雷公藤甲素对IgA肾病患者外周血单个核细胞炎性反应及凋亡的影响

目的 探讨IgA肾病(IgAN)患者外周血单个核细胞（PBMC）炎性因子分泌及自身凋亡状态，以及雷公藤甲素(TP) 对其调节的相关机制。 方法 取IgAN患者（n=29）及健康人（n=16）外周血，分别通过酶联免疫吸附试验（ELISA）和硝酸还原酶法（Griess法）检测血浆肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）和一氧化氮(NO)表达情况。体外培养IgAN患者PBMC，以不同浓度TP刺激PMBC，四唑盐比色间接法（MTT）检测TP对细胞生长的抑制效应。根据MTT结果，分别分为IgAN患者PBMC组、植物血球凝集素（PHA，10 mg/L，促细胞分裂原）模型组和PHA（10 mg/L）+TP（12.5 μg/L、25 μg/L）处理组。ELISA法检测上清液中TNF-α、IL-6的分泌情况；Griess法检测上清液中NO含量；流式细胞仪分析细胞凋亡率；RT-PCR法、Western印迹法检测PBMC中B细胞淋巴瘤/白血病-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、半胱天冬蛋白酶（caspase）-9、caspase-3基因和蛋白表达变化。 结果 IgAN患者血浆TNF-α（131.57±50.61） ng/L比（30.24±18.93） ng/L，P < 0.01]，IL-6（76.36±25.21） ng/L比（35.08±16.59） ng/L，P < 0.01]和NO（46.36±12.93） μmol/L比（26.61±10.87） μmol/L，P < 0.01]均显著高于健康人，PBMC凋亡率亦显著高于健康人（16.88±5.66）%比（8.67±3.87）%，P < 0.01]。TP可抑制IgAN患者PBMC分泌 TNF-α、IL-6 和NO，诱导其凋亡；也可抑制Bcl-2表达，增加Bax、caspase-9、caspase-3表达。 结论 IgAN患者体内PBMC处于高度活化状态，并且凋亡率增高。TP能有效抑制IgAN患者PBMC的炎性活化状态，并可能通过线粒体途径改变Bcl-2蛋白家族抗凋亡因子和促凋亡因子平衡而诱导IgAN患者PBMC凋亡。

Effect of triptolide on the regulation of inflammatory response and apoptosis in PBMC from IgA nephropathy patients

Objective To investigate the inflammatory response and apoptosis of peripheral blood mononuclear cells (PBMCs) and their regulation by triptolide (TP) in IgA nephropathy (IgAN) patients. Methods Blood 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.Results The 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.