E-64d对大鼠视网膜缺血再灌注损伤中Calpain和Caspase-3表达的调控作用

Calpain and Caspase-3 expression in retina of ischemia/reperfusion injury and E-64d treatment in rat

目的探讨E-64d(半胱氨酸蛋白酶抑制剂及钙激活中性蛋白酶抑制剂)在大鼠视网膜缺血再灌注损伤(RIRI)中对Calpain/Caspase-3表达的调节作用。方法将80只SD大鼠随机分为正常组、视网膜缺血再灌注组、对照组和E-64d治疗组,并分为1、3、6、24、72h5个时间段。通过前房穿刺加压法制成RIRI动物模型,采用Western-blot以及RT-PCR方法测定在大鼠RIRI模型中的Calpian、Caspase-3蛋白和mRNA表达情况。结果缺血再灌注24hm-Calpain、Caspase-3蛋白的表达缺血再灌注组较正常组上升,而E-64d组表达与正常组相似,比缺血再灌注组下降。E-64d能下调视网膜缺血再灌注后m-Calpain/Calpastatin的mRNA比值,在24h与缺血组有显著统计学差异。结论E-64d能降低视网膜缺血再灌注时m-Calpain、Caspase-3蛋白的表达,调控m-Calpain和Calpastatin的mRNA比值。

Objective Retinal ischemia/reperfusion damage is a common pathologic procedure in many ocular diseases. Research showed that retinal hypoxia statue and retinal ischemia/reperfusion damage can activate Calpain and Caspase, or the critical enzyme induced cell apoptosis. Present paper was to investigate the dynamic expression of Calpain and Caspase-3 in rat retina with ischemic/reperfusion injury（RIRI） and after E-64 d treatment. Methods Retinal ischemia and reperfusion model was induced in the right eyes of 40 Sprague-Dawley （SD） rats with increasing the intraocular pressure way by elevating the perfusion container to 150 cm for 1 hour and following the reperfusion by removing the container. E-64d was injected intravitreously（5 μl of 100μmol/L） immediately after RIRI in 20 eyes of models. The normal salt solution was injected into vitreous cavity in 20 eyes from normal rats as positive control group and 20 normal rat eyes were as normal control group. Experimental eyes of SD rats were enucleated at 1,3,6,24 and 72 hours for the detection of Calpain and Caspase-3 using Western-blot and RT-PCR. Results Expression of Calpain and Caspase-3 protein was significantly increased at the 24 th hour after RIRI in ischemia/reperfusion model group compared with normal control group （ P 〈 0. 05 ） , and E-64d treatment group showed a decrease of Calpain and Caspase-3 protein expression in comparison with ischemia/reperfusion model group （P 〈 0. 05 ）. The ratio of mcalpain/Calpastatin mRNA reached peak at the 24 th hour after the ischemia/reperfusion injury,and E-64d treatment decreased the ratio of the mcalpain/Calpastatin mRNA. The statistical difference were found in mcalpain/Calpastatin mRNA ratio in 24 hours between ischemia group and control group or ischemia group and E-64d group （ P 〈 0. 05 ）. Conclusion E-64d can downregulate the protein expression of Calpain and Caspase-3 and attenuate the ratio of mRNA of m- Calpain/Calpastatin.