Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
J Neurosci Res. 2010 Aug 1;88(10):2155-64. doi: 10.1002/jnr.22375.
Intracerebral hemorrhage-associated tissue damage is triggered by blood-derived serine proteases such as thrombin. In addition, our previous studies have suggested that mitogen-activated protein (MAP) kinases contribute to intracerebral hemorrhage- and thrombin-induced striatal tissue damage in vivo. Here we addressed the mechanisms of MAP kinase involvement in thrombin cytotoxicity in rat corticostriatal slice culture, focusing on striatal tissue damage. Thrombin induced apoptotic nuclear condensation and fragmentation in striatal cells, which was suppressed by DEVD-CHO, a caspase-3 inhibitor. DEVD-CHO also prevented shrinkage of the striatal tissue induced by thrombin. Phagocytotic activity may be involved in tissue deterioration, because a phagocytosis inhibitor (cytochalasin D) and an inhibitor of phagocytosis of apoptotic cells (O-phospho-L-serine) suppressed shrinkage of the striatal tissue. OX42 immunostaining revealed that apoptosis-like microglial cell death was induced only when thrombin treatment was combined with application of inhibitors of MAP kinase/extracellular signal-regulated kinase kinase (PD98059), p38 MAP kinase (SB203580), or c-Jun N-terminal kinase (SP600125). Thrombin-induced increase in the number of microglia was also prevented by these inhibitors of MAP kinase pathways. We also found that thrombin-induced production of tumor necrosis factor (TNF)-alpha was inhibited by PD98059, SB203580, and SP600125. Finally, thrombin-induced neuronal apoptosis and shrinkage of the striatal tissue were significantly inhibited by anti-TNF-alpha neutralizing antibody. These results suggest that MAP kinases contribute to thrombin-induced striatal damage by supporting survival of activated microglia, which induce neuron death by producing TNF-alpha and cause tissue shrinkage by phagocytosing apoptotic cells.
脑出血相关的组织损伤是由血液来源的丝氨酸蛋白酶如凝血酶触发的。此外，我们之前的研究表明，丝裂原激活蛋白（MAP）激酶有助于体内脑出血和凝血酶诱导纹状体组织损伤。在这里，我们研究了 MAP 激酶参与凝血酶细胞毒性的机制，重点是纹状体组织损伤。凝血酶诱导纹状体细胞的凋亡核浓缩和碎裂，这被 caspase-3 抑制剂 DEVD-CHO 抑制。DEVD-CHO 也防止了凝血酶诱导的纹状体组织收缩。吞噬作用可能参与组织恶化，因为吞噬作用抑制剂（细胞松弛素 D）和凋亡细胞吞噬抑制剂（O-磷酸-L-丝氨酸）抑制了纹状体组织的收缩。OX42 免疫染色显示，只有当凝血酶处理与 MAP 激酶/细胞外信号调节激酶激酶（PD98059）、p38 MAP 激酶（SB203580）或 c-Jun N-末端激酶（SP600125）抑制剂联合应用时，才会诱导类似凋亡的小胶质细胞死亡。这些 MAP 激酶途径的抑制剂也阻止了凝血酶诱导的小胶质细胞数量增加。我们还发现，凝血酶诱导的肿瘤坏死因子（TNF）-α的产生也被 PD98059、SB203580 和 SP600125 抑制。最后，抗 TNF-α中和抗体显著抑制了凝血酶诱导的神经元凋亡和纹状体组织收缩。这些结果表明，MAP 激酶通过支持激活的小胶质细胞的存活来促进凝血酶诱导的纹状体损伤，这些小胶质细胞通过产生 TNF-α诱导神经元死亡，并通过吞噬凋亡细胞导致组织收缩。
