小檗碱通过破坏线粒体功能选择性诱导淋巴瘤细胞的凋亡

目的研究小檗碱(BBR)对T淋巴瘤细胞凋亡的影响及机制。方法正常T淋巴细胞、T淋巴瘤细胞、Jurkat细胞用BBR 0,10,20和40μmol·L^-1处理24 h,多柔比星(Dox)40μmol·L^-1为阳性对照;Jurkat p0细胞用BBR 0和40μmol·L^-1处理24 h。流式细胞术检测细胞凋亡率,Seahorse XF24细胞代谢分析仪和H2DCFDA活性氧探针检测细胞氧消耗率(OCR)和活性氧(ROS)水平。CellTiter-Glo发光法细胞活力检测试剂盒检测细胞ATP水平,试剂盒测线粒体呼吸链复合物Ⅰ,Ⅱ,Ⅳ和Ⅴ的活性。Western印迹法检测细胞内胱天蛋白酶3、Bcl-2、Bax、NF-κB抑制蛋白激酶α/β(IKKα/β)、磷酸化IKKα/β(p-IKKα/β)、NF-κB抑制因子α(IκBα)、p-IκBα和P65蛋白表达水平。结果BBR 40 mmol·L^-1明显增加T淋巴瘤细胞和Jurkat细胞凋亡率(P<0.01),胱天蛋白酶3和Bax蛋白表达水平显著上调(P<0.01),Bcl-2表达显著下调(P<0.01),但对正常T淋巴细胞的凋亡并无影响;BBR 40 mmol·L^-1明显降低Jurkat细胞的OCR及线粒体呼吸链复合物Ⅰ活性(P<0.01),增加ROS水平(P<0.01),降低ATP水平(P<0.01),但不影响线粒体缺陷型淋巴瘤Jurkat p0细胞呼吸链和凋亡;BBR 40μmol·L-1明显下调Jurkat细胞中NF-κB通路相关蛋白p-IKKα/β/IKKα/β、p-IκBα/IκBβ和核内P65表达(P<0.01)。结论BBR通过破坏线粒体功能选择性诱导淋巴瘤T细胞的凋亡,可能与抑制NF-κB通路有关。

Berberine selectively induces apoptosis of lymphoma cells by disrupting mitochondrial function

OBJECTIVE To investigate the effect of berberine(BBR)on apoptosis of T lymphoma cells.METHODS Normal T lymphocytes,T lymphoma cells and Jurkat cells were treated with BBR 0,10,20 and 40μmol·L^-1 and doxorubicin(Dox)40μmol·L^-1(positive control)for 24 h.Jurkat p0 cells were treated with BBR 0,40μmol·L^-1 for 24 h.The apoptotic rate was detected by flow cytometry.Oxygen consumption rate(OCR)and reactive oxygen species(ROS)levels were measured by the Seahorse XF24 cell metabolism analyzer and the H2DCFDA reactive oxygen probe.Cell ATP levels were measured by the cell Titer-Glo luminescence cell viability assay kit.The activity of mitochondrial respiratory complexes Ⅰ,Ⅱ,Ⅳ and Ⅴ was measured by the mitochondrial complex Ⅰ,Ⅱ,Ⅳ and Ⅴ activity assay kits.The expression levels of caspase 3,Bcl-2,Bax,NF-κB inhibitory protein kinaseα/β(IKKα/β),phosphorylated IKKα/β(p-IKKα/β),NF-κB inhibitorα(IκBα),p-IκBαand P65 protein were determined by Western blotting.RESULTS BBR 40 mmol·L^-1 significantly increased the apoptosis rate of T lymphoma cells of patients and lymphoma Jurkat cells(P<0.01),up-regulated the expressions of caspase 3 and Bax protein level(P<0.01),and down-regulated the expression of the anti-apoptotic protein Bcl-2(P<0.01),but had no effect on the apoptosis of T lymphocytes.BBR 40μmol·L^-1 significantly decreased OCR and mitochondrial respiratory chain complex Ⅰ activity in Jurkat cells(P<0.01),increased ROS levels(P<0.01),and decreased ATP levels(P<0.01),but without any effect on mitochondrial-deficient lymphoma Jurkat p0 cells;BBR 40μmol·L^-1 significantly down-regulated the expressions of NF-κB pathway-related proteins p-IKKα/β/IKKα/β,p-IκBα/IκBαand nuclear P65 in Jurkat cells(P<0.01).CONCLUSION BBR selectively induces apoptosis of lymphoma cells by disrupting mitochondrial function,which may be related to inhibition of NF-κB pathway.