Abstract

We had previously reported that neuroinflammation and memory impairment associated with intracerebroventricular streptozotocin (ICV STZ) injection in rats was due to glial activation and modulation of the N-methyl-D-aspartate (NMDA) receptor function. However, the exact role of the NMDA receptor and the molecules associated with downstream calcium ion signaling in STZ-induced astroglial activation is not known. Thus, in the present study, Memantine (an NMDA receptor antagonist) and Ibuprofen (an anti-inflammatory drug) were used as the pharmacological tool to investigate the molecular mechanisms involved in STZ-induced astroglial inflammation. We have studied the effect of STZ (100 μM) treatment for 24 h on NMDA receptor subunits (NR1, NR2A, and NR2B) expression and its associated calcium ion regulated molecules calcium/calmodulin-dependent protein kinase II subunit α (CaMKIIα), cyclic AMP-response element-binding (CREB) protein, Calpain, and Caspase 3. We have found a significant increase in the expression of NR1, NR2B, Calpain, and Caspase 3 expression, whereas a decrease in the level of NR2A, CaMKIIα, and CREB protein expression after 24 h of STZ treatment. These results indicate that STZ altered the NMDA receptor subunit expression and its downstream calcium (Ca²⁺) ion signaling molecules. We have also found that both Memantine (5 µM) and Ibuprofen (200 μM) significantly prevented the STZ-induced change in CaMKIIα, CREB, Calpain, and Caspase 3 expressions in C6 astrocytoma cells. Interestingly, only Memantine (and not Ibuprofen) was able to prevent the changes in NMDA receptor subunit expression in STZ-treated astrocytoma cells. STZ treatment also increased the level of glial fibrillary acidic protein (GFAP), tumor necrosis factor-alpha (TNF-α), inducible nitric oxide synthase (iNOS), and decreased the level of interleukin-10 (IL-10), indicating inflammatory condition, which was restored by both Memantine and Ibuprofen. These results suggest that both Memantine and Ibuprofen exert anti-inflammatory effect against STZ-induced astroglial activation and neuroinflammation via modulation of NMDA receptor-associated downstream calcium signaling cascade. However, only Memantine (not Ibuprofen) was able to revert STZ-induced changes in NMDA receptor subunit expression.

Graphic abstract

中文翻译：

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美金刚和布洛芬预处理通过调节 NMDA 受体相关下游钙离子信号传导对链脲佐菌素诱导的星形胶质细胞炎症发挥抗炎作用

摘要

我们之前曾报道，与大鼠脑室内链脲佐菌素 (ICV STZ) 注射相关的神经炎症和记忆障碍是由于神经胶质激活和 N-甲基-D-天冬氨酸 (NMDA) 受体功能的调节。然而，NMDA 受体和与下游钙离子信号传导相关的分子在 STZ 诱导的星形胶质细胞激活中的确切作用尚不清楚。因此，在本研究中，美金刚（一种 NMDA 受体拮抗剂）和布洛芬（一种抗炎药）被用作研究 STZ 诱导的星形胶质细胞炎症的分子机制的药理学工具。我们研究了 STZ（100 μM）处理 24 小时对 NMDA 受体亚基（NR1、NR2A、和 NR2B) 表达及其相关的钙离子调节分子钙/钙调蛋白依赖性蛋白激酶 II 亚基 α (CaMKIIα)、环 AMP 反应元件结合 (CREB) 蛋白、钙蛋白酶和半胱天冬酶 3。我们发现NR1、NR2B、Calpain 和 Caspase 3 的表达，而 STZ 处理 24 小时后 NR2A、CaMKIIα 和 CREB ​​蛋白表达水平降低。这些结果表明 STZ 改变了 NMDA 受体亚基表达及其下游钙离子（Ca²⁺) 离子信号分子。我们还发现，美金刚 (5 µM) 和布洛芬 (200 µM) 均能显着阻止 STZ 诱导的 C6 星形细胞瘤细胞中 CaMKIIα、CREB、钙蛋白酶和 Caspase 3 表达的变化。有趣的是，只有美金刚（而非布洛芬）能够阻止 STZ 处理的星形细胞瘤细胞中 NMDA 受体亚基表达的变化。STZ 治疗还增加了胶质纤维酸性蛋白 (GFAP)、肿瘤坏死因子-α (TNF-α)、诱导型一氧化氮合酶 (iNOS) 的水平，并降低了白细胞介素 10 (IL-10) 的水平，表明炎症美金刚和布洛芬都恢复了这种状况。这些结果表明，美金刚胺和布洛芬均通过调节 NMDA 受体相关下游钙信号级联反应对 STZ 诱导的星形胶质细胞激活和神经炎症发挥抗炎作用。然而，只有美金刚（不是布洛芬）能够恢复 STZ 诱导的 NMDA 受体亚基表达变化。

图形摘要