左旋盐酸去甲基苯环壬酯对MPTP诱导的SH-SY5Y细胞损伤的保护作用(英文)

Neuroprotective effect of l-demethyl phencynonate hydrochloride against MPTP-induced SH-SY5Y cell injury

目的探究M4受体选择性拮抗剂左旋盐酸去甲基苯环壬酯(R-DM8021)对1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)诱导人多巴胺能神经母细胞瘤(SH-SY5Y)细胞损伤的保护作用。方法预先给予不同浓度R-DM8021(1×10^(-9),1×10^(-8),1×10^(-7),1×10^(-6)mol·L^(-1))孵育SH-SY5Y细胞1 h后,加入MPTP1 mmol·L^(-1)共孵育24 h,MTT比色法检测细胞存活率,流式细胞仪检测细胞内活性氧(ROS)含量,TUNEL法观察细胞凋亡形态,Western蛋白印迹法检测细胞凋亡相关蛋白Bax和胱天蛋白酶3前体蛋白表达水平。结果 MTT结果显示,与细胞对照组相比,MPTP 1 mmol·L^(-1)作用SH-SY5Y细胞24 h,细胞存活率显著降低至(44.5±2.7)%(P<0.01);与MPTP组相比,预先给予R-DM8021(1×10^(-9),1×10^(-8),1×10^(-7),1×10^(-6)mol·L^(-1))均可逆转MPTP对SH-SY5Y细胞的损伤作用,细胞存活率分别提高至(80.5±4.7)%,(71.5±8.1)%,(74.4±6.5)%和(61.3±3.2)%(P<0.01)。流式细胞检测结果显示,与细胞对照组相比,MPTP 1 mmol·L^(-1)作用SH-SY5Y细胞24 h,细胞内ROS含量显著升高至129±22(P<0.01);与MPTP组相比,预先给予R-DM8021(1×10^(-9),1×10^(-8),1×10^(-7),1×10^(-6)mol·L^(-1))显著降低MPTP诱导的胞内ROS释放,细胞内ROS含量降低至84±12,86±15,86±6和88±8(P<0.05)。TUNEL检测显示,与细胞对照组相比,MPTP 1 mmol·L^(-1)作用SH-SY5Y细胞24 h,贴壁细胞染色质皱缩,核膜裂解,并产生大量凋亡小体;与MPTP组相比,预先给予R-DM8021 1 nmol·L^(-1)可显著改善MPTP诱导的细胞凋亡,凋亡小体显著减少。Western蛋白印迹结果显示,与MPTP组相比,R-DM8021 1 nmol·L^(-1)可显著抑制MPTP诱导SH-SY5Y细胞凋亡,降低Bax和胱天蛋白酶3前体蛋白的表达(P<0.01)。结论 R-DM8021能有效对抗MPTP诱导的SH-SY5Y细胞损伤,其机制与提高细胞存活率,降低胞内ROS含量,抑制细胞凋亡有关。

OBJECTIVE To investigate the effect of l-demethyl phencynonate hydrochloride(RDM8021)against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced injury in SH-SY5 Y cells.METHODS SH-SY5 Y cel s were pre-treated with R-M8021(1×10-9,1×10-8,1×10-7 and 1×10-6 mol·L-1)for 1 h and co-administeted with MPTP(1 mmol·L-1)for 24 h.Cell viability was analyzed by MTT assay,the production of reactive oxygen species(ROS)was detected by flow cytometric analysis,cell apoptosis morphology was detected by TUNEL staining,while the expressions of Bax and pro-caspase 3 were evaluated by Western blotting.RESULTS MPTP 1 mmol·L-1 significantly decreased cell survival to(44.5±2.7)%(vs control,P<0.01).R-DM8021(1×10-9,1×10-8,1×10-7 and 1×10-6 mol·L-1)reversed MPTP-induced cell injury and increased cell survival to(80.5±4.7)%,(71.5±8.1)%,(74.4±6.5)%and(61.3±3.2)%(vs MPTP,P<0.01).MPTP 1 mmol·L-1 significantly increased ROS production to 129±22(vs control,P<0.01),while R-DM8021(1×10-9,1×10-8,1×10-7 and 1×10-6 mol·L-1)inhibited MPTP-induced ROS production and reduced ROS production to 84±12,86±15,86±6 and 88±8(vs MPTP,P<0.05).According to TUNEL staining,MPTP-induced cell apoptosis was characterized by mitochondrial swelling and irregularly scattered chromatin.Cells pretreated with R-M8021 1 nmol·L-1 could significantly reverse MPTPinduced morphological apoptotic changes and TUNEL-positive cells.According to Western blotting,R-DM8021 1 nmol·L-1 protected MPTP-induced SH-SY5 Y cell apoptosis morphology and reduced the expressions of Bax and pro-caspase 3(vs MPTP,P<0.01).CONCLUSION R-DM8021 can effectively protect SH-SY5 Y cell injury induced by MPTP treatment,and the mechanisms are related to improvement of cell against survival,reduction of intracellular ROS levels and apoptosis.