高糖诱导的视网膜新生血管形成中NLRP3炎症小体与自噬的关系研究

Relationship between NLRP3 inflammasome and autophagy in high-glucose induced retinal angiogenesis

目的探讨在高糖诱导的视网膜新生血管形成过程中,核苷酸结合寡聚化结构域样受体蛋白3(nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)炎症小体与自噬之间的关系。方法根据不同干预方式将人视网膜微血管内皮细胞(human retinal microvascular endothelial cells,HRMEC)随机分为三组高糖组、3-甲基腺嘌呤(3-methyladenine,3-MA)+高糖组、CY-09+高糖组。高糖组在M199培养基中加入30 mmol·L^-1葡萄糖溶液处理48 h;3-MA+高糖组和CY-09+高糖组分别先用5 mmol·L^-1的3-MA和10 mmol·L^-1的CY-09处理2 h,然后更换培养基再加入30 mmol·L^-1葡萄糖溶液处理48 h。分别采用MTT、Transwell及Matrigel法检测细胞活力、细胞迁移和管腔形成情况,比较三组细胞活力、细胞迁移数和管腔形成数;采用Western blot法检测三组细胞NLRP3炎症小体信号通路的关键蛋白NLRP3、凋亡相关颗粒样蛋白(ASC)、Caspase-1及自噬标志性蛋白LC3-II、LC3-I、Beclin-1的表达并对比;采用Ad-mCherry-GFP-LC3B腺病毒转染细胞观察自噬流的情况。结果高糖组、3-MA+高糖组、CY-09+高糖组HRMEC细胞活力分别为(153.56±1.46)%、(115.33±3.26)%和(116.10±1.66)%,细胞迁移数分别为(117.33±7.23)个、(70.00±2.00)个和(71.67±2.52)个,细胞管腔形成数分别为(88.33±2.08)个、(61.33±1.53)个和(62.00±3.00)个。与高糖组相比,3-MA+高糖组及CY-09+高糖组HRMEC的细胞活力、细胞迁移数和管腔形成数均明显减少(均为P<0.001),而3-MA+高糖组与CY-09+高糖组间差异均无统计学意义(均为P>0.05)。与高糖组相比,3-MA+高糖组及CY-09+高糖组细胞NLRP3、ASC、Caspase-1、Beclin-1的蛋白相对表达量和LC3-II/LC3-I比值均明显降低(均为P<0.05),细胞内自噬流明显减弱。结论抑制NLRP3炎症小体与自噬均可有效抑制高糖诱导的视网膜新生血管形成,且二者在其中可能发挥相互正向调控的作用。

Objective To investigate the relationship between nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome and autophagy in high-glucose induced retinal angiogenesis.Methods Human retinal microvascular endothelial cells(HRMECs)were divided into the high-glucose group,3-methyladenine(3-MA)+high-glucose group and CY-09+high-glucose group.The cells in the high-glucose group were cultured in the M199 medium adding 30 mmol·L^-1 glucose for 48 hours,the cells in the 3-MA+high-glucose group were pretreated with 5 mmol·L^-13-MA for 2 hours and then exposed to 30 mmol·L^-1 glucose for 48 hours,and the cells in the CY-09+high-glucose group were pretreated with 10 mmol·L^-1 CY-09 for 2 hours and then exposed to 30 mmol·L^-1 glucose for 48 hours.The vitality,migration and tube formation of HRMECs were detected by MTT,transwell and matrigel assay,respectively.The vitality,number of migrated cells and tube formation of HRMECs were statistically compared.The expression of proteins in NLRP3 inflammasome signaling pathway including NLRP3,apoptosis-associated speck like protein(ASC)and cysteiny aspartate-specific protease-1(Caspase-1),as well as microtubule-related protein 1 light chain 3(LC3-II,LC3-I)and Beclin-1 protein contents in the cells were detected by Western blot.Autophagy flux was then observed by adenovirus transfection with Ad-mCherry-GFP-LC3B.Results In the high-glucose group,3-MA+high-glucose group and CY-09+high-glucose group,the vitality of HRMECs was(153.56±1.46)%,(115.33±3.26)%and(116.10±1.66)%,respectively;the number of migrated HRMECs was(117.33±7.23)cells,(70.00±2.00)cells and(71.67±2.52)cells,respectively;the number of tube formation was(88.33±2.08)tubes,(61.33±1.53)tubes and(62.00±3.00)tubes,respectively.Compared with the high-glucose group,the cell viability,the number of cell migration,and tube formation were significantly reduced in the 3-MA+high-sugar group and the CY-09+high-sugar group(all P<0.0001),while there was no statistically significant difference between the 3-MA+high-sugar group and the CY-09+high-sugar group(P>0.05).Compared with the high-glucose group,the relative expression levels of the NLPRP3,ASC,Caspase-1,and Beclin-1 proteins as well as LC3-II/LC3-I ratios were significantly reduced in the 3-MA+high-glucose group and the CY-09+high-glucose group(both P<0.05),and intracellular autophagic flow was significantly reduced.Conclusion Inhibition of NLRP3 inflammasome and autophagy can effectively inhibit retinal angiogenesis induced by high glucose,and they may play a positively regulatory role in each other.