姜黄素对高氧诱导肺泡Ⅱ型上皮细胞损伤的保护作用及机制
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摘要
目的探讨姜黄素对高氧诱导肺泡Ⅱ型上皮细胞(AEC2)损伤的保护作用,以及β-catenin/p300相互作用与姜黄素保护AEC2的关系。方法采用分离培养的AEC2建立高氧诱导的细胞损伤模型。培养AEC2细胞,随机分为对照组、高氧组、姜黄素组(分为高、中、低剂量3个亚组)、IQ-1(β-catenin/p300相互作用抑制剂)组、p300siRNA组、siRNA阴性对照组。药物处理24h后,CCK-8法和细胞计数板检测AEC2细胞增殖活力和细胞总数,定量PCR检测AEC2、肺泡Ⅰ型上皮细胞(AEC1)的标记基因SP-C和AQP5mRNA表达量,流式细胞仪检测AQP5、SP-C阳性细胞百分比,Western blot检测β-catenin、p300蛋白表达量。结果与对照组比较,高氧组AEC2细胞增殖活力、细胞计数、SP-C mRNA表达以及AEC2百分比均明显减少(P<0.01),而AEC1标记基因AQP5mRNA表达、AEC1百分比、AEC2向AEC1转分化的中间型细胞百分比则明显增加(P<0.01),β-catenin、p300蛋白表达量明显增加(P<0.01)。姜黄素组、IQ-1组和p300siRNA组AEC2细胞增殖活力、细胞计数、SP-C mRNA表达以及AEC2百分比均明显增加(P<0.01),而AQP5mRNA表达、AEC1百分比、转分化中间型细胞百分比均减少(P<0.01),β-catenin、p300蛋白表达量明显减小(P<0.01)。结论姜黄素对高氧诱导培养AEC2损伤具有保护作用,其机制与抑制β-catenin/p300相互作用有关。
Objective To investigate the protective effect of curcumin on hyperoxia-induced injury of alveolar typeⅡ epithelial cells(AEC2)and the relationship between beta-catenin/p300 and the protective effect of curcumin.Methods The models of hyperoxia-induced cellular injury were established in the primary cultured AEC2.AEC2 were randomly divided into 8 groups including the control group,hyperoxia model group,curcumin treatment group which was subdivided into the high-dose,medium-dose and low-dose groups,IQ-1(β-catenin/p300 interaction inhibitor)group,p300 siRNA group and siRNA negative control group.After 24 hours of the drug treatment,the proliferation activity of AEC2 was detected by CCK-8 assay and the total amount of AEC2 cells was measured by blood cell counting board.The mRNA expression of the marker genes SP-C for AEC2 and AQP5 for alveolar type I epithelial cells(AEC1)was detected by quantitative Polymerase Chain Reaction(qPCR),the percentage of the AQP5 and SP-C positive cells was detected by flow cytometry,and the protein expression ofβ-catenin and P300 was detected by Western blot.Results When compared with the control group,there was a significant decrease in the AEC2 proliferation,total cell amount,mRNA expression of the AEC2 marker gene SP-C and percentage of AEC2(P<0.01)and a significant increase in the mRNA expression of the AEC1 marker gene AQP5,percentage of AEC1 and percentage of the transdifferentiated intermediate cells from AEC2 to AEC1,and protein expression ofβ-catenin and p300(P<0.01).In contrast,the AEC2 proliferation activity,cell count,SP-C mRNA expression and AEC2 percentage increased significantly(P<0.01)while the AQP5 mRNA expression,AEC1 percentage,percentage of the transdifferentiated intermediate cells,and protein expression ofβ-catenin and p300 decreased significantly(P<0.01)in the curcumin treatment group,IQ-1 group and p300 siRNA group.Conclusion Curcumin can protect AEC2 against the hyperoxia-induced injury in vitro,and the mechanism is related to its inhibition of the interaction betweenβ-catenin and p300.
Objective To investigate the protective effect of curcumin on hyperoxia-induced injury of alveolar typeⅡ epithelial cells(AEC2)and the relationship between beta-catenin/p300 and the protective effect of curcumin.Methods The models of hyperoxia-induced cellular injury were established in the primary cultured AEC2.AEC2 were randomly divided into 8 groups including the control group,hyperoxia model group,curcumin treatment group which was subdivided into the high-dose,medium-dose and low-dose groups,IQ-1(β-catenin/p300 interaction inhibitor)group,p300 siRNA group and siRNA negative control group.After 24 hours of the drug treatment,the proliferation activity of AEC2 was detected by CCK-8 assay and the total amount of AEC2 cells was measured by blood cell counting board.The mRNA expression of the marker genes SP-C for AEC2 and AQP5 for alveolar type I epithelial cells(AEC1)was detected by quantitative Polymerase Chain Reaction(qPCR),the percentage of the AQP5 and SP-C positive cells was detected by flow cytometry,and the protein expression ofβ-catenin and P300 was detected by Western blot.Results When compared with the control group,there was a significant decrease in the AEC2 proliferation,total cell amount,mRNA expression of the AEC2 marker gene SP-C and percentage of AEC2(P<0.01)and a significant increase in the mRNA expression of the AEC1 marker gene AQP5,percentage of AEC1 and percentage of the transdifferentiated intermediate cells from AEC2 to AEC1,and protein expression ofβ-catenin and p300(P<0.01).In contrast,the AEC2 proliferation activity,cell count,SP-C mRNA expression and AEC2 percentage increased significantly(P<0.01)while the AQP5 mRNA expression,AEC1 percentage,percentage of the transdifferentiated intermediate cells,and protein expression ofβ-catenin and p300 decreased significantly(P<0.01)in the curcumin treatment group,IQ-1 group and p300 siRNA group.Conclusion Curcumin can protect AEC2 against the hyperoxia-induced injury in vitro,and the mechanism is related to its inhibition of the interaction betweenβ-catenin and p300.
引文
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[22]Buckley S,Barsky L,Weinberg K,et al.In vivo inosine protects alveolar epithelial type 2cells against hyperoxiainduced DNA damage through MAP kinase signaling[J].Am J Physiol Lung Cell Mol Physiol,2005,288(3):L569-L575.
[23]Fu H M,Zhang T S,Huang R W,et al.Calcitonin generelated peptide protects type II alveolar epithelial cells from hyperoxia-induced DNA damage and cell death[J].Exp Ther Med,2017,13(4):1279-1284.
[24]吴建魏,刘洁,谭利斌,等.姜黄素预处理对肺缺血再灌注损伤大鼠的影响[J].新中医,2018,50(1):7-10.
[25]张先明,杨睿,刘庆,等.姜黄素对机械通气相关性肺损伤的保护作用及其机制研究[J].重庆医学,2016,45(22):3034-3037.
[26]Rieger M E,Zhou B Y,Solomon N,et al.P300/β-catenin interactions regulate adult progenitor cell differentiation downstream of WNT5a/Protein kinase C(PKC)[J].J Biol Chem,2016,291(12):6569-6582.
[2]Chen C M,Hwang J,Chou H C,et al.Tn(N-acetyl-dgalactosamine-O-serine/threonine)immunization protects against hyperoxia-induced lung injury in adult mice through inhibition of the nuclear factor kappa B activity[J].Int Immunopharmacol,2018,59:261-268.
[3]Chen Y,Lu Z J,Yang Y,et al.Suppression of plasminogen activator inhibitor-1 by inhaled nitric oxide attenuates the adverse effects of hyperoxia in a rat model of acute lung injury[J].Thromb Res,2015,136(1):131-138.
[4]Entezari M,Javdan M,Antoine D J,et al.Inhibition of extracellular HMGB1 attenuates hyperoxia-induced inflammatory acute lung injury[J].Redox Biol,2014,2:314-322.
[5]Hoffman A M,Ingenito E P.Alveolar epithelial stem and progenitor cells:Emerging evidence for their role in lung regeneration[J].Curr Med Chem,2012,19(35):6003-6008.
[6]Qin S,Chen M,Ji H,et al.miR?21?5p regulates type IIalveolar epithelial cell apoptosis in hyperoxic acute lung injury[J].Mol Med Rep,2018,17(4):5796-5804.
[7]Hou A N,Fu J H,Shi Y Y,et al.Decreased ZONABexpression promotes excessive transdifferentiation of alveolar epithelial cells in hyperoxia-induced bronchopulmonary dysplasia[J].Int J Mol Med,2018,41(4):2339-2349.
[8]Sasaki T,Kahn M.Inhibition ofβ-catenin/p300interaction proximalizes mouse embryonic lung epithelium[J].Transl Respir Med,2014,2:8.
[9]Malleske D T,Hayes D J,Lallier S W,et al.Regulation of human airway epithelial tissue stem cell differentiation byβ-catenin,P300,and CBP[J].Stem Cells,2018,36(12):1905-1916.
[10]吴建魏,黄觉生,谭利斌,等.姜黄素预处理对肺缺血再灌注损伤模型大鼠炎性及抗炎性细胞因子的影响[J].广州中医药大学学报,2018,35(4):668-672.
[11]解东兴,史妍,李萍,等.姜黄素对急性肺损伤大鼠肺组织诱导型一氧化氮合酶和内皮型一氧化氮合酶表达的影响[J].解剖学杂志,2016,39(6):678-681.
[12]张明,唐晶晶,谢颖颖,等.姜黄素对慢性阻塞性肺疾病大鼠肺泡上皮细胞内质网应激的影响[J].西安交通大学学报(医学版),2018,39(3):361-365.
[13]王小玲,韦红,张钰,等.姜黄素对600mL/L氧致新生大鼠肺损伤保护作用的探讨[J].四川大学学报(医学版),2014,45(2):225-229,244.
[14]Dobbs L G,Gonzalez R,Williams M C.An improved method for isolating type II cells in high yield and purity[J].Am Rev Respir Dis,1986,134(1):141-145.
[15]汪鸿,常立文,卢红艳,等.高氧对胎鼠肺泡Ⅱ型上皮细胞Notch受体的影响[J].中华急诊医学杂志,2008,17(11):1158-1162.
[16]Nielsen S,King L S,Christensen B M,et al.Aquaporins in complex tissues.II.Subcellular distribution in respiratory and glandular tissues of rat[J].Am J Physiol,1997,273(5Pt 1):C1549-C1561.
[17]Mason R J,Kalina M,Nielsen L D,et al.Surfactant protein C expression in urethane-induced murine pulmonary tumors[J].Am J Pathol,2000,156(1):175-182.
[18]Crapo J D,Barry B E,Foscue H A,et al.Structural and biochemical changes in rat lungs occurring during exposures to lethal and adaptive doses of oxygen[J].Am Rev Respir Dis,1980,122(1):123-143.
[19]Budinger G R S,Mutlu G M,Urich D,et al.Epithelial cell death is an important contributor to oxidant-mediated acute lung injury[J].Am J Respir Crit Care Med,2011,183(8):1043-1054.
[20]Pagano A,Barazzone-Argiroffo C.Alveolar cell death in hyperoxia-induced lung injury[J].Ann N Y Acad Sci,2003,1010:405-416.
[21]Altemeier W A,Sinclair S E.Hyperoxia in the intensive care unit:Why more is not always better[J].Curr Opin Crit Care,2007,13(1):73-78.
[22]Buckley S,Barsky L,Weinberg K,et al.In vivo inosine protects alveolar epithelial type 2cells against hyperoxiainduced DNA damage through MAP kinase signaling[J].Am J Physiol Lung Cell Mol Physiol,2005,288(3):L569-L575.
[23]Fu H M,Zhang T S,Huang R W,et al.Calcitonin generelated peptide protects type II alveolar epithelial cells from hyperoxia-induced DNA damage and cell death[J].Exp Ther Med,2017,13(4):1279-1284.
[24]吴建魏,刘洁,谭利斌,等.姜黄素预处理对肺缺血再灌注损伤大鼠的影响[J].新中医,2018,50(1):7-10.
[25]张先明,杨睿,刘庆,等.姜黄素对机械通气相关性肺损伤的保护作用及其机制研究[J].重庆医学,2016,45(22):3034-3037.
[26]Rieger M E,Zhou B Y,Solomon N,et al.P300/β-catenin interactions regulate adult progenitor cell differentiation downstream of WNT5a/Protein kinase C(PKC)[J].J Biol Chem,2016,291(12):6569-6582.