Padgaonkar V A, Lin L R, Leverenz V R, Rinke A, Reddy V N, Giblin F J
Eye Research Institute of Oakland University, Rochester, MI 48309-4480, USA.
Exp Eye Res. 1999 Apr;68(4):493-504. doi: 10.1006/exer.1998.0630.
Previous studies have shown that treatment of guinea pigs with hyperbaric oxygen (HBO) produces certain changes in the lens nuclei of the animals which are typical of those occurring during aging. These include an increase in nuclear light scattering (NLS), elevation in levels of oxidized thiols, loss of water-soluble protein and damage to nuclear membranes. The present study investigated the effect of HBO-treatment in vivo on lens cytoskeletal proteins and MIP26 which are also known to undergo alteration with age. Young (2-month-old) and old (18-month-old) guinea pigs were treated 15 and 30 times with HBO (3 times per week with 2.5 atmospheres of 100% oxygen for 2.5 hr periods). SDS-PAGE and Western blotting showed that HBO-treatment of the older animals accelerated the age-related loss of five nuclear cytoskeletal proteins including actin, vimentin, ankyrin, alpha-actinin and tubulin, compared to levels present in age-matched controls (effects on spectrin and the beaded filaments were not investigated in this study). Treatment of the young animals with HBO produced losses which were primarily associated with concentrations of the nuclear alpha- and beta-tubulins; these cytoskeletal proteins were observed to be most sensitive to the induced oxidative stress, and were affected earliest in the study. Disulfide-crosslinking, rather than proteolysis, appeared to be the main cause of the HBO-induced cytoskeletal protein loss (elevated levels of calcium, which might have induced proteolysis, were not found in the experimental nuclei). Loss of MIP26 was observed only in the older guinea pigs treated 30 times with HBO; both disulfide-crosslinking and degradation to MIP22 were associated with the disappearance. Thus, nuclear MIP26 was susceptible to oxidative stress, but less so than the cytoskeletal proteins, particularly the tubulins. No cortical effects on either MIP26 or the cytoskeletal proteins were observed under any of the treatment protocols. No direct link was observed between an HBO-induced increase in NLS (observed in both the young and old animals using slit-lamp biomicroscopy) and losses of either MIP26 or the cytoskeletal proteins. The appearance of HBO-induced nuclear opacity without any change in the levels of nuclear sodium, potassium or calcium is similar to that observed previously for human senile pure nuclear cataracts. The results provide additional evidence that molecular oxygen can enter the nucleus of the lens and promote age-related events. The observed effects on MIP26 and the cytoskeletal proteins are indicative of an increased level of lens nuclear oxidative stress in the HBO model, possibly a precursor to nuclear cataract.
先前的研究表明，用高压氧（HBO）处理豚鼠会使其晶状体核产生某些变化，这些变化是衰老过程中典型的变化。这些变化包括核光散射（NLS）增加、氧化硫醇水平升高、水溶性蛋白质损失以及核膜损伤。本研究调查了体内HBO处理对晶状体细胞骨架蛋白和MIP26的影响，已知这些蛋白也会随着年龄的增长而发生改变。对年轻（2个月大）和年老（18个月大）的豚鼠进行15次和30次HBO处理（每周3次，在2.5个大气压的100%氧气环境中处理2.5小时）。SDS-PAGE和蛋白质免疫印迹分析表明，与年龄匹配的对照组相比，对年老动物进行HBO处理加速了包括肌动蛋白、波形蛋白、锚蛋白、α-辅肌动蛋白和微管蛋白在内的五种核细胞骨架蛋白与年龄相关的损失（本研究未调查对血影蛋白和串珠状细丝的影响）。对年轻动物进行HBO处理产生的损失主要与核α-和β-微管蛋白的浓度有关；这些细胞骨架蛋白被观察到对诱导的氧化应激最敏感，并且在研究中最早受到影响。二硫键交联而非蛋白水解似乎是HBO诱导的细胞骨架蛋白损失的主要原因（在实验核中未发现可能诱导蛋白水解的钙水平升高）。仅在接受30次HBO处理的老年豚鼠中观察到MIP26的损失；二硫键交联和降解为MIP22都与MIP26的消失有关。因此，核MIP26易受氧化应激影响，但比细胞骨架蛋白，尤其是微管蛋白的敏感性低。在任何处理方案下，均未观察到对MIP26或细胞骨架蛋白的皮质效应。在HBO诱导的NLS增加（使用裂隙灯生物显微镜在年轻和年老动物中均观察到）与MIP26或细胞骨架蛋白的损失之间未观察到直接联系。HBO诱导的核混浊出现而核钠、钾或钙水平无任何变化，这与先前在人类老年性纯核性白内障中观察到的情况相似。这些结果提供了额外的证据，表明分子氧可以进入晶状体核并促进与年龄相关的事件。观察到的对MIP26和细胞骨架蛋白的影响表明HBO模型中晶状体核氧化应激水平增加，这可能是核性白内障的先兆。
