NF-E2相关因子2对糖尿病模型小鼠视网膜内细胞及血管的保护作用

Protective effects of NF-E2-related factor 2 on retinal cells and blood vessels in diabetic mice

目的探讨NF-E2相关因子2(Nrf2)对糖尿病视网膜细胞及血管的保护作用。方法 8周龄雄性Nrf2^(+/+)(野生型)和Nrf2^(-/-)C57BL/6小鼠60只随机分为模型组和对照组,共4组,每组各15只。模型组小鼠腹腔注射链脲佐菌素(STZ,45mg/kg),连续注射5 d;对照组小鼠注射同体积的枸橼酸缓冲液。模型组和对照组小鼠初次注射后每周测空腹血糖及体重,至第10周取视网膜。采用免疫印迹法检测视网膜内Nrf2激活的下游蛋白血红素加氧酶-1(HO-1)的表达情况;免疫荧光染色检测具有多种剪接形式的RNA结合蛋白(RBPMS)阳性的视网膜节细胞(RGCs)及胆碱乙酰转移酶(Ch AT)阳性的无长突细胞(ACs)并计数;采用过碘酸-希夫和苏木素染色,观察视网膜内无细胞毛细血管并进行定量分析。结果 STZ诱导1周后,野生型和Nrf2^(-/-)模型组小鼠血糖急剧升高,体重开始显著下降,随着周龄的增长体重无明显增加;对照组小鼠血糖值无明显升高,体重呈正常缓慢增长趋势。STZ诱导糖尿病10周后,RBPMS和Ch AT染色发现,Nrf2~(^(-/-))糖尿病小鼠视网膜内RGCs和ACs数量显著降低(P<0.05);过碘酸-希夫和苏木素染色显示,Nrf2^(-/-)糖尿病小鼠视网膜内出现无细胞毛细血管;免疫印迹法显示,野生型小鼠模型组中Nrf2激活的下游蛋白HO-1表达升高。结论 Nrf2对糖尿病视网膜细胞及血管具有保护作用,其可能是通过诱导HO-1上调发挥作用。

Objective To investigate the protective effects of NF-E2 related factor 2 （Nrf2） on the retinal cells and blood vessels in diabetic mice. Methods Sixty 8-week-old male Nrf2＋/＋ （wild type） and Nrf2-/- C57BL/6 mice were randomly divided into the model group and control group. The mice in the model group were intraperitoneally injected with streptozotocin （STZ, 45mg/kg） for 5 days, and those in the control group were injected with the same volume of citric acid buffer. Fasting blood glucose and body weight were measured every week in both groups after the initial injection. The retina was harvested at the tenth-week. The expression of heine oxygenase-1 （ HO-1 ） which was one of the downstream proteins activated by Nrf2 in the retina was detected by Western blotting. Immunofluorescent staining was used to detect the expression of RNA binding protein with multiple splicing （RBPMS）and choline acetyl transferase （CHAT）, which specifically marks the retinal ganglion cells （RGCs） and amacrine cells （ACs）, respectively. Acellular capillaries in the retina were observed by using periodic acid-Schiff and hematoxylin staining. Results At 1 week after STZ- injection, the fasting blood glucose of the model group including wild type and the Nrf2 -/- mice were increased rapidly, accompanied by significantly decreased body weight. The blood glucose levels in the control group were not significantly increased, and the body weight showed a slow growth trend. At 10 weeks after STZ-injection, the number of RGCs and ACs Nrt2-/- in the retina of diabetic mice were significantly decreased （P 〈 0.05 ）. RGCs and ACs Nrf2 -/- which were demonstrated by RBPMS and ChAT immunofluorescent staining, respectively. Periodic acid-Schiff and hematoxylin staining showed that the number of acellular capillaries were significantly increased in Nrf2-/- mouse retina after STZ injection. Western blotting showed HO-1 expression was increased in wild-type mice after STZ injection. Conclusion Nrf2 has a protective effect on diabetic retinal ceils and blood vessels, which may be mediated through the induction of HO-1.