Gastrodia Elata Attenuates Inflammatory Response Caused By Rheumatoid Arthritis By Inhibiting The NF-?B Pathway

Background Rheumatoid arthritis(RA)a chronic and generalized autoimmune disease characterized by joint inflammatory and bone destruction which occured around the world.So far,its pathogenesis has not been fully confirmed until now and also clinically also lack of effective treatments for the disease.Western medicine treatment often use hormones,non-steroidal anti-inflammatory drugs and immunosuppressive drugs to alleviate the development of RA,while there are a lot of side effects after using for a long time.Gastrodia elata(GE),which belongs to the Orchidaceae family,is a traditional C hinese herbal medicine and mainly distributed in the mountainous areas of Southwest China,Many previous studies have demonstrated that GE possesses anti-oxidative,anti-inflammatory,anti-tumor and anti-asthmatic activities,it is widely used in the history of C hinese medicine.Some reseach shows that GE can attenuates inflammatory response in BV-2 cells,while the effect of GE on inflammatory response caused by rheumatoid arthritis remains largely unknown.Fibroblast-like synoviocytes(FLS)is a specialized cell type located in synovial joints,which play crucial roles in the development of rheumatoid arthritis,its hyperplasia can affect the absorption of nutrients in articular cartilage and bones.Thus,FLS is often used as a cell model for in vitro studies of RA.Tumor necrosis factor-?(TNF-?)is a pro-inflammatory cytokine produced by monocytes/macrophage.It was reported that TNF-a is abundant in the RA synovium,it can regulate the signal pathway and then induce the expression of inflammatory factors(such as IL-6,IL-8)which result in thet inflammatory responses in RA.NF-?B is an important transcription factor to regulate the expression of various inflammatory factors.Through the reseach of NF-?B,we can study the pathogenesis of GE in inflammatory reaction of RA.Objective In this study,we built the CIA mouse model induced by chicken type II collagen to detect joint swelling and cartilage destruction of mice,as well as the level of antibody and the expression of related inflammatory factors with different concentrations of GE.By building the RA-FLS cells model to investigate the effect of different concentrations of GE on cell viability,cell proliferation,TNF-?-induced cell inflammatory response,apoptosis and NF-?B activity.By studying the activity of NF-?B,the expression of inflammatory factors and apoptosis conducted by PDTC and GE,in order to understand the underlying molecular mechanism of GE,and lay the foundation of the clinical treatment of RA.Method(1)Construction of Chicken Type II Collage n-induced CIA mice model,to reseach the pharmacological mechanism of GE from the whole animal level.The treatment group were injected with 5?g/m L and 10?g/m L of GE at 21 d,and then the effects of different concentrations of GE on the joint swelling of CIA mice were observed every 3 days.In addition,Micro-CT was used to scan the joints of each group,and the degree of cartilage injury and bone mineral density were recorded.We used ELISA to detect levels of Ig G,IL-1?,IL-6 and IL-8,and q RT-PCR to detect levels of MMP-3 and MMP-13 after treatment with GE.(2)Construction of RA-FLS cells model,to reseach the effect of GE on TNF-?-induced inflammatory response from the cell level.MTT assay was used to determine the effect of different concentrations of GE(0~20?g/m L)on the activity and the proliferation of RA-FLS cells.The expression of TNF-?-induced IL-1?,IL-6,IL-8,MMP-3 and MMP-13 was analyzed by ELISA and q RT-PCR,apoptosis of RA-FLS cells was analyzed by flow cytometry,and the expression of NF-?B-related transcription factor was analyzed by Western blot at 24 h after GE treatment.(3)The expression of inflammatory factors is regulated by signaling pathways,to understand whether GE can regulate the inflammatory response by inhibiting the NF-?B pathway in rheumatoid arthritis fibroblast-like synoviocytes,we introduce NF-?B signaling pathway inhibitor as a positive control in this experiment to study the anti-inflammation mechanism of GE.The FLS cells were divided into blank group,NC group,PDTC group,GE group and PDTC + GE group.After conducted by GE and PDTC for 24 h,the expression of NF-?B-related transcription factor was detected by Western blot,the levels of IL-1?,IL-6,IL-8,MMP-3 and MMP-13 was analyzed by q RT-PCR,and the apoptosis of FLS cells was analyzed by flow cytometry.Results(1)Mice joint swelling results showed that the joint swelling gradually increased of the model group after the end of modeling,GE can relieve joint swelling significantly(P<0.05),in which high dose group had better effect on swelling than low dose group.Micro-CT scan revealed that the joints of the model group showed a certain degree of erosion,the joint space was significantly reduced and bone density decreased,the mice of the model group were the most severely damaged.GE treatment can significantly alleviate the phenomenon of joint damage,in which GE high-dose had better effect on joint damage.ELISA assay showed that levels of Ig G2 a,Ig G2 b,TNF-?,IL-1?,IL-6 and IL-8 were significantly higher than those of control group,GE can significantly attenuates the expression of Ig G and inflammatory factors(P<0.05),and high-dose group had stronger inhibitory effect.q RT-PCR assay showed that the expression of MMP-3 and MMP-13 were higher than control group,GE can attenuates the expression of MMP-3 and MMP-13(P < 0.05),and high-dose group had stronger inhibitory effect.(2)MTT assay showed that the activity and proliferation of RA-FLS cells were significantly inhibited in GE treatment group,inhibitory effect was strengthened with increase of concentration while concentration of GE was 1~10?g/m L.ELISA assay showed that levels of IL-1?,IL-6 and IL-8 were significantly higher than those of control group after induced by TNF-?.GE can significantly attenuates the expression of inflammatory factors(P<0.05),in which GE high-dose had stronger inhibitory effect.Westernblot and q RT-PCR assay showed that the expression of MMP-3 and MMP-13 were higher than control group,GE can attenuates the expression of MMP-3 and MMP-13(P<0.05),and high-dose group had stronger inhibitory effect.The results of flow cytometry showed that the apoptotic rate was significantly decreased after induced by TNF-?(P <0.05),GE can promote apoptosis,and GE high-dose group had stronger induction than low-dose.(3)Western blot results showed that the level of p-p65 was significantly increased and I?B? was decreased in NC group(P<0.05),the expression of p-p65 was attenuated and I?B? was induced in PDTC group,GE group and PDTC+GE group,in which inhibitory effect of GE on p-p65 and induction on I?B? was stronger than that of PDTC group,PDTC+GE group was most obvious than other groups.q RT-PCR assay showed that the level of IL-1b?IL-6?IL-8?MMP-3?MMP-13 m RNA was significantly increased in NC group(P<0.05),while those expression were attenuated in PDTC group,GE group and PDTC+GE group,in which inhibitory effect of GE was stronger than PDTC group,PDTC+GE group was most obvious than other groups.The results of flow cytometry showed that the apoptotic rate was significantly decreased in NC group(P <0.05),GE and PDTC can promote apoptosis,while the induction of GE was more significant than PDTC group,PDTC+GE group was most obvious than other groups.Conclusion GE can effectively relieve the damage of joints and arthritis,inhibit the activity and proliferation of RA-FLS cells and induce apoptosis.In addition that GE can attenuates expression of inflammatory factor by inhibiting the NF-?B pathway and then al eviate the inflammatory response of RA patients.