Tetramethylpyrazine Suppresses Lipid Accumulation Via Up-regulation The Atp-binding Cassette Transporters And Down-regulation Scavenger Receptors In Macrophages

Objective: Tetramethylpyrazine(TMP),the predominant active ingredient in Rhizoma Ligustici Wallichii(chuanxiong),possesses anti-proliferative properties and apoptosis-inducing activity in different cancer cell types.TMP is effective in the treatment of atherosclerosis by preventing endothelial dysfunction,regulating lipid level in plasma,attenuation of oxidative stress,inhibiting inflammation.Previous studies demonstrated that foam cell formation is the key event in the pathogenesis of atherosclerosis.The process of oxidized low-density lipoprotein(ox-LDL)uptake and cholesterol efflux affects foam cell formation.Scavenger receptors(SRs)on macrophage membrane,such as class A scavenger receptor(SR-A)and the cluster of differentiation 36(CD36),are responsible for the uptake of ox-LDL.Efflux of intracellular lipid occurs primarily via a process called reverse cholesterol transport(RCT),Several membrane proteins,such as ATP-binding cassette transporter A1(ABCA1),ABCG1,and SR-B type ?(SR-B?),has been shown to play a critical role in RCT pathway.PI3K/Akt signaling is integral to the uptake of modified lipoproteins and cholesterol efflux in various cells.It has been suggested that blockage of p38 MAPK signaling is involved in TMP-invoked inhibition of inflammation in endothelialcells.Nevertheless,studies on the effects and molecular mechanism by which TMP mediates lipid accumulation in macrophage-derived foam cells are not well documented.This study intends to investigate the effect of TMP and its underlying mechanism in RAW264.7 cells and apolipoprotein E-deficient(ApoE-/-)mice.Methods:(1)RAW264.7 cells were cultured with TMP for 24 h,then cell viability was examined by MTT;the lipid accumulation was measured by Oil Red O staining;fluorescent probe DiI-ox-LDL was used to detect ox-LDL uptake,cholesterol efflux was determined using fluorometer;the changes of ABCA1,ABCG1,SR-B?,SR-A and CD36 were assessed by Western blotting.(2)RAW264.7 cells were pretreated with LY294002(a PI3K/Akt inhibitor)or SB203580(an inhibitor of p38 MAPK)for 1 h and then co-incubated with TMP for additional 24 h,then the changes of ATP-binding cassette transporters,SRs,Akt phosphorylation(p-Akt),p38phosphorylation(p-p38),ox-LDL uptake and cholesterol efflux were examined.(3)To detect the protein stability of ABCA1 and ABCG1 in macrophages after treatment with TMP.(4)ApoE-/-mice were treated with TMP by gastric gavages,then aortas were collected and the protein expression of ABCA1,ABCG1,SR-B?,SR-A and CD36 were detected by western blotting.Results:(1)TMP treatment decreased ox-LDL uptake,increased cholesterol efflux and attenuated intracellular lipid accumulation;TMP significantly inhibited the expression of SR-A andCD36,while increasing the expression of ABCA1 and ABCG1.(2)Inhibition of PI3K/Ak and p38 MAPK activation enhanced the TMP-mediated expression of SRs and ATP-binding cassette transporters,and the TMP-mediated effects on ox-LDL uptake,cholesterol efflux and lipid accumulation in RAW264.7 cells.(3)TMP up-regulated the protein stability of ABCA1 without affecting ABCG1.(4)The progress of atherosclerosis in ApoE-/-mice was retarded by TMP,and TMP also reduced the expression of CD36 and SR-A,increased the expression of ABCA1 and ABCG1 in vivo.Conclusions: These findings suggest that TMP can down-regulate SRs and up-regulate ATP-binding cassette transporters via PI3K/Akt and p38 MAPK signaling,thus suppressing lipid accumulation in macrophages.