猪肝星状细胞内质网应激模型的建立及对泛素化的影响

doi:10.16431/j.cnki.1671-7236.2020.02.010

摘要/Abstract

摘要： 试验对衣霉素（tunicamycin，TM）处理猪肝星状细胞的浓度和培养时间进行筛选，以期成功构建内质网应激（endoplasmic reticulum stress，ERS）模型，并在此基础上探究ERS下猪肝星状细胞泛素化的变化。试验采用浓度为0、1、2、5、10和15 μg/mL的TM处理猪肝星状细胞，培养2、4、8、16、24和36 h。通过检测细胞抑制率对TM浓度和培养时间进行初筛；通过检测细胞周期及ERS标志基因和蛋白表达对TM浓度和培养时间进行终选，以确定是否成功构建ERS模型，同时，在ERS下检测猪肝星状细胞泛素化相关基因的表达变化。结果表明：由细胞抑制率检测结果初步确定TM浓度5 μg/mL、培养8或24 h后有可能出现ERS。5 μg/mL TM在培养8或24 h时，ERS标志基因表达均极显著上调（P < 0.01）。在培养8 h时，ERS标志蛋白中仅ATF4显著上调（P < 0.05），细胞周期中仅G2/M期细胞比例显著下降（P < 0.05）；在培养24 h时，ERS标志蛋白均显著上调（P < 0.05），细胞周期在G1期出现阻滞，并导致S期和G2/M期细胞比例极显著下降（P < 0.01）。以上结果说明，5 μg/mL TM培养24 h可成功构建细胞ERS模型。在ERS下，细胞泛素化相关基因UBA2和UBE2E的表达均极显著升高（P < 0.01）。综上，猪肝星状细胞经5 μg/mL TM处理24 h，可成功建立ERS模型，并启动泛素化机制。

关键词: 衣霉素; 猪肝星状细胞; 内质网应激; 泛素化

Abstract: This study was conducted to screen the different concentrations and culture time of tunicamycin (TM) in porcine immortalized hepatic stellate cell,in order to model endoplasmic reticulum stress (ERS) successfully and explore the changes of ubiquitination under ERS.Porcine hepatic stellate cell was treated with TM with concentrations of 0,1,2,5,10 and 15 μg/mL,respectively,and cultured for 2,4,8,16,24 and 36 h.TM concentration and culture time were initially screened by detection of cell inhibition rate,and finally selected by detection of cell cycle and ERS marker genes and proteins expression,in order to model ERS successfully.In addition,the expression of ubiquitin related genes in porcine liver stellate cells under ERS were detected.The results showed that 5 μg/mL TM culture for 8 or 24 h might model ERS via detection of cell inhibition rate.ERS marker gene expressions were all extremely significantly upregulated by 5 μg /mL TM,cultured for 8 or 24 h (P < 0.01).In terms of ERS marker protein expression,only ATF4 increased significantly (P < 0.05),and only G2/M phase of cell proportion in cell cycle significantly decreased at 8 h of culture (P < 0.05).At 24 h of culture,ERS marker proteins were significantly increased (P < 0.05),and cell cycle arrest occurred in G1 phase,leading to extremely significant declines in cell proportion in S phase and G2/M phase (P < 0.01).The result indicated that ERS model was successfully constructed treated by 5 μg/mL TM for 24 h.Ubiquitin related genes UBA2 and UBE2E expressions were extremely significantly increased (P < 0.01).In conclusion,the ERS model was successfully established and ubiquitylation mechanism was initiated by cultured with 5 μg/mL TM for 24 h in porcine hepatic stellate cell.

Key words: tunicamycin; porcine hepatic stellate cell; endoplasmic reticulum stress; ubiquitination

中图分类号:

Q256

辛海瑞, 张闯, 郝月, 顾宪红. 猪肝星状细胞内质网应激模型的建立及对泛素化的影响[J]. 中国畜牧兽医, 2020, 47(2): 399-406.

XIN Hairui, ZHANG Chuang, HAO Yue, GU Xianhong. A Establishment of Endoplasmic Reticulum Stress Model of Porcine Hepatic Stellate Cell and Its Effect on Ubiquitination[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(2): 399-406.

参考文献

[1] RON D,WALTER P.Signal integration in the endoplasmic reticulum unfolded protein response[J].Nature Reviews Molecular Cell Biology,2007,8(7):519-529.
[2] ZHU P,ZUO Z,ZHENG Z,et al.Aflatoxin B (1) affects apoptosis and expression of death receptor and endoplasmic reticulum molecules in chicken spleen[J].Oncotarget,2017,8(59):99531-99540.
[3] YUAN S,WU B,YU Z,et al.The mitochondrial and endoplasmic reticulum pathways involved in the apoptosis of bursa of Fabricius cells in broilers exposed to dietary aflatoxin B1[J].Oncotarget,2016,7(40):65295-65306.
[4] GUO H,CUI H,PENG X,et al.Nickel chloride (NiCl2) induces endoplasmic reticulum (ER) stress by activating UPR pathways in the kidney of broiler chickens[J].Oncotarget,2016,7(14):17508-17519.
[5] CUI Y,HAO Y,LI J,et al.Chronic heat stress induces immune response,oxidative stress response,and apoptosis of finishing pig liver:A proteomic approach[J].International Journal of Molecular Sciences,2016,17(5):393.
[6] 方帅,赵博.泛素化及相关疾病研究进展[J].生物化学与生物物理进展,2017,44(5):377-384. FANG S,ZHAO B.Ubiquitination in diseases[J].Progress in Biochemistry and Biophysics,2017,44(5):377-384.(in Chinese)
[7] 王花枝,姜迪譞,胡瑞成,等.内质网相关性降解与呼吸系统疾病[J].国际呼吸杂志,2014,34(7):555-560. WANG H Z,JIANG D X,HU R C,et al.Endoplasmic reticulum-associated degradation and respiratory system disease[J].International Journal of Respiratory,2014,34(7):555-560.(in Chinese)
[8] WU Y,LI X,JIA J,et al.Transmembrane E3 ligase RNF183 mediates ER stress-induced apoptosis by degrading Bcl-xL[J].Proceedings of the National Academy of Sciences,2018,115(12):E2762-E2771.
[9] 戎志斌,孙珂焕,黄飞娟,等.柴胡疏肝散含药血清对衣霉素诱导NG108-15神经元内质网应激模型泛素基因表达的影响[J].深圳中西医结合杂志,2016,26(6):1-3. RONG Z B,SUN K H,HUANG F J,et al.The inhibition on neuron apoptosis and mechanism research for tunicamycin induced NG108-15 endoplasmic reticulum stress by Chaihushugansan[J].Shenzhen Journal of Integrated Traditional Chinese and Western Medicine,2016,26(6):1-3.(in Chinese)
[10] 李旭.ROS依赖的内质网应激-自噬反应在醉茄素A诱导人胰腺癌细胞凋亡中的作用研究[D].武汉:华中科技大学,2013. LI X.The role of ROS-dependent endoplasmic reticulum stress and autophagy pathway in the apoptosis induced by withaferin A in human pancreatic cancer cells[D].Wuhan:Huazhong University of Science & Technology,2013.(in Chinese)
[11] SPRATT D E,WALDEN H,SHAW G S.RBR E3 ubiquitin ligases:New structures,new insights,new questions[J].Biochemical Journal,2014,458(3):421-437.
[12] BRUNING A,MATSINGOU C,BREM G J,et al.Inhibin beta E is upregulated by drug-induced endoplasmic reticulum stress as a transcriptional target gene of ATF4[J].Toxicologya and Applied Pharmacology,2012,264(2):300-304.
[13] LERNER A G,UPTON J P,PRAVEEN P V,et al.IRE1alpha induces thioredoxin-interacting protein to activate the NLRP3 inflammasome and promote programmed cell death under irremediable ER stress[J].Cell Metabolism,2012,16(2):250-264.
[14] BROWN M,STRUDWICK N,SUWARA M,et al.An initial phase of JNK activation inhibits cell death early in the endoplasmic reticulum stress response[J].Journal of Cell Science,2016,129(12):2317-2328.
[15] GU X,LI K,LAYBUTT D R,et al.Bip overexpression,but not CHOP inhibition,attenuates fatty-acid-induced endoplasmic reticulum stress and apoptosis in HepG2 liver cells[J].Life Sciences,2010,87(23-26):724-732.
[16] 李雅楠.Calpain-2介导内质网应激诱导HSC凋亡在肝纤维化中的作用[D].唐山:华北理工大学,2018. LI Y N.Role of Calpain-2 in endoplasmic reticulum stress-induced HSC apoptosis in hepatic fibrosis[D].Tangshan:North China University of Science and Technology,2018.(in Chinese)
[17] HERNÁNDEZ G V,HILSCHER M,ROZENFELD R,et al.Endoplasmic reticulum stress induces fibrogenic activity in hepatic stellate cells through autophagy[J].Journal of Hepatology,2013,59(1):98-104.
[18] 王铭.衣霉素对肝星状细胞凋亡的调控作用[D].唐山:华北理工大学,2017. WANG M.The regulation of tunicamycin on the apoptosis of hepatic stellate cell[D].Tangshan:North China University of Science and Technology,2018.(in Chinese)
[19] HERNANDEZ G V,FRIEDMAN S L.Pathogenesis of liver fibrosis[J].Annual Review of Phytopathology,2011,6(1):425-456.
[20] NISHA V M,ANUSREE S S,PRIYANKA A,et al.Apigenin and quercetin ameliorate mitochondrial alterations by tunicamycin-induced ER stress in 3T3-L1 adipocytes[J].Applied Biochemistry and Biotechnology,2014,174(4):1365-1375.
[21] ZHANG L J,LI Z Q,YANG Y P,et al.Tunicamycin suppresses cisplatin-induced HepG2 cell apoptosis via enhancing p53 protein nuclear export[J].Molecular and Cellular Biochemistry,2009,327(1-2):171-182.
[22] GROOTJANS J,KASER A,KAUFMAN R J,et al.The unfolded protein response in immunity and inflammation[J].Nature Reviews Immunology,2016,16(8):469-484.
[23] CARLISLE R E,BRIMBLE E,WERNER K E,et al.4-Phenylbutyrate inhibits tunicamycin-induced acute kidney injury via CHOP/GADD153 repression[J].PLoS One,2014,9(1):e84663.
[24] ZHU G,LEE A S.Role of the unfolded protein response,GRP78 and GRP94 in organ homeostasis[J].Journal of Cellular Physiology,2015,230(7):1413-1420.
[25] 孙美涛,梅雯,王唯斯,等.MTERF3过表达对脑胶质瘤U251细胞线粒体基因表达及细胞增殖和迁移的影响[J].实用医药杂志,2019,36(5):454-459. SUN M T,MEI F,WANG W S,et al.Influence of MTERF3 overexpression on mitochondrial gene expression as well as the proliferation and migration of cells in brain glioma U251 cells[J].Practical Journal of Medicine & Pharmacy,2019,36(5):454-459.(in Chinese)
[26] LI L,TAN H,GU Z,et al.Heat stress induces apoptosis through a Ca (2)(+)-mediated mitochondrial apoptotic pathway in human umbilical vein endothelial cells[J].PLoS One,2014,9(12):e111083.
[27] 包文华.内质网应激对蛋白酶体抑制剂诱导乳腺癌MCF-7细胞自噬的作用[D].长春:吉林大学,2016. BAO W H.Effect of endoplasmic reticulum stress on breast cancer MCF-7 cell autophagy induced by proteasome inhibitor[D].Changchun:Jilin University,2016.(in Chinese)
[28] CYBULSKY A V.The intersecting roles of endoplasmic reticulum stress,ubiquitin-proteasome system,and autophagy in the pathogenesis of proteinuric kidney disease[J].Kidney International,2013,84(1):25-33.