红景天苷通过IL-17A抑制TRAF6/NLRP3信号通路改善Müller细胞的功能

Protective Function of Salidroside on the Müller Cell Through IL-17A by Down-Regulation of the TRAF6/NLRP3 Signal Pathway

目的探讨红景天苷(salidroside,SAL)对高糖培养的视网膜Müller细胞异常激活的保护作用及其机制。方法采用含10%胎牛血清,1%青-链霉素的DME/F12(1∶1)培养基培养Müller细胞。培养条件为37℃,5%CO_(2)。用25、50、75、100、150 mmol·L^(-1)葡萄糖浓度培养Müller细胞,根据CCK-8实验考察细胞活力确定75 mmol·L^(-1)葡萄糖为最佳浓度。实验分为对照组、HG组、HG+SAL组、HG+IL-17A组、HG+SAL+IL-17A组和HG+IL-17A+siRNA-TRAF6组共6组。通过ELISA检测Müller细胞表达IL-17A水平,通过ROS检测试剂盒检查细胞内ROS水平,MDA检测试剂盒检测细胞内MDA含量,SOD检测试剂盒检测SOD活力。细胞免疫荧光实验检测IL-17A和IL-17RA在细胞内的定位表达情况。Western blotting检测IL-17A、IL-17RA、TRAF6、NLRP3、GFAP、VEGF的蛋白表达水平。结果与对照组相比,HG组Müller细胞表达IL-17A水平明显升高,ROS和MDA含量显著升高,SOD酶活力明显降低,IL-17A、IL-17RA、TRAF6、NLRP3、GFAP、VEGF蛋白表达水平明显升高(P<0.05)。给予SAL后,与HG组相比,Müller细胞表达IL-17A水平显著下降,ROS和MDA含量显著降低,SOD酶活力明显升高,IL-17A、IL-17RA、TRAF6、NLRP3、GFAP、VEGF蛋白表达水平明显下降(P<0.05)。当给予IL-17A活性分子刺激后,相应指标又继续升高(P<0.05),Müller细胞表现异常激活状态。通过细胞免疫荧光实验表明IL-17A和IL-17RA定位表达在细胞质和细胞膜上。结论SAL能改善高糖培养的Müller细胞功能,其可能的机制是通过下调IL-17A表达,进而抑制TRAF6/NLRP3信号通路。

OBJECTIVE To investigate the protective effect of salidroside(SAL)against abnormal activation of retinal Müller cells cultured in high glucose and its mechanism.METHODS Müller cells were cultured in DME/F12(1:1)medium containing 10%fetal bovine serum and 1%penicillin-streptomycin at 37℃and 5%CO_(2).CCK-8 kit was used to detect cell viability among a serials of glucose concentration 25,50,75,100,150 mmol·L^(-1) and 75 mmol·L^(-1) glucose was determined to be the optimal concentration.The tests were divided into 6 groups:Control group,HG group,HG+SAL group,HG+IL-17A group,HG+SAL+IL-17A group,and HG+IL-17A+siRNA-TRAF6 group.Müller cells were tested for the expression of IL-17A levels by ELISA,intracellular ROS levels were examined by ROS assay kits,MDA assay kits detected the intracellular MDA level,and SOD activity by SOD assay kit.Cell immunofluorescence assay was performed to detect the localized expression of IL-17A and IL-17RA in Müller cells.Western blotting detected the protein expression levels of IL-17A,IL-17RA,TRAF6,NLRP3,GFAP,VEGF.RESULTS Compared with the control group,Müller cells in the HG group expressed significantly higher levels of IL-17A,significantly higher levels of ROS and MDA,significantly lower SOD enzyme activity,and significantly higher levels of IL-17A,IL-17RA,TRAF6,NLRP3,GFAP and VEGF protein expression(P<0.05).After the administration of SAL,Müller cells expressed significantly lower levels of IL-17A,significantly lower levels of ROS and MDA,significantly higher SOD enzyme activity,and significantly lower levels of IL-17A,IL-17RA,TRAF6,NLRP3,GFAP and VEGF protein expression compared with the HG group·2352·Chin J Mod Appl Pharm,2024 September,Vol.41,No.17(P<0.05).When IL-17A active molecules were given stimulation,the corresponding indexes continued to rise again(P<0.05),and Müller cells showed abnormal activation status.The cellular immunofluorescence assay showed that IL-17A and IL-17RA were localized and expressed in the cytoplasm and cell membrane.CONCLUSION SAL improves the function of Müller cells cultured with high glucose,and the possible mechanism is related to the inhibition of the TRAF6/NLRP3 signaling pathway through the down-regulation of IL-17A expression.