Effects Reaserch Of Xinfeng Capsules On Inproving Cardiopulmonary Function Of Rheumatoid Arthritis And The Research On Its Mechanism Based On Keap1-Nrf2/Are Pathway

1ObjectiveTo research changes of cardiopulmonary function in Rheumatoid arthritis (RA) patients and rats. Based on Xin’an medical theory, evaluate the protective effect of XFC, investigate the mechanism for improving cardiopulmonary function in RA.2Methods2.1Clinical Research2.1.1100cases of RA patients which choosed from Anhui Provincial Hospital of Chinese Medicine were randomly divided into observer group (XFC,50cases) and control group (LEF,50cases). To observe changes of age, gender, course, quality of life, mental health and other laboratory indicators of100cases RA patients.2.1.2Using ultrasonic beckoning diagnostic instrument to detect RA patients’cardiac function. The measured parameters of heart function including Left ventricular posterior wall(LVPW), Left ventricular end-diastolic dimension(LVDd), Left ventricular ejection fraction(LVEF), Fraction Shortening(FS%), level of early diastolic peak flow velocity (E), atrial peak flow velocity (A) and E/A.Using spirometer to detect RA patients’pulmonary function. The measured parameters of lung function including Forced vital capacity(FVC), Forced expiratory volume in one second(FEV1), FEV1/FVC, Peak expiratory flow(PEF), Maximal mid-expiratory flow(MEF25-75), Maximal mid-expiratory flow velocity in pulmonary vital capacity50%(MEF50), Maximal mid-expiratory flow velocity in pulmonary vital capacity25%(MEF25). 2.1.3To analyze the improvement of cardiopulmonary functionin RA, compare DAS28, ACR20\50\70, SF36, SAS, SDS scores, level of markers of oxidative stress(ROS,SOD,MDA,GSH), expression frequency of BTLA and other cytokines(IL-1β IL-17、IL-35、IFN-γ), and related laboratory parameters (ESR, etc.) before and after treatment. Using statistical software of SPSS17.0to analyze the relationship between those indexes.2.2Experimental research2.2.160SPF male SD rats,150±20g in weight, were randomly divided into those five groups:normal control group (NC), model control group (MC), Xinfeng Capsule research group (XFC), leflunomide control group (LEF), Astragalus polysaccharide control group (APS), with12in each group. Except the NC group, each rat was injected in the right rear paw intradermally with Freund’s complete adjuvant to induce inflammation and thus obtain AA model. The rats were given the dose from the19th days after inflammation, and the dosage was equivalent to10times the clinical dosage., The dosage of each group was as follows:NC group and MC group:saline was given(9mg/lml/100g); XFC group:(0.034g/lml/100g); APS group:(1mg/1ml/100g); LEF group:(0.5mg/1ml/100g); all the rats were continuously dosed once per day for30days.2.2.2To observe the activity state and behavior of AA rats, measure body mass, paw swelling and arthritic index, cardiopulmonary function, markers of oxidative stress(ROS,RNS,SOD,MDA,GSH,TRX,T-AOC),cytokines(IL-18、IL-35、IFN-γ、TNF-α), expression of Keapl, maf, Nrf2mRNA in heart and lung tissues, expression of HO-1, Y-GCS protein in heart tissue and Keapl, MAF, Nrf2protein in lung tissue were observed. To analyze the effects of XFC on AA rats.3Results3.1Clinical studies3.1.1Changes of cardiac function parameters and correlation study in RA patients Compared with the normal people, the parameters of cardiac function in RA were significantly decreased. Experiment results shows that68cases got abnormal cardiac function parameters, accounting for the80.95%of tested RA patients. The most prone to abnormal parameters was E/A, followed were E, A, EF, SV etc.. EF, E, E/A were decreased significantly in RA patients, while A was increasing (P<0.05or P<0.01). In correlation analysis, E was negatively correlated with age, disease curative effect evaluation score while A was positively correlated. E/A was negatively correlated with age, symptom score and the therapeutic efficacy assessment integration.3.1.2Changes of pulmonary function parameters and correlation study in RA patientsCompared with the normal people, the parameters of pulmonary function in RA were significantly decreased. Experiment results shows that45cases got abnormal pulmonary function parameters, accounting for the68.2%of tested RA patients.The most prone to abnormal parameters was PEF, followed were FEV1/FVC、FEF50、MVV、FEF25etc.. FVC, FEV1, FEV1/FVC, PEF, MEF25-75, MEF50, MEF25were decreased significantly in RA patients(P<0.05or P<0.01).In correlation analysis, FVC was negatively correlated to course, integral value of systemic symptoms and signs; FEV1were negatively correlated with age and course, MEF50were negatively correlated with age (P<0.05or P<0.01).3.1.3Changes of serum markers of oxidative stress and cytokine in RA patientsAfter treatment, compared with the normal, serum oxidative stress material ROS, MDA were increased significantly, while antioxidant as SOD, GSH were decreased significantly in RA patients (P<0.05or P<0.01). Compared with normal, the serum pro-inflammatory cytokines like IL-1β, IL-17were highly increased, while anti-inflammatory cytokines like IL-35, IFN-γ were decreased in RA patients (P<0.05or P<0.01). Significant efficiency on GSH, IL-35, IFN-γ in XFC group was obviously higher than that in LEF group.3.1.4Changes of serum BTLA and related laboratory parameters in RA patients Compared with the normal, CD19+BTLA+Bcell, CD24+BTLA+Bcell were lower in RA patients than those in normal group, and RF, CRP, ESR were significantly higher than normal group (P<0.05or P<0.01).3.1.5Effect of XFC in RA patientsAfter treatment, compared with control group, the total effective rate of treatment group was86%in XFC group, in control group the total effective rate was78%. There were no significant difference in the total effective rate, but the effective rate of treatment was significantly higher in XFC group than that in control group (P<0.05).3.1.6Effect of XFC on cardiac function in RA patientsAfter treatment, the parameters of cardiac function were improved effectively in RA patients. Compared with before treatment, XFC can obviously improve the EF%, FS%, E, E/A; and the effect on improving the E, E/A were significantly better than the control group (P<0.05or P<0.01).3.1.7Effect of XFC on pulmonary function in RA patientsAfter treatment, the parameters of pulmonary function were improved effectively in RA patients. Compared with before treatment, XFC can obviously improve the PEF, FVC, FEV1, MEF25-75, MEF50, MEF25(P<0.05or P<0.01); and the effect has no significant different with the control group.3.1.8Effect of XFC on serum markers of oxidative stress and cytokine in RA patientsCompared with untreatment, XFC can obviously improve the peripheral blood SOD, GSH levels, reduce ROS, MDA level in RA patients after treatment. And in the aspect of reducing the level of MDA, the effect is better than the control group (P<0.05or P<0.01).Compared with untreatment, XFC can significantly improve serum IL-35, IFN-levels, reduce the level of IL-1β, IL-17. And in the elevated IL-35, IFN-γ, curative effect is better than that of control group (P<0.05or P<0.01).3.1.9Effect of XFC on BTLA and related laboratory parameters in RA patients Compared with untreatment, the expression frequency of BTLA were increased after treatment, RF, Hs-CRP, ESR were significantly reduced. XFC can significantly increase serum level of CD19+BTLA+Bcell, CD24+BTLA+Bcell in RA patients, the curative effect is better than that of control group (P<0.05).3.1.10Effect of XFC on scoring form of clinical symptoms and signs, the quality of life in RA patientsCompared with untreatment, XFC can significantly decrease the cores of clinical symptoms and signs (P<0.01). XFC can significantly increase the scores of General health(GH), Role limitation due to physical problems (RP), Body Pain(BP), Social functioning (SF), Mental health (MH), reduce scores of SAS, SDS (P<0.05or P<0.01).3.2Experimental results3.2.1Effect of XFC on body mass, paw swelling and AI in AA ratsPrior to administration, compared with the NC group, body weight was reduced in MC rats, and paw swelling degree, AI were significantly higher than NC rats (P<0.01).After treatment, compared with MC group, XFC can significantly increase the body mass, reduce paw swelling and AI (P<0.05or P<0.01).3.2.2Effect of XFC on articular pathology in AA ratsCompared with NC group, the synovium in MC rats can found a large number of inflammatory cells infiltration, and multiple vasculars hyperplasis in synovial layered, fibroplasia, macrophage-like type-A cell hyperplasia.etc.After treatment, the inflammatory cells infiltration, and multiple vasculars hyperplasis in synovial layered were decreased. Compared with the control group, the XFC group’s synovial cells mitochondrial swelling reduced obviously.3.2.4Effect of XFC on cardiopulmonary function in AA ratsCompared with NC group, MC group HR, HI, LVSP, LVEDP increased,±p/dtmax decreased significantly (P<0.05, P<0.01). Compared with MC group, XFC can reduce HR, HI, LVSP, LVEDP increase±dp/dtmax significantly (P<0.05, P<0.01).Compared with NC group, MC group FEV1, FEF50, FEF75, PEF decreased significantly (P<0.05). FVC and MMF had no significant change (P>0.05). Compared with the MC group, XFC can increase FVC, FEF25, FEF50, FEF75, MMF, PEF; decrease FEV/FVC (P<0.05, P<0.01).3.2.5Effect of XFC on cardiopulmonary pathology in AA ratsMyocardium fabric structure in NC rats was clear, did not appear obvious pathological changes; MC group:myocardial can find generally myocardial fibers edema, separated, broadening. Longitudinal section of the visible portion can see the myocardial fibers were in a "branch-like" disordered arrangement. Transverse section of the visible portion can see the myocardial fibers were in a crowded "vortex-like" arrangement. The myocardial fibers in XFC group were arranged in-regular array, myocardial fiber edema were eased, connective tissue hyperplasia and fatty degeneration, occasionally, fibroblast proliferation were improved.Lung tissue structure of NC rats was clear, the alveolar structure was regular, did not appear obvious pathological changes; lung tissue of MC rats alveolar structure was irregular, alveolar atrophy or disappear, part of lung substantive changes, pulmonary septal thickening, inflammatory cell infiltration could be seen in the stroma. The alveolar structure in XFC treated rats were obviously much regular than MC group, part of alveolar were atrophy, deformation, and made a presentation of pulmonary consolidation, pulmonary septal were thicken, neutrophils and other inflammatory cell infiltration in the stroma.3.2.4Effect of XFC on serum markers of oxidative stress and cytokine in AA ratsCompared with NC group, MC group cytokines IL-18, TNF-a increased significantly in serum, while IFN-γ, IL-35decreased significantly (P<0.05or P<0.01). Compared with MC group, XFC can effectively decrease the expression of IL-18, TNF-α, and increase the expression of IFN-γ, IL-35(P<0.05or P<0.01). Compared with LEF group, XFC group have a higher levels of IL-18, IFN-y, IL-35; a lower levels of TNF-a; compared with APS group, XFC group have a higher levels of IFN-y, IL-35; a lower levels of IL-18, TNF-a (P<0.05).Compared with NC group, MC group sera peroxidation index MDA, ROS expression was significantly elevated, and SOD, T-AOC which can reflect the antioxidant capacity decreased significantly (P<0.05or P<0.01). Compared with MC group, the expression of MDA, ROS in the treatment group were significantly decreased, while SOD, T-AOC were significantly increased (P<0.05or P<0.01). Compared with LEF group, SOD, MDA, ROS, T-AOC expression were higher in XFC group(P<0.05); compared with APS group, SOD, T-AOC were higher in XFC group, MDA, ROS expression were lower(P<0.05).3.2.5Effect of XFC on the expression of oxidative stress protein of heart and lung tissues in AA ratsCompared with MC group, XFC group decreased expression of ROS, RNS protein, increased the expression level of GSH, TRX protein (P<0.05or P<0.01); compared with LEF control group, GSH, RNS, ROS were higher in XFC group (P<0.05or P<0.01); compared with APS control group, GSH, TRX were higher in XFC group (P<0.05).Compared with MC group, XFC group decreased expression of RNS protein, increased the expression level of ROS, GSH, TrX protein (P<0.05or P<0.01); compared with the LEF control group, RNS, GSH expression was lower in XFC group, ROS, TRX was significantly higher; compared with APS control group, ROS, RNS, GSH, TrX were higher in XFC group.3.2.6Effect of XFC on Keapl-Nrf2/ARE pathway related mRNA expression of heart and lung tissues in AA ratsCompared with group NC, Keap1, MAF, Nrf2mRNA were increasingly expressed in heart tissue of rats MC (P<0.01); compared with the MC group, XFC can recover the expression of Keap1, MAF, Nrf2mRNA(P<0.01); compared with LEF control group, the expression of Nrf2mRNA was higher in XFC group(P<0.05); compared with APS group, the heart of rats, the expression of MAF XFC Keap1than in the LEF group, the expression of NrGmRNA was higher than that in LEF group (P<0.05).Compared with NC group, MC group rats has no significant change in Keapl, but the expression of MAF, Nrf2mRNA were decreased significantly in lung tissue (P<0.05or P<0.01); compared with MC group, XFC group have expression of Keap1, MAF, Nrf2mRNA all increased, Nrf2mRNA increased obviously (P<0.05or P<0.01); compared with LEF group, Keap1, Nrf2expression was lower in XFC group, Maf was higher (P<0.05or P<0.01); compared with APS group, the expression of MAF, Keapl, Nrf2mRNA were higher in XFC group (P<0.05or P<0.01).3.2.7Effect of XFC on Keapl-Nrf2/ARE pathway related protein expression of heart and lung tissues in AA ratsCompared with group NC, MC group HO-1, γ-GCS protein were highly expressed in heart tissue (P<0.01or P<0.05). Compared with MC group, XFC group HO-1, y-GCS protein were decreased (P<0.05or P<0.01). Expression of HO-1, y-GCS in LEF group also decreased (P<0.05).Compared with NC group, the expression of Keapl, MAF, Nrf2protein in lung tissue of MC group were increased(P<0.01or P<0.05). Compared with MC group, the expression of Keap1, MAF, Nrf2protein decreased in XFC group (P<0.05). Compared with LEF control group, MAF, Nrf2expression was higher in XFC group, Keap1was lower (P<0.05); compared with APS group, XFC group, Keapl, MAF expression was higher in XFC group (P<0.05).4. Conclusion4.1RA disease easy to cause cardiopulmonary function declineCardiopulmonary function decline are prevalent exist in RA patients. Cardiopulmonary function changes were closely related with Synovial inflammation and systemic symptoms and physical performance, anxiety, depression, laboratory parameters(RF, hs-CRP, ESR, BTLA, et al., which suggested that RA not only can cause pain and swelling of joint, but also declination of cardiopulmonary function, and finally lead to physical and psychological barriers.4.2The mechanism of XFC improving cardiopulmonary function of RA4.2.1Emphasize the overall concept of TCM, improve the pathological injury of heart and lung tissueXFC can reduce inflammation of synovium and heart, lung tissue, regulate the immune response, reduce the infiltration of inflammatory cells, improve the structure of synovial, heart, lung tissue, so as to improve the joint inflammation, improve cardiaopulmonary function.4.2.2Enhance the antioxidant capacity, improve the state of oxidation, reduce oxidative stress injury of heart and lung tissueXFC can significantly improve the RA patients peripheral blood antioxidant SOD, GSH level, reduce free radicals and oxidation reduction product ROS, MDA level, improve the ability of anti-oxidation, promote free radical scavenging, reducing the oxidative stress injury of heart and lung tissue. XFC can also improve AA rats’ peripheral blood SOD, T-AOC, decrease MDA, ROS level. XFC could increase the expression of GSH, TRX protein in heart and lung tissues of AA rats, decrease ROS, RNS content. XFC can regulate oxidative stress balance, improve the antioxidant capacity, so as to improve the function of heart and lung.4.2.3Keep the balance of cytokine network, inhibit humoral immunity, reduce the immune and inflammatory injury of heart and lung tissueXFC can up regulate BTLA expression in peripheral blood of RA patients, raise expression of CD19+BTLA+Bcell, CD24+BTLA+Bcell, inhibit humoral immune responses mediated by B cells. XFC can also significantly increased anti-inflammatory cytokines IL-35, IFN-γ, reduce pro-inflammatory cytokine IL-1β, IL-17expression in RA patients, inhibit the inflammatory reaction, reduce the immune injury of heart and lung tissue. XFC could increase the suppression levels of IL-35, IFN-levels, reduce IL-18, TNF-α in AA rats. XFC can regulate the balance of cytokine network, reduce tissue inflammatory injury, so as to improve the function of heart and lung.4.2.4Regulat Keapl-Nrf2-ARE signaling pathway, recover the steady state of oxidative stress in heart and lung tissueXFC can regulate the excessive activation of Keap1-Nrf2/ARE pathway in heart and lung tissue in AA rats, improve the highly oxidative stress state in tissue, restore normal antioxidant capacity, reduce oxidative stress injury of heart and lung tissues, thereby protecting the cardiopulmonary function.