| 340 | 0 | 76 |
| 下载次数 | 被引频次 | 阅读次数 |
目的:研究五子衍宗丸对全反式维甲酸(atRA)诱导的神经细胞凋亡途径与泛素-蛋白酶体系统(UPS)的关系。方法:将大鼠肾上腺嗜铬细胞瘤细胞(PC12)随机分为空白对照组、模型对照组、五子衍宗丸125μg/mL组、MG132蛋白酶体抑制剂组。采用atRA诱导PC12细胞凋亡模型；采用CCK-8法测定细胞活力；流式细胞术检测细胞凋亡率；高通量RNA-seq测序技术对细胞总RNA测序，筛选差异表达基因，运用趋势分析找出目的趋势模块，对目的趋势模块进行GO富集分析和KEGG通路富集分析，筛选凋亡和泛素相关基因。再次以atRA诱导PC12细胞凋亡模型，实时荧光定量PCR(RT-qPCR)法检测凋亡基因细胞周期检测点激酶2(Chek2)、极光激酶B(Aurkb)、半胱天冬酶4(Caspase4)和泛素相关基因Polo-like激酶(Pik1)、泛素结合酶E2C(Ube2C)、同源蛋白3(Trib3)mRNA的表达。结果:五子衍宗丸细胞活力显著增加(P<0.01),细胞凋亡率降低(P<0.01);目的趋势模块GO富集分析和KEGG分析中，五子衍宗丸＿下调＿上调与MG132＿下调、五子衍宗丸＿上调＿下调与MG132＿上调显著富集的GO条目和KEGG通路有多数重合，筛选出的凋亡和泛素相关基因在PPI和共表达网络分析中连接度较高，且两个分析结果中共有的基因有Chek2、Aurkb、Caspase4等凋亡相关基因，Pik1、Ube2c、Trib3等泛素相关基因。既包含这些基因中的凋亡相关基因又包含这些基因中的泛素相关基因的KEGG通路有细胞周期，流体剪切力与动脉粥样硬化等通路；RT-qPCR结果显示，五子衍宗丸125μg/mL组凋亡相关基因、Trib3、Chek2、Aurkb mRNA的表达下调(P<0.05或P<0.01);MG132组凋亡相关基因、Trib3、Chek2、Aurkb mRNA的表达上调(P<0.05);MG132+五子衍宗丸组对于凋亡相关基因和UPS相关基因表达的影响与MG132组的趋势相反(P<0.05)。结论:五子衍宗丸能够抑制atRA诱导的PC12细胞凋亡，对神经元样细胞具有一定的保护作用。其机制与泛素有一定的关系，可能是通过调控Chek2、Aurkb、Birc5、Pik1、Ube2c、Aurka、Caspase4 mRNA的表达实现。
Abstract：Objective:To study the relationship between ubiquitin-proteasome system(UPS) and all-trans retinoic acid(atRA)-induced neuronal apoptosis induced by Wuzi Yanzong Pills(五子衍宗丸,WYP). Methods:Rat adrenal pheochromocytoma cells(PC12) were randomly divided into a normal control group, model control group, 125 μg/mL WYP group, and MG132 proteasome inhibitor group. PC12 cells were treated with atRA to induce an apoptosis model. The cell viability was measured by the CCK-8 method, and the apoptosis rate in each group was detected by flow cytometry. High-throughput RNA-Seq sequencing technology was used to sequence the total RNA of cells and screen the differentially expressed genes. GO enrichment analysis and KEGG pathway significant enrichment analysis were performed on the target trend modules to screen apoptosis-and ubiquitin-related genes. The atRA was used to induce a PC12 cell apoptosis model. Real-time fluorescent quantitative PCR(RT-qPCR) was used to detect the mRNA expression of apoptosis genes, including cell cycle checkpoint kinase2(Chek2) and aurora kinase B(Aurkb),recombinant raspase 4(Caspase4),as well as ubiquitin-related genes, such as Polo-like kinasel(Pik1),recombinant ubiquitin conjugating enzyme E2C(Ube2C),and tribbles pseudokinase3(Trib3). Results:WYP significantly enhanced the cell viability(P<0.01) and reduced the apoptosis rate(P<0.01). In target trend module, GO enrichment analysis, and KEGG analysis, the GO items and KEGG pathways significantly enriched with MG132＿down-regulation and MG132＿down-regulation in Wuziyanzongwan＿up-regulation and MG132＿up-regulation overlapped in most of the GO items and KEGG pathways. The screened apoptosis-and ubiquitin-related genes were highly connected in PPI and co-expression network analysis. The two analysis results showed that there were apoptosis-related genes such as Chek2, Aurkb,and Caspase4,and ubiquitin-related genes such as Pik1, Ube2c, and Trib3. KEGG pathways involving both apoptosis-related genes and ubiquitin-related genes, included cell cycle, fluid shear stress, atherosclerosis, and other pathways. The RT-qPCR results showed that WYP down-regulated the expression of apoptosis-related genes and Trib3 mRNA(P<0.05,P<0.01) and up-regulated the expression levels of Chek2 and Aurkb mRNA(P<0.05). MG132 up-regulated the expression of apoptosis-related genes and Trib3 mRNA(P<0.05) and down-regulated the expression levels of Chek2 and Aurkb mRNA(P<0.05). The effect of MG132+atRA+WYP on the expression of apoptosis-related genes and UPS-related genes was consistent with the trend of the MG132+atRA group(P<0.05). Conclusion:WYP can inhibit the apoptosis of PC12 cells induced by atRA and has a protective effect on neuron-like cells. The mechanism may be related to ubiquitin via regulating the mRNA expressions of Chek2, Aurkb,Birc5,Pik1,Ube2c,Aurka, and Caspase4.
[1]Kirby R S,Browne M L.Putting birth defects surveillance data to action[J].Birth Defects Res,2021,113(2):115-116.
[2]Lee S Y,Papanna R,Farmer D,et al.Fetal Repair of Neural Tube Defects[J].Clin Perinatol,2022,49(4):835-848.
[3]Kancherla V.Neural tube defects:a review of global prevalence,causes,and primary preventions[J].Childs Nerv Syst,2023,39(7):1703-1710.
[4]Engelhardt D,Martyr C,Niswander L.Pathogenesis of neural tube defects:The regulation and disruption of cellular processes underlying neural tube closures[J].WIREs Mech Dis,2022,14(5):e1559.
[5]毛宏梅，石丽丽，霍军生.个性化叶酸补充预防神经管缺陷[J].卫生研究，2023,52(4):676-681.
[6]Wolujewicz P,Steele J,Kaltschmidt J,et al.Unraveling the complex genetics of neural tube defects:From biological models to human genomics and back[J].Genesi,2021,59(11):e23459.
[7]Huang W,Huang T,Liu Y,et al.Nuclear factor I-C disrupts cellular homeostasis between autophagy and apoptosis via miR-200b-Ambra1 in neural tube defectss[J].Cell Death Dis,2021,13(1):17.
[8]Wu Y,Wang F,Reece E,et al.Curcumin ameliorates high glucose-induced neural tube defects by suppressing cellular stress and apoptosis[J].Am J Obstet Gynecol,2015,212:802-808.
[9]Wang J,Xiao Y,Hsu C W,et al.Yap and Taz play a crucial role in neural crest-derived craniofacial development[J].Development,2016,143(3):504-515.
[10]LIU Y S,GU H,HUANG T C,et al.miR-322 treatment rescues cell apoptosis and neural tube defect formation through silencing NADPH oxidase 4[J].CNS Neurosci Ther,2020,26(9):902-912.
[11]CHEN N,XU J,ZHANG X,et al.Effect of Notch1 on neural tube defects and neural stem cell differentiation induced by alltrans retinoic acids[J].Mol Med Rep,2021,23(3):220.
[12]LI L,XI H M,LU H,et al.Combination of Ethacrynic Acid and ATRA Triggers Differentiation and/or Apoptosis of Acute Myeloid Leukemia Cells through ROS[J].Anticancer Agents Med Chem,2024,24(6):412-422.
[13]LIANG C,QIAO G,LIU Y,et al.Overview of all-trans-retinoic acid (ATRA) and its analogues:Structures,activities,and mechanisms in acute promyelocytic leukaemia[J].Eur J Med Chem,2021,220:113451.
[14]LIU J,ZHOU R,HE Q,et al.Calmodulin kinase II activation of mitogen-activated protein kinase in PC12 cell following all-trans retinoic acid treatment[J].Neurotoxicology,2009,30(4):599-604.
[15]袁莉刚，赵兴绪，刘英.泛素-蛋白酶体通路在神经系统中的生物学功能[J].中国兽医科学，2007,(5):444-448.
[16]杭薇，樊慧杰，李艳荣，等.五子衍宗丸对帕金森病小鼠脑组织中神经营养因子及凋亡相关基因表达的影响[J].解放军医学杂志，2023,48(2):168-174.
[17]樊慧杰，李艳彦，王永辉，等.五子衍宗丸对NTDs胎鼠神经管细胞凋亡的影响[J].中国实验方剂学杂志，2014,20(9):143-146.
[18]樊慧杰，柴智，解军，等.五子衍宗丸对ATRA诱导的神经管畸形防治作用初探[J].中成药，2014,36(5):1054-1056.
[19]李瑞雪.基于转录组测序技术研究五子衍宗丸对NTDs细胞模型凋亡途径的调控作用[D].山西中医药大学，2020.
[20]WANG X,YANG C,RU Y,et al.An optimal combination of five main monomer components in Wuzi Yanzong Pill that prevents neural tube defects and reduces apoptosis and oxidative stress[J].Ethnopharmacol,2023,2023:116540.
[21]WANG X,YANG C,LI Y,et al.Wuzi Yanzong Pill protects neural tube defects by activating PI3K/Akt signaling pathwayl[J].Int J Dev Neurosci,2023,83(5):417-430.
[22]陈望强，马健雄，江雪娟，等.五子衍宗丸对生精障碍大鼠生精功能的保护作用研究[J].中药材，2020,43(12):3010-3016.
[23]刘红娟，吴德玲，童小慧，等.五子衍宗丸干预线粒体通透性转换孔抑制精子凋亡的机制[J].中国实验方剂学杂志，2020,26(21):34-39.
[24]ZENG K,WAN Y,LIAO L,et al.Identification and function analysis of neuroprotective targets group of modified Wuzi Yanzong pill[J].China journal of Chinese materia medica,2017,42(19):3656-3660.
[25]Nie J,Shen Y,Roshd Y M,et al.Polystyrene nanoplastics exposure caused defective neural tube morphogenesis through caveolae-mediated endocytosis and faulty apoptosis[J].Nanotoxicology,2021,15(7):885-904.
[26]柴智，刘海宇，朱婷婷，等.细胞凋亡途径在神经管畸形发生机制中的研究进展[J].世界中西医结合杂志，2014,9(3):312-314.
[27]Asmamaw M D,Liu Y,Zheng Y C,et al.Skp2 in the ubiquitin-proteasome system:A comprehensive review[J].Med Res Rev,2020,40(5):1920-1949.
[28]LI Y,LI S,WU H.Ubiquitination-Proteasome System (UPS) and Autophagy Two Main Protein Degradation Machineries in Response to Cell Stress[J].Cells,2022,11(5):851.
[29]Graffeo R,Rana H Q,Conforti F,et al.Moderate penetrance genes complicate genetic testing for breast cancer diagnosis:ATM,CHEK2,BARD1 and RAD51D[J].Breast,2022,65:32-40.
[30]Garlapati C,Joshi S,Bhattarai S,et al.PIK1 and AURKB phosphorylate survivin differentially to affect proliferation in racially distinct triple-negative breast cancer[J].Cell Death Dis,2023,14(1):12.
[31]Chauhan M,Modi P K,Sharma P.Aberrant activation of neuronal cell cycle caused by dysregulation of ubiquitin ligase Itch results in neurodegeneration.Cell Death Dis,2020,11(6):441.
[32]Wandel M P,Kim B H,Park E S,et al.Guanylate-binding proteins convert cytosolic bacteria into caspase-4 signaling platforms[J].Nat Immunol,2020,21(8):880-891.
[33]Oliveira A M,Ross J S,Fletcher J A.Tumor suppressor genes in breast cancer:the gatekeepers and the caretakers[J].Am J Clin Pathol,2005,124 Suppl:S16-28.
[34]ZHOU W,WANG X.Aurkb regulates neuronal survival and synaptic plasticity in mouse hippocampus[J].Neuroscience Letters,2020,729,135016.
[35]WANG S,YANG X,LI G.Inhibition of Caspase4 activity suppresses apoptosis and improves cardiac function after myocardial infarction[J].Journal of Molecular and Cellular Cardiology,2019,134,79-88.
[36]Kalous J,Aleshkina D.Multiple Roles of PIK1 in Mitosis and Meiosis[J].Cells,2023,12(1):187.
[37]刘俊文，杨向东，王仁，等.蛋白酶体抑制剂MG132诱导人巨噬细胞THP-1凋亡[J].中国动脉硬化杂志，2004,(3):271-274.
[38]郭芳，屈顺林，孙文清，等.蛋白酶体抑制剂MG132诱导人血管平滑肌细胞凋亡及其机制研究[J].中国临床药理学与治疗学，2009,14(5):487-492.
基本信息:
DOI：10.13412/j.cnki.zyyl.2026.07.004
中图分类号:R285.5
引用信息:
[1]茹意,李瑞雪,谢良骐,等.五子衍宗丸通过调控泛素-蛋白酶体系统抑制反式维甲酸诱导的PC12细胞凋亡[J].中药药理与临床,2026,42(07):12-23.DOI:10.13412/j.cnki.zyyl.2026.07.004.
基金信息:
国家中医药管理局青年岐黄学者培养项目(编号：国中医药人教函[2022]256号); 山西省重点科技创新人才团队专项(编号：202304051001019); 山西省重点国别科技合作项目(编号：202204041101002); 山西省卫健委中医药重点研究室建设专项(编号：zyyyjs2024022);山西省卫健委中医药创新团队建设专项(编号：zyytd2024022); 山西省青年拔尖人才支持计划项目(编号：晋组办字[2019]35号文); 山西省医学重点科研项目(编号：2023XM018); 山西中医药大学科技创新团队建设项目(编号：2022TD1013)
2025-07-14
2025-07-14
2025-07-14