Studies Of Aspirin On Inhibiting The Rupture Of Rabbit's Atherosclerotic Plaque And Its Mechanisms

Objective: To investigate whether aspirin might increase the atherosclerotic plaque stability, and its possible mechanisms in vivo and in vitro, and the aspirin concentration exerting the best effect.Methods: In vivo, the hyperlipidemic atherosclerotic model was generated in male New Zealand rabbits that were given high fat diet and abrasion of the abdominal aorta. These rabbits were then treated with aspirin 5-20mg/kg for 4 weeks. At experimental end, the plaques were evoked into rupture by drugs. Areas of thrombosis on atherosclerotic aorta were determined by image analysis, morphologic character of plaque rupture was determined by light microscope, platelet aggregation induced by ADP or arachidonic acid was determined with turbidimetric method, the protein expression of macrophages was detected by immunohistochemistry, and the mRNA expression of COX-2 and MMP-2 was determined by hybridization in situ, respectively. In vitro, THP-1 cells were cultured and then differentiated to macrophages by PMA. Then, cells were incubated in RPMI 1640 plus aspirin 12.5-200μg/ml for 24h, respectively. The levels of MMP-2, MMP-9, PGE2 and PGD2 in supernatant were measured with ELISA, the mRNA expression of MMP-2, MMP-9, PPARα, PPARγ, COX-1, COX-2, mPGES-1 and L-PGDS were examined with RT-PCR, the protein expression of PPARα, PPARγ, NF-κB, mPGES-1 and L-PGDS was detected by Western blot, respectively.Results: (1) Aspirin at doses of 5-10mg/kg was able to inhibit thrombosis on abdominal aorta of rupture of atherosclerotic plaque, and the effect of 5mg/kg was better.(2) Aspirin could inhibit foam cell formation and aggregation in plaque, and keep the integrity of fiber cap and shoulder area. (3) Aspirin could inhibit platelet aggregation induced by ADP or arachidonic acid, but the effect in 5mg/kg or 10 mg/kg groups was better than that in 20mg/kg group.(4) Aspirin could decrease the number of macrophages obviously in 5mg/kg or 10mg/kg group, inhibit COX-2 mRNA expression in 10mg/kg or 20mg/kg group, and also significantly decrease the MMP-2 mRNA expression in 5 mg/kg group in atherosclerotic plaque.(5) Aspirin could notably decrease the levels of MMP-2 and MMP-9 in supernatant and the mRNA expression of MMP-2 and MMP-9 in macrophages derived from THP-1 cells.(6) Aspirin could inhibit the protein expression of NF-κB via the activation of PPARα/γmRNA and protein expression. After pretreatment with PPARα/γantagonists, aspirin-decreased MMP-9 mRNA expression and release were significantly weakened. Inversely, PPARα/γagonists could decrease the MMP-9 mRNA expression and release.(7) Aspirin also could decrease the PGE2 release to inhibit PGE2-dependent MMPs expression via the inhibition of COX-1 and COX-2 mRNA expression, mPGES-1 mRNA and protein expression. Addtionally aspirin also could increase L-PGDS mRNA and protein expression to increase PGD2 content, and 15d-PGJ2, a metabolite of PGD2, could inhibit MMP-9 expression and release.(8) All of these effects in vitro were the better when concentration of aspirin was at 50μg/ml.Conclusion: Aspirin was able to increase the stability of atherosclerotic plaque, and this effect was associated with reduction of macrophages and MMP expression in atherosclerotic plaque. The latter mechanisms might include the reduction of NF-κB protein expression via the elevation of PPARα/γmRNA and protein expression, and the inhibition of COX-mPGES-1/PGE2 pathway to reduce the PGE2-dependent MMP-2 and MMP-9 expression and release. Aspirin at a concentration of 50μg/ml might exert the best effects in vitro, and this might be related to the effect of aspirin on the ratio of mPGES-1/L-PGDS.