Yuqian Wang1, Yuxia Qin2, Tingwen Guo3, Chuanxi Tang2, Liyun Liu2, Dianshuai Gao2. 1. Department of Neurology, The Affiliated Huai'an Hospital of Xuzhou Medical University, Huai'an, China. 2. Department of Neurobiology, Xuzhou Key Laboratory of Neurobiology, Xuzhou Medical University, Xuzhou, China. 3. Department of Neurosurgery, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, China.
Abstract
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
BACKGROUND: 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. METHODS: 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. RESULTS: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. CONCLUSIONS: 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.