The Research Of Effect And Mechanism Of Leech Enzymatic Extraction On The Foam Cell Of Atherosclerosis

Lecch is often used in the treatment of cardiovascular disease in clinical, as a remedy for blood stasis and traumatic. Our lab had acquired low molecular leech enzymatic extraction through controlled enzymolysis technology with anticogulant, prevention and treatment of stroke, and resistance to atherosclerosis. This study was based on the previous. We constructed foam cells and observed the inhibition of leech enzymatic extraction for foam cell formation, and further explored its action mechanism. The main works were as follows:1. Induced foam cell formationUsing different concentrations of oxLDL induced RAW246.7macrophage cell line form foam cell, and oil red O dye and cholesterol content determination methods were used to identification the formation of foam cells. With the increase of oxLDL dose, the percent of foam cell formation was increasing, when the concentration of oxLDL was80ug/ml, intracellular CE/TC>50%, and granules of oil red O staining in intracellular was accumulated, so the optimum concentration of oxLDL was80ug/ml. Since oxLDL was an oxidative lipoprotein cholesterol, not only increase the lipids, also could cause oxidative damage to cells. With the increase of concentration of oxLDL, part of the cell nucleus appeared chromatin condensation by Hoechst33342dye. To investigate the oxLDL oxidative damage, mitochondrial transmembrane potential detection by Flow cytometry instrument showed the decrease after oxLDL treatment. OxLDL could make DNA oxidizative fracture, produce500bp DNA fragments in the DNA ladder. The results showed that the80ug/ml was optimum to induce macrophage form foam cell, and oxLDL could damage foam cell in some extent.2. The effect of leech enzymatic extraction on foam cellsThe red granules in cells were decreased significantly after leech enzymatic extraction treatment by oil red O staining, and intracellular TC, FC and CE content were also decreased, with the CE/TC<50%, the results showed that leech enzymatic extraction could reduce the lipid content in the foam cells, and inhibite the formation of foam cells. Hoechst33342dye found the percent of nucleus deformation was decrease; mitochondrial transmembrane potential decline had been inhibited in a certain degree; DNA ladder showed DNA fragmentation was less. The above results showed that the leech enzymatic extraction had inhibitory effect on the formation of foam cells, and anti-oxLDL oxidative damage.3. The mechanism of inhibition of leech enzymatic extraction on foam cell formationMDC staining results found that cell fluorescent particles were increased after leech enzymatic extraction treatment with foam cells, but after treated with autophagy inhibitor3-MA, fluorescent particles were decrease. Western blot showed the ratio of LC3â…¡/LC3I was rise after leech enzymatic extraction treatment; The ratio was decrease after treated with3-MA and the oil red O staining granules and cholesterol level was increase after3-MA treatment. At the same time, according to the results of mitochondrial transmembrane potential and DNA ladder after3-MA treatment, the effect of inhibiting membrane potential decrease was restrained, segments of DNA fragments were increase, the above results showed that the leech enzymatic extraction inhibite foam cell formation and oxLDL oxidative damage was through the process of autophagy.ACAT1, ABCA1and LXRa transcription results showed that leech enzymatic extraction could not influence the transcription level of ABC A1and LXRa. But leech enzymatic extraction could raise the level of AC ATI, and the effect was restrained after3-MA treatment. The above results demonstrated that the leech enzymatic extraction could not influence the cholesterol efflux, but could increase the AC ATI transcription level, this process was associated with autophagy process. The effect of leech enzymatic extraction was dependent on autophagy further confirmed.The experimental results displayed that leech enzymatic extraction could effectively reduce the lipid content in cells, and inhibition foam cell formation, and also protect the cell from oxidative damage. The mechanism was dependent on the intracellular autophagy. Thus this study provided theoretical basis and guidance for leech clinical application.