Therapeutical Effect Of Fengshining Capsules On Collagen-induced Arthritis In Rats And Its Mechanism

Rheumatoid arthritis (RA) is a autoimmune disease characterized by inflammation and proliferation of synovial tissue resulting in bone and cartilage destruction.The pathogenesis of RA involving the activation of t-lymphocytes and is closed related with oxidative stress, deactivated mitochondrial function which regulated the apoptosis process.In recent years, studies have shown that reactive oxygen species (ROS) played an important role in the prevention and treatment of RA. In many organizations, ROS-induced oxidative stress could damage mitochondria respiratory chain, inducing mitochondrial injury and cell apoptosis and death.But it is known that for tumor-like proliferation of synovial tissue, inducing the expression or biological activity of ROS, will become an important pathological aspects that curbing the excessive proliferation of RA synovial cell, which will also be in favour of prevention and treatment of RA. Fengshining capsule (FSN) is mainly for the treatment of rheumatoid arthritis casued by cold-dampness and blood stasis blocking collaterals, has been confirmed its significantly effect through more than10years clinical application. Early experimental studies have shown that FSN had a wide range of pharmacological effects such as anti-inflammatory, analgesic and immune regulation.Although FSN has a good anti-RA prospects, its mechanisms on anti-inflammatory, molecular of immune regulation are not yet cleared, especially if its anti-RA mechanism has some relationship of inducing the excessive proliferation of synovial cell apoptosis, so far is not yet clear. Therefore, a series of studies were focused on the subject to explore the FSN in the regulating immune function and promoting the molecular mechanism of synovial cells apoptosis. Results proved that FSN could regulate the secretion of inflammatory factors and reinstate Th1/Th2, CD4+/CD8+balance.Meanwhile, it could increase expression of reactive oxygen species in inducing apoptosis of synovial cells via mitochondrial pathway, which probably is a new resistance mechanisms of FSN for RA.Objective:To enunciate the FSN possible molecular mechanism of the anti-inflammatory and immune regulation effects from the foot swelling, bone of CIA rat index and pathologic histological changes and cell factors and t-lymphocyte subsets. Bovine type Ⅱ collagen-induced rats model were prepared. The apoptosis-promoting effects of the FSN on CIA rat synovial cell was observated and its possible mechanism was investigaged on generation of reactive oxygen species, mitochondria function, Bcl-2, caspase.Method:The CIA model in rats was induced by Freund’s complete adjuvant. Therapeutic treatment of interagastric administration FSN (0.33,0.66,1.32g·kg-1,30d), tripterygium glycosides (0.03g·kg-1) were given as positive control. Foot swelling of CIA rats was measured with foot volume method. Arthritis index was used to evaluate the degree of joint damage of rat arthritis. Body weight, spleen and thymus weights were measured. Histopathological changes of joints were observed under the light microscope. Joint X ray films were taken. ELIS A method was used for detecting the content of serum TNF-a, IL-1, IFN-y, IL-4and IL-10. The level of t-lymphocyte subsets were detected with flow cytometry. Transmission electron microscopic was used to observate the ultrastructure of rat synovial cells. Hydroxyl free radicals were detected with Fe2++H2O2system.Synovial ATP content, activity of caspase3and caspase-9were assayed using UV spectrophotometric detection.ELISA was used to assay synovial Cytc levels in the supernatant. VEGF, Bcl-2, Bax expression in synovial tissues were detected by immunohistochemical assay. Apoptosis-related gene expression levels of caspase-3, caspase-9mRNA were detected by RT-PCR assay.Bradford protein concentration assay was used to detect synovial membrane protein concentration. The expression changes of synovial caspase-3, caspase-9p35, VDAC1, AIF were detected by Western blot assay.Results:1. FSN played a treatment role on CIA rats. Its anti-inflammatory and immunomodulatory effects were related with reducing levels of Inflammatory Cytokines(IL-1, TNF-a, IFN-y)secreted by Th1cell, increasing the content of anti-inflammatory cytokine (IL-4, IL-10) secreted by Th2cells and restoring CD4+/CD8+balance. In addition, in vascular micro-environment, it was confirmed that high expression VEGF protein in CIA rats were decreased on varying degrees after FSN medication intervention, which further inhibited the growth of synovial cells.FSN (0.66,1.32g· kg-1) could significantly suppress the CIA rat foot swelling and relieve joint pain, multiple arthritis symptom, as well as eased the decreased weight of CIA rats. Articular mild hyperplasia, inflammatory cell infiltration in the synovium and synovial tissue reduction of neovascularization, vascular proliferation of associated mitigation, occasionally a small amount of fiber, bone and cartilage damage mitigation were observed under light microscopic observation. FSN could reduce IL-1, TNF-a, IFN-y levels secreted by Thl cells on varying degrees and increase IL-4, IL-6, IL-10levels secreted by Th2cells. FSN was proved to lower CIA rat thymus index. Streaming results also showed that FSN could restore CD4+/CD8+ratio in CIA rats, reducing the level of CD4+T lymphocyte.2. FSN raised the level of ROS expression in CIA rats synovial tissueROS content of synovial tissue were reduced in CIA rats. The increasing ROS expression of FSN (0.33,0.66,1.32g· kg-1, ig,d30) in drug delivery was indicated that FSN risen ROS level of CIA rat was one of its features on regulating mitochondrial membrane permeability transition pore open, ATP levels in synovial cells, mitochondrial transmembrane potential, affecting the synovial apoptosis process, suppressing immune hyperthyroidism and playing treatment role.3. FSN induced the synovial cells apoptosis by affecting the signal transduction pathwaysThe basic structure of synovial cells in CIA rats is returned to normal after FSN in vivo delivery and FSN was able to contain abnormal hyperplasia of interstitial collagen fibers, as well as improved mitochondrial swelling or voided denatured state. The result of Western-blot analysis demonstrated that FSN could promote the expression of VDAC1rising.The result of ultraviolet spectrophotometry analysis showed that FSN significantly decreased the ATP content in drug group. The supernatant of Synovial tissue homogenate was detected that FSN (0.66g·kg-1) Cytc emissions were clearly increased.Immunohistochemical results showed that as the dose increasing, Bcl-2positive expression of synovial cells in FSN drug delivery decreased, had a downward trend.Meanwhile,Bax expression of positive cells significantly increased.The result of RT-PCR, Western-blot analysis confirmed that the expression of caspase-3, caspase-9p35, AIF markedly increased in the CIA synovial tissue of FSN intervention group. The caspase enzymes experimental data also stated that caspase-3, caspase-9activity in FSN had a notable difference between before and after medication.Conclusion:1.FSN has significantly improve effect on inflammation of multiple joints in CIA rats.FSN might inhibit proinflammatory cytokines IL-1, CIA rats TNF-α, IFN-γ secreted, promoting anti-inflammatory cytokines(IL-4, IL-6, IL-10) formated, reducing CD4+/CD8+and suppressing the expression of VEGF protein.That may be the key which FSN regulate hyperthyroidism immune function in CIA rats.2. FSN could promote the apoptosis via caspase-dependent, AIF pathway and curb the excessive proliferation of synovium by regulating ROS generation in vivo to affect synovial mitochondrial function, the Th cell differentiation to play its therapeutic effect.3. Mitochondrial features change led to synovial cells apoptosis participating RA pathology physiological change in FSN control group,such as:Under the stimulation of ROS, mitochondrial promote VDAC1channel open, affecting the opening of MPTP and the escaping of ATP energy matter, resulting in inhibition of the mitochondrial respiratory chain, loss of membrane potential, release of Cytc and AIF,which triggered apoptosis in two ways:one is a Bcl-2family and Caspase enzymes dependented participation way, activating caspase-9and the downstream of caspase-3, leading to apoptosis. Another is multiplex polymerase pathway regulating by AIF. The anti-apoptotic factor Bcl-2and apoptosis factor Bax protein through combinating with VDAC1regulate the apoptosis process. Therefore, mitochondrial dysfunction may play an important role in synovial apoptosis.