Effects of tripterygium hypoglaucum(Levl.)Hutch on in vitro proliferation and apoptosis of synoviocytes from patients with rheumatoid arthritis

Runming ZENG; Shixin DU; Jie WU; Shaowei LUO; Dongxin LIU; Hu WANG

ABSTRACT

BACKGROUND:

Tripterygium hypoglaucum(Levl.)Hutch (THH) has been accurately confirmed a good clinical therapeutic effect for rheumatoid arthritis(RA).However.functionaI mechanism of THH remains unclearly.

OBJECTIVE:

To investigate the effect of THH on proliferation and apoptosis of macrophage-like synoviocytes and fibroblast-like synoviocytes from patients with RA and to explore its dose-effect relation.DESIGN.TIME AND

SETTING:

In vitro cytology grouping controlled observation was performed in the Center Laboratory of the First Clinical College of Shantou University and the Laboratory for Cellular Biology of Southern MedicaI University from August 2003 to June 2007.MATERIALSSynovium was obtained from 6 patients with RA and 3 normaI synovial samples.All samples sourced from patients admitted in Department of Orthopaedics.the First Clinical College of Shantou University and in Spinal and Joint Surgery of Nanfang Hospital.The water extracts of THH contained 0.667 g/mL crude drug.

METHODS:

Synovial cells were isolated by digesting synovial tissue with collagenase.CD68+and CD68-synoviocytes were sorted from synovial cells by Dynabeads(magnetic celI sorting)from the 2~(nd)-4~(th) passages of synoviocyes.After 72 hours incubation,all groups of isolated synoviocytes were cultured in culture medium containing the flnaI concentrations of 0 mL/L,5 mL/L,1 0 mL/L and 20 mL/L of THH for 24 and 48 hours,respectively,and the common culture medium was sewed as the control. MAIN OUTCOME

MEASURES:

The morphology of synoviocytes were observed by inverted phase contrast microscope and studied by scanning electron microscopy(SEM).In vitro cell growth was assessed by the MTT assay,and synoviocytes apoptosis was evaluated using temlinaI deoxynucleotidyl transferase biotin-dUTP nick end labeling(TUNEL)assay and a flow cytometry.

RESULTS:

CD68~+ synoviocytes were appeared macrophage-like and CD68~- synoviocytes were exhibited fibroblast-like.After incubated with THH,some synoviocytes presented the volume of cells deflated,progressively destruction of cell membrane,microvillus or pseudopodium tended to be decreased even disappeared,and apoptosis bodies appeared.When incubated with 2% THH,proliferation inhibiUon and inducing apoptosis could be observed;it showed that THH had the cytotoxic effect on synoviocytes.The effect of proliferation inhibition and inducing apoptosis on CD68-and CD68+synoviocytes from RA patients was more significantly than that from normal arthritis (P<0.01).When incubated with 1%THH.proliferation inhibition and inducing apoptosis on synoviocytes from RA patients also could be found(P<0.01),but not found on normal synoviocytes(P>0.05).Compared with the negative controI group.proliferation inhibition was no significant difference On all groups with the low dose (≤5 mL/L) of THH(P>0.05).Furthermore,the effect of proliferation inhibition and inducing apoptosis on synoviocytes decreased in a dose-dependent and time-dependent manner.The inhibition rate of synoviocytes also could be found positively correlated with apoptosis rate aftat treatment of THH(r=0.497,P<0.01).CONCLUSlONTHH have cytotoxic effect on rheumatoid synoviocytes and normal synoviocytes.it can inhibit macrophage-like synoviocytes and fibroblast-like synoviocytes from RA on proliferation and induce them to apoptosis.In the some contraction of THH.proliferation Inhibition and inducing apoptosis can be occur on synoviocytes from RA but not on normal synoviocytes.