Fuxinfang improves hypoxia-induced injury of human aortic endothelial cells by regulating c-Fos-NR4A1-p38 pathway

Abstract

To explore the molecular mechanism of Fuxinfang for improving injury of human aortic endothelial progenitor cells (HAECs). Serum samples were collected from male SD rats treated with Fuxinfang (n=8) or saline (n= 5). HAECs cultured in normoxia or hypoxic condition (2% O2) were treated with serum from normal rats or with diluted serum (1% and 10%) from rats treated with Fuxinfang. The differentially expressed genes (DEGs) between Fuxinfang-treated and control cells were detected using high-throughput sequencing to screen the target DEGs that participated in arterial endothelial cell injury and underwent changes in response to both hypoxia and Fuxinfang treatment. AmiGo and String databases were used to infer the interactions among the target genes, and the expressions of the genes were analyzed in HAECs with different treatments using enzyme-linked immunosorbent assay (ELISA) and Western blotting. HAECs cultured in hypoxia did not show obvious changes in cell morphology or expressions of hypoxia-related factors in response to treatment with 1% or 10% serum from Fuxinfang-treated rats. The results of high-throughput sequencing showed a total of 7134 DEGs (4205 up-regulated and 2929 down-regulated genes) in HAECs in hypoxia model group and 762 DEGs (305 upregulated and 457 down-regulated genes) in Fuxinfang-treated HAECs. Analysis of AmiGo and String databases and the constructed protein-protein interaction network identified c-Fos, NR4A1, and p38MAPK as the target genes. The results of ELISA and Western blotting showed that the expressions of c-Fos, NR4A1, p38MAPK and pp38MAPK increased significantly in cells with hypoxic exposure (P < 0.05); treatment with the serum containing Fuxinfang significantly reduced the expression levels of c-Fos, NR4A1 and p-p38MAPK in hypoxic HAECs in a concentration-dependent manner (P < 0.05). The serum from Fuxinfang-treated rats can concentration-dependently inhibit the expressions of the DEGs occurring in hypoxia. Fuxinfang improves hypoxic injuries of HAECs possibly by down-regulating the expression of c-Fos to inhibit NR4A1 expression and suppressing hypoxia-induced p38 phosphorylation.