葛根素缓解镉致大鼠神经细胞线粒体结构损伤和Pink1_Parkin介导的线粒体自噬.pdf

-0276-06ChineseVeterinaryScience网络首发时间：2 0 2 3-10-2 72024,54(02):276-281中国兽医科学DOI:10.16656/j.issn.1673-4696.2024.0028中图分类号：S856.9文献标志码：A文章编号：16 7 3-46 96（2 0 2 4)0 2-葛根素缓解镉致大鼠神经细胞线粒体结构损伤和Pink1/Parkin介导的线粒体自噬糜晓霞1,徐明畅2.3,王莉2.3,闫俊琳2.3,袁燕2.3*（1.仪征市农业技术综合服务中心，江苏仪征211400;2.扬州大学兽医学院，江苏扬州225009;3.江苏高校动物重要疫病与人兽共患病防控协同创新中心，江苏扬州225009)摘要：为研究葛根素(Pur）在镉（Cd)致大鼠神经细胞线粒体结构损伤和Pink1/Parkin介导的线粒体自噬中的作用，用10 0 umol/L葛根素预处理PC12细胞和大鼠大脑皮质神经元1h，再与10 mol/L单独或联合处理12 h;24只SD大鼠随机分为对照组、葛根素组、镉组、镉与葛根素共处理组，葛根素组、镉与葛根素共处理组大鼠每日以每千克体重2 0 0 mg的葛根素（羧甲基纤维素钠溶解）灌胃，对照组、镉组大鼠每日用相同剂量羧甲基纤维素钠灌胃，持续7 周；从第4周开始,组、镉与葛根素共处理组大鼠在灌胃的同时自由饮用7 5mg/L镉水溶液，持续4周。透射电子显微镜观察线粒体超微结构，免疫印迹法检测Pinkl、Pa r k in和LC3I蛋白表达。结果显示,与对照组相比，镉组大鼠神经细胞线粒体踏断裂、模糊，线粒体基质内出现空泡，线粒体内Pinkl、Pa r k i n 和LC3II蛋白表达量显著升高(P0.05)；与镉组相比，镉与葛根素共处理组大鼠神经细胞线粒体崂排列较规整，基质内空泡化现象减轻，线粒体内Pinkl和Parkin蛋白表达量显著降低(P0.05），大鼠大脑皮质神经元和大鼠大脑皮质线粒体内LC3II蛋白表达量显著降低（P0.05）。结果表明，葛根素可缓解镉诱导的大鼠神经细胞线粒体结构损伤和Pink1/Parkin介导的线粒体自噬。关键词：镉；葛根素；大鼠神经细胞；线粒体结构损伤；线粒体自噬Puerarin alleviates cadmium-induced mitochondrial structural damageand Pink1/Parkin-mediated mitophagy in neuronal cells of ratsMI Xiaoxia,XU Mingchang3,WANG Li23,YAN Junlin?23,YUAN Yan2.3(1.Agricultural Technology Comprehensive Service Center of Yizheng,Yizheng 211400,China;2.College of VeterinaryMedicine,Yangzhou University,Yangzhou 225009,China;3.Jiangsu Co?innovation Center for Prevention and Control ofImportant Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)Abstract:To study the role of puerarin(Pur)in cadmium(Cd)-induced mitochondrial structural damageand Pinkl/Parkin-mediated mitophagy in neuronal cells of rats,PC12 cells and rat cerebral corticalneuron were pretreated with 100 mol/L Pur for 1 h,and then treated with 10 mol/L Cd alone or in combinationfor 12 h.24 SD rats were randomly divided into a control group,a Pur group,a Cd group,and a Cd+Pur group.The rats in the Pur group and Cd+Pur group underwent gavage of Pur(dissolved in sodium carboxymethylcellulose)at a dose of 200 mg/kg body weight daily,while the rats in the control group and Cd group weregiven the same dose of sodium carboxymethyl cellulose by gavage every day for 7 weeks.Rats in the Cdgroup and Cd+Pur group were given drinking water containing 75 mg/L Cd for 4 weeks from the 4th week of收稿日期：2 0 2 3-0 9-2 2；修回日期：2 0 2 3-10-2 5基金项目：江苏省自然科学基金项目（BK20221372）；高等学校学科创新引智计划资助项目（D18007）；江苏高校优势学科建设工程资助项目(PAPD)作者简介：糜晓霞（197 3-），女，江苏仪征人，兽医师，E-mail：mi x i a o x i a 7 3 12 6.c o m。*通讯作者：袁燕，教授，博士，主要从事动物营养代谢病与中毒病研究,E-mail:。277第2 期糜晓霞等：葛根素缓解镉致大鼠神经细胞线粒体结构损伤和Pink1/Parkin导的线粒体自噬gavage.The ultrastructure of mitochondria was observed by transmission electron microscope,and theprotein levels of Pinkl,Parkin and LC3 II were detected by Western-blot.The results showed that comparedwith the control group,the mitochondrial crest was broken and blurred,and vacuoles appeared in themitochondrial matrix,and the protein levels of Pink1,Parkin and Lc3 II in mitochondria were signifi-cantly increased(P0.05)in the Cd group.Compared with the Cd group,the arrangement of mitochondrialcristae was more regular,the degree of vacuolization in the mitochondrial matrix was alleviated,andthe protein levels of Pinkl and Parkin in mitochondria were significantly decreased(P0.05)in the Cd+Pur group,and the levels of Lc3 II proteins in the mitochondria of rat cerebral cortical neurons and ratcerebral cortex were significantly decreased(P0.05）；不同字母表示组别之间差异显著（P0.05）,C d 组大鼠神经细胞线粒体内Pinkl、Parkin和LC3II蛋白表达水平显著升高（P0.05)；与Cd组相比,Cd+Pur组大鼠神经细胞线粒体内Pinkl、Pa r k i n 蛋白表达水平显著降低（P0.05），大鼠大脑皮质神经元和大鼠大脑皮质线粒体内LC3II蛋白表达水平显著下降(P0.05)；不同字母表示组别之间差异显著（P0.05),while the groups labeled with different letters aresignificantly different(P0.05).The same as below.ConPur3CdPink1-66kuCd+PurCParkin-55ku2bbCabLC3 I-17kuabbTa-14kuaabaLC3 IIaCOXIV-15ku0CdCl2(mol/L)001010Pink1ParkinLC3 IIPur(mol/L)01000100图5Pur与Cd对大鼠大脑皮质神经元线粒体自噬相关蛋白表达水平的影响Figure 5Effects of Pur and Cd on the expression of mitophagy-related proteins in rat cerebral cortical neuron280第54卷中国兽医科学ConPurPink1-66kuCdCd+PurbParkin-55ku2-TbbCababaLC3 I-17kuaT-14kuaaaLC3 II1aCOXIV-15kuCdCl2(mol/L)007575Pink1ParkinLC3 IIPur(mol/L)02000200图6 Pur与Cd对大鼠大脑皮质线粒体自噬相关蛋白表达水平的影响Figure 65Effects of Pur and Cd on the expression of mitophagy-related proteins in cerebral cortices of rats3讨论神经元的存活很大程度上取决于线粒体的完整性和功能性，神经元内、外环境的改变均可引起线粒体结构和功能的异常。由于线粒体生理学的复杂性、应激条件的相关性以及不同病理的影响，线粒体对镉引起的中毒反应值得探究。线粒体是镉毒性的重要靶点，镉可缩短并减少线粒体的数量1。研究表明，镉可诱导大鼠原代肾小管上皮细胞线粒体断裂、膜破坏及基质模糊12 。本课题组前期研究也发现，镉可诱导大鼠大脑皮质神经元和大脑皮质线粒体损伤9,13。本研究结果显示，可诱导PC12细胞、大鼠大脑皮质神经元和大鼠大脑皮质线粒体断裂、模糊，线粒体基质内出现空泡，与上述研究结果一致。线粒体自噬可选择性地识别和消除受损的线粒体，是线粒体质量控制中的一个重要过程。大部分的线粒体自噬都由Pink1/Parkin介导。当线粒体在内部或外部应激下发生去极化损伤时,Pinkl在去极化的线粒体中稳定存在,并募集胞浆中Parkin转移到受损线粒体,Parkin泛素化线粒体外膜相关蛋白，并与自噬体膜LC3结合共同促进损伤的线粒体经自噬途径而被清除2 。大量研究表明，多种外源刺激（铅、砷、锑和乙醇）可诱导小鼠肝、肾组织线粒体自噬4-16 。Wei等17 的研究发现，镉可诱导小鼠脑、肾组织线粒体自噬。本课题组前期研究也表明，镉通过激活Pink1/Parkin和Nix通路诱导大鼠大脑皮质神经元线粒体自噬，导致线粒体质量下降4。本研究结果显示，镉显著促进大鼠神经细胞线粒体内Pinkl、Pa r k in 和LC3II蛋白的表达，说明镉可诱导大鼠神经细胞线粒体发生自噬。葛根是中国传统的药食同源植物，近年来被广泛用于制备药物和功能性食品。葛根素是葛根提取物中的主要活性成分，可发挥神经保护作用8-9。研究发现，葛根素可缓解大鼠肝组织线粒体结构损伤18 。另有研究表明,葛根素可减轻大鼠肾小管上皮细胞线粒体超微结构损伤，恢复线粒体通透性转换孔周期性开放，并可通过改善线粒体膜电位来逆转镉诱导的ATP耗竭19,表明葛根素可修复线粒体结构和功能。笔者所在课题组前期研究发现，葛根素可缓解镉致大鼠大脑皮质线粒体自噬41。本研究结果显示，葛根素可缓解镉诱导的大鼠神经细胞线粒体超微结构损伤，并能显著抑制镉致大鼠神经细胞Pink1、Pa r k i n、LC3I I 蛋白表达水平升高，表明葛根素可缓解镉致大鼠神经细胞线粒体结构损伤及Pink1/Parkin介导的线粒体自噬，提示葛根素可能是通过修复损伤的线粒体发挥神经保护作用，综上所述，葛根素可缓解镉诱导的大鼠神经细胞线粒体结构损伤和Pink1/Parkin介导的线粒体自噬，该研究结果可为临床上葛根素防控镉的神经细胞毒性损伤作用提供理论依据参考文献(References)1MENZIES R A,GOLD PH.The turnover of mitochondriain a variety of tissues of young adult and aged ratsJ.The Journal of Biological Chemistry,1971,246(8):2425-2429.2GREENE A W,GRENIER K,AGUILETA M A,et al.Mitochon-drial processing peptidase regulates PINKl pro-cessing,import and Parkin recruitment J.EMBOReports,2012,13(4):378-385.3WANG T,ZHU Q,CAO B,et al.Cadmium induces mitophagyvia AMP-activated protein kinases activation in aPINK1/Parkin-dependent manner in PC12 cells J.Cell 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