Study On Isolation,Purification And Mechanism Of Hirudin Peptide With Activity Of Inhibiting Macrophage Migration

At present,cardiovascular and cerebrovascular diseases have become a very high mortality rate in the world.Among them,atherosclerosis is considered to be the pathological basis of a variety of cardiovascular and cerebrovascular diseases.Modern studies have shown that inflammation inhibition is a new way to treat atherosclerosis(Atherosclerosis,AS).In the immune system,macrophages are the primary cells in AS,and play an important role in the development of AS.If we could inhibit the migration of macrophages to the site of onset of AS,it will be possible for early prevention and treatment and delay the progression of AS with little influence on the immune system.Leech is a traditional Chinese medicine,which has the function of expelling blood and removing blood stasis,which is mainly used in the prognosis of cardiovascular disease.Our previous study confirmed that the HE can reduce the number of macrophages in plaques of ApoE-/-mice,also has significant inhibitory effect on the formation and development of AS.On this basis,the migration of macrophages to endothelial layer in AS development process plays a key role.In this study,we extracted and purified a hirudo peptide which can inhibit the migration of macrophages,and then explored its mechanism.The main research contents are as follows:1 HP was obtained by anion exchange column chromatography and gel column chromatography under the condition of active trackingWe constructed cell migration model with tranwell chambers with LPS-induced Raw264.7 macrophages.HE were obtained by enzymatic treatment of hirudo powder.5 peak components were got via Q Sepharose strongly basic anion exchange chromatography with activity detection based on tranwell assay.The activity of HE31 can inhibit the migration.Then Superdex 30 gel column chromatography was used,followed by Superdex peptide gel column chromatography.HP obtained by Sephadex G10 gel column chromatography.The results of transwell assay and scratch test showed that HP had the activity of inhibiting LPS-induced migration of Raw264.7 macrophages.2.HP plays an important role in inhibiting macrophage migration via MAPK pathwayIn order to investigate the effect of HP on inhibiting the migration activity of LPS induced macrophage,the combination of HP with 50%inhibitors of MAPK,JNK,p38 ERK,NF-?B and Src respectively or HP alone were detected by transwell on the migration of macrophages induced by LPS.The results showed that the inhibitory effect of superposition does not appear in HP with inhibitors,suggesting that HP plays an active role in macrophage migration may be through p38 MAPK,JNK,has nothing to do with the ERK,NF-?B and Src pathway.3.HP inhibits the activity of macrophage migration by affecting the transcription of MEKK4 and ASK2,Since p38 MAPK and JNK belong to MAPK pathway kinases,detection of p38 and JNK upstream kinase MKK4,MLK3,MEKK3/4,ASK 1/2 transcription levelusing were done via PCR.Results showed that HP may regulate the activity of each kinase to inhibit macrophage migration via modulating transcription of MEKK4 and ASK2 in MAPK pathway.Thus,the inhibition activity of macro experiments is explainedIn summary,a leech peptide HP was isolated and purified from the leech for the first time.We confirmed that HP affects p38 MAPK and JNK through its effect on transcription of MEKK4 and ASK2 in MAPK pathway,thereby affecting the downstream p38 MAPK and JNK.Early prevention of AS in the presence of minimal impact on immune cells is very important for prevention and cure of AS.It is of great theoretical and practical significance for the further development and utilization of Hirudo to provide a new treatment strategy and strategy for early prevention and treatment of AS.