ChREBP在糖尿病大鼠视网膜中的表达及作用机制研究

Expression and Molecular Mechanism of ChREBP in the Retina of Diabetic Rats

目的观察转录因子碳水化合物反应元件结合蛋白(carbohydrate response element binding protein,ChREBP)在糖尿病(diabetes mellitus,DM)大鼠视网膜组织中的表达变化,探索其在糖尿病性视网膜病变(diabetic retinopathy,DR)中的作用分子机制。方法采用随机数字表法将40只雄性SD大鼠分为DM组和对照组,每组20只。DM组给予一次性腹腔注射1%链脲佐菌素(streptozocin,STZ)(60 mg/kg)建立DM模型;对照组注射等量的柠檬酸盐缓冲液。造模后每月监测大鼠血糖变化,3月后麻醉处死。获取大鼠眼球行冰冻切片,分别利用苏木精-伊红(hematoxylin-eosin,HE)染色和免疫荧光染色观察两组大鼠视网膜结构的病理变化并检测ChREBP在视网膜中的分布变化。利用实时荧光定量聚合酶链式反应(quantitative real-time-polymerase chain reaction,qPCR)及Western blot检测大鼠视网膜组织中ChREBP和其下游靶基因硫氧还蛋白相互作用蛋白(thioredoxin-interacting protein,TXNIP)的表达变化。结果与对照组相比,DM组血糖浓度显著升高(P<0.05),同时生长缓慢,体质量偏低(P<0.05)。HE染色显示在3月时DM组已经出现DR早期的病理变化。免疫荧光染色显示ChREBP在DM组大鼠的视网膜组织中表达量较对照组增加,部分蛋白分布由胞质转移至胞核。qPCR检测显示DM组大鼠视网膜组织中ChREBP和TXNIP的mRNA表达水平较对照组显著升高(P均<0.05)。Western blot检测显示ChREBP蛋白表达水平较对照组表达显著增加(P<0.05)。结论在DM大鼠视网膜组织中,ChREBP的表达增加并出现核转移。ChREBP可能通过调节下游分子TXNIP的表达参与DR的发生发展。

Objective To observe the expression of carbohydrate response element binding protein(ChREBP)in retina of diabetes mellitus(DM)rats,and investigate its possible molecular mechanism in diabetic retinopathy(DR).Methods A total of 40 male SD rats were randomly divided into DM group and control group,with 20 in each.The DM model was established by intraperitoneal injection of 1%streptozocin(STZ)(60 mg/kg)in the DM group;the control group was injected with the same volume of citrate buffer.Blood glucose was monitored monthly after modeling,and the rats were excuted after 3 months.The eyeball of rats was obtained for frozen section,and the pathological changes of retinal structure and the distribution of ChREBP in the retina of the two groups were observed by hematoxylin-eosin(HE)staining and immunofluorescence staining.The expression of ChREBP and its downstream target gene thioredoxin-interacting protein(TXNIP)in the retinal tissue of the rats in the two groups was detected by quantitative real-time-polymerase chain reaction(qPCR)and Western blot.Results Compared with the control group,the concentration of blood glucose in the DM group significantly increased(P<0.05),while the growth was slow and the body weight was lighter(P<0.05).The results of HE staining showed the early pathological changes of DR appeared in DM group in the 3rd month.Immunofluorescence staining showed that the expression of ChREBP in the DM group was higher than that in the control group,and part of protein was transferred from the cytoplasm to the nucleus.qPCR showed that the mRNA expression levels of ChREBP and TXNIP in the retina of the DM group were significantly higher than those in the control group(all P<0.05).Western blot showed that the expression level of ChREBP protein significantly increased compared with the control group(P<0.05).Conclusion The expression of ChREBP significantly increases and nuclear metastasis is observed in the retinal tissue of DM rats,which indicates that ChREBP may be involved in the occurrence and development of DR by regulating the expression of its downstream molecule TXNIP.