Up-regulation of thioredoxin system by puerarin inhibits lipid uptake in macrophages.

Cellular oxidative stress promotes lipid accumulation in macrophages during atherogenesis. Puerarin is a natural isoflavone with beneficial effects against oxidation and atherosclerosis. In this study, we investigated the effects of puerarin on lipid uptake and explored the underlying molecular regulation. We found puerarin up-regulated thioredoxin-1 (Trx1) and Trx reductase-1 (TrxR1) expression; it increased TrxR1 activity, cellular thiols contents and decreased oxidized form of Trx1, thus inhibiting cellular ROS generation. Confocal microscope and flow cytometry analysis showed fluorescence labeled Dil-oxLDL uptake was dramatically inhibited by puerarin in RAW264.7 cells as well as in primary bone marrow derived macrophages and peritoneal macrophages. The effects were reversed when Trx1 was inhibited by treatment with Trx1 inhibitor PX-12 or Trx1 siRNA. We also found scavenger receptors such as SR-A and Lox-1, but not CD36 were involved in the Trx1-mediated lipid uptake inhibition. Moreover, measurements of foam cell accumulation and ROS production in sections of aortic roots showed those were reduced by puerarin but raised when additional treatment with PX-12 or Trx1 siRNA in apoE-/- mice, which demonstrates the lipid uptake reduction by puerarin requires Trx1 inhibition in vivo. In addition, we analyzed the upstream regulation and found puerarin induced Nrf2 activity; cooperation between Nrf2 and ATF4 facilitated the puerarin effects. PERK phosphorylation was detected to be increased by puerarin, while PERK inhibition reduced cellular Trx1, TrxR1, nuclear Nrf2 and ATF4. Altogether, puerarin modulates PERK/Nrf2 that coordinates with ATF4 to active Trx1, which causes SR-A and Lox-1 reduction and lipid uptake inhibition in macrophages. This suggests Trx1 could be an effective target by puerarin in the prevention of atherosclerosis.