Effect of Kurozu on hippocampal gene expression profiles in SAMP8

Kurozu is a traditional Japanese rice vinegar. During fermentation and aging of the Kurozu liquid in an earthenware jar over 1 year, solid residue called Kurozu Moromi is produced. In the present study, we evaluated whether concentrated Kurozu or Kurozu Moromi could ameliorate cognitive dysfunction in the senescence accelerated P8 mouse. Senescence accelerated P8 mice were fed 0.25% (w/w) concentrated Kurozu or 0.5% (w/w) Kurozu Moromi for 4 or 25 weeks. Kurozu suppressed cognitive dysfunction and amyloid accumulation in the brain, while Kurozu Moromi showed a tendency to ameliorate cognitive dysfunction, but the effect was not significant. We hypothesize that concentrated Kurozu has an antioxidant effect, however, the level of lipid peroxidation in the brain did not differ in senescence accelerated P8 mice. DNA microarray analysis indicated that concentrated Kurozu increased HSPA1A mRNA expression, a protein that prevents protein misfolding and aggregation. The increase in HSPA1A expression by Kurozu was confirmed using quantitative real-time PCR and immunoblotting methods. Therefore, the suppression of amyloid accumulation by concentrated Kurozu may be associated with HSPA1A induction. However, concentrated Kurozu could not increase HSPA1A expression in mouse primary neurons, suggesting it may not directly affect neurons. Ten-times concentrated Kurozu (CK) was made from Kurozu liquid (Sakamoto Kurozu, Fukuyama, Kagoshima, Japan) by repeated vacuum distillation. The CK diet included 0.25% (w/w) CK in CE-2 basic rodent diet (Nihon CLEA, Tokyo, Japan). Senescence resistance (R1) and senescence accelerated P8 (P8) mice were purchased from Japan SLC (Shizuoka, Japan). Mice were housed at 25±2°C with 55±10% humidity on a 12-h light/dark cycle (lighting time 08:00-20:00). All mice were housed in independent cages and had free access to food and water. All procedures were compliant with the guidelines of the Kagoshima University Animal Ethics Committee (A10030). Ten-week old R1 mice (n=16) were fed a control CE2 diet and P8 mice were divided into three groups as follows: control CE2 diet group (n=9), KM diet group (n=9) or CK diet group (n=9). Feeding of the experimental diet started from 12 weeks of age until sacrificed. All mice were sacrificed under anesthesia at 17 weeks old (4 months old). The left side of the hippocampus region was excised from brains of 4 mice selected at random in each group, and then subjected to microarray analysis.黑醋（Kurozu）是日本传统米醋。在陶罐中对黑醋液开展1年以上的发酵陈化时，会产生名为黑醋醪（Kurozu Moromi）的固体残渣。本研究旨在评估浓缩黑醋与黑醋醪是否能够改善快速衰老P8小鼠（senescence accelerated P8 mouse）的认知功能障碍。实验中，将快速衰老P8小鼠分为两组，分别以0.25%（w/w）浓缩黑醋或0.5%（w/w）黑醋醪饲喂，饲喂时长设为4周或25周。结果显示，浓缩黑醋可抑制小鼠脑内认知功能障碍与淀粉样蛋白积累，而黑醋醪虽表现出改善认知功能障碍的趋势，但该效果未达到统计学显著性。我们推测浓缩黑醋具有抗氧化作用，但快速衰老P8小鼠脑内的脂质过氧化水平并无显著差异。DNA微阵列（DNA microarray）分析结果显示，浓缩黑醋可上调HSPA1A的mRNA表达水平，该蛋白可防止蛋白质错误折叠与聚集。通过实时荧光定量PCR（quantitative real-time PCR）与免疫印迹（immunoblotting）实验，验证了黑醋对HSPA1A表达的上调作用。因此，浓缩黑醋对脑内淀粉样蛋白积累的抑制作用可能与HSPA1A的诱导表达有关。但浓缩黑醋无法在小鼠原代神经元中上调HSPA1A的表达，提示其可能不会直接作用于神经元。通过反复真空蒸馏法，由日本鹿儿岛县福山坂本黑醋液制备得到10倍浓缩黑醋（CK）。CK饲料是以日本东京Nihon CLEA公司生产的CE-2基础啮齿动物饲料为基底，添加0.25%（w/w）的CK制备而成。衰老抵抗系R1小鼠与快速衰老P8小鼠均购自日本静冈县的Japan SLC公司。小鼠饲养环境温度控制为25±2℃，相对湿度为55±10%，采用12小时光暗循环（光照时段为08:00-20:00）。所有小鼠均单笼饲养，可自由采食与饮水。所有实验操作均符合鹿儿岛大学动物伦理委员会（Kagoshima University Animal Ethics Committee）的相关指南（审批编号：A10030）。10周龄的R1小鼠（n=16）饲喂对照CE2饲料；P8小鼠则分为3组：对照CE2饲料组（n=9）、黑醋醪饲料组（n=9）与CK饲料组（n=9）。实验饲料的饲喂从小鼠12周龄开始，直至安乐死取材。所有小鼠均在17周龄（即4月龄）时经麻醉后安乐死取材。从每组中随机选取4只小鼠的脑组织，切除其海马左侧区，用于后续的微阵列分析。