ABSTRACT
ObjectiveToinvestigatetheinflammatoryresponseandapoptosisof peripheral blood mononuclear cells(PBMCs) and their regulation by triptolide(TP) in IgA nephropathy(IgAN) patients.MethodsBlood samples were collected from 29 IgAN patients and 16 healthy individuals.TNF-α and IL-6 concentrations were measured by ELISA and NO concentration by Griess reagent in the plasma of samples.PBMCs were isolated from IgAN patients and cultured in vitro,and subsequently activated by PHA(10 mg/L).The cytotoxicity of different TP concentrations was assayed by MTT and two non-toxic concentrations (12.5 μg/L or 25.0 μg/L)were selected for treatment.TNF-α,IL-6 and NO concentrations were measured in the culture media collected from PBMCs cultures activated by PHA (10 mg/L) and treated with TP (12.5 μg/L or 25.0 μg/L).The PHA-activated,TP-treated cells apoptotic rate was analyzed by FACS using Annexin V-FITC staining.The expression of Bcl-2,Bax,caspase-9 and caspase-3 were detected by RT-PCR and Western blotting from lyses of PHA-activated with or without TP-treated cells.ResultsThe serum concentrations of TNF-α[(131.57±50.61) ng/L vs(30.24±18.93) ng/L,P＜0.01],IL-6[(76.36±25.21) ng/L vs(35.08±16.59) ng/L,P＜0.01] and NO[(46.36±12.93) μmol/Lvs (26.61 ±10.87) μmol/L,P＜0.01] were significantly increased in IgAN patients compared to healthy individuals.PBMCs viability in culture decreased after TP treatment in a dose-dependent manner.TP also inhibited TNF-α,IL-6 and NO levels in the media of PHA-activated PBMCs in culture and induced PBMCs apoptosis.The expression of Bcl-2 decreased markedly and Bax,caspase-9 and caspase-3 increased significantly after TP treatment (all P＜0.05).Conclusions The PBMCs from IgAN patients are in a highly activated state,and have a high apoptotic rate.TP treatment induces benificial effects in IgAN patients by inhibiting the activation of PBMCs by activating pro-apoptotic pathway.