Effect Of Glycyrrhetinic Acid On The Expression Of Inflammatory Factors In Fibroblast-like Synovial Cells From Collagen Induced Arthritis (CIA-FLS)

[Aims]To observe the features of fibroblast-like synovial cells from collagen induced arthritis in rats (CIA-FLS), and determine the effects of glycyrrhetinic acid (GA) on CIA-FLS cell TNF alpha and interleukin-1βexpression in vitro.[Methods]1. The CIA was established by subcutaneous injection with typeⅡcollagen and incomplete Freud's adjuvant to rats. And then evaluated the reliability of CIA rat with rheumatoid arthritis from the arthrositis index, serology, pathology and imageology aspects.2. Synovium tissues obtained from CIA rats and normal rats respectively were cultured by tissue culture method. FLS was identified by inverted phase contrast microscop and immunocytochemical staining. Cell proliferation of FLS was assayed by Cell Counting Kit-8 (CCK-8) method. Then cell growth curves were drawn. ELISA method was used to detect the levels of TNF-αand IL-1βin Cell culture supernatant fluid.3. The inhibitory rates of CIA-FLS proliferation were assayed by CCK-8 method after CIA-FLS were treated with different concentrations of different Treatment Group respectively for 3 days, and IC10 was tested respectively. CIA-FLS were treated with MTX, GA and GA/MTX combinations with concentration of IC10 for different time (1d,3d,5d). Real time qPCR method was used to detect the expression of TNF-αand IL-1βmRNA in CIA-FLS. ELISA method was used to detect the levels of TNF-αand IL-1βin Cell culture supernatant fluid.[Results]1. Lose of weights, swelling of paws involving the ankle, foot, and digits in CIA rats were observed. We got 7 of 14 CIA rat model successfully (AI≥4). At the 4th week since first injection, X-ray photograph and pathological techniques showed typical chronic synovial inflammation with bone erosion. The levels of TNF-αand IL-1βin serum were both elevated in CIA rats'serum compared with the normal ones (P<0.05).2. FLS after the 3rd generations showed typical morphological characters under inverted phase contrast microscope. FLS appeared as spindle-shaped, with oval nucleus which located of the center of the cell, clear nucleolus. Immunohistoehemical staining of CIA-FLS showed Vimentin+/CK+/CD68-staining. Doubling time of CIA-FLS was about 5 days investigated by CCK-8 assay, and N-FLS was 7 days. The levels of TNF-αand IL-1βin serum were both elevated in CIA-FLS Cell culture supernatant fluid compared with N-FLS (P<0.05).3. CCK-8 method tested the IC10 of GA, MTX and GA/MTX combinations were 28uM,0.3uM and 12uM GA+0.13uM MTX respectively for 3 days.Real time qPCR method detected the expression of TNF-αand IL-1βmRNA in CIA-FLS. dl:MTX, GA and GA/MTX combinations did not result obvious down-regulation of TNF-αand IL-1βmRNA (P>0.05); d3:MTX, GA and GA/MTX combinations resulted down-regulation of TNF-αand IL-1βmRNA (P<0.05), no differience between each other (P>0.05); d5:All of MTX, GA and GA/MTX combinations suppressed the expression of TNF-αand IL-1βmRNA (P<0.05), and obvious differience between each other (P〈0.05), in order was GA/MTX combinations, MTX and GA (P<0.05). All treatment groups were effected in time-dependent manner (P<0.05).ELISA method was used to detect the levels of TNF-αand IL-1βin Cell culture supernatant fluid. And we got the similar results with the PCR.[Conclusions]1. GA/MTX combinations, MTX, GA suppressed the expression of TNF-αand IL-1βmRNA in successively intension and time-dependent manner.2. GA/MTX combinations may have synergism or the summation action.