The pathogenesis and treatment of cerebral ischemia based on the mechanism of cytoskeletal changes

从细胞骨架变化机制探讨脑缺血的发病机制及治疗

• 批准号: 07457357
• 负责人: SAKABE Takefumi
• 金额: $ 3.26万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07457357/
• 关键词: cerebral ischemia; ischemic neuronal death; cytoskeleton; calpain; calpastatin; fodrin; calpain inhibitor-1; hypothermia; 細胞内情報伝達; カルパイン; カルパスタチン; 細胞骨格蛋白; フォドリン

IntroductionIncrease in intracellular calcium (Ca^<2+>) has been speculated to play a pivotal role in the progress of ischemic brain damage. Calpain, calcium-dependent neutral protease, and its endogenous inhibitor calpastatin, may be involved in the pathogenesis, by influencing the breakdown of fodrin, a major constituent of the membrane skeleton in the central nervous system (CNS). We examined the activity of calpain/calpastatin and the proteolysis of alpha-and beta-subunits of fodrin during ischemia in the rat.Methods and ResultsDecapitated heads of rats were incubated at 37ﾟC for 0-40 min. The proteolysis of fodrin in the homogenates were analyzed by Western blotting. The 100 000xg supernatant was applied to a DEAE-cellulose column, and the amount of m-calpain in the 0.4 M NaCl eluate was measured by spectrophotometry as the release of trichloroacetic acid-soluble peptides from azocasein in the presence of 5 mM Ca^<2+>. Calpastatin activity in the heat treated 100 000xg supernatant … More was determined by measuring the inhibition of bovine lung m-calpain.Degradation of alpha-and beta-fodrin were progressively increased with ischemia time. The amount of m-calpain showed no significant changes, and calpastatin activity decreased significantly in 40 minute-period of ischemia.When the heads of rats were incubated at 33ﾟC for 40 min, breakdown product (150-kD fagment) was decreased significantly comparing to that from the samples of normothermia (37ﾟC).In the brain subjected to 30 minute-forebrain ischemia, the amount of calpain was significatnly decreased, and the breakdown of alpha-fodrin was significantly increased during and 60 min after ischemia.Pretreatment with calpain inhibitor-1 (CAI) (iv.) showed the trend inhibition of calpain activation and significant attenuation of the breakdown of alpha-fodrin.Zymography, applicable to smaller samples, showed the regional variation of the m-calpain activation and breakdown of fodrin, which were marked in the hippocampus during (15min) and (60min) after ischemia.ConclusionsThe results suggest that ischemia induces the proteolysis of alpha-and beta-fodrin chiefly through an activation of m-calpain. Hypothermia and CAI provide substantial protective effect by modifying the calpain activity and hence the breakdown of the cytoskeleton of CNS. Less

细胞内钙离子（Ca^<2+>）的升高在缺血性脑损伤的发生发展中起着重要作用。钙蛋白酶，钙依赖性中性蛋白酶，及其内源性抑制剂钙蛋白酶抑制剂，可能参与的发病机制，通过影响fodrin，在中枢神经系统（CNS）的膜骨架的主要成分的分解。我们研究了钙蛋白酶/钙蛋白酶抑制素的活性和胞衬蛋白的α-和β-亚基在大鼠缺血过程中的蛋白水解。方法和结果断头大鼠的头在37 ℃孵育0-40 min。胞衬蛋白的蛋白水解在匀浆中进行了分析，通过Western印迹。将100 000 × g上清液加到DEAE-纤维素柱上，用分光光度法测定0.4 M NaCl缓冲液中m-钙蛋白酶的含量，即在5 mM Ca^2+存在下，从偶氮酪蛋白中释放出的三氯乙酸可溶性肽。热处理的100 000 xg上清液中的钙蛋白酶抑制蛋白活性 ...更多信息 α-和β-胞衬蛋白的降解随着缺血时间的延长而逐渐增加。脑缺血40 min时，m-calpain含量无明显变化，calpastatin活性明显下降，33 ℃孵育40 min时，脑组织中m-calpain的降解产物在前脑缺血30分钟的脑中，缺血60 min时，钙蛋白酶含量明显减少，α-胞衬蛋白降解明显增加，用钙蛋白酶抑制剂1（CAI）预处理（iv.）显示钙蛋白酶激活的趋势抑制和α-胞衬蛋白分解的显著衰减。适用于较小样品的酶谱法显示m-钙蛋白酶激活和胞衬蛋白分解的区域变化，结论脑缺血主要通过激活海马内的m-胞衬蛋白和β-胞衬蛋白来诱导α-胞衬蛋白和β-胞衬蛋白的水解，并可能通过激活海马内的m-胞衬蛋白和β-胞衬蛋白来诱导α-胞衬蛋白和β-胞衬蛋白的水解。钙蛋白酶低温和CAI通过改变钙蛋白酶活性，从而破坏CNS的细胞骨架，提供实质性的保护作用。少

项目成果

Sakabe T.et al.: "Regional variation of the calpain activation and breakdown of fodrin during and after forebrain ischemia." Stroke. (in preparation).

Sakabe T.et al.：“前脑缺血期间和之后钙蛋白酶激活和胞质蛋白分解的区域变化。”

Sakabe T.et al:"Ischemia induces proteolysis of calspectin through an increase in m-calpain activation and calpastatin down-regulation in the rat brain." Journal of Cerebral Blood Flow and Metabolism. (in preparation).

Sakabe T.等人：“缺血通过大鼠大脑中 m-钙蛋白酶激活的增加和钙蛋白酶抑制剂的下调来诱导钙蛋白酶解。”

Sakabe T.et al:"Regional variation of the calpain activation and breakdown of fodrin during and after forebrain ischemia." Stroke. (in preparation).

Sakabe T.et al：“前脑缺血期间和之后钙蛋白酶激活和胞质蛋白分解的区域变化。”