Department of Pathology II, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan.
In Vivo. 2011 Jul-Aug;25(4):617-23.
A single systemic administration of N-methyl-N-nitrosourea (MNU) causes retinal degeneration involving photoreceptor cell death within 7 days. MNU-induced photoreceptor cell death is due to apoptosis, and is a reliable animal model for human retinitis pigmentosa. The purpose of this study was to elucidate the involvement of calpain-mediated autophagy, as well as apoptosis on the cell death cascade caused by MNU and to evaluate the efficacy of calpain inhibitor SNJ-1945.
Seven-week-old BALB/c mice were left untreated or received an intraperitoneal (i.p.) injection of MNU. The MNU-exposed mice received an i.p. injection of SNJ-1945 or vehicle alone (distilled water containing 0.5% carboxymethyl cellulose) 3 h prior to MNU and once daily thereafter until sacrifice. Eyes were examined histologically, histochemically, and morphometrically to analyze the photoreceptor cell ratio and retinal damage ratio. The retinal expression of caspase-3, microtubule-associated protein light chain 3 (LC3), autophagy-related protein 5 (Atg5), and α-spectrin was determined by Western blot analysis.
During the 72-h period after MNU exposure, the caspase-3 expression increased and the LC3 and Atg5 expression decreased, indicating increased levels of apoptosis and decreased levels of autophagy, as compared with the MNU-unexposed control mouse retina. MNU-induced photoreceptor cell death was caused by increased calpain activation as measured by α-spectrin proteolysis products, while SNJ-1945 ameliorated photoreceptor cell death by blocking calpain activation and restoring basal autophagy.
Calpain activation is involved in MNU-induced photoreceptor cell death, and calpain inhibition effectively restored photoreceptor cell autophagy and photoreceptor cell death in mice.
单次全身给予 N-甲基-N-亚硝脲（MNU）可导致视网膜变性，在 7 天内涉及光感受器细胞死亡。MNU 诱导的光感受器细胞死亡是由于细胞凋亡，并且是人类色素性视网膜炎的可靠动物模型。本研究的目的是阐明钙蛋白酶介导的自噬以及凋亡在 MNU 引起的细胞死亡级联中的作用，并评估钙蛋白酶抑制剂 SNJ-1945 的疗效。
7 周龄 BALB/c 小鼠未处理或接受腹腔内（i.p.）注射 MNU。MNU 暴露的小鼠在接受 MNU 前 3 小时和此后每天一次接受 i.p.注射 SNJ-1945 或单独的载体（含 0.5%羧甲基纤维素的蒸馏水），直到处死。通过组织学、组织化学和形态计量学检查分析光感受器细胞比例和视网膜损伤比例。通过 Western blot 分析确定视网膜中 caspase-3、微管相关蛋白轻链 3（LC3）、自噬相关蛋白 5（Atg5）和α- spectrin 的表达。
在 MNU 暴露后的 72 小时期间，与 MNU 未暴露的对照小鼠视网膜相比，caspase-3 表达增加，LC3 和 Atg5 表达减少，表明凋亡水平增加，自噬水平降低。MNU 诱导的光感受器细胞死亡是由钙蛋白酶激活增加引起的，如α- spectrin 蛋白水解产物所示，而 SNJ-1945 通过阻断钙蛋白酶激活和恢复基础自噬来改善光感受器细胞死亡。
钙蛋白酶激活参与 MNU 诱导的光感受器细胞死亡，钙蛋白酶抑制通过恢复光感受器细胞自噬和光感受器细胞死亡而有效恢复 MNU 诱导的光感受器细胞死亡。
