叔丁基对苯二酚对糖尿病视网膜病变大鼠视网膜新生血管生成及HIF-1α/VEGF通路蛋白的影响

Effects of tert-butyl hydroquinone on retinal neovascularization and HIF-1α/VEGF pathway proteins in diabetic retinopathy rats

目的探讨叔丁基对苯二酚(TBHQ)对糖尿病视网膜病变大鼠视网膜新生血管生成及缺氧诱导因子-1α(HIF-1α)/血管内皮生长因子(VEGF)通路蛋白的影响。方法通过单次腹腔注射链脲佐菌素建立30只DR模型大鼠,然后将其按照随机数字表法分为DR模型组(腹腔注射30 mg/kg浓度为3%的无水乙醇)、TBHQ低剂量组(腹腔注射30 mg/kg溶解于3%无水乙醇的TBHQ)和TBHQ高剂量组(腹腔注射40 mg/kg溶解于3%无水乙醇的TBHQ),各10只,另取10只大鼠作为正常对照组(腹腔注射30 mg/kg浓度为3%的无水乙醇),连续治疗5周。苏木精-伊红染色法观察4组大鼠眼球血管,并对其内皮细胞核数目进行计数;蛋白质印迹法检测4组DR大鼠视网膜组织HIF-1α/VEGF通路相关蛋白[HIF-1α、VEGF、细胞间黏附分子-1(ICAM-1)、核因子E2相关因子2(Nrf2)]及凋亡蛋白(Bcl-2、Bax)的表达;生化试剂盒检测4组大鼠血清氧化应激指标[脂质氧化(MDA)、谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)]水平。结果与正常对照组相比,DR模型组大鼠眼球血管细胞数量、视网膜组织中HIF-1α、VEGF、ICAM-1、Nrf2、Bax蛋白相对表达量以及血清MDA含量均显著增加,而视网膜Bcl-2蛋白相对表达量和血清GSH-Px含量和SOD活性均显著降低,差异均有统计学意义(P<0.05);与DR模型组相比,TBHQ低剂量组和TBHQ高剂量组视网膜Bcl-2蛋白相对表达量和血清GSH-Px含量和SOD活性均显著增加,而眼球血管细胞数量、视网膜组织中HIF-1α、VEGF、ICAM-1、Nrf2、Bax蛋白相对表达量以及血清MDA含量均显著降低,差异均有统计学意义(P<0.05);与TBHQ低剂量组相比,TBHQ高剂量组Bcl-2蛋白相对表达量、GSH-Px含量和SOD活性均显著增加,而血管细胞数量和Bax蛋白相对表达量均显著降低(P>0.05),其余指标两组比较差异无统计学意义(P>0.05)。结论TBHQ可抑制糖尿病视网膜病变大鼠视网膜新生血管生成和视网膜细胞凋亡,并提升机体抗氧化能力,其作用机制可能与调控HIF-1α/VEGF通路蛋白表达相关,且可能存在一定剂量依赖。

Objective To investigate the effects of tert-butyl hydroquinone(TBHQ)on retinal neovascularization and hypoxia-inducible factor-1α(HIF-1α)/vascular endothelial growth factor(VEGF)pathway proteins in diabetic retinopathy rats.Methods Thirty DR model rats were established by a single intraperitoneal injection of streptozotocin,then they were divided into DR model group(intraperitoneal injection of 30 mg/kg of absolute ethanol at a concentration of 3%)and TBHQ low-dose group(intraperitoneal injection of 30 mg/kg of TBHQ dissolved in 3%absolute ethanol)and TBHQ high-dose group(intraperitoneal injection of 40 mg/kg TBHQ dissolved in 3%absolute ethanol)according to the random number table,10 each.Another 10 rats were taken as normal control group(abdominal injection of 30 mg/kg of absolute ethanol at a concentration of 3%).After 5 weeks of continuous treatment,Hematoxylin-Eosin staining was used to observe the blood vessels of the eyes of each group of rats,and count the number of endothelial cell nuclei;Western blotting was used to detect the expression of HIF-1α/VEGF pathway related proteins[HIF-1α,VEGF,intercellular cell adhesion molecule-1(ICAM-1),nuclearfactor-E2-relatedfactor2(Nrf2)]and apoptosis proteins(Bcl-2,Bax)in the retina of DR rats in each group;The biochemical kit was used to detect the serum oxidative stress indicators[malondialdehyde(MDA),glutathione peroxidase(GSH-Px),superoxide dismutase(SOD)]levels of rats in each group.Results Compared with the normal control group,the number of blood vessel cells in the eyeball,the relative expression of HIF-1α,VEGF,ICAM-1,Nrf2,Bax protein in the retinal tissue and the serum MDA content in the DR model group were increased significantly,whlie the relative expression of retinal Bcl-2 protein,serum GSH-Px content and SOD activity were all significantly reduced,the differences were statistically significant(P<0.05);Compared with the DR model group,the relative expression of Bcl-2 protein,serum GSH-Px content and SOD activity in the large retina of the TBHQ low-dose and TBHQ high-dose groups were significantly increased,whlie the number of blood vessel cells in the eyeball,the relative expression of HIF-1α,VEGF,ICAM-1,Nrf2,Bax protein in the retina tissue,and the serum MDA content were all significantly reduced,the differences were statistically significant(P<0.05);Compared with the low-dose TBHQ group,the relative expression of Bcl-2 protein,GSH-Px content and SOD activity in the high-dose TBHQ group were increased significantly,whlie the number of vascular cells and the relative expression of Bax protein were significantly reduced,the differences were statistically significant(P>0.05).There was no significant difference in the other indicators between the two groups(P>0.05).Conclusion TBHQ can inhibit retinal neovascularization and retinal cell apoptosis in diabetic retinopathy rats,and enhance the body's antioxidant capacity.Its mechanism of action may be related to the regulation of HIF-1α/VEGF pathway protein expression,and may be dose-dependent.