依达拉奉对视网膜脱离模型大鼠早期感光细胞自噬的调控作用

Regulatory effect of edaravone on the photoreceptor autophagy at the early stage of experimental retinal detachment in rats

目的研究氧自由基清除剂依达拉奉对视网膜脱离(RD)模型大鼠早期视网膜自噬的调控及感光细胞的保护作用。方法取51只成年雄性Sprague-Dawley大鼠进行RD造模,另取24只大鼠作为PBS注射组。造模组采用右眼视网膜下注射0.5%透明质酸钠的方法制作RD动物模型。将造模成功大鼠按照随机数字表法随机分为RD模型组和依达拉奉治疗组。依达拉奉治疗组造模后连续给予3 mg/kg依达拉奉腹腔内注射,每天2次,PBS注射组和RD模型组腹腔注射等容量0.9%氯化钠溶液。分别于造模后1、3、7 d处死动物并取材。收集眼内液并检测总超氧化物歧化酶(T-SOD)活性及丙二醛(MDA)含量。Western blot法检测视网膜组织中超氧化物歧化酶2(SOD2)、核因子E2相关因子2(Nrf2)、自噬相关基因4(Atg4)、微管相关蛋白质1轻链3(LC3)蛋白表达。全眼球石蜡切片TUNEL染色分析感光细胞凋亡情况。结果所有RD模型眼视网膜脱离范围均>60%。不同时间点各组眼内液中MDA含量和T-SOD活性总体比较,差异均有统计学意义(MDA:F分组=385.513,P<0.01;F时间=13.021,P<0.01.T-SOD:F分组=48.865,P<0.01;F时间=7.700,P=0.003);与PBS注射组相比,造模后1、3、7 d RD模型组和依达拉奉治疗组眼内液MDA含量显著升高,T-SOD活性显著降低,差异均有统计学意义(均P<0.05);与RD模型组比较,造模后1、3、7 d依达拉奉治疗组MDA含量显著降低,T-SOD活性显著升高,差异均有统计学意义(均P<0.05)。与PBS注射组相比,RD模型组和依达拉奉治疗组造模后1、3、7 d视网膜中SOD2和Nrf2蛋白相对表达量显著升高,造模后1、3 d视网膜中Atg4、LC3B-Ⅱ/LC3B-Ⅰ相对表达量显著升高,差异均有统计学意义(均P<0.05);与RD模型组比较,依达拉奉治疗组造模后1、3、7 d视网膜中SOD2相对表达量显著升高,造模后1、3 d视网膜中Nrf2蛋白相对表达量显著升高,造模后3 d视网膜中Atg4、LC3B-Ⅱ/LC3B-Ⅰ蛋白相对表达量显著升高,差异均有统计学意义(均P<0.05)。PBS注射组各时间点未见明显阳性染色细胞,RD模型组造模后1、3和7 d均可见感光细胞TUNEL阳性染色细胞,视网膜caspase-3水平较PBS注射组显著升高,依达拉奉治疗组造模后1、3、7 d感光细胞凋亡指数及视网膜caspase-3水平均较RD模型组显著降低,差异均有统计学意义(均P<0.05)。结论采用依达拉奉每天2次腹腔内注射能明显提高RD大鼠视网膜的抗氧化能力,调控视网膜自噬,减轻RD早期感光细胞凋亡的发生。

Objective To investigate the effect of edaravone,a free radical scavenger,on the regulation of retinal autophagy and the protection of photoreceptor cells at the early stage of experimental retinal detachment(RD)in rats.Methods Fifty-one adult male Sprague-Dawley rats were used for RD model establishment,and another 24 rats were served as PBS injection group.The RD model was established via subretinal injection of 0.5%sodium hyaluronate into the right eye of the rats and the rats with successful modeling were randomly divided into RD model group and edaravone treatment group.The rats in the edaravone treatment group were given edaravone of 3 mg/kg intraperitoneally,twice a day after modeling,and the rats in the PBS injection group and RD model group were given equal volume of normal saline.Rats were sacrificed on the 1st day,3rd day and 7th day following modeling.The T-superoxide dismutase(T-SOD)activity and malondialdehyde(MDA)content in the intraocular fluid was detected.The expression levels of superoxide dismutase 2(SOD2),nuclear factor E2-related factor 2(Nrf2),autophagy related gene 4(Atg4),microtubule-associated protein 1 light chain 3B(LC3B)and other proteins in retinal tissue were identified by Western blot analysis.TUNEL staining was performed on paraffin sections of the whole eyeball to analyze the apoptosis of photoreceptor cells.The study protocol was approved by an Ethics Committee of Xi'an Fourth Hospital(No.2016016).The use and care of animals complied with the Regulations on the Administration of Experimental Animals.Results The RD area was more than 60%in rat eyes of RD model.There were significant differences in MDA content and T-SOD activity among different groups at various time points(MDA:F group=385.513,P<0.01;F time=13.021,P<0.01.T-SOD:F group=48.865,P<0.01;F time=7.700,P=0.003).Compared with the PBS injection group,the MDA concentration was significantly increased and the T-SOD activity was significantly decreased in the RD group and edaravone treatment group on the 1st,3rd and 7th day after modeling(all at P<0.05).The MDA concentration was significantly reduced and the T-SOD activity was significantly elevated in the edaravone treatment group on the 1st,3rd and 7th day after modeling in comparison with those of the RD group(all at P<0.05).Compared with the PBS injection group,the relative expression levels of SOD2 and Nrf2 proteins were significantly increased in the RD group and edaravone treatment group on the 1st,3rd and 7th day after modeling(all at P<0.05),and Atg4 and LC3B-Ⅱ/LC3B-Ⅰwere significantly increased on the 1st,3rd and 7th day after modeling(all at P<0.05).The expression level of SOD2 in the edaravone treatment group was significantly higher than that in the RD group on the 1st,3rd and 7th day after modeling(all at P<0.05),and the expression level of Nrf2 was significantly increased in the edaravone treatment group on the 1st and 3rd day after modeling compared with that of the RD group(both at P<0.05),and the expression levels of Atg4 and LC3B-Ⅱ/LC3B-Ⅰwere significantly increased in the edaravone treatment group on the 3rd day after modeling in comparison with those of the RD group(both at P<0.05).No significant TUNEL positive cells were observed in PBS injection group at all time points,and TUNEL positive cells were observed on the 1st,3rd and 7th day after modeling in the RD group,and the expression level of caspase-3 in the RD group was significantly increased in comparison with that of the PBS injection group(P<0.05).The apoptosis of photoreceptor cells and the expression level of caspase-3 in edaravone treatment group were significantly decreased in comparison with those of the RD group on the 1st,3rd and 7th day after modeling(all at P<0.05).Conclusions The intraperitoneal injection of edaravone,twice a day,can significantly improve the antioxidant capacity of the retina after experimental RD in rats,regulate retinal autophagy and reduce the apoptosis of photoreceptor cells in early-stage RD.