Vascular Protection of TPE-CA on Hyperhomocysteinemia-induced Vascular Endothelial Dysfunction through AA Metabolism Modulated CYPs Pathway

Abstract

A high concentration of homocysteine (Hcy) in plasma induces vascular endothelial dysfunction, and it may ultimately accelerate the development of cardiovascular diseases (CVDs). Although several B vitamins have been clinically applied for hyperhomocysteinemia (HHcy) treatment, the outcomes are not satisfied due to their limited therapeutic mechanism. Hence, in order to improve the curative effect, development of new effective therapeutic strategies should be put on the agenda. Total phenolic extracts of Citrus aurantium L. (TPE-CA) is a naturally obtained phenolic mixture, mainly containing flavones, flavanones and their glycosyl derivatives, flavonols, polymethoxyflavones and coumarins. Previous reports indicated that bioactive phenolic compounds possessed potent vascular protective effects and regarded as a protective agent against CVDs. Intriguingly, the exact mechanism underlying the suppressed effects of TPE-CA on HHcy could assist in revealing their therapy on CVDs. Here, the multi-targeted synergistic mechanism of TPE-CA on HHcy-induced vascular endothelial dysfunction was uncovered in a deduced manner. TPE-CA treatment exhibited an obvious superiority than that of B vitamins treatment. Network pharmacology was employed to identify the interrelationships among compounds, potential targets and putative pathways. Further experimental validation suggested that the treatment of TPE-CA for HHcy could not only effectively reduce the Hcy level in plasma through up-regulating transsulfuration pathway in Hcy metabolism, but also restore the HHcy-induced vascular endothelial dysfunction by activating cytochrome P450 enzymes (CYPs) epoxygenase signal cascades and inhibiting CYPs hydroxylase signal cascades in arachidonic acid (AA) metabolism.

Keywords: Arachidonic acid metabolism; CYPs signal pathway; Hyperhomocysteinemia; Total phenolic extracts of Citrus aurantium L.; Vascular endothelial dysfunction.

Figure 1

Effects of TPE-CA and B vitamins on the body, heart and liver weight of rats.

Figure 2

Effects of TPE-CA and B vitamins on Hcy level and the release of vasoactive mediators in rat's plasma. (A) The concentrations of Hcy. (B) The concentrations of Ang Ⅱ. (C) The concentrations of NO. (D) The concentrations of ET-1.

Figure 3

Vascular histopathology analysis (Magnification: 200×). (A) Normal group. (B) H-Met group. (C) TPE-CA group. (D) B vitamins group. The marked histopathological characteristic in each group was indicated by arrowhead. Scale bar = 10μm.

Figure 4

Effects of TPE-CA and B vitamins on the pivotal proteins of Hcy metabolism. (A) The relative expression of MS. (B) The relative expression of MTHFR. (C) The relative expression of BHMT. (D) The relative expression of CBS. Protein expression levels were normalized to GAPDH.

Figure 5

Network pharmacology-based analysis of 32 active compounds contained in TPE-CA. (A) The Component-Target-Pathway network. The green circles, gray circles, blue round rectangle, red round rectangle and purple quadrangle corresponded to compounds with potential targets, compounds without potential targets, candidate targets, screened candidate targets and putative pathways, respectively. (B) The enrichment analysis of 38 target proteins by STRING database. (C) The enrichment analysis of target proteins related diseases by DIVAD database.

Figure 6

Effects of TPE-CA on the balance of PGI₂ / TXA₂ and PGIS / TXAS. (A) The relative expression of PGIS and TXAS. (B) The concentration of PGI₂ and TXA₂. #p<0.05 vs Normal group, ##p<0.01 vs Normal group, ###p<0.001 vs Normal group. *p<0.05 vs H-Met group, **p<0.01 vs H-Met group, ***p<0.001 vs H-Met group.

Figure 7

Effects of TPE-CA on the CYPs epoxygenase and CYP hydroxylase signal transduction of AA metabolism. (A) The relative expression of NOX2, CYP2C11, sEH, CYP4A and eNOS. (B) The concentration of ACE. (C) The concentration of 14, 15-EET. (D) The concentration of 20-HETE. #p<0.05 vs Normal group, ##p<0.01 vs Normal group, ###p<0.001 vs Normal group. *p<0.05 vs H-Met group, **p<0.01 vs H-Met group, ***p<0.001 vs H-Met group.

Figure 8

The schematic diagram of therapeutic mechanism of TPE-CA for HHcy-induced vascular endothelial dysfunction. TPE-CA treatment for HHcy could not only effectively reduce the Hcy level in plasma through up-regulating transsulfuration pathway in Hcy metabolism, but also restore the HHcy-induced vascular endothelial dysfunction by activating CYPs epoxygenase signal transduction pathway and inhibiting CYPs hydroxylase signal transduction pathway. Promotion: Inhibition: Promotion by TPE-CA: Inhibition by TPE-CA: