硼替佐米通过干预Parkin介导的线粒体自噬导致神经毒性的研究  

Bortezomib interferes with Parkin-mediated mitochondrial autophagy leading to neurotoxicity

作　　者：曹慧琴[1] 韦玮[2] 张雯[1] 崔兴[3] CAO Huiqin;WEI Wei;ZHANG Wen;CUI Xing(Department of Hematology,Affiliated Hospital of Yan′an University,Yan′an 716000,China;Department of Neurosurgery,Affiliated Hospital of Yan′an University,Yan′an 716000,China;Department of Hematology&Oncology,The Second Affiliated Hospital of Shandong University of Traditional Chinese Medicine,Jinan 250011,China)

机构地区：[1]延安大学附属医院血液科,陕西延安716000 [2]延安大学附属医院神经外科,陕西延安716000 [3]山东中医药大学第二附属医院肿瘤血液科,济南250011

出　　处：《延安大学学报（医学科学版）》2023年第4期17-22,34,共7页Journal of Yan'an University:Medical Science Edition

基　　金：山东省重点研发计划(2019GSF108162)。

摘　　要：目的 探讨硼替佐米神经毒性的具体机制。方法 观察硼替佐米干预下大鼠嗜铬细胞瘤PC12细胞的活性。检测胱天蛋白酶3(Cleaved-caspase-3),B淋巴细胞瘤-2/B淋巴细胞瘤-2相关X蛋白(B-cell lymphoma-2/BCL2-Associated X,Bcl-2/Bax),自噬膜型LC3/胞浆型LC3(LC3Ⅱ/LC3Ⅰ),P62蛋白,Parkin蛋白,线粒体内膜转位酶23蛋白(Mitochondrial Endomembrane Transposase 23 Protein,TIM23)的表达。透射电镜下观察线粒体超微结构和自噬泡,荧光显微镜下观察硼替佐米作用下细胞的自噬情况,进一步验证硼替佐米调控Parkin功能。构建动物模型,进一步证实硼替佐米对坐骨神经线粒体自噬的影响。结果 硼替佐米(50 nmol/L)作用下,PC12细胞的增殖率显著下降,电镜下自噬泡数目减少,线粒体结构毁坏严重,LC3Ⅱ/LC3Ⅰ表达水平显著下降,而P62表达水平显著升高,LC3免疫荧光明显减弱。线粒体标志物Parkin、TIM23蛋白表达水平显著降低,伴有线粒体结构的破坏。过表达Parkin后,硼替佐米对PC12细胞的毒性明显降低。动物实验结果,硼替佐米可以导致小鼠动物行为学异常,坐骨神经纤维形态不完整,可见明显的脱髓鞘,坐骨神经免疫组化显示LC3和Parkin表达水平显著降低。结论 硼替佐米可通过降低Parkin表达,抑制其介导的线粒体自噬通路,从而抑制PC12细胞的增殖导致神经毒性。Objective To investigate the inhibitoryside effect mechanism of bortezomib on nerve cell systemproliferation.Methods The proliferation of rat pheochromocytoma PC12 cells was observed after bortezomib treatment.The cleaved caspase-3,B-cell lymphoma-2/BCL2-Associated X(Bcl-2/Bax),LC3Ⅱ/LC3Ⅰ,P62,Parkin,mitochondrial endomembrane transposase 23 protein(TIM23)expression were detected.The ultrastructure of mitochondria and autophagic vesicles were observed by transmission electron microscope,and autophagy of cells was observed by fluorescence microscope.A response experiment was designed to further verify that bortezomib could regulate Parkin function.Animal models were constructed to further confirm the effect of bortezomib on sciatic nerve mitochondrial autophagy.Results Bortezomib(50 nmol/L)significantly reduced the proliferation rate of PC12 cells,reduced the number of autophagic vesicles under electron microscope,severely damaged mitochondrial structure,significantly decreased the expression level of LC3Ⅱ/LC3Ⅰ,significantly increased the expression level of P62,significantly decreased the immunofluorescence level of LC3,significantly decreased the expression level of Parkin,TIM23 protein and immunofluorescence.The toxicity of bortezomib to PC12 cells was significantly reduced in response to Parkin overexpression.Animal experiments have confirmed that bortezomib can cause abnormal behavior of mice,pathological abnormalities like incomplete morphology of the sciatic nerve fibers and marked demyelination,and the expression levels of LC3 and Parkin were significantly reduced by immunohistochemistry of sciatic nerve.Conclusion Bortezomib can inhibit the proliferation of PC12 cellsinduce neurotoxicity by decreasing the expression of Parkin and inhibiting its pathway-mediated mitochondrial autophagy.

关 键 词：硼替佐米 PARKIN 线粒体自噬 化疗 周围神经病变 

分 类 号：R551.3[医药卫生—血液循环系统疾病]