Effect Of Xiao Wu Gui Granule On The CIA Rats And Proliferation Of Its Fibroblast-like Synoviocytes In Vitro

BackgroundRheumatoid arthritis (RA) is a common chronic inflammation in joint tissues as the main performance, characterized by articular synovial inflammation, autoimmune disease, its pathogenesis is mainly characterized by synovial hyperplasia of cells, inflammatory cells infiltration, lining layer thickening, pannus formation, which in turn cause the destruction of cartilage and bone tissue, resulting in joint deformities and loss of function. Clinical manifestations for the affected joint pain, swelling and function drops, pathological changes are continuous, iterative process. RA disease distribution is global, the incidence of0.26～0.5%in our country, can happen at any age, incidence increased with age also gradually to increase. Women more commonly, peaks between the ages of45～50. RA course of lingering, repeated, high morbidity, almost all of RA patients have different degrees of joint function is lost, leading to serious social problems and economic burden.The pathogenesis of RA is complex, there are a variety of immune cells and cytokines involved in, the main pathological features of synovial cell hyperplasia, gathered a large number of inflammatory cells, such as T cells, plasma cells and macrophages, thickening of the lining layer, eventually pannus formation, excessive proliferation synovial tissue and vascular direct destruction of articular cartilage and bone. RA synovial tissue is the most important composition of fibroblast-like synoviocytes(FLS), which originated in the interstitial cells, besides participating in articular cartilage degradation and bone erosion, also secrete a variety of inflammatory factors indirectly involved in bone destruction process,including IL-1, IL-6, IL-10, TNF alpha, transforming growth factor (TGF)-β, matrix metalloproteinases (MMPs), etc, become the direct effect of joint inflammation and tissue destruction, plays an important role in the occurrence and development of RA.RA patients for Thl and cytokines predominate, proinflammatory factor TNF-a, IL-1and angiogenic factors related to VEGF and MMPs in serum significantly increased in RA patients.Youn in RA patients was detected in serum and joint fluid such as TNF-α levels are significantly higher than in patients with osteoarthritis (OA), and normal person. Using soluble TNF-a receptor fusion protein and monoclonal antibody blocking TNF-α, can effectively reduce RA disease activity in patients, control of bone destruction. RA activation of inflammatory cells in the secretion of inflammatory factor at the same time, also secrete large amounts of MMPs, RA and angiogenesis, except related to VEGF is closely related to the biological activity of MMPs. MMPs is a kind of protease activity depends on the metal zinc ions, it is in the form of zymogen secretion, after activation involved in the degradation of collagen and other extracellular matrix and influence the angiogenesis.11-10is a kind of natural immune inhibitor, it can effectively inhibit the T cells and antigen-presenting cells, inhibit the release of TNF alpha, IL-1and the production of MMPs, promoting TNF-α, IL-1receptors, and thus play a protective role of cartilage and bone cells.RA belongs to the category of traditional Chinese medicine-bi syndrome. Of occurrence are mainly caused by a lack of vital qi, feel the wind, cold, wet, heat caused by the evil. Internal cause is the foundation of lack of vital qi, is the cause of internal factors, such as evil invaded again, and make the muscles, joints obstruction.From the above pathogenesis characteristics, treatment of the disease its expelling wind and cold-dispelling dehumidification, Qi and activating blood circulation. Xiao Wu Gui decoction come from’Qian Jin Yao Fang’-Xiao Xu Ming decoction,and’Jing Gui Yao Lue’-Wu Tou decoction and Gui Zhi Shao Yao Zhi Mu decoction,is the development of national famous professor Chen Baotian, our department of clinical application for more than30years, a large number of cases of proven experience has good curative effect. From the perspective of modern medicine, the party has anti-inflammatory, analgesic, immune function. It is clinically used in RA treatment and has made certain curative effect, but its detailed mechanism has yet to clear. Methotrexate (MTX) is a cytotoxic agent, can inhibit the humoral and cellular immunity, is currently the first-line treatment for RA, it through a variety of mechanisms play a role in RA therapy, such as inhibiting the secretion of TNF-a, IL-1, IL-12, reduce the activity of MMPs, this paper chooses MTX as positive control drug. CIA animal model is composed of a type II collagen induced immune inflammatory models, mainly for the multiple terminal arthritis, pathological changes mainly proliferative synovitis, the articular cartilage damage and bone destruction, mononuclear cells into and continue to exist in the synovial membrane, and has high titer IgG antibody at the collagen type Ⅱ.CIA animal model are both cellular immunity and humoral immunity, is recognized as a classic animal model to the pathogenesis of RA and one of the ideal animal models of RA treatment drugs. Study the effect of Xiao Wu Gui granule on the CIA rats and proliferation of its fibroblast-like synoviocytes (CIA-FLS) in vitro is helpful to further explore mechanism of Xiao Wu Gui granule in the treatment of RA.Objectives1. The evaluation the therapeutic effect of Xiao Wu Gui granules on arthritis in CIA rats and the related cytokine expression.2. To investigate the effect of Xiao Wu Gui granule drug-containing serum on proliferation of CIA-FLS in vitro and the related cytokine secretion.Methods1laboratory animals82SPF female SD rats (70for the CIA rat, CIA-FLS access and the CIA model evaluation;12for Xiao Wu Gui granule drug-containing serum access), weight of200g±20g, provided by experimental animal research center, southern medical university (license number:NO.440210192). 2Experimental drugXiao Wu Gui granules main components:ephedra, cassia twig, angelica, Codonopsis, Radix Paeoniae Alba, Rhizoma Chuanxiong, gypsum, dried ginger, Radix Astragali, Radix Aconiti, Rhizoma Anemarrhenae, windproof, aconite, Cortex Moutan, baikal skullcap root, licorice root. Methotrexate tablets (Shanghai Xinyi pharmaceutical factory), methotrexate powder (sigma).3Research methods3.1Effect of Xiao Wu Gui granule on the CIA rats and the related cytokine expression.The chicken type Ⅱ collagen rat model of CIA was established, using the arthritis index (AI) score and pathological inflammatory severity scoring system for assessment. The CIA rats were randomly divided into model success into5groups,8rats in each group, and began to22d, with high/medium/low dose Xiao Wu Gui granules in distilled water (2ml/d) and MTX (0.9mg/kg/w), distilled water blank (2ml/d) was infused into the stomach of each group CIA rats, continuous21d. Each medicine intervention after CIA, every7d, scoring method of recording arthritis disease incidence and severity of AI. Intervention21d, pathological score of knee joint in rats with inflammation pathological grading system, ELISA to detect the rat peripheral blood the molecular level (TNF-α, IL-10).3.2Effect of Xiao Wu Gui granule drug-containing serum proliferation of CIA-FLS in vitro and the related cytokine secretion.Isolation and culture of CIA-FLS in vitro tissue culture, cell morphology was observed under the light microscope, immunohistochemical staining for vimentin cells were identified and cell purity calculation. The experiment was divided into blank control group, MTX group (0.3μmol/L) and high/medium/low dose of Xiao Wu Gui granule drug-containing serum group5groups, respectively interference CIA-FLS for24h,48h,72h, proliferation is determined by MTT method and ELISA to detect the cell supernatant level of TNF-a and MMP-1.4Statistical methodsUsing SPSS13.0statistical software, measuring data with mean±standard deviation, according to normal distribution measurement data is compared between groups using single factor analysis of variance, two two compared with bonferroni test; Non-normal distribution measurement data using multiple nonparametric test, P <0.05for the difference was statistically significant.Results1. Effect of Xiao Wu Gui granule on the CIA rats and the related cytokine expression.CIA model21d, rat paw joints including ankle obvious swelling,76%(44/58) of rat AI score≥4, the successful rat model of the overall AI score X=6.68±0.77. The high/medium/low dose of Xiao Wu Gui granule group and MTX group intervention CIA rats7d,14d,21d, the AI scores decreased significantly, there was statistical significance compared with the model group (p<0.05);The high/medium/low dose of Xiao Wu Gui granule group and MTX group intervention CIA rats21d, the pathological score decreased, reducing peripheral blood serum TNF-α level, increase the level of IL-10, there was statistical significance compared with the model group (p<0.05).2. Effect of Xiao Wu Gui granule drug-containing serum proliferation of CIA-FLS in vitro and the related cytokine secretion.The high/medium dose drug-containing serum and MTX suppressed the CIA-FLS proliferation and reduced the TNF-a and MMP-1level at24,48,and72h after the exposure (p<0.05). The low dose drug-containing serum suppressed the CIA-FLS proliferation and reduced the TNF-a level at48and72h after the exposure (p<0.05).The low dose drug-containing serum reduced the MMP-1level at24,48,and72h after the exposure (p<0.05).Conclusion1. The Xiao Wu Gui granule effectively reduce the CIA rats of AI score and pathological grading, shows that the model has a therapeutic effect on CIA rats.2. The Xiao Wu Gui granule effectively reducing CIA rats peripheral blood serum TNF-a level, increase the level of IL-10, this could be one of its therapeutic.3. The Xiao Wu Gui granule drug-containing serum can inhibit CIA-FLS proliferation, and CIA-FLS secdreted TNF-α and MMP-1, this could be one of its therapeutic.