Effect Of Bizhongxiao Decoction In The Peripheral Blood Mononuclear Cells Of Patients With Actived Rheumatoid Arthritis By Proteomic Methods

Objective:(1) To find the different proteins association with active rheumatoid arthritis (RA) by comparing the expression levels of proteins in the peripheral blood mononuclear cells (PBMCs) of healthy control individuals to those of patients with rheumatoid arthritis using a proteomics approach. And to investigate the useful and new clues for elucidating the pathogenesis of rheumatoid arthritis.(2) To elucidate the mechanism of bizhongxiao decoction (BZXD) in treating rheumatoid arthritis by analyzing the differential expression proteins of PBMCs of patients with rheumatoid arthritis treated with BZXD.Methods:(1) 18 patients with early rheumatoid arthritis (RA) on active phase were divided into BZXD-treated group (BZXDG) and methotrexate with nimesulide tablets group (MTXG). Furthermore, 9 healthy people were selected taken as healthy controls.(2) Morning stiffness time,grasp force,tender joint count,tender joint count index,swollen joint count, swollen joint count index, erthrocyte sedimentation rate (ESR), C-reactive protein,(CRP), rheuatoid factor (RF) of the patients were compared and observed before treatment and 3 months after the treatment.(3) PBMCs were isolated from blood by using Lymphozytes separation medium. The total protein was extracted from the peripheral blood mononuclear cells.The total protein from either RA patients or normal ones was prepared by means of immobilized pH gradient based on two-dimensional gel electrophoresis. After Coomassie brilliant blue G250 staining, gel-image analysis was performed by using PDQuest. The differentially expressed proteins were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALD I-TOF-MS). Then ApoA-I was validated by reverse transcription polymerase chain reaction (RT-PCR).Results:(1) The results showed that after 3-month treatment,symtoms and signs,such as morning stiffness time, tender joint count,tender joint count index,swollen joint count, swollen joint count index and grasp force of patients in both groups had been greatly improved (P<0.01 or P<0.05), but BZXD had a better effect than MTX. ESR, CRP and RF decreased significantly in both groups after treatment(P<0.01 or P<0.05), ESR, CRP in BZXDG decreased more than those in MTXG(P<0.05). In BZXG the obviously efficient rate was 77.8%, the total effective rate was 88.9%, while in MTXG was 55.6% and 77.8% respectively.(2) 2-DE patterns of PBMCs from controls, before and 3 months after the treated RA patients were presented. Gel-image analysis reveals that there are 23 differential protein spots. 15 of total 18 differential protein spots were successfully identified by MALDI-TOF-MS, of which 8 proteins were up-regulated in RA patients, and 7 proteins were down-regulated as compared with control. After 3-month treatment , Compared with MTXG, 6 proteins in differential expression are more obvious in BZXDG.(3) Differential expression protein identification and by mass spectrometry and protein functional classification: 15 more than 2 folds differential expression protein spots were identified. The functions of these proteins involve in metabolism, energy generation, transportation, antioxidation, sigal transduction and cytoskeleton etc according to information provided in NCBI and MSDB database.Among them,there are some proteins closely related to rheumatoid arthritis,such as apolipoprotein A-I actin, beta Talin 1 ATP synthase phosphatidylethanolamine-binding protein Prohibitin Ras related protein Heterogeneous nuclear ribonucleoprotein K .(4) Result of differential expression protein : The verified result of ApoA-I by RT-PCR is consistent with the proteome result.Conclusion:(1) Some of these differentially expressed proteins are shown in the PBMCs of patients with rheumatoid arthritis, which may play a potential role in the pathogenesis of RA. Protein expression profiles of RA in the PBMCs can be regulated by BZXD.(2) The verified result of ApoA-I by RT-PCR is identical to the proteome result. It indicats that BZXD may have therapeutical effect on RA via regulating the protein expression such as ApoA-I in PBMCs.(3) It is indicated that BZXD can improve symptoms and signs of patients with RA and has a better effect on acute phase reaction than MTX, and has no obvious side effect.