NLRP3炎症小体在迷迭香酸抑制视网膜色素上皮细胞衰老中的作用  

The Role of NLRP3 Inflammasome in Rosmarinic Acid-Mediated Inhibition of Retinal Pigment Epithelial Cell Senescence

作　　者：陈琳[1] 李蓉[1] 邢春涛 姚国敏[1] CHEN Lin;LI Rong;XING Chuntao;YAO Guomin(The First Affiliated Hospital of Xi'an Medical University,Xi'an 710077,China)

机构地区：[1]西安医学院第一附属医院,西安710077

出　　处：《中国中医眼科杂志》2023年第9期807-812,共6页China Journal of Chinese Ophthalmology

基　　金：陕西省科学技术厅社会发展项目(2021SF-157)。

摘　　要：目的研究迷迭香酸(RA)对过氧化氢(H2O2)诱导的人视网膜色素上皮细胞ARPE-19衰老模型的影响,并探讨NOD样受体蛋白3(NLRP3)炎症小体在其中的作用。方法分别用0、20、40、80μmol/L的RA预处理ARPE-19细胞48 h后,加入200μmol/L的H2O2诱导细胞衰老模型,细胞计数试剂-8(CCK-8)法检测细胞活性,筛选RA的最佳剂量。将ARPE-19细胞分为对照组(CG)、模型组(MG)、RA组(80μmol/L),再次按照上述方法处理细胞。衰老相关β-半乳糖苷酶(SA-β-gal)法检测细胞衰老,Western Blot法检测细胞周期依赖性蛋白激酶抑制因子2A、1A(P16、P21)、NLRP3、凋亡相关颗粒样蛋白(ASC)、半胱氨酸天冬氨酸特异性蛋白酶-1(Caspase-1)表达。结果(1)RA最佳剂量:80μmol/L的RA干预后ARPE-19细胞活性最高,作为最佳剂量。(2)细胞衰老:与CG组比较,MG组ARPE-19细胞衰老率增加(t=8.642,P=0.000);与MG组比较,RA组ARPE-19细胞衰老率降低(t=7.585,P=0.000),差异均有统计学意义。(3)NLRP3炎症小体通路关键蛋白:与CG组比较,MG组ARPE-19细胞P16、P21、NLRP3、ASC、Caspase-1表达升高(tP16=8.483,tP21=8.111,t_(NLRP3)=6.976,t_(ASC)=11.550,t_(Caspase)-1=7.300,均P=0.000);与MG组比较,RA组ARPE-19细胞P16、P21、NLRP3、ASC、Caspase-1表达降低(tP16=4.342,P=0.004;tP21=4.580,P=0.005;t_(NLRP3)=4.022,P=0.007;t_(ASC)=4.243,P=0.005;t_(Caspase)-1=4.434,P=0.004),差异均有统计学意义。结论RA预处理可以减轻H2O2诱导的ARPE-19细胞衰老,该效应可能与RA下调H2O2刺激下NLRP3炎症小体的表达有关。OBJECTIVE To investigate the effects of rosmarinic acid(RA)on H2O2-induced senescence in adult retinal pigment epithelial cells(ARPE-19)and explore the involvement of NOD-like receptor protein 3(NLRP3)inflammasome in this process.METHODS ARPE-19 cells were pretreated with RA at concentrations of/0,20,40,and 80μmol/L for 48 h,followed by induction of cell senescence using 200μmol/L H2O2.Cell viability was assessed using the Cell Counting Kit-8(CCK-8)assay to determine the optimal dosage of RA.ARPE-19 cells were divided into control group(CG),model group(MG),and RA group(80μmol/L),and treated accordingly.Senescence-associatedβ-galactosidase(SA-β-gal)staining was performed to assess cellular senescence,while Western Blot was used to measure the expression of cell cycle-dependent kinase inhibitors 2A and 1A(P16,P21),NLRP3,apoptosis-associated speck like protein(ASC),and cysteiny aspartatespecific protease-1(Caspase-1).RESULTS(1)Optimal dosage of RA:Treatment with 80μmol/L RA resulted in the highest cell viability in ARPE-19 cells,thus considered as the optimal dosage.(2)Cell senescence:Compared to the CG,the senescence rate of ARPE-19 cells in the MG increased significantly(t=8.642,P=0.000),while the RA group showed a significant reduction in cell senescence compared to the MG(t=7.585,P=0.000).(3)Key proteins in the NLRP3 inflammasome pathway:Compared to the CG,the expression levels of P16,P21,NLRP3,ASC,and Caspase-1 were elevated in the MG(tP16=8.483,tP21=8.111,t_(NLRP3)=6.976,t_(ASC)=11.550,t_(Caspase)-1=7.300,all P=0.000)in ARPE-19 cells.Conversely,the RA group showed decreased expression of P16,P21,NLRP3,ASC,and Caspase-1 compared to the MG(tP16=4.342,P=0.004;tP21=4.580,P=0.005;t_(NLRP3)=4.022,P=0.007;t_(ASC)=4.243,P=0.005;t_(Caspase)-1=4.434,P=0.004)in ARPE-19 cells.CONCLUSIONS Pretreatment with RA can alleviate H2O2-induced senescence in ARPE-19 cells,and this effect may be associated with the downregulation of NLRP3 inflammasome expression under H2O2 stimulation.

关 键 词：迷迭香酸 视网膜色素上皮细胞 细胞衰老 NLRP3炎症小体 

分 类 号：R774.1[医药卫生—眼科]