Immunochemical characterization of tripterygium wilfordii (triptolide, triptonide and synthetic racemic derivatives) immunosuppressive properties in allogenic mixed lymphocyte reaction

Abstract

Tripterygium wilfordii (TW) has been used as immunosuppressant inorgan transplantation to prolong graft survival attributing to its anti-inflammatory and immunomodulating properties. Because of poten-tial cytotoxicity and unwanted side effects, practical applications ofthe TW compounds are not widely accepted in clinical settings. Fur-thermore, the molecular basis of TW active component responsiblefor immunomodulatory activity has not been fully elucidated. Thepresent study aims to characterize the functional constituents that areconferring the immunosuppressive effect in the established allogenicrat mixed lymphocyte reaction (MLR). High-responder inbred strainsof Lewis and Dark Agouti (DA) rats were used, and their splenocyteswere cocultured in triplicates at 2 × 105cells per well. After addingvariable concentrations of TW active components (triptolide, trip-tonide and their synthetic racemic derivatives) for 48 hours, the cellswere pulsed with 1 µCi [3H] methylthymidine per well for 16 hours.The proliferative response was determined by measuring the incor-poration by direct β counting. Cells grown with Concanavalin A wereused as positive control. Cells grown with or without FK506 wereused as controls. The final concentration of DMSO has no effect onthe growth of cells. Results were expressed as mean ± SD and evalu-ated by one-way ANOVA (Dunnett’s test). Significant values wereconsidered at the level of P < 0.05. Our present findings showed thatthe γ-butyrolactone group was essential to the pharmacologic prop-erties of TW active components that were studied. In addition, theIC50observed in the T59 (racemic derivative of triptolide) and trip-tonide was significantly elevated as compared to the correspondingtriptolide. The data implied that spatial arrangement of the hydroxylgroup in the triptolide compound was important to the immunosup-pressive function of TW on lymphocyte proliferative response. Col-lectively, a potent but less toxic TW active constituent may bedesigned to enhance its clinical applicability in organ transplantationand/or immune modulation of inflammatory reactions.(Supported by Research Grants Council of Hong Kong: HKU7280/02M)