Apigenin inhibits the TNFα-induced expression of eNOS and MMP-9 via modulating Akt signalling through oestrogen receptor engagement

Apigenin is a naturally occurring plant flavone with strong anti-oxidant and anti-inflammatory activity. While the anticancer properties of Apigenin have been extensively studied, little is known about its effects on endothelial dysfunction. We investigated the effects of Apigenin in EAhy926 endothelial cells exposed to TNF alpha by evaluating the expression of eNOS and MMP-9, two key molecules in endothelial dysfunction. MMP-9 activity was measured by gel zymography. Western blot analysis was performed to analyze eNOS expression and signal transduction. Treatment with Apigenin (50 mu M) counteracted the TNF alpha-induced expression of eNOS and MMP-9 and the TNF alpha- triggered activation of Akt, p38MAPK and JNK signalling suggesting that multiple signalling pathways are involved in mediating the protective effects of Apigenin on endothelial function. To better understand the molecular mechanisms underlying the protective effects of Apigenin, we used a pharmacological approach with specific inhibitors. The use of an Akt inhibitor mimicked the inhibitory effects of Apigenin on eNOS and MMP-9 expression, suggesting that eNOS and MMP-9 induction by TNF alpha depends on Akt activation. The TNF alpha-induced expression of MMP-9 was also affected by the JNK inhibitor SP600125. No effect on eNOS and MMP-9 expression was observed in the presence of the p38MAPK inhibitor SB203580 or the ERK 1/2 inhibitor PD98059. Pretreatment with 'classic' (ER alpha and ER beta) or 'non classic' (GPR30) oestrogen receptor (ER) inhibitors (ICI182,780 and PTX, respectively) counteracted the ability of Apigenin to decrease the TNF alpha-triggered activation of the Akt pathway. Consistently, the use of both ER inhibitors reversed the inhibitory effects of Apigenin on the TNF alpha-induced expression of eNOS and, to a lesser extent, MMP-9. We can conclude that Apigenin exerts its inhibitory effect on the TNF alpha-induced expression of eNOS and MMP-9 through the Akt signalling inhibition generated by ER activation. Oestrogen signalling has been implicated in protection from cardiovascular disease. Therefore, having regard to its ability to bind to ERs, Apigenin may be considered an oestrogen-like molecule to potentially be used against the onset and progression of vascular diseases associated with endothelial dysfunction.