The Molecular Pathway Analysis Of Porcine Vascular Endothelial Cells Inflammation Induced By ApoCIII

Vascular endothelial cell(VEC) is the main component of vascular endothelium. VEC can maintain the integrity of the vascular intima, and the function of the blood vessels. In normal physiological conditions, VEC can regulate the relaxation and contraction of the blood vessels, blood coagulation and anticoagulation, cell adhesion, the proliferation and migration of the vascular smooth muscle cell. In pathological conditions, VEC may participate in the inflammation and the formation of the thrombosis. The activated VEC can secrete large amounts of inflammatory cytokines, and regulate the function of the inflammatory cells, and thus participate in the inflammatory process.Apolipoprotein CIII(Apo CIII), a 79-amino acid peptide, is synthesized in the liver and small intestine. Apo CIII mainly resides on the surface of chylomicron and VLDL. Apo CIII can inhibit the activity of lipoprotein lipase and the metabolism and clearance of the hepatic triglyceride-rich lipoproteins, and finally caused vascular restenosis and atherosclerosis through a series of biological effects. In this study, we simulated the process of Apo CIII triggering VEC inflammation by adding Apo CIII to the cultured VEC in vitro. Then we explored the key inflammatory cytokines and molecular pathways of Apo CIII triggering cellular inflammation by deep-sequencing.First, we isolated and cultured the porcine aortic VEC. And then we examined the m RNA and protein expression levels of different concentrations of Apo CIII on adhesion molecules(VCAM-1 and ICAM-1), and pro-inflammatory cytokines(IL-6 and TNF-α) by RT-q PCR and ELISA, to screen the optimal concentration of Apo CIII. The morphology of VEC changed from polygon to spindle after adding Apo CIII. The results showed that VCAM-1, ICAM-1 m RNA levels, and IL-6, TNF-α protein levels were significantly increased when the concentration of Apo CIII was 50 μg / ml.Subsequently, we screened the differential expression genes induced by Apo CIII using the high-throughput sequencing technology, and verified the sequencing results by RT-q PCR. The results showed 647 significant differential expression genes between the treatment and control group, including 390 up-regulated genes and 257 down-regulated genes. Pathway analysis showed that the differential expression genes is related to the immune response and inflammation pathways. There are 21 genes involved in MAPK pathway.Finally, we examined the effects of Apo CIII on the ERK1/2 and p38 MAPK phosphorylation by Western blot, and detected the expression levels of IL-6 and TNF-α with the addition of MAPK inhibitors and Apo CIII in VEC by ELISA. The results of Western blot showed that Apo CIII promote the ERK1/2 and p38 MAPK phosphorylation. The results of ELISA suggested that the inhibitor of ERK1/2 and p38 MAPK decreased the protein expressing levels of IL-6 and TNF-α. These findings provide a theoretical basis for further understanding the molecular mechanisms of the VEC inflammation by Apo CIII.