鱼藤酮对大鼠星形胶质细胞CaMKⅡ基因和蛋白表达的影响

The toxic effects of rotenone on CaMK Ⅱ in primary cultured astrocytes

目的研究鱼藤酮对大鼠原代培养星形胶质细胞钙/钙调素依赖性蛋白激酶Ⅱ(CaMKⅡ)活性的影响。方法MTT法检测染毒星形胶质细胞活力,激光共聚焦结合Fluo-4荧光法动态测定细胞内游离钙浓度([Ca2+]i),采用RT-PCR技术检测CaMKⅡα和CaMKⅡβmRNA的表达,Western-blot技术观察磷酸化CaMKⅡ蛋白活性的变化。结果1.0和2.0μmol/L鱼藤酮染毒时星形胶质细胞活力明显降低,染毒星形胶质细胞[Ca2+]i呈浓度依赖性升高,CaMKⅡα亚基mRNA明显下调(P<0.05),CaMKⅡ蛋白活性显著降低(P<0.01)。结论鱼藤酮染毒星形胶质细胞内游离钙浓度的升高,CaMKⅡ基因表达和蛋白活性显著降低,可能是其诱导神经细胞变性损伤的重要原因之一。

Objective To study the toxic effects of rotenone on the activity of CaMKⅡ in primary cultured astrocytes. Methods MTT assay and morphological observation were used to evaluate the toxicity of rotenone on astrocytes, intracellular calcium concentrations were measured by confocal laser scanning microscopy and Fluo-4 fluorescence dyeing techniques, the expression of CaMK Ⅱ α and CaMK Ⅱ β mRNA were detected by RT-PCR, and the activity of phosphory-CaMK Ⅱ protein was measured by Western blot. Results The viability of cultural cells was significantly decreased after treated with 1.0 and 2. 0 μmol/L rotenone, rotenone was showed to increase intracellular calcium concentration in a dose-dependent manner, the expression of CaMK Ⅱ αmRNA decreased significantly in 1.0 and 2. 0 μmol/L rotenone intoxication groups （ P 〈 0. 05）, but the expression of phosphory-CaMK Ⅱ enhanced obviously （ P 〈 0. 01 ）. Conclusions The elevation of astrocytes[ Ca^2＋ ] i and the inhibition of CaMK Ⅱ activity induced by rotenone may be the main reason of its neurotoxicity on CNS.