MTX Combined With CTX By RORγt Mediated Effects On CIA Mice Th17/Treg Cell

Objective:MTX combined with CTX can block the RORγt collaborative signal channel, and then adjust the balance of Th17/Treg cells, inhibiting the inflammatory reaction.Methods:Male DBA1 mice with collagen induced arthritis model in 40, after the success of the model according to the different drugs were randomly divided into 4 groups: CIA model group(saline), MTX group(1.5mg-kg-1-3d-1), CTX group(30 mg-kg-1-10d-1) and MTX+CTX group(the combined group, MTX:1.5Mg-kg-1-3d-1, CTX:30g-kg-1-10d-1).At the same time not selected mice model of 10 rats as normal control group. The first immunization began after 3 weeks of administration, the mice were treated continuously in the process of right hind ankle joint swelling and arthritis index measurement(arthritis index, AI) score. At eleventh weeks after administration, into sections, HE staining,observe the change of pathology of synovial tissue, and detected by FCM Th17 and Treg cells and the ratio of detection; the treatment group RORγt and Foxp3 m RNA and the ratio of using RT-PCR method.Results:After 11 weeks after treatment in the treatment group, AI, joint swelling joint pathology was improved than the CIA group, there was significant difference between the groups was statistically significant; joint swelling degree difference compared to the CIA group(P <0.05), compared with CTX, MTX single drug group, there is no statistically significant difference. There was statistical significance with group AI score difference compared tothe CIA group(P < 0.05), compared with CTX, MTX single drug group, there is no statistically significant difference.After 11 weeks after administration of flow cytometry in Th17 cells after cultured mouse spleen Naive T cells proportion, CIA group than in the control group accounted for the proportion of Th17 cells increased significantly, the difference was statistically significant(P < 0.05); the treatment group compared with CIA group, the percentage of Th17 cells decreased, there statistically significant difference(P < 0.05); combined with CTX, MTX group compared with the single drug group decreased Th17 cells, there is no statistically significant difference(P < 0.05).By 11 weeks after the administration of flow cytometry in Treg cells after culture of mouse spleen Naive T cells proportion, CIA group than in the control group accounted for the proportion of Treg cells decreased significantly, the difference was statistically significant(P < 0.05); the treatment group compared with the CIA group of Treg cells decreased, there is no statistically significant difference.Th17/Treg can be found in CIA group compared with normal control group, Th17/Treg was significantly increased, the difference was statistically significant(P< 0.05); the treatment group Th17/Treg decreased significantly compared with CIA group, the difference was statistically significant(P < 0.05); CTX, MTX combined group and single drug group decreased compared to Th17/Treg, there is no statistically significant difference(P < 0.05).The expression level of RT-PCR 11 weeks after treatment were detected in Th17 cells after differentiation of ROR gamma t m RNA, CIA group and normal control group RORγt the proportion of m RNA increased significantly, the difference was statistically significant(P < 0.05); the treatment group compared with the CIA group RORγt m RNA proportion has decreased statistically significant difference(P < 0.05); combined with CTX, MTX group compared with the single drug group decreased RORγt m RNA, there is no statistically significant difference(P < 0.05).The expression level of RT-PCR 11 weeks after treatment were detected in Th17 cells after differentiation of Foxp3 m RNA, CIA group compared to the control group theproportion of Foxp3 m RNA decreased significantly, the difference was statistically significant(P< 0.05); the treatment group compared with CIA group, Foxp3 m RNA decreased, the difference is statistically significantRORγt/Foxp3 m RNA can be found in CIA group than normal control group ROR gamma t/Foxp3 m RNA significantly increased, the difference was statistically significant(P < 0.05); combined group compared with the CIA group RORγt/Foxp3 m RNA significantly decreased, the difference was statistically significant(P < 0.05); CTX, MTX single drug group compared with CIA group, RORγt/Foxp3 m RNA decreased, the difference there is no statistically significant(P < 0.05).Conclusions:That MTX combined with CTX is better than single medication can effectively inhibit RORγt signal down channel, so as to control the immune inflammatory cells increased Th17, adjust the balance of Th17/Treg cells.