| Objectives:In order to investigate the effect of genistein(gen) on the secretion of RANKL/OPG secreted by rats with collagen induced arthritis (CIA) rats and fibroblast-like synoviocytes.Subject:fibroblast-like synoviocytes、The rats with CIAMethods:Sixty Sprague-Dawley rats were randomly divided into six groups, with10rats in each group, respectively:normal group,model group, low-dose genistein(Gen) treatment group, moderate-dose Gen treatment group,high-dose Gen treatment group and methotrexate(MTX) group(n=10). Model group and all treatment groups were induced CIA model in SD rats immunized by subcutaneous injection of collagen type Ⅱ combined with complete Freund’s adjuvant(CFA).On the second day after the injection, lml of suspension of different concentration of gen(50μM,100μM,200μM, once a day for35days)and MTX(0.1mg/ml body weight,once a week)were administered by oral gavage respectively.The rats in normal group and model group were administrated with normal saline in the same volume. The arthritis was scored once a week with the arthritis index(AI) and hind paw swelling. After the treatment for5weeks, the rats were sacrificed, and sera were immediately separated from3ml of blood samples collected from the abdominal veins in serum tubes.The plasma concentrations of OPG、RANKL were determined with enzyme-linked immunosorbent assay (ELISA).The expression of CD29, CD90of human rheumatoid arthritis FLS was detected by Flow cytometry. The experience was divided into four groups:control group, TNF agroup, TNF a+Gen group and Gen group. Western blot were used to detect the expression of estrogen receptor a(ER a), RANK, RANKL, OPG. Immunofluorescence were used to detect ER a.Results:1. Joint swelling of the CIA rats which established with collagen type II combined with complete Freund’s adjuvant were observed apparently three days later.AI score and hind paw swelling were shown significant differences between the model group and the control group, indicating that the model of CIA rats was constructed successfully.2. The level of RANKL serum was significantly higher in the model group than that in control group (p<0.05). Gen can down-regulate of RANKL. Expression levels of OPG in the model group is the lower than that in the control group (p<0.05); Gen can up-regulation of OPG Gen had positive effects on OPG which is higher than MTX, but did not reach the level of the control group.3. Western Blotting showed that Gen can significantly up-regulate the expression of OPG and RANKL relative to control group(p<0.05); but had no effect on RANK and ER a. The statistical results showed that the ratio of OPG/RANKL in Gen group was increased. Compared with control group, Result from the immunofluorescence also showed that most of the ER a was tranfered into the nucleus.Conclusions:1. Gen can down-regulate RANKL in serum of CIA rats, and can down-regulate OPG and ratio of OPG/RANKL, which suggest that Gen may influence the RANKL/OPG/RNAK pathway, which can delay bone destruction.2. Gen can influence the RA-FLS cells RANKL, OPG. Gen can improve the ratio of OPG/RANKL of RA-FLS cells. Therefore, Gen may influence OPG/RANKL balance, which may exert their inhibitory effect of RA bone destruction.3. Gen had no effect on the RA-FLS expression of ER a, but it can influence ER a into the nucleus, which sugest that Gen may influence ERα to regulate the OPG/RANKL/RANK pathway. |
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