The Effect And Related Cell Signaling Pathway Caused By LPC And SPC Through The GPR4 Receptor

Objective To explore the effect on HUVEC proliferation and the related cell signaling pathway caused by LPC and SPC through the G protein-coupled receptor4.Methods HUVEC cells were transfected with the expression plasmids pEFneo-GPR4 (containing the GPR4 receptor gene) by the lipofectamineTM 2000, and the neomycin-resitant(G418 sulfate at 800μg/ml for HUVEC cells )stable cell lines were established. The expression of GPR4 receptor was detected by RT-PCR. The cell viability was measured by MTT assay. The structure changes of the HUVEC caused by lysophosphatidylcholine or sphingosylphosphorylcholine were observed by AO dyes. The expression of pro-caspase 3,p-JNK,p-Akt and MMP-2 influenced by lysophosphatidylcholine or sphingosylphosphorylcholine through GPR4 receptor was detected by western blot.Results The HUVEC cell line stably expressing the GPR4 receptor was established. After treatment with 0.5-8μmol/l LPC for 16 h, MTT assay showed that LPC decreased the HUVEC cell viability, and the OD value (570nm) was: 1.50±0.136; 1.45±0.050;1.42±0.208;1.36±0.191;1.32±0.122;1.08±0.189 respectively. On the contrary, SPC (0.25, 0.5,1.0μmol/l) increased the cell viability, and the OD value (570nm) was: 1.08±0.089;1.15±0.097;1.21±0.096;1.30±0.11 respectively. LPC caused some impairment to the HUVEC with some morphological changes. However, the SPC did not cause evident morphological changes .The expression of the pro-caspase 3 protein was promoted by LPC at the lower concentration 0.5μmol/l, but the expression was inhibited at the higher concentrations. LPC activated the JNK kinase and inhibited the expression of the MMP-2 protein. The expression of the p-Akt and MMP-2 protein were promoted by SPC at the lower concentration 0.25-0.5μmol/l, but the expression was inhibited and restored the basal level almostly at the higher concentrations. SPC inhibited the expression of the pro-caspase 3 protein .Conclusion 1. LPC decreased the cell viability at the HUVEC. LPC decreased the expression of the pro-caspase 3 protein , activated the JNK kinase and inhibited the expression of the MMP-2 ; 2. SPC increased the HUVEC viability, promoted the expression of the p-Akt and MMP-2 protein at the lower concentration, but the expression was inhibited and restored the basal level almostly at the higher concentration. SPC inhibited the expression of the pro-caspase 3 protein .