对比剂诱导巨噬细胞来源外泌体对肾小管上皮细胞的作用及甘草酸苷的干预效应

The effect of contrast agent-induced macrophage-derived exosomes on renal tubular epithelial cells and the intervention effect of glycyrrhizin

目的探讨对比剂诱导巨噬细胞来源外泌体对肾小管上皮细胞的作用及甘草酸苷的干预效应。方法分别用PBS、碘海醇、碘海醇+甘草酸苷刺激THP-1细胞,获取3种外泌体(NC-Exo、CIN-Exo、CIN-GL-Exo)。透射电镜观察外泌体的形态;蛋白免疫印迹(Western blot)检测CD63、TSG101、calnexin的表达;荧光标记法检测外泌体的摄取。将HK-2细胞随机分为4组:Con组、NC-Exo组、CIN-Exo组、CIN-GL-Exo组。Western blot检测3种外泌体中高迁移率族蛋白1(HMGB1)表达量,以及4组细胞中凋亡相关蛋白及HMGB1/Toll样受体4(TLR4)/核因子κB(NF-κB)信号通路相关蛋白的表达;AnnexinⅤ-FITC/PI双染法检测4组细胞的凋亡率;实时荧光定量PCR检测4组细胞炎性因子[白细胞介素(IL)-1β、IL-6、肿瘤坏死因子(TNF)-α]mRNA的表达。结果 THP-1细胞来源的外泌体为圆形或类圆形小囊泡,表达标志物蛋白CD63、TSG101,而不表达内质网蛋白calnexin。标记DiR荧光素的外泌体可被HK-2细胞摄取。CIN-Exo中HMGB1表达量高于NC-Exo和CIN-GL-Exo(P<0.05)。CIN-Exo组的细胞凋亡率以及Bax、Cleaved caspase-3表达量高于Con组、NC-Exo组、CIN-GL-Exo组,而Bcl-2表达量低于其他3组(P<0.05)。CIN-Exo组细胞中IL-1β、IL-6、TNF-αmRNA水平,以及HMGB1、TLR4、NF-κB表达量高于其他3组(P<0.05)。结论对比剂可通过巨噬细胞来源外泌体诱导肾小管上皮细胞凋亡和炎症反应,可能与外泌体HMGB1水平上调有关,甘草酸苷可抑制该途径而发挥保护效应。

Objective To explore the effect of contrast agent-induced macrophage-derived exosomes on renal tubular epithelial cells and the intervention effect of glycyrrhizin.Methods THP-1 cells were stimulated with PBS,iohexol and iohexol+glycyrrhizin,respectively,and three kinds of exosomes(NC-Exo,CIN-Exo,CIN-GL-Exo)were obtained.Transmission electron microscopy was used to observe the morphology of exosomes.Western blot assay was used to detect the expression levels of CD63,TSG101 and calnexin.Fluorescent labeling method was used to detect the uptake of exosomes.HK-2 cells were randomly divided into 4 groups:Con group,NC-Exo group,CIN-Exo group and CIN-GL-Exo group.Western blot assay was used to detect the expression levels of HMGB1 in three exosomes,the expression levels of apoptosis-related proteins and HMGB1/TLR4/NF-κB signaling pathway related proteins in 4 groups of cells.AnnexinⅤ-FITC/PI double staining method was used to detect the apoptosis rate of cells.Quantitative real-time PCR was used to detect the mRNA expression of inflammatory factors[interleukin(IL)-1β,IL-6,tumor necrosis factor(TNF)-α]in 4 groups of cells.Results The exosomes derived from THP-1 cells were round or round-like small vesicles,which expressed the marker protein CD63 and TSG101,but not the endoplasmic reticulum protein calnexin.DiR-labeled exosomes could be taken up by HK-2 cells.The expression level of HMGB1 was higher in CIN-Exo than that of NC-Exo and CIN-GL-Exo(P<0.05).The apoptosis rate and the expression levels of Bax and Cleaved caspase-3 were higher in CIN-Exo group than those in Con group,NC-Exo group and CIN-GL-Exo group,while the expression level of Bcl-2 was lower than that in the other three groups(P<0.05).The mRNA levels of IL-1β,IL-6 and TNF-α,and the expression levels of HMGB1,TLR4 and NF-κB were higher in CIN-Exo group than those in the other three groups(P<0.05).Conclusion Contrast agents can induce apoptosis and inflammation of renal tubular epithelial cells through macrophage-derived exosomes,which may be related to the upregulation of exosome HMGB1 levels.Glycyrrhizin can inhibit this pathway and exert a protective effect.