| Rheumatoid arthritis is a chronic, inflammatory and tissue-specific autoimmune disease with the global incidence about 1%. It’s also a common disease with high disability rate and difficult to cure. The pathogenesis of RA is very complex and not fully understand as yet. Generally, it’s related to environmental factors, genetic factors and autoimmune disorders. Research has shown that many cells and molecules involved in joint inflammation and tissue destruction. What’s more, The activation of T cells was considered as the key point in the development of RA. CD4+T cells as an important component of effector T cells was involved in the every parts of immune response and played a key role in immune regulation. Conventional wisdom holds that Th1 cell-mediated immune response starts the occurrence of RA and the imbalance of Th1/Th2 plays an important role in the pathogenesis of RA. With the discovery of Th17 cell, research shows that Th17 cell also plays a significant role during the process of RA. At present, nonsteroidal anti-inflammatory drugs, antirheumatic drugs andbiological agents are often used in treating RA in clinical. However, therapeutic efficacy of these drugs is not significant and with obvious side effects. Diosgenin is one of the effective components which is extracted from Rhizoma Dioscoreae Nipponicae. that is commonly used in the treatment of back pain and sprains. Modern pharmacological studies show that diosgenin has the function of anti-tumor, hypolipidemic and.immunoregulation as well as insignificant side effect. This research is aimed to study the effect of diosgenin on CD4+T cell by establishing CIA mouse model and further discuss the treatment mechanism of RA.Objective:To discuss the effects of diosgenin on expression of Th1, Th2, Th17 cells and its specific transcription factors in vitro, then clarify the immune regulation mechanism of diosgenin on CIA mouse model and provide some experimental basis for the treatment of RA.Methods:The 75 DBA1/J mice was immunized with the prepared type collagenⅡ and each mouse was intradermal injected 0.1 m L in tail root. On the 21 st day after the first immunization, to establish the CIA mouse model by strengthening the immunization as the same method. On the 28 th day after the first immunization, to select the CIA mice model according to AI index, then excise the inguinal lymph nodes of them under aseptic condition, made it into single cell suspension and test drug toxicity in order to determine medical concentration with CCK-8. The experiment were divided into five groups including control group(adding IL-2-stimulated lymphocytes), model group(adding IL-2 and collagen-stimulated lymphocytes), diosgenin high dose group(adding IL-2 and collagen-stimulated lymphocytes and high dose of diosgenin), diosgenin medium dose group(adding IL-2 and collagen-stimulated lymphocytes and medium dose of diosgenin), diosgenin low dose group(adding IL-2 and collagen-stimulated lymphocytes and low dose of diosgenin).The percentage of Th1, Th2 and Th17 cells were detected by flow cytometry. The expression of the specific transcription factor T-bet, GATA3 and RORγt were observed by real-time PCR.Results: 1 Test drug toxicity and the inhibition rate to lymphocyte proliferation by CCK-8 1.1 After the CIA mice lymphocytes were cultured 48 h, the survival rate of diosgenin groups(3.125-25 μmol/L) was more than 70%. It showed that diosgenin(3.125-25 μmol/L) had no significant cytotoxicity on T lymphocytes. 1.2 After the CIA mouce lymphocytes were cultured 48 h, compared with the stimulating group(P<0.05), the drug group had certain inhibit effect tolymphocytes. The inhibition rates of diosgenin groups(3.125, 6.25, 12.5, 25, 50 μmol/L) were 0.083, 0.215, 0.498, 0.722, 0.905 respectively. According to the results of inhibition rates, determine the dose of diosgenin in the high, medium, low dose groups were 6.25, 12.5, 25 μmol/L respectively. 2 Detected the percentage of Th1, Th2, Th17 cells by flow cytometry Compared with the control group, the ratio of Th1 cells in model group was significantly improved(P<0.05). Compared with the model group, the ratio of Th1 cells in diosgenin high dose group and medium dose group were significantly decreased(P<0.05), but in diosgenin low dose group it was no significant difference(P>0.05). Compared with the diosgenin low dose group, the ratio of Th1 cells in diosgenin high dose group and diosgenin medium dose group were reduced(P<0.05). Compared with the diosgenin medium dose group, the ratio of Th1 cells in diosgenin high dose group was no significant difference(P>0.05). Compared with the control group, the ratio of Th2 cells in model group was significantly higher(P<0.05).Compared with model group, the ratio of Th2 cells in diosgenin groups were no significant difference(P>0.05). Compared with the control group, the ratio of Th17 cells in model group was significantly higher(P<0.05). Compared with model group, the ratio of Th17 cells in diosgenin high dose group and medium group were significantly decreased(P<0.05), but in diosgenin low group it was no significant difference(P>0.05), Compared with the diosgenin low dose group, the ratio of Th17 cells in diosgenin high dose group and diosgenin medium dose group were reduced(P<0.05), Compared with the diosgenin medium dose group, the ratio of Th17 cells in diosgenin high dose group was no significant difference(P>0.05). 3 Detect expression of specific transcription factor T-bet, GATA3, RORγt by Real-time PCR Compared with the control group, in model group T-bet increased significantly(P<0.05). Compared with the model group, T-bet reduced in diosgenin high dose group(P<0.05), but in diosgenin low dose group and diosgenin medium dose group there were no significant difference(P> 0.05)Compared with the diosgenin low dose group, T-bet was reduced in the diosgenin high dose group(P<0.05), but in the diosgenin medium dose group it was no significant difference(P>0.05). Compared with the control group, GATA-3 was much elevated in model group(P<0.05), but in diosgenin groups there were no significant difference compared with model group(P>0.05). Compared with the control group, RORγt was obvious increased in model group(P<0.05), however, it significantly reduced in diosgenin high dose group than model group(P<0.05), but in diosgenin low dose groups and diosgenin medium dose groups there were no significant difference(P>0.05), Compared with the diosgenin low dose group, RORγt was reduced in the diosgenin high dose group(P<0.05), but in the diosgenin medium dose group it was no significant difference(P>0.05).Conclusion:1 Diosgenin had a obvious inhibition on Th1 and Th17 cells of CIA model mice in vitro. It also could improve the percentage, of Th2 cells, but the effect is not significant. 2 The effect of Diosgenin on CD4+T cells is probably based on the effect of diosgenin on the specific transcription factors of CD4+T cell. |
PDF Full Text Request