| Rheumatoid arthritis is a chronic autoimmune disease which is characterized by erosive arthritis and the resulting articular cartilage and bone destruction. Without proper treatment, the illness will progressively worse, eventually leading to joint deformity.CD4+T cells are an important component of immune system and play an important role in the occurrence and development of diseases. CD4+ T cells can be divided into Th1,Th2, Th17 and Treg cells in accordance with their different functions. Early studies suggest that the incidence of RA is mainly associated with the Th1 cells, But recent studies have found that, but recent studies have found that, Th1, Th2, Th17 and Treg cells are closely related to the incidence of RA.Dioscorea nipponica often used as drugs. Folks always dip it into the wine or decocted it, to treat backache and numbness. Dioscin which is isolated from Dioscorea nipponica is steroidal saponins, and it is an important basic raw materials for producing steroid drugs. Dioscin is considered to be the main active ingredient in many traditional Chinese medicine,such as Diaoxinxuekang capsules and weiaoxin tablets. Modern pharmacological studies confirmed that dioscin has a variety of biological activity.To be sure, dioscin is a promising drug.Objective:To observe the influence and specific regulatory mechanism of dioscin on CD4+ T cell subsets on chicken type Ⅱ collagen-induced arthritis in mice, initially clear what mechanism dioscin to produce a therapeutic effect by and provide a theoretical basis for further research and clinical application of dioscin.Methods:A total of 72 DBA1/J mice were divided into four groups(control group, model group, dioscin group and triptolide group) randomly. Prepared the type Ⅱ collagen emulsion by mixing the chick type Ⅱ collagen with Freund’s complete adjuvant. The mice in model group, dioscin group and triptolide group were immunized by this type Ⅱ collagen emulsion under aseptic condition. Each mouse is injected intracutaneously with 0.1 mL in tail root and the same procedure repeated on the 21 st day after the first immunization. The control group was injected with saline as described above. Starting from the 22 nd day of the first immunization, dioscin group was given suspension of dioscin 100 mg /kg/d.Triptolide group was given suspension of triptolide according to the ratio of body surface of human and mouse(By triptolide meter 17 μg/kg/d).The mice of model group and control group were given equal volume of solvent. The mice were killed in the thirty-five days after the first immunization,take lymph nodes in the groin under sterile condition.The percentage of Th1, Th2, Th17 and Treg cells were detected by flow cytometry.The contents of p-STAT1, p-STAT3, p-STAT4, p-STAT5, p-STAT6 and SOCS3 were detected by Western Blot Assay.The expression of the transcription factor T-bet, RORγt, Foxp3 and GATA3 were observed by real-time PCR technique.Results:1 Changes of body weight, paw swelling and AI valueWeight of mice in the control group growed steady,while the model group decreased significantly because arthritis(P<0.05). Compared to control group,dioscin group decreased significantly(P<0.05),but there was no significant difference between model group and dioscin group(P> 0.05). triptolide group was similar to the dioscin group.Footpad thickness of mice in the control group had no significant change during the experiment,stabilizing at around 1.40 mm. While the model group reached 1.99 mm in the 35 th day after immunized, consistent with the severity of arthritis. There had significant difference compared with the control group(P<0.05). Footpad thickness of mice in the dioscin group increased less compared with the model group, the difference was statistically significant(P<0.05). Footpad thickness of mice in the triptolide group increased least and reached only 1.72 mm in the 35 th day after immunized. Compared with the model group, difference was statistically significant(P<0.05). There was no significant difference between triptolide group and dioscin group(P>0.05).Control group showed no swelling and AI value has been to 0 during the experiment.While AI index of model group reached 11.84 in the 35 th day after immunized, compared with the control group the difference was statistically significant(P<0.05). AI index of dioscin group significantly improved compared to the model group(P<0.05).AI index of triptolide group was smallest and the symptom was lightest. There was no significant difference between triptolide group and dioscin group(P> 0.05).2 Percentage of Th1, Th2,Th17 and Treg cells detected by flow cytometryCompared with the control group, the ratio of Th1 cells in model group was significantly higher(P<0.05)。The ratio of Th1 cells was significantly lower in dioscin group compared to the model group(P<0.05).Control group compared with the model group, the ratio of Th2 cells showed no difference(P>0.05).Dioscin group was higher than model group(P<0.05). Compared with the control group, the ratio of Th17 cells in model group were significantly higher(P<0.05).Dioscin group was significantly lower than model group. The ratio of Treg cells showed no difference between control group and model group(P>0.05). Dioscin group was significantly higher than model group(P<0.05).3 western blot detection of transcription factor p-STAT1, p-STAT3, p-STAT4, p-STAT5, p-STAT6 and SOCS3 contentCompared with the control group, p-STAT1 in model group was significantly higher(P<0.05). P-STAT3 increased significantly in model group compared with the control group(P <0.05).In dioscin group p-STAT3 decreased significantly(P<0.05) and triptolide group decreased less(P<0.05). Compared with the control group, p-STAT4 in model group was significantly higher(P<0.05). In dioscin group p-STAT4 decreased significantly(P<0.05) and triptolide group decreased more(P<0.05). P-STAT5 in model group increased significantly compared with the control group(P<0.05).Dioscin group increased significantly compared with the model group(P<0.05),and triptolide group increased less(P<0.05).Compared with the control group,p-STAT6 significantly increased in dioscin group(P<0.05).SOCS3 increased in all the other group compared with the control group.SOCS3 showed no difference between dioscin group and model group(P>0.05),but triptolide group was significantly higher(P<0.05) than model group.4 Real-time PCR technology to detect expression of transcription factor T-bet, GATA3, RORγt, Foxp3Compared with the control group, T-bet was significantly higher in model group(P<0.05). Compared control group to model group GATA-3 showed no difference(P> 0.05),but dioscin group and triptolide group was significantly higher than model group(P<0.05) and dioscin group increased more. Compared with the control group,RORγt increased significantly in model group.Dioscin group and triptolide group decreased compared with model group(P<0.05). Foxp3 was significantly higher(P<0.05) in model group compared with the control group.Conclusion:Dioscin can regulate differentiation of CD4+ T cell subset by adjusting key transcription factors and then affects the balance of Th1/Th2 and Th17/Treg cells,which have an effect on collagen-induced arthritis in mice. It is expected to develop a new drug to treat rheumatoid arthritis. |
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