| In recent years,the incidence and mortality of cardiovascular diseases have been increasing significantly in the world,and the prevention and treatment of cardiovascular diseases is facing severe challenges.Atherosclerosis(AS)is a chronic vascular inflammatory disease,which has been regarded as the main pathological basis for the development of cardiovascular diseases.Immunology suggests that the migration of macrophages to the intima of the blood vessels is an important step in the development of AS.If we could inhibit the migration of macrophages to inflammatory blood vessels,it will be possible for early prevention and treatment of AS with little impact on the immune system.Leech is a traditional Chinese medicine with many biological functions such as anti-coagulation,anti-thrombotic and anti-inflammatory,and has been widely used in the treatment of various cardiovascular diseases.Our previous study found that Hirudo extraction(HE)can significantly reduce the area of AS plaques in aorta of ApoE-/-mice and the number of macrophages in plaques.We speculate that the mechanism of its effect may be related to inhibition of macrophage migration to inflammatory vascular intima.Therefore,this study was designed to purify a leech peptide that could inhibit the migration of LPS-induced RAW264.7 cells and explored the mechanisms underlying this effect.The main experimental contents and results are as follows:1.Isolation,purification and synthesis of HE4-1In this experiment,HE was prepared from Whitmania pigra Whitman by enzymatic hydrolysis.Transwell results showed that HE can inhibit the migration of LPS-induced RAW264.7 cells in a dose dependent manner.Subsequently,we used Q Sepharose FF exchange column chromatography,Superdex 30,Superdex peptide and G10 gel column chromatography to separate and purify HE.Through activity tracking,we finally obtained a peptide with the activity of anti-migration,and named it as HE4-1.The results of HPLC and mass spectrometry showed that HE4-1 had a purity of 98%,a molecular weight of 142.9.294 Da,and an amino acid sequence of EAGSAKELEGDPVAG.We also found that 800 ?g/mL synthetic peptide HE-D could also inhibit the migration of LPS-induced RAW264.7 cells.2.Study on the mechanism of HE-D inhibiting macrophage migrationPresent studies have shown that JNK and p38 signaling pathways are involved in the regulation of macrophages migration.In this study,potential mechanisms of HE-D on cell migration in response to LPS stimulation in RAW264.7 cells were investigated by Western blot.The results showed that 800 ?g/mL HE-D could significantly inhibit the expression of phosphorylated protein of JNK and p38,suggesting that HE-D can inhibit the migration of macrophages via JNK and p38 signaling pathways.3.Study on the effect of HE on the biological function of RAW264.7 cellsMacrophages play a key role in the immune response of humans.When exogenous microorganisms invade the human body,macrophages can regulate the inflammatory response by phagocytizing,lysing bacteria and releasing pro-inflammatory factors.In this study,the effects of HE4-1 and HE-D on the immune function of macrophages were examined by neutral red phagocytosis assay,lysozyme activity assay and ELISA assay.Our results showed that 0-800 ?g/mL HE4-1 and HE-D had no significant effect on macrophage phagocytosis,lysozyme activity and secretion of IL-1,IL-6 and IL-12,but 400,800 ?g/mL HE4-1 and HE-D can promote the secretion of TNF-a.In summary,a leech pepetide named HE4-1 was purified from Whitmania pigra Whitman,which can inhibit the migration of LPS-induced RAW264.7 cells through JNK and p38 signaling pathways.In addition,HE4-1 and HE-D had no significant effect on macrophage phagocytosis,lysozyme activity and IL-1,IL-6,IL-12 secretion.This topic may provide new theoretical support for the application of traditional Chinese medicine leeches in the early prevention and treatment of AS. |
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