姜黄素对大鼠视网膜缺血/再灌注损伤时内质网应激的影响

Effect of Curcumin on Endoplasmic Reticulum Stress during Retinal Ischemia/Reperfusion Injury in Rats

目的:探讨姜黄素对大鼠视网膜缺血/再灌注损伤(RIRI)时内质网应激(ERS)的影响。方法:选取清洁级SpragueDawley(SD)雄性大鼠96只,采用随机数字表法分为3组(n=32):对照组(C组)、缺血/再灌注组(I/R组)和姜黄素组(CUR组)。I/R组和CUR组采用前房灌注法使眼内压升高而制备大鼠RIRI模型,缺血60 min,再灌注24 h后结束实验。于缺血前60 min时,CUR组腹腔注射姜黄素100 mg/kg,C组和I/R组腹腔注射等容量生理盐水。各组于再灌注24 h时处死8只大鼠,取视网膜组织,光镜下观察病理学改变;采用TUNEL法检测视网膜组织细胞凋亡情况并计算凋亡指数(AI)。3组于再灌注24 h时处死8只大鼠,取视网膜组织,电镜下观察大鼠视网膜组织超微结构改变。3组于再灌注24h时处死8只大鼠,取视网膜组织,逆转录-聚合酶链式反应(RT-PCR)检测大鼠视网膜组织中CCAAT增强子结合蛋白(C/EBP)同源蛋白(CHOP)、活化的转录因子4(ATF4)和X-盒结合蛋白-1(XBP1)mRNA表达。3组于再灌注24 h时处死8只大鼠,取视网膜组织,蛋白免疫印迹法(Western Blot)检测大鼠视网膜组织中、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)及含半胱氨酸的天冬氨酸蛋白水解酶3(caspase-3)的蛋白表达,计算Bcl-2/Bax比值。结果:与C组比较,I/R组大鼠视网膜组织XBP-1、ATF4和CHOP mRNA表达明显上调(P<0.05);与I/R组比较,CUR组大鼠视网膜组织XBP-1、ATF4和CHOP mRNA表达明显下调(P<0.05)。与C组比较,I/R组大鼠视网膜组织CHOP、Bax和caspase-3蛋白表达升高,Bcl-2蛋白表达及Bcl-2/Bax比值均下降,与C组比较,差异均有统计学意义(P<0.05),CUR组大鼠视网膜组织CHOP、Bax和caspase-3蛋白表达下降,Bcl-2蛋白表达及Bcl-2/Bax比值均升高,与I/R组比较,差异均有统计学意义(P<0.05)。与C组比较,I/R组大鼠视网膜组织出现形态结构及超微结构损伤,AI值升高(P<0.05)。与I/R组比较,CUR组大鼠视网膜组织形态结构及超微结构损伤均减轻,AI值降低(P<0.05)。结论:姜黄素可减轻大鼠RIRI,其机制可能与抑制ERS介导的细胞凋亡有关。

Objective： To investigate the effect of curcumin on endoplasmic reticulum stress（ ERS） retinal ischemia/reperfusion injury（ RIRI） in rats. Methods： A total of 96 Sprague-Dawley（ SD） male rats were randomly divided into normal control group（ C group）,ischemia/reperfusion group（ I/R group） and curcumin group（ CUR group）,with 32 rats in each group. The rat model of RIRI was established by using anterior chamber eannulafion to elevate intra-ocular pressure above systolic pressure for 60 minutes,and the test was finished after 24 h for reperfusion. Curcumin（ 100 mg/kg） was injected into the abdominal cavity 60 min before ischemia in CUR group,and the same dose of 0. 9% normal saline was injected into the abdominal cavity at the same time points in C group and I/R group,respectively. In order to take the retinal tissue of rats,I/R model was established successfully and then eight rats were sacrificed 24 h after reperfusion,the changes of pathology of retinal tissue of rat were observed after conventional hematoxylin-eosin（ HE） staining,and the cell apoptosis was detected by TUNEL method and the apoptosis index（ AI） of retinal ganglion was calculated. Eight rats were sacrificed 24 h after reperfusion and retinal tissue was collected,and the ultrastructural changes of retinal tissue of rats were observed by transmission electron microscope. Eight rats were sacrificed 24 h after reperfusion and retinal tissue was collected,and the expressions of CCAAT-enhancer binding protein homologous protein（ CHOP）,activation of transcription factors（ ATF4） and X-4 box binding protein 1（ XBP1） mRNA in retinal tissue were detected by reverse transcription-polymerase chain reaction（ RT-PCR）. Eight rats were sacrificed 24 h after reperfusion and retinal tissue was collected,and the expressions of CHOP,B-cell lymphoma-2（ Bcl-2）,Bcl-2 associated X protein（ Bax） and cysteinyl aspartate specific proteinase 3（ caspase-3） proteins in retinal tissue were measured by Western Bolt,and the ratio of Bcl-2 to Bax was calculated. Results： Compared with C group,the expressions of XBP1,ATF4 and CHOP mRNA of retinal tissue were significantly increased（ P 〈 0. 05） in I/R group. Compared with I/R group,the expressions of XBP1,ATF4 and CHOP mRNA of retinal tissue were significantly decreased（ P 〈 0. 05） in CUR group. Compared with C group,the expressions of CHOP,Bax and caspase-3 proteins were significantly higher（ P 〈 0. 05）,while the expression of Bcl-2 protein and the ratio of Bcl-2 to Bax were both lower（ P 〈 0. 05） in I/R group.Compared with I/R group,the expressions of CHOP,Bax and caspase-3 protein were significantly lower（ P 〈 0. 05）,while the expression of Bcl-2 protein and the ratio of Bcl-2 to Bax were both higher（ P 〈 0. 05） in CUR group. Compared with C group,the structure and the ultrastructure of retinal tissue of rats were more significantly injured,and AI was higher（ P 〈 0. 05） in I/R group. Compared with I/R group,the injuries of the structure and the ultrastructure of retinal tissue of rats were distinctly alleviative,and AI was lower（ P 〈 0. 05） in CUR group. Conclusion： Curcumin can significantly reduce RIRI in rats,and the mechanism may be related to alleviate ERS related cell apoptosis of retina.