橙皮素调节Keap1/Nrf2/ARE信号通路对D-半乳糖诱导的白内障大鼠氧化应激损伤的影响

Impact of Hesperetin on D-Galactose-Induced Oxidative Stress Injury in Ratswith Cataract by Regulating Keap1/Nrf2/ARE Signaling Pathway

目的:探究橙皮素对D-半乳糖诱导的白内障大鼠氧化应激损伤的影响以及调节Keap1/Nrf2/ARE信号通路的机制。方法:将大鼠随机分为假手术组、模型组、阳性对照组、橙皮素低、高剂量组、橙皮素高剂量+Nrf2抑制剂ATRA组,除假手术组,其余组均通过皮下注射200mg/kg的D-半乳糖建立白内障大鼠模型。阳性对照组灌胃复明胶囊(30mg/kg),橙皮素低、高剂量组分别灌胃橙皮素(50mg/kg、150mg/kg),橙皮素高剂量+ATRA组先灌胃橙皮素(100mg/kg),再腹腔注射ATRA(10mg/kg)。裂隙灯观察双眼晶状体组织浑浊程度;HE观察各组晶状体上皮组织形态学变化;TUNEL法检测晶状体上皮组织细胞凋亡率;ELISA测定晶状体中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)的活性;Western blotting检测晶状体中Keap1、Nrf2、HO-1蛋白的表达水平。结果:与假手术组相比,模型组大鼠模型组晶状体上皮组织出现细胞排列紊乱、水肿、间隙增大等病变;晶状体浑浊程度、上皮细胞凋亡率、Keap1、细胞质Nrf2蛋白表达升高,SOD、CAT、GSH-Px活性、细胞核Nrf2、HO-1蛋白表达均降低(P<0.05)。与模型组相比,阳性对照组和橙皮素低、高剂量组大鼠晶状体上皮损伤减轻;晶状体浑浊程度、上皮细胞凋亡率、Keap1、细胞质Nrf2蛋白表达降低,SOD、CAT、GSH-Px活性、细胞核Nrf2、HO-1蛋白表达均升高(P<0.05)。Nrf2抑制剂ATRA可显著减弱高剂量橙皮素对白内障大鼠晶状体损伤的改善作用。结论:橙皮素可减轻D-半乳糖诱导的白内障大鼠氧化应激损伤,可能通过抑制Keap1表达,促进Nrf2/ARE通路活化实现。

Objective:To explore the impact of hesperetin on D-galactose-induced oxidative stress injury in rats with cataract and its mechanism of regulating Keap1/Nrf2/ARE signaling pathway.Methods:Seventy-two SPF Wistar male rats were randomly separated into sham operation group,model group,positive group,low and high dose hesperetin groups,and high dose hesperetin+Nrf2 inhibitor ATRA group.Cataract rat models were established by subcutaneous injection of 200mg/kg of D-galactose in all groups except the sham-operation group.The positive control group was treated with fuming capsule(30mg/kg),the low and high dose groups were treated with hesperetin(50mg/kg,150mg/kg)respectively,and the high dose+ATRA group was treated with hesperetin(100mg/kg)first,and then ATRA(10mg/kg)was injected intraperitoneally.Slit lamp was used to observe the degree of opacity of the lens tissue in both eyes;HE was applied to observe the morphological changes of lens epithelium in each group;TUNEL method was applied to detect the apoptosis rate of lens epithelial tissue;ELISA was applied to measure superoxide dismutase(SOD),catalase(CAT),and glutathione peroxidase(GSH-Px)activities in the lens;Western blotting was applied to detect the expression levels of Keap1,Nrf2,and HO-1 proteins in the lens.Results:Compared with the sham-operation group,the lens epithelial tissue of the model group had disordered cell arrangement,edema,and enlarged gaps,the degree of lens opacity,epithelial cell apoptosis rate,Keap1 and cytoplasmic Nrf2 protein expression increased(P<0.05),SOD,CAT,GSH-Px activity,nuclear Nrf2,HO-1 protein expressions decreased(P<0.05).Compared with the model group,the damage of the lens epithelium of the rats in the positive group and the low and high-dose hesperetin groups was alleviated,the degree of lens opacity,epithelial cell apoptosis rate,Keap1 and cytoplasmic Nrf2 protein expression decreased(P<0.05),SOD,CAT,GSH-Px activity,nuclear Nrf2,HO-1 protein expressions increased(P<0.05).The Nrf2 inhibitor ATRA could greatly attenuate the ameliorating effect of high-dose hesperetin on lens damage in cataract rats.Conclusion:Hesperetin can alleviate D-galactose-induced oxidative stress injury in cataract rats,which may be achieved by inhibiting the expression of Keap1 and promoting the activation of Nrf2/ARE pathway.