Mechanism Of Lysophosphatidic Acid Induced Apoptosis Of Neuronal PC12 Cells

Objective:1.To investigate the molecular mechanisim of lysophosphatidic acid induced apoptosis of neuronal PC12 cells.2.To determine the roles of LPA receptor(LPA1and LPA2)inhibitors and MAPK(ERK1/2、p38 and JNK)inhibitors in LPA induced apoptosis of neuronal PC12 cells.3.To suggest effective interventions that can block lysophosphatidic acid induced apoptosis of neuronal PC12 cells.Methods:The nerve growth factor differentiated neuronal PC12 cells were cultured in 96-well plates 、24-well plates、 6-well plates or Petri dishes 6 cm in diameter.PC12 cells were treated with three ways.The first way of treatment:PC12 cells were treated with various concentration of LPA(0μM 、20μM、40μM、60μM)for 24 hours and then were used for further experiments;the second way of treatment: PC12 cells were treated with LPA(60μM)for various time(0h、6h、12h、24h)and then were used for further experiments;the third way of treatment:PC12 cells were pretreated with dmso(vehicle)、LPA1 inhibitor(AM095,5μM)、LPA2 inhibitor(LPA2 antagonist1,5μM)、ERK1/2 inhibitor(U0126,5μM)、p38 inhibitor(SB203580,8μM)and JNK inhibitor(SP600125,10μM)for 2h and then all groups were subjected to 60μM LPA for 24 h respectively and finally were used for further experiments.Further experiments:CCK-8 was used to determine the cell viability of PC12 cells;TUNEL staning was used to detect the apoptosis of PC12 clls;Rhodamine 123 staning was used to check the mitochondrial membrane potential of PC12 cells;Western Blot was used to investigate the levels of Bcl2 and the.ratio of Bcl2/Bax;RT-q PCR was used to determine the Bcl2 m RNA levels of PC12 cells.Results:1.LPA decreased the cell viability and mitochondrial membrane potential of neuronal PC12 cells while increased the apoptotic index of neuronal PC12 cells in a concentration-dependent/time-dependent manner.2.LPA downpregulated the Bcl2 m RNA levels 、Bcl2 protein levels and Bcl2/Bax ratio of PC12 cells while increased the translocation of Bax from the cytosol to the mito chondria of PC12 cells in a concentration-dependent/time-dependent manner.3.Both LPA receptor(LPA1 and LPA2)inhibitors and MAPK(ERK1/2、p38 and JNK)inhibitors could block LPA induced decrease in cell viability of PC12 cells.Among them,LPA2 inhibitor、p38inhibitor and JNK inhibitor had given the better results while LPA1 inhibitor and ERK1/2 inhibitor were least effective.4.Both LPA receptor(LPA1 and LPA2)inhibitors and MAPK(ERK1/2、p38 and JNK)inhibitors could block LPA induced decrease in mitochondrial membrane potential and increase in apoptotic index of PC12 cells.Among them,LPA2inhibitor、ERK1/2 inhibitor、p38 inhibitor and JNK inhibitor were more effective while LPA1 inhibitor was least effective.5.Both LPA receptor(LPA1 and LPA2)inhibitors and MAPK(ERK1/2、p38 and JNK)inhibitors could block LPA induced decrease in Bcl2 m RNA levels、Bcl2protein levels and Bcl2/Bax ratio and increase in translocation of Bax from the cytosol to the mitochondria of PC12 cells.Among them,LPA2 inhibitor、ERK1/2inhibitor、p38 inhibitor and JNK inhibitor had worked better while LPA1 inhibitor was least effective.Conclusion:1.LPA downregulated the levels of Bcl2 m RNA and furtherly reduced the anti-apoptotic protein Bcl2 protein levels and Bcl2/Bax ratio resulting in translocation of Bax from the cytosol to the mitochondria,which led to mitochondrial dysfunctions and finally increased the apoptotic ratio of PC12 cells.2.Both LPA receptor(LPA1 and LPA2)inhibitors and MAPK(ERK1/2、p38 and JNK)inhibitors could block LPA induced decrease in Bcl2 m RNA levels and furtherly alleviated the reduce in Bcl2 protein levels and Bcl2/Bax ratio,which led to blcokage of translocation of Bax from the cytosol to the mitochondria and finally reduced apoptosis of PC12 cells as a result of attenuated mitochondrial dysfunctions.Among them,LPA2 inhibitor、ERK1/2 inhibitor、p38inhibitor and JNK inhibitor had given the better results while LPA1 inhibitor was least effective.3.LPA decreased the cell viability of PC12 cells in a dose-dependent/time-dependent manner,while LPA receptor(LPA1 and LPA2)inhibitors and MAPK(ERK1/2、p38 and JNK)inhibitors blocked this effect.In this paper,cell viability had roughly a negative correlation with apoptic rate.However,this paper suggested that cell viability seems to be an index that is determined by many factors.For example,under my experimental condition ERK1/2 inhibitor obviously blocked LPA induced apoptosis of PC12 cells while its ability of bolocking the decrease in cell viability induced by LPA was very limited.