The Effect Of Aspirin On The Expression Of MMP-9 And Its Mechanisms In TNF- α-induced RAW264.7 Cells

Atherosclerosis(AS) is the pathological base of the development of acute coronary syndrome(ACS) which was serious harm to human health. It was estimated that 15.4 million patients have suffered cardiovascular disease and AS. Inflammatory invasion is involved in the development of AS, which caused that activated macrophages could secret matrix metalloproteinases(MMPs) in the atherosclerotic plaques. MMPs could virtually degrade every component of the extracellular matrix(ECM) which is contributed to increase the morbidity and mortality of cardiovascular disease. MMP-9, one family of the MMPs, not only can excessively degrade the type IV collage of basement membrane but also promote inflammation and inscrease vascular smooth muscle cell proliferation and migration, which lead to the atherosclerotic plaque instability and blood clots. Macrophage-secreted-MMP-9 plays an important role in the development of AS and plaque instability. Therefore, it is a new drug target that inhibits the expression of MMP-9 in atherosclerotic inflammatory cells to prevent the atherosclerotic development. Aspirin, a non-steroidal anti-inflammation drug, has been known for its important effect in prevention of vascular complications of atherosclerosis. The research indicated that aspirin inhibited the vascular inflammation, COX-2 activity and NF-κB p65 nuclear trascripitional expression in patients with atherosclerosis patients. The study showed that the stability of atherosclerotic plaque was related to the inhibitory effect of aspirin on MMP-9 expression. However, the mechanism was not full clear. Mouse peritoneal macrophages cell RAW264.7 was choosed in the present study to further investigate the effect of aspirin on MMP-9 and explore the possible mechanisms. Objective: To investigate the effect of aspirin on MMP-9 in TNF-α-induced RAW264.7 cells and its possible mechanisms. 1. The effect of aspirin on the expression and activity of MMP-9 in TNF-α-induced RAW264.7 cells RAW264.7 cells were pretreated with aspirin( 75, 150, 300,600μM) for 1h, then incubated with TNF-α(10ng/ml)for another 24 h. MTT was used to evaluate the cytotoxic effect of aspirin on RAW264.7 cells. Western blot and Q-RT-PCR were used to detect the protein expression and gene expression of MMP-9. The effect of aspirin on MMP-9 activation was analyzed by gelatin zymography. These results showed that different concentration of aspirin has no cytotoxic effect on RAW264.7 cells. Aspirin effectively inhibits MMP-9 expression and activity in a dose-dependent manner in TNF-α-stimulated RAW264.7 cells. 2. The effect of aspirin on the phosphorylated protein expression of MAPK signal pathway and the expression of MMP-9 after inhibiting the MAPK signal pathway RAW264.7 cells were pretreated with aspirin(600μM) for 1h, then incubated with TNF-α(10ng/ml) for another 60min、 30min、20min and 10 min. Western blot was used to detect the protein expression of P-ERK1/2、 ERK1/2、 P-JNK、 JNK、P-p38 and p38. These results showed that aspirin inhibited the phosphorylated protein expression of ERK1/2、JNK and p38 at 20min、60min and 30 min respectively. The expression of non-phosphorylated ERK1/2、JNK and p38 were significantly unchanged. Inhibiting the MAPK signal pathway including ERK1/2, P38 and JNK with the specific inhibitions PD98059(10μM)、SB203580(10μM) and SP600125(20μM). Cells were pretreated with these inhibitors for 1h, then incubated with TNF-α(10ng/ml)for another 24 h. Western blot and QRT-PCR were used to examine the protein expression and gene expression of MMP-9. These results showed that the expression of MMP-9 was significantly decreased in the presence of specific inhibitors of MAPK signal pathway. 3. The effect of aspirin on the nuclear protein expression of NF-κBp65 and the expression of MMP-9 after inhibiting the NF-κB signal pathway. RAW264.7 cells were pretreated with aspirin(75, 150, 300 and 600μM) for 1h, then incubated with TNF-α(10ng/ml) for another 1h. Western blot was used to detect the expression of the nuclear protein expression of p65. These results showed that the nuclear protein expression of p65 was strongly inhibited by aspirin. Cells were pre-incubated with PDTC, a specific inhibitor of NF-κB, for 1h, then incubated with TNF-α(10ng/ml)for anther 1h. Western blot and QRT-PCR were used to detect the protein expression and gene expression of MMP-9. The results showed that the protein and gene expression of MMP-9 was significantly decreased after treating with PDTC inhibitor. Conclusions: Aspirin has an inhibitory effect on the expression of MMP-9 and no cytotoxic effect on cells. Moreover, the effect of aspirin on the expression of MMP-9 in TNF-α-induced RAW264.7 cells may be involved with mediating the MAPK and NF-κB signal transduction pathway.