苦参碱对PC12细胞的毒性及线粒体损伤机制

Cytotoxicity and mitochondrial injury mechanism of matrine on PC12 cells

目的研究苦参碱(MT)对大鼠肾上腺嗜铬细胞瘤细胞(PC12细胞)的毒性作用及其机制。方法用MT 2,4和8 mmol·L-1分别作用PC12细胞8,16,24和48 h,MTT法测定细胞存活率。PC12细胞经上述浓度的MT作用24 h后,采用Hoechst33342荧光染色法观察细胞形态变化,采用羟胺法和硫代巴比妥酸(TBA)法分别检测PC12细胞内超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量,流式细胞术检测细胞凋亡率及活性氧(ROS)水平,JC-1染色法检测细胞线粒体膜电位(MMP)的改变,Western蛋白印迹法检测胱天蛋白酶原3、胱天蛋白酶原9、活化的胱天蛋白酶3、Bax和Bcl-2蛋白水平。结果随着作用时间及浓度的增加,MT对PC12细胞的抑制作用逐渐增强。MT作用24 h后,与正常对照组相比,MT 2,4和8 mmol·L-1组的细胞数减少,出现染色质凝集和部分破裂的现象,且给药组细胞凋亡率均显著增加(P<0.01);细胞内ROS和MDA含量显著增加(P<0.05,P<0.01),SOD活性显著降低(P<0.01),且MMP降低;细胞内胱天蛋白酶原9、胱天蛋白酶原3和Bcl-2蛋白水平显著下降(P<0.01,P<0.05),活化的胱天蛋白酶3和Bax蛋白水平显著上升(P<0.05,P<0.01);且Bcl-2/Bax比值显著降低(P<0.01)。结论 MT对PC12细胞具有一定毒性作用,其机制可能与细胞内活性氧堆积而引起线粒体途径诱导细胞凋亡有关。

OBJECTIVE To study the toxicological effect of matrine（MT） on PC12 cells and the mechanism of mitochondrial damage. METHODS After treatment with MT 2, 4 and 8 mmol·L^-1 for 8,16, 24 and 48 h, respectively, the viability of PC12 cells was measured with methylthiazolyltetrazolium assay. PC12 cells were treated with MT 2, 4 and 8 mmol·L^-1 for 24 h, before the morphological changes were observed by Hoechst33342 staining. The superoxide dismutase（SOD） activity and malondialdehyde（MDA） content in cells were detected using hydroxylamine method and thiobarbituric acid method,apoptosis rate and reactive oxygen species（ROS） of PC12 cells were detected with flow cytometry,mitochondrial membrane potential（MMP） was detected with JC^-1 staining, and the expressions of procaspase 3, procaspase 9, cleaved-caspase 3, Bax and Bcl-2 were detected with Western blotting.RESULTS MT inhibited the growth of PC12 cells in a time-and concentration-dependent manner. After being treated with MT for 24 h, the nuclei of PC12 cells in MT groups showed chromatin agglutination and partial rupture to different degrees, and compared with normal control group, the apoptosis rates of MT 2, 4 and 8 mmol· L^-1 groups were significantly increased（P〈0.01）. Intracellular ROS and MDA levels increased（P〈0.05, P〈0.01）, SOD activity decreased（P〈0.01）, and MMP decreased. The expressions of procaspase 9, procaspase 3 and Bcl-2 were significantly decreased（P〈0.01, P〈0.05）, the expressions of cleaved-caspase 3 and Bax were significantly increased（P〈0.05, P〈0.01）, and the ratio of Bcl-2/Bax was significantly decreased（P〈0.01）. CONCLUSION MT has toxic effect on PC12 cells and induces apoptosis through ROS mediated mitochondrial pathway.