CAJ | 학술논문

目的探讨胰岛素样生长因子1(IGF-1)通过JAK/STAT信号转导通路对左旋多巴诱导的多巴胺能神经元凋亡的保护作用.方法应用100μg/L β神经生长因子(β-NGF)将体外常规培养的PC12细胞诱导分化为多巴胺能神经元.MTT比色法筛选左旋多巴的神经损伤浓度和IGF-1的神经保护浓度(150μmol/L、100nmol/L),实验分为4组:(1)左旋多巴+IGF-1组;(2)左旋多巴+IGF-1+AG490组,AG490(10 μmol/L)在给予左旋多巴和IGF-1前20 min加人;(3)左旋多巴组;(4)对照组.Western blotting检测4组细胞P-JAK2、P-STAT3蛋白表达,Hoechst33258染色和流式细胞仪分别检测细胞凋亡和凋亡率.结果 Westen blotting法检测显示对照组和左旋多巴组P-JAK2、P-STAT3蛋白表达阴性,而左旋多巴+IGF-1组和左旋多巴+IGF-1+AG490组表达阳性.左旋多巴+IGF.1+AG490组P.JAK2、P.STAT3表达强度低于左旋多巴+IGF.1组,差异有统计学意义(P<0.05):Hoechst33258染色结果显示左旋多巴组细胞核固缩及碎裂最明显,较多凋亡小体形成,而左旋多巴+IGF-1组与左旋多巴+IGF-1+AG490组仅有少量凋亡小体形成.流式细胞仪检测显示4组多巴胺能神经元凋亡率不同,差异有统计学意义(F=180.991,P=0.000);与对照组比较,另3组细胞凋亡率均增高,差异有统计学意义(P<0.05);其中左旋多巴组细胞凋亡率最高,其次为左旋多巴+IGF-1+AG490组、左旋多巴+IGF-1组.结论大剂量左旋多巴对多巴胺能神经元具有毒性作用.IGF-1对左旋多巴诱导的神经细胞毒性作用呈保护作用,JAK2/STAT3信号转导通路参与了此过程.
목적 탐토이도소양생장인자1(IGF-1)통과JAK/STAT신호전도통로대좌선다파유도적다파알능신경원조망적보호작용.방법 응용100μg/L β신경생장인자(β-NGF)장체외상규배양적PC12세포유도분화위다파알능신경원.MTT비색법사선좌선다파적신경손상농도화IGF-1적신경보호농도(150μmol/L、100nmol/L),실험분위4조:(1)좌선다파+IGF-1조;(2)좌선다파+IGF-1+AG490조,AG490(10 μmol/L)재급여좌선다파화IGF-1전20 min가인;(3)좌선다파조;(4)대조조.Western blotting검측4조세포P-JAK2、P-STAT3단백표체,Hoechst33258염색화류식세포의분별검측세포조망화조망솔.결과 Westen blotting법검측현시대조조화좌선다파조P-JAK2、P-STAT3단백표체음성,이좌선다파+IGF-1조화좌선다파+IGF-1+AG490조표체양성.좌선다파+IGF.1+AG490조P.JAK2、P.STAT3표체강도저우좌선다파+IGF.1조,차이유통계학의의(P<0.05):Hoechst33258염색결과현시좌선다파조세포핵고축급쇄렬최명현,교다조망소체형성,이좌선다파+IGF-1조여좌선다파+IGF-1+AG490조부유소량조망소체형성.류식세포의검측현시4조다파알능신경원조망솔불동,차이유통계학의의(F=180.991,P=0.000);여대조조비교,령3조세포조망솔균증고,차이유통계학의의(P<0.05);기중좌선다파조세포조망솔최고,기차위좌선다파+IGF-1+AG490조、좌선다파+IGF-1조.결론 대제량좌선다파대다파알능신경원구유독성작용.IGF-1대좌선다파유도적신경세포독성작용정보호작용,JAK2/STAT3신호전도통로삼여료차과정.
Objective To explore the protective effect of insulin-like growth factor 1 (IGF-1) on apoptosis of dopaminergic neurons induced by L-dopa via JAK/STAT signaling pathway. Methods PC12 cells were induced to differentiate into dopaminergic neurons with 100 μg/L β-NGF; MTT assay was employed to identify the changes in the viability of PC12 cells following L-dopa treatment at 0, 10,20, 50, 100, 150 and 200 μmol/L, and the different concentrations of IGF-1 at 0, 10, 25, 50 and 100 nmol/L with the same concentration of L-dopa (150 μmol/L); Western blotting was used to detect the levels of P-JAK2/P-STAT3 in PC12 cells treated with PBS (controls), L-dopa, L-dopa+IGF-1 and L-dopa+IGF-1+AG490 for 24 h, and then the apoptosis rate was assessed by flow cytometry and Hchest33258 staining. Results Western blotting showed that the expressions of P-JAK2 and P-STAT3 were detected in the L-dopa+IGF-1 and L-dopa+IGF-1+AG490 treatment groups but not in the control group or L-dopa treatment group; the expression of P-STAT3 in the L-dopa+IGF-1+AG490 treatment group was obviously lower than that in the L-dopa+IGF-1 treatment group (P<0.05). Hchest33258 staining indicated that L-dopa treatment group had the most obvious karyopyknosis and karyorrhexis,much more apoptotic bodies than the L-dopa+IGF-1 and L-dopa+IGF-1+AG490 treatment groups. Flow cytometry showed that the apoptosis rate was significantly different among the 4 groups (F=180.991,P=0.000): as compared with the control group, the other 3 groups had a higher apoptosis rate (P<0.05);L-dopa treatment group (38.13 ±2.54 %) enjoyed the highest level, followed by L-dopa+IGF-1 +AG490treatment group (25.60±1.30 %) and L-dopa+IGF-1 treatment group (20.17±1.54 %). Conclusion L-dopa has toxic effect on PC12 cells; IGF-1 could protect the PC12 cells from the neurotoxic effect of L-dopa and JAK2/STAT3 signaling pathway is activated in this procedure.