Lipidomics Study On Endothelial Cell Injury And Protection Mechanism By Medicine Under Oxidative Stess

The study of phospholipids metabolism changes in oxidative stress induced endothelial cells (ECs) is important to elicit the link between endothelium dysfunction and cerebral/cardio vascular diseases. In this research, we set exogenous H2O2 induced human unbilic vein endothelial cell line EA.hy926 as the model, and applied lipidomics with signal transduction strategy together to explore the underlying mechanisms of tetramethylpyrazine (TMP) and butylidenephthalide (BP) protection of endothelial cells against oxidative stress.In this research, we establish the phospholipids profile of EA.hy926 cell using high-throughput LC/ESI/MSn techniques. Eight main classes (PG, PE, PC, PI, PS, PA, LPC and LPE) including more than 200 species of phospholipids was detected and analyzed qualitatively and quantitatively. By multi-statistic analysis, we found the phospholipids with long carbon chain, polyunsaturated fatty acids (PUFAs) or ether link in sn-1 position (ether phospholipids) take priorities in oxidative degradation; meanwhile, phospholipase A2 was activated to generated a great amount of lysophospholipids including LPC and LPE. These above two main findings were responsible to the injury of ECs.Our results showed that with TMP pretreatment, content of PCs, especially the PCs with long carbon chain, PUFAs or ether PCs in injured cells was largely increased and, contents of LPC and LPE were decreased. These results indicated the antioxidation activity and inhibition of PLA2 effect of TMP. Beside of these properties, BP also had inhibitory effect over PI-PLC and consequentially increased PI contents. TMP and BP showed extraordinary abilities in protection of ECs for its antioxidation of phospholipids and alteration of phospholipase activation properties.Since PLA2 was one of the key factors in our results, we add PD98059 and ETYA, inhibitors of MAPK pathway and of COX, LOX pathway which were supposed to be the upstream and downstream signal of PLA2, to the injury model. With multi-statistic analysis, we find similarity in phospholipids metabolism pattern between ETYA and TMP pretreated ECs. However, ETYA showed stronger preference over ether PC than PCs with long carbon chain or polyunsaturated fatty acids, which indicated that the antioxidation activity of ETYA was not as good as TMP. Similarity in phospholipids metabolism pattern between BP and PD98059 pretreated ECs was also found on the basis of inhibitory effect on PI-PLC activation; however, the long carbon chain PSs were synthesized with PD98059 pretreatment to induce apoptotic death in ECs.Fluorescent substrate was utilized to prove the activation of PLA2 and generation of LPC; western blot method was employed to show the phosphorylation of cPLA2 in acute oxidative injury of ECs in which existed cross-talk between phosphorylated cPLA2 and ERK1/2. Based on these above findings, a phospholipids and phospholipase based signal transduction model was established to summarize our basic findings in this research.