Pulmonary fibroblast proliferation were inhibited by XFC and TPT at a dose-dependent manner.However,pulmonary fibroblast proliferation were significantly inhibited at 24h treating with H-XFC and 5nmol/L TPL.Therefore,H-XFC and5nmol/L TPL is the optimal stimulation concentration.3.2.4 XFC inhibited pulmonary fibroblast secrete IL-6,ColⅠα,ColⅢandα-SMA by regulating FAK/Calpain signal pathway Compared with control group,the level of IL-6,ColⅠα,ColⅢandα-SMA was incresed in MC group treating with TGF-β1（P<0.01）.Compared with MC group,the level of IL-6,ColⅠα,ColⅢandα-SMA was decresed in H-XFC and 5nmol/LTPL group（P<0.01）.Compared with only treating with H-XFC or 5nmol/LTPL,the level of IL-6,ColⅠα,ColⅢandα-SMA was decresed in the group treating with 20uM FAK inhibiter and H-XFC or the group treating with 20uM FAK inhibiter and 5nmol/LTPL（P<0.01 or P<0.05）,while the level of IL-6,ColⅠα,ColⅢandα-SMA was incresed in the group treating with 50uM Calpain inhibiter and H-XFC or the group treating with50uM Calpain inhibiter and 5mmol/LTPL（P<0.01）.Compared with 20uM FAK inhibiter group,the level of IL-6,ColⅠα,ColⅢ and α-SMAwas incresed in 50uM Calpain inhibiter group（P<0.01）.3.2.5 The effect of XFC on FAK/Calpain signal pathway in pulmonary fibroblasts.Compared with control group,the protein expression of FAK and p-FAK were incresed,Calpain1 and Calpain2 were decreased in MC group treating with TGF-β1（P<0.01;P<0.01）.Compared with MC group,the protein expression of FAK and p-FAK were decresed,Calpain1 and Calpain2 were increased in H-XFC and5mmol/LTPL group（P<0.01;P<0.01）.Compared with the group only treating with H-XFC or 5nmol/LTPL,the protein expression of FAK and p-FAK were decresed,Calpain1 and Calpain2 were increased in the group treating with 20uM FAK inhibiter and H-XFC or the group treating with 20uM FAK inhibiter and 5nmol/LTPL（P<0.01 or P<0.05）,while the protein expression of FAK and p-FAK were incresed,Calpain1 and Calpain2 were decreased in the group treating with 50uM Calpain inhibiter and H-XFC or the group treating with 50uM Calpain inhibiter and 5nmol/LTPL（P<0.01）.Compared with 20uM FAK inhibiter group,the protein expression of FAK and p-FAK were incresed,Calpain1 and Calpain2 were decreased in 50uM Calpain inhibiter group（P<0.01）.3.2.6 XFC inhibited platelet activation to improve lung fibrosis by regulating FAK/Calpain signal pathway Compared with the group only treating with 50ng/mlTGF-β1,the proliferation rate of pulmonary fibroblasts,the levels of IL-6,ColⅠαmRNA and ColⅢmRNA were significantly increased in the group co-cultured with platelets and lung fibroblasts（P<0.01）.After treating with H-XFC,the proliferation rate of pulmonary fibroblasts,the levels of IL-6,ColⅠαmRNA and ColⅢmRNA were significantly decreased（P<0.01）.4.Conclusion 4.1 There are platelet activation and lung function injury in AA rats,and platelet activation can further aggravate lung function injury.The molecular mechanism is thatbecause of stimulating with inflammation,then appearing the imbalance of cytokine network,activates the FAK signaling pathway,inhibits activation of the Calpain pathway,leads to platelet activation and lung function parameters decreased,finally results in lung function damage.In addition,platelet activation products circulation into the lung tissue,will lead to further lung function damage.4.2 XFC can improve the degree of toe swelling and arthritis index of AA rats,inhibit platelet activation,and improve lung function injury,and has an similar effect of FAK inhibitors and Calpain pathway,inhibit activation of FAK signaling pathway.The molecular mechanism is that XFC can suppress the immune inflammatory response,restore the balance of cytokine network,inhibit activation of FAK signaling pathway,promote activation of Calpain signaling pathway,inhibit platelet activation,finally improved lung function injury in AA rats.