Wang Yuqian, Qin Yuxia, Guo Tingwen, Tang Chuanxi, Liu Liyun, Gao Dianshuai
Department of Neurology, The Affiliated Huai'an Hospital of Xuzhou Medical University, Huai'an, China.
Department of Neurobiology, Xuzhou Key Laboratory of Neurobiology, Xuzhou Medical University, Xuzhou, China.
Dev Neurosci. 2018;40(2):134-144. doi: 10.1159/000487853. Epub 2018 Apr 13.
Studies have shown that astrocytes play an important role in a variety of biological processes, so damage to astrocytes can cause a series of related diseases. Glial cell line-derived neurotrophic factor (GDNF) has always been considered a protective factor for dopamine neurons. However, it remains unclear whether GDNF has a protective effect on glial cells, especially astrocytes. In this study, we put forward the hypothesis that a high concentration of GDNF in the microenvironment of astrocytes exerts an inhibitory effect on the apoptosis of astrocytes by DNA-damaging reagents.
We isolated, purified, and identified primary astrocytes from neonate rats. Astrocytes were exposed to mitoxantrone (MTN, a DNA-damaging compound) for 24 h. The effects of MTN on astrocytes were tested by Hoechst 33342 staining, CCK-8 assay, and flow cytometry assay. One of the concentrations of MTN was applied to construct an apoptotic model of astrocytes. The astrocytes were then treated with GDNF together with a selected concentration of MTN for 24 h. The cell viability, cell nucleus morphology, and apoptosis ratio of the cells was assessed by Hoechst 33342 staining, CCK-8 assay, and flow cytometry assay, respectively. RNA sequencing (RNA-Seq), quantitative PCR analysis, and KEGG pathway mapping were performed to examine the genes involved in the procedure. Finally, Western blot analysis was applied to confirm the expression levels of the proteins of interest.
Hoechst 33342 staining revealed a one-tenth change in the percentage of Hoechst-positive cells after the addition of 500 ng/mL GDNF combined with 1,000 nM MTN for 24 h. The viability of the cells treated the same as described above was 1.4-fold that of the control group. Flow cytometry assays indicated that the apoptotic rates were 17.67, 8.67, and 4.34% for 0, 200, and 500 ng/mL GDNF, respectively. Birc2, Birc3, and Gadd45b were linked to the antiapoptotic process induced by GDNF in astrocytes. Western blot analysis confirmed the elevated expression of Birc2 and Gadd45b.
Our studies revealed that GDNF has a noticeable antiapoptotic effect on gene-injured astrocytes. This may provide critical clues for the treatment of a series of diseases in which damaged astrocytes are involved.
研究表明，星形胶质细胞在多种生物学过程中发挥着重要作用，因此星形胶质细胞受损会引发一系列相关疾病。胶质细胞源性神经营养因子（GDNF）一直被认为是多巴胺能神经元的保护因子。然而，GDNF对胶质细胞尤其是星形胶质细胞是否具有保护作用仍不清楚。在本研究中，我们提出一个假设，即星形胶质细胞微环境中高浓度的GDNF可通过DNA损伤试剂对星形胶质细胞的凋亡产生抑制作用。
我们从新生大鼠中分离、纯化并鉴定原代星形胶质细胞。将星形胶质细胞暴露于米托蒽醌（MTN，一种DNA损伤化合物）24小时。通过Hoechst 33342染色、CCK-8检测和流式细胞术检测MTN对星形胶质细胞的影响。应用MTN的其中一个浓度构建星形胶质细胞凋亡模型。然后将星形胶质细胞与选定浓度的MTN一起用GDNF处理24小时。分别通过Hoechst 33342染色、CCK-8检测和流式细胞术检测细胞活力、细胞核形态和细胞凋亡率。进行RNA测序（RNA-Seq）、定量PCR分析和KEGG通路映射以检查该过程中涉及的基因。最后，应用蛋白质印迹分析来确认目标蛋白的表达水平。
Hoechst 33342染色显示，加入500 ng/mL GDNF与1000 nM MTN共同处理24小时后，Hoechst阳性细胞百分比变化了十分之一。按上述相同方法处理的细胞活力是对照组的1.4倍。流式细胞术检测表明，0、200和500 ng/mL GDNF处理组的凋亡率分别为17.67%、8.67%和4.34%。Birc2、Birc3和Gadd45b与GDNF在星形胶质细胞中诱导的抗凋亡过程相关。蛋白质印迹分析证实了Birc2和Gadd45b的表达升高。
我们的研究表明，GDNF对基因损伤的星形胶质细胞具有显著的抗凋亡作用。这可能为治疗一系列涉及星形胶质细胞受损的疾病提供关键线索。
