The Mechanism Of The Protective Effect Of Glycine Receptor Cells

Objectives:The aims of this study were to determine the downstream pathways of the cytoprotective effect of glycine mediated by glycine receptor, which started to elucidate the molecular mechanism of glycine against ATP depletion and provide theoretical reasons to discover new cytoprotective agents.Methods:This study first used respiratory chain inhibitor with glucose-free medium to mimesis the ATP depletion model. MDCK (Madin-Darby canine kidney) cells were divided into two groups, which were ATP depletion group and Glycine protection group. Western blot was used to detect the phosphorylation activity of ERK1/2, p38MAPK, JNK1/2 and AKT. The LDH release assay was measured to assess the cell death with suppression of ERK1/2 (PD98059) and AKT (LY294002). The pcDNA3. 1-GlyRα1 plasmid was constructed and transfected into HEK293 (Human embryonic kidney 293) cells with lipofectin. The stable transfectants were screened by G418. The stable cell lines and wild-type HEK293 cells were divided into ATP depletion group and Glycine protection group. Western blot was used to detect the phosphorylation activity of ERK1/2, p38MAPK, JNK1/2 and AKT. siRNA2 which down regulated GlyRα1 most efficiently was used to transfected into MDCK cells. After 48 hours, MDCK cells was ATP depleted. Western blot was used to detect the protein expression levels of the GlyRα1 and the phosphorylation activity of ERK1/2, p38MAPK, JNK1/2 and AKT.Results:MDCK cells were ATP depleted. After 15min, 30min, 1h and 2h, the phosphorylation activity of the ERK1/2 of Glycine protection group increased more than ATP depletion group, p38MAPK decreased, AKT increased, and JNK1/2 had no changes.The inhibitor PD98059 and LY294002 both could reduce the cytoprotective effect of glycine. The stable GlyRα1-expressed HEK293 cells and wide-type HEK293 cells were ATP depleted. After 1 hour, in stable cell lines, the phosphorylation activity of the ERK1/2 of Glycine protection group increased more than ATP depletion group, p38MAPK decreased, AKT increased, and JNK1/2 had no changes. Then there were no difference between ATP depletion group and Glycine protection group in wide-type HEK293 cells.siRNA2 was transfected into MDCK cells. After 48h, the expression of GlyRα1 was down regulated by 60%. Then after 1 hour's ATP depletion, in control group, the phosphorylation activity of ERK1/2 and AKT of Glycine protection group increased compared with ATP depletion group, p38MAPK decreased, and JNK1/2 had no changes. Otherwise in interferent group, there were no difference between ATP depletion group and Glycine protection group.Conclusion:ERK1/2, p38MAPK and AKT participated into the cytoprotective effect of the GlyR. Glycine binding with GlyR triggered a serial reaction to increase the phosphorylation activity of ERK1/2 and AKT to promote the cell proliferation and increase the cell survival probability, and to decrease the phosphorylation activity of p38MAPK to inhibit cell death in order to protect the ATP depleted cells.