Differential PARP Cleavage: An Indication of Heterogeneous Forms of Cell Death and Involvement of Multiple Proteases in the Infarct of Focal Cerebral Ischemia in Rat
Differential PARP Cleavage: An Indication of Heterogeneous Forms of Cell Death and Involvement of Multiple Proteases in the Infarct of Focal Cerebral Ischemia in Rat
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DOI：
10.1007/s10571-009-9348-8
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发表时间：
2009-06-01
影响因子：
4
通讯作者：
Babu, Phanithi Prakash
中科院分区：
文献类型：
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作者：
Chaitanya, Ganta Vijay;Babu, Phanithi Prakash
目的聚腺苷二磷酸核糖聚合酶（PARP）是一种核修复酶，广泛参与多种生理和病理过程。在本研究中，我们想检查PARP的状态和各种细胞死亡蛋白酶参与细胞凋亡和非凋亡形式的细胞死亡在短暂局灶性脑缺血大鼠模型中的作用。这些蛋白酶的活化可导致PARP片段的产生，所述PARP片段可被视为病理学中所涉及的特定蛋白酶的特异性特征片段，从而导致细胞死亡的类型。结果Western blot和免疫组化分析显示，在缺血组织中，钙蛋白酶、组织蛋白酶-b、半胱氨酸天冬氨酸蛋白酶-3和颗粒酶-b被激活，它们作用于PARP并切割PARP产生特异性信号片段。甲酚紫染色显示存在凋亡和坏死细胞死亡。此外，我们观察了AIF和gra-b与PARP的双重免疫荧光和免疫共沉淀实验中的相互作用。结论在甲酚紫染色中，钙蛋白酶、组织蛋白酶-b、caspase-3和颗粒酶-b的激活与细胞凋亡或坏死相关。PARP特征片段的出现给出了关于病理学中特定蛋白酶参与的清楚概念。特征片段如89-和50-kDa的出现表明病理学中涉及凋亡和坏死细胞死亡。AIF和gra-b与PARP的进一步相互作用也表明在局灶性脑缺血的病理过程中涉及细胞死亡的非凋亡模式。
Aim Poly (ADP-ribose) polymerase (PARP) is a nuclear repair enzyme whose role is widely depicted in various physiological and pathological processes. In the present study, we wanted to check the status of PARP and the role of various cell death proteases involved in apoptotic and non-apoptotic forms of cell death during transient focal cerebral ischemia in rat model. The activation of these proteases can result in the production of PARP fragments which can be treated as specific signature fragments to the particular protease involved in the pathology and hence the type of cell death. Results In the ischemic samples, we observed activation of calpain, cathepsin-b, caspase-3, and granzyme-b which were known to act on and cleave PARP to produce specific signature fragments by Western blot and immunohistochemical analysis. Cresyl violet staining showed the presence of apoptotic and necrotic cell deaths. Further we observed interaction of AIF and gra-b with PARP in double immunofluorescence and co-immunoprecipitation experiments. Conclusion Activation of calpains, cathepsin-b, caspase-3, and granzyme-b correlated with either apoptotic or necrotic cell deaths in cresyl violet staining. The appearance of PARP signature fragments gives a clear idea on the involvement of particular protease in the pathology. Appearance of signature fragments like 89- and 50-kDa indicates the involvement of apoptotic and necrotic cell death in the pathology. Further interaction of AIF and gra-b with PARP also indicates the involvement of non-apoptotic modes of cell death during the pathology of focal cerebral ischemia.