漆黄素对高糖诱导视网膜Müller细胞氧化应激炎症反应的作用

Effect of Fisetin on Oxidative Stress Inflammation in Retinal Müller Cells Induced by High Glucose

目的探讨漆黄素(fisetin,FIS)对高糖诱导的视网膜Müller细胞的作用及其机制。方法采用100 mmol/L葡萄糖模拟体外高糖环境,实验分为4组:对照组、高糖组、漆黄素+高糖组、漆黄素+高糖+TCBN(Akt/Gsk3β信号通路抑制剂)组。通过CCK8法检测Müller细胞增殖活力;硫代巴比妥酸比色法测定丙二醛(malondialdehyde,MDA)含量,黄嘌呤氧化酶法测定超氧化物歧化酶(superoxide dismutase,SOD)活性;DCFH-DA荧光探针检测细胞内活性氧(reactive oxygen species,ROS)水平;JC-1法检测线粒体膜电位(mitochondrial membrane potential,MMP);免疫荧光检测线粒体膜通透性转换孔(membrane permeability transition pore,mPTP)及IL-1β、IL-6、TNF-α表达;实时荧光定量PCR检测IL-1β、IL-6、TNF-αmRNA表达;Western Blot检测炎症因子及信号通路蛋白表达。结果与高糖组相比,漆黄素组降低MDA含量,提高SOD活力,明显抑制ROS产生,抑制mPTP开放,稳定MMP(P<0.05);与漆黄素组相比,TCBN组明显阻断了漆黄素的这种作用。免疫荧光和qPCR结果发现,与高糖组相比,漆黄素组能明显降低IL-1β、IL-6、TNF-α炎症因子表达水平(P<0.05);与漆黄素组相比,TCBN组各炎症因子表达增加(P<0.05)。同时Western Blot结果发现,与高糖组相比,漆黄素组能明显增加p-Akt、p-Gsk3β蛋白表达(P<0.05);而TCBN组p-Akt、p-Gsk3β蛋白表达减少(P<0.05)。结论漆黄素通过降低细胞内MDA含量、增加SOD活性、抑制ROS产生、mTPT开放来维持MMP稳定,减少炎症因子表达从而减轻氧化应激损伤,这种作用与激活Akt/Gsk3β信号通路有关。

Objective To investigate the effect and mechanism of fisetin on retinal Müller cells induced by high glucose.Methods 100 mmol/L glucose was used to simulate the hyperglycemic environment in vitro.The experiment was divided into four groups:control group,HG group,FIS+HG group,and FIS+HG+TCBN(Akt/Gsk3βsignaling pathway inhibitor)group.The proliferation activity of Müller cells was detected by CCK8;the content of malondialdehyde(MDA)was determined by thiobarbituric acid colorimetry and the activity of superoxide dismutase(SOD)was determined by xanthine oxidase method;intracellular reactive oxygen species(ROS)were detected by DCFH-DA fluorescence probe;mitochondrial membrane potential was detected by JC-1 method;the expression of IL-1β,IL-6,TNF-αand mPTP were detected by immunofluorescence;the expression of IL-1β,IL-6,TNF-αmRNA was detected by real-time fluorescence quantitative PCR;Western Blot was used to detect the expression of inflammatory factors and signaling pathway proteins.Results Compared with HG group,FIS group decreased MDA content,increased SOD activity,inhibited ROS production,inhibited mPTP opening and stabilized MMP(P<0.05);compared with FIS group,TCBN group significantly blocked this effect.Immunofluorescence and qPCR results showed that compared with HG group,the expression level of IL-1β,IL-6,TNF-αwas significantly decreased in the FIS group(P<0.05);compared with HG group,the expression of p-Akt,p-Gsk3βwas significantly increased in FIS group(P<0.05);the expression of p-Akt and p-Gsk3βdecreased in TCBN group(P<0.05).Conclusion Fisetin can maintain the stability of MMP by decreasing the content of MDA in cells,increasing the activity of SOD and inhibiting the ROS generation of mTPT opening,reducing the expression of inflammatory factors and alleviating oxidative stress injury,which is related to the activation of Akt/Gsk3βsignaling pathway.