Suemori Shinsuke, Shimazawa Masamitsu, Kawase Kazuhide, Satoh Masahiko, Nagase Hisamitsu, Yamamoto Tetsuya, Hara Hideaki
Department of Biofunctional Molecules, Gifu Pharmaceutical University, Gifu, Japan.
Invest Ophthalmol Vis Sci. 2006 Sep;47(9):3975-82. doi: 10.1167/iovs.06-0275.
To clarify the functional role of metallothionein (MT) in retinal damage in mice deficient in both MT-I and -II (MT-I/-II-deficient mice [C57BL/6J background]) and wild-type (C57BL/6J) mice and MT induction (zinc sulfate [ZnSO4] and 1alpha, 25-dihydroxyvitamin D3 [Vit. D3]).
Retinal, cell damage was induced by intravitreous injection of N-methyl-D-aspartate (NMDA; 40 nmol/eye). Retinal MT-I, -II, and -III mRNA expression was monitored by real-time reverse-transcription-PCR of total retinal RNA from eyes injected or not injected with NMDA. In wild-type mice, MT-I and -II immunohistochemistry was performed (with antibody that recognizes both proteins) 12 and 24 hours after intravitreous NMDA injection. To examine the involvement of induced retinal MT, ZnSO4 (10 nmol/eye) or Vit. D3 (0.2 or 2 ng/eye) was intravitreously injected 24 hours before NMDA injection in wild-type or MT-I/-II-deficient mice, and ganglion cell layer (GCL) cell loss and inner plexiform layer (IPL) thinning were evaluated 7 days after the NMDA injection. The protective effect of Vit. D3 was assessed against the RGC-5 cell death induced by oxidative stress (using buthionine sulfoximine [BSO] to deplete glutathione in combination with glutamate to inhibit cystine uptake).
In wild-type mice, MT-II mRNA expression was time-dependently elevated by NMDA (5.9 and 7.4 times versus the nontreated control at 4 and 12 hours, respectively, after injection), with the normal level being regained within 24 hours. In contrast, MT-I and -III showed persistent decreases (to <50% control) from 4 to 24 hours. In wild-type mice, MT-like immunoreactivity was increased in the inner retina (GCL and IPL) 12 and 24 hours after NMDA injection. At 7 days after NMDA injection in MT-I/-II-deficient mice (versus wild-type mice), GCL cell loss was increased, but IPL thickness was not different. Pretreatment with ZnSO4 or Vit. D3 increased inner retinal MT-like immunoreactivity 24 hours after NMDA injection and significantly attenuated NMDA-induced GCL cell loss in wild-type mice, but ZnSO4 pretreatment did not protect against such cell loss in MT-I/-II-deficient mice. In vitro, Vit. D3 pretreatment (100 nM) reduced BSO+glutamate-induced RGC-5 cell death.
These findings suggest that MT, especially MT-II, protects against retinal neuron damage, by acting as an endogenous antioxidant.
阐明金属硫蛋白（MT）在缺乏MT-I和MT-II的小鼠（MT-I/-II缺陷小鼠[C57BL/6J背景]）、野生型（C57BL/6J）小鼠以及MT诱导（硫酸锌[ZnSO4]和1α,25-二羟基维生素D3[维生素D3]）过程中对视网膜损伤的功能作用。
通过玻璃体内注射N-甲基-D-天冬氨酸（NMDA；40 nmol/眼）诱导视网膜细胞损伤。通过对注射或未注射NMDA的眼睛的总视网膜RNA进行实时逆转录聚合酶链反应，监测视网膜MT-I、-II和-III mRNA表达。在野生型小鼠中，玻璃体内注射NMDA后12小时和24小时进行MT-I和-II免疫组织化学检测（使用识别这两种蛋白的抗体）。为了研究诱导的视网膜MT的作用，在野生型或MT-I/-II缺陷小鼠中，于NMDA注射前24小时玻璃体内注射ZnSO4（10 nmol/眼）或维生素D3（0.2或2 ng/眼），并在NMDA注射后7天评估神经节细胞层（GCL）细胞丢失和内网状层（IPL）变薄情况。评估维生素D3对氧化应激诱导的RGC-5细胞死亡的保护作用（使用丁硫氨酸亚砜胺[BSO]消耗谷胱甘肽并联合谷氨酸抑制胱氨酸摄取）。
在野生型小鼠中，NMDA使MT-II mRNA表达呈时间依赖性升高（注射后4小时和12小时分别是未处理对照的5.9倍和7.4倍），24小时内恢复至正常水平。相比之下，MT-I和-III在4至24小时持续下降（降至对照的<50%）。在野生型小鼠中，NMDA注射后12小时和24小时，视网膜内层（GCL和IPL）中MT样免疫反应性增加。在MT-I/-II缺陷小鼠中，NMDA注射后7天（与野生型小鼠相比），GCL细胞丢失增加，但IPL厚度无差异。ZnSO4或维生素D3预处理可使NMDA注射后24小时视网膜内层MT样免疫反应性增加，并显著减轻野生型小鼠中NMDA诱导的GCL细胞丢失，但ZnSO4预处理对MT-I/-II缺陷小鼠的这种细胞丢失无保护作用。在体外，维生素D3预处理（100 nM）可减少BSO+谷氨酸诱导的RGC-5细胞死亡。
这些发现表明，MT，尤其是MT-II，作为内源性抗氧化剂可保护视网膜神经元免受损伤。
