The Effects Of Leech Extracts On The Expression Of MMP-2,9, And 13 In PMA Stimulated THP-1 Cells And Its Mechanism Of Action

Due to the aging of population, unhealthy dietary style, lack of exercises and environmental pollution, cardiovascular disease has become a leading cause of morbidity and mortality worldwide. Atherosclerosis is the important pathologic basis of cardiovascular disease. As a kind of chronic inflammatory disease, the main pathological symptom of atherosclerosis is the gradual formation of lipid plaque in the lumen side of arterial wall, with a fibrous cap adhering above it. Plaque rupture could lead to myocardial infarction, stroke, peripheral vascular disease and a series of complications.Leech (Whitmania pigra Whitman) is a traditional Chinese medicine, commonly used to eliminate blood stasis. According to Chinese pharmacopoeia, leech can be applied to treat amenorrhea, stroke, hemiplegia and traumatic injury. Our previous study has proven leech extracts (LE) could inhibit the progression of atherosclerosis. Lots of research showed matrix metalloproteinases (MMPs) may play an important role in atherosclerosis. On account of our study basis and research trends, this subject aimed at illuminating the effect of LE on MMPs expression in PMA induced THP-1 cells and its mechanism. The experimental process and results are as follows:1. LE inhibited the invasion ability of PMA-induced THP-1 cellsCell model was established by stimulating THP-1 with PMA. PMA treatment could induce this round suspension cell type to form flat adherent macrophages which express more MMPs. Then, this model is used to study cell invasion ability with a 0.8 μm Transwell assay. To be more specific, cells are cultured on upper side of the Transwell which has been pretreated with Matrigel to simulate extracellular matrix. So cell invasion ability can be estimated based on the amount of cells migrated to the lower side of the Transwell by decomposing Matrigel. Experimental results showed PMA treatment enhanced THP-1 invasion ability and LE could inhibit this effect.2. LE could suppress the MMPs expression and activity in PMA induced THP-1 cellsCell supernatant was collected to detect MMP-2 and MMP-9 secretion by using gelatin zymography. Western blotting and RT-PCR were applied to study the MMP-9 and MMP-13 expression. Experimental results showed LE could weaken the MMPs secretion and expression induced by PMA in THP-1 cells.3. LE inhibited MMPs expression by suppressing the EMMPRIN and MAPK signaling pathwayEMMPRIN is a MMPs inducer located on the surface of cells. It can promote the expression of MMPs in macrophages. Immunofluorescence and western blotting were used to assess the EMMPRIN expression in PMA induced THP-1 cells. Results showed EMMPRIN level was suppressed by LE treatment. Moreover, research also revealed that LE suppressed EMMPRIN and MMPs expression by inhibiting the MAPK signaling pathway in PMA induced THP-1 cells.In conclusion, LE could inhibit cell invasion ability by reducing the MMPs expression in macrophages. This powerfully explains the anti-atherosclerosis effect of LE which has been demonstrated in our previous animal experiments. Also this study provides a new theoretical support for the leech therapy in treating atherosclerosis.