Yan Wenzhe, Peng Youming, Liu Chan, Ye Muyao, Chen Xian, Peng Xiaofei, He Liyu, Wang Jiayi, Liu Hong, Liu Fuyou, Zhang Guangsen
a Department of Nephrology , The Second Xiangya Hospital, Central South University, Hunan Key Lab of Kidney Disease and Blood Purification , People's Republic of China and.
b Department of Hematology/Institute of Molecular Hematology , the Second XiangYa Hospital, Central South University , People's Republic of China.
Ren Fail. 2016;38(1):109-16. doi: 10.3109/0886022X.2015.1096730. Epub 2015 Oct 13.
This study aimed to evaluate the extent of apoptosis of tonsillar mononuclear cells (TMCs) derived from patients with IgA nephropathy (IgAN) and the effects of triptolide (TP) on the apoptosis of these TMCs.
TMCs were isolated from tonsillar tissues of patients with IgAN or chronic tonsillitis (control group). Rates of TMCs apoptosis were measured by annexin V-fluorescein isocyanate (FITC)/propidium iodide (PI)-labeled flow cytometry (FCM). Expression levels of Bcl-2 family proteins were quantified by immunohistochemistry of fixed tonsillar sections and Western blot analyzes of TMCs lysates. TMCs from IgAN patients were treated 10, 20, or 30 ng/mL TP for 24 h and then evaluated for viability by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, for the percentage of apoptotic cells by FCM, and for the relative expression levels of Bcl-2 family proteins by Western blot analysis.
Compared to TMCs from the control group, TMCs from the IgAN group demonstrated lower rates of apoptosis, higher expression levels of the anti-apoptosis proteins Bcl-2 and Bcl-xL, and lower expression levels of the pro-apoptosis protein Bax. Treatment of IgAN patient-derived TMCs with 10, 20, or 30 ng/mL TP for 24 h suppressed the viability and promoted the apoptosis of TMCs in a dose-dependent manner. Western blot analysis revealed a TP dose-dependent decrease in Bcl-2 and Bcl-xL expression levels, in parallel with increased Bax protein levels.
TMCs from IgAN patients may be in a state of inhibited apoptosis mediated by Bcl-2 family proteins, which may be reversed by TP treatment.
本研究旨在评估IgA肾病（IgAN）患者扁桃体单个核细胞（TMCs）的凋亡程度以及雷公藤甲素（TP）对这些TMCs凋亡的影响。
从IgAN患者或慢性扁桃体炎患者（对照组）的扁桃体组织中分离TMCs。采用膜联蛋白V-异硫氰酸荧光素（FITC）/碘化丙啶（PI）标记的流式细胞术（FCM）检测TMCs的凋亡率。通过固定扁桃体切片的免疫组织化学和TMCs裂解物的蛋白质印迹分析对Bcl-2家族蛋白的表达水平进行定量。将IgAN患者的TMCs用10、20或30 ng/mL的TP处理24小时，然后通过3-（4,5-二甲基-2-噻唑基）-2,5-二苯基-2H-四氮唑溴盐（MTT）法评估细胞活力，通过FCM评估凋亡细胞百分比，并通过蛋白质印迹分析评估Bcl-2家族蛋白的相对表达水平。
与对照组的TMCs相比，IgAN组的TMCs凋亡率较低，抗凋亡蛋白Bcl-2和Bcl-xL的表达水平较高，促凋亡蛋白Bax的表达水平较低。用10、20或30 ng/mL的TP处理IgAN患者来源的TMCs 24小时，以剂量依赖的方式抑制了TMCs的活力并促进了其凋亡。蛋白质印迹分析显示TP剂量依赖性降低Bcl-2和Bcl-xL的表达水平，同时Bax蛋白水平升高。
IgAN患者的TMCs可能处于由Bcl-2家族蛋白介导的凋亡抑制状态，TP处理可能会逆转这种状态。
