The Pharmacological Mechanism Of Leech Enzymolysis Extract And Icariin In Inhibiting Atherosclerosis Development

ObjectiveCardiovascular diseases rank first on the list of cause of death in elder people all over the world. This affects the living quality of patients gravely. Around 9.4 million people die from cardiovascular diseases, especially in low and middle income countries.In 2008, 17.3 million people die from cardiovascular diseases, and this accounted for 30% of global death. As predicted this number will raise to 23.3 million in 2030. And cardiovascular diseases will be the single first cause of death. Atherosclerosis (AS) is common in clinical. It is also regarded as the pathological fundament of some kinds of cardiovascular diseases such as coronary heart disease (CHD), cerebrovascular disease(CVD) and thrombus.One of the most well-known hypothesis of AS is endothelial dysfunction hypothesis. Endothelial dysfunction descripts the improper activation of endothelium.It takes endothelial dysfunction as the first step of AS initiation. Modified LDL permeates into intima from the gap of endothelium. And the improper activation of endothelial cells expresses more adhesion molecules such as E-selectin, P-selectin,ICAM-1, VCAM-1 and so on. Over expressed adhesion molecules lead to easier adhesion of blood cells (such as monocytes) to endothelium. This is followed by monocytes migrating into intima,which is driven by chemokines released from endothelial cells and other inflammatory cells. After migration into intima,monocytes can also proliferate and differentiate into macrophages with the action of colony stimulating factor (CSF). The macrophages devour modified LDLs and other lipid particles by phagocytosis. And overloading of cholesterol turns them into foam cells.Deposition of macrophages, foam cells and lipid particles, which makes the earliest stage of AS, the fatty streak. As this progress goes on, more and more cells and lipid particles deposit in intima, and the fatty streak grows into bigger lesion. Meanwhile,macrophages and foam cells release cytokines and chemokines to recruit more inflammatory cells and smooth muscle cells (SMCs) into intima. This makes the lesion more complex and complicated. Macrophages, foam cells and apoptosis cell debris and lipid particles form the core of a plaque. And SMCs proliferate and release extracellular matrix (ECM) to form the cap of a plaque that covering the core. Complicated plaques can be classified into two types according to their thickness of cap, stable plaque and unstable plaque. Stable plaque has a thick cap and is not tending to rupture. It will grow with time and get larger. This results in a narrower inner lumen which leads to ischemia of related tissues and organs. Unstable plaque has a thinner cap and is easier to rupture which results in a thrombus.Medicine leech is a traditional Chinese medicine. It is used for thousands of years.As the traditional Chinese medicine records it plays a role of blood-breaking and blood stasis removing. Although it is well accepted that medicine leech has an anti-thrombus effect, its application in non-thrombus diseases is not well explained.leech enzymolysis extract (LEE) is prepared by enzymolysis. The effect of LEE in atherosclerosis is not known.This study is to illuminate the anti-atherosclerosis effect of LEE with animal cellular and molecular experiments.Methods(1) ApoE knockout mice were used as model animals to investigate whether LEE can inhibit progression of atherosclerosis.ApoE knockout mice were fed with high cholesterol diet for 12 weeks. At age of 18 weeks, ultrasound was taken to confirm that animals had atherosclerotic plaque formation. The animals were sorted according to their weight,and divided into seven groups with a random number table:Blank control group: do not give any drugs, still continue to give high cholesterol feed;leech enzymolysis extract (LEE) group: given 0.02g/kg, 0.1g/kg and 0.5g/kg of LEE;Icariin group: given 30mg/kg and 60mg/kg of icariin;Simvastatin group: given 10mg/kg simvastatin.Mice was continuely fed for 12 weeks, at age of 30 weeks, the anatomy was carried out.(2) Ultrasound biomicroscopy to evaluate the effect of LEE on atherosclerosisUBM were carried out before sacrifice. The plaque formation at the aortic arch was measured and the intravascular diameter of the aortic arch was evaluated to evaluate the anti-atherosclerosis effect of the drugs.(3) Frozen tissue slice of oil red O staining to evaluate the effect of the drugs on atherosclerosisTake the mouse heart and make frozen tissue sections. The oil red O staining method shows the plaque size of the aortic root to evaluate the anti- atherosclerosis effect of the drug.(4) Frozen tissue sections of MOMA-2 staining to evaluate the effect of the drugs on atherosclerosisThe MOMA-2 antibody was used to show the infiltration of mononuclear macrophages in the aorta to evaluate the anti- atherosclerosis effect of drugs and the possible mechanism.(5) ELISA was used to detect the cytokines associated with the migration and migration of monocytes and macrophages in mouse serum.(6) Construction of TNF-?-induced endothelial cell dysfunction model.The role of LEE in endothelial cells was studied by using TNF-?-stimulated endothelial cell activation to establish a cell model of endothelial cell dysfunction.(7) Adhesion test to verify the inhibitory effect of LEE on abnormal activation of endothelial cells.(8) migration experiments to verify the inhibitory effect of LEE on abnormal activation of endothelial cells.(9) Western blot was used to detect the cytokines associated with endothelial cell adhesion and migration.(10) to detect LEE inhibition of endothelial cell abnormal activation of NF?B activation and nuclear transfer effect.(11) to detect LEE inhibition of MAPK signaling pathway in the relevant protein p38, ERK, JNK phosphorylation.(12) qPCR was used to detect the expression of CX3CR1 and CX3CL1 in mice aortic wall of Icariin group.(13) Western blot was used to detect the expression of CX3CR1 and CX3CL1 in the mice vessel wall of Icariin group.(14) Gene chip expression profile analysis of Icariin on LPS - stimulated macrophages RAW264.7 gene expression differences.(15) qPCR was used to detect the expression of CX3CR1 and CX3CL1 in LPS -stimulated macrophages RAW264.7.(16) Western blot was used to detect the expression of CX3CR1 and CX3CL1 in the RAW264.7 macrophages stimulated by Icariin.(17) migration assay confirmed that Icariin could inhibit the migration of macrophage RAW264.7.Results(1) UBM observe and measure the ID/OD ratio of aortic arch, LEE (0.1g/kg and 0.5g/kg) and icariin (30mg/kg and 60mg/kg) group has a larger ID/OD ratio compared to control group.(2)Oil red O staining results shows that LEE (0.5g/kg) and icariin (30mg/kg and 60mg/kg) has a smaller ratio of plaque area to the aortic cross.(3)MOMA-2 staining results shows that LEE (0.1g/kg and 0.5g/kg) and icariin(60mg/kg) group has less infiltration of monocytes and macrophages in AS plaque.(4) ELISA was used to detect the levels of VCAM-1, ICAM-1, CX3CR1 and CX3CL1 in serum of ApoE null mice, the dose level of LEE (O.1g/kg) and high dose level (0.5g/kg) Significantly reduced the levels of VCAM-1 and ICAM-1 in serum, but had no significant effect on CX3CR1 and CX3CL1. The high dose level of Icariin(60mg/kg) could significantly decrease the levels of CX3CR1 and CX3CL1 in the serum of mice, but had no significant effect on VCAM-1 and ICAM-1.(5) The endothelial cell dysfunction model was established by using TNF-? to establish endothelial cell dysfunction model. The transcription level of eNOS induced by different TNF-? concentration was detected by PCR with eNOS transcription. The induction concentration of TNF-? was lOng / ml , While the LEE treatment dose of 200?g/ml.(6) Compared with the control group, TNF-? could significantly increase the adhesion of THP-1 to endothelial cells, while LEE could decrease the adhesion rate and inhibit the adhesion of THP-1 to endothelial cells.(3) Compared with the control group, TNF-? could significantly induce the migration of THP-1 to endothelial cells in the Transwell assay, while LEE could inhibit the migration of THP-1 to endothelial cells.(8) Western blot was used to detect ICAM-1 and chemokine MCP-1, which were expressed by adhesion and migration in endothelial cells. The results showed that TNF-? could significantly induce the expression of ICAM-1 and MCP-1 in endothelial cells,while LEE could decrease the expression of ICAM-1 and MCP-1.(9) The NF-?B activation and the nuclear transfer of p65 subunit were detected by fluorescence staining and Western blot. The results showed that TNF-? could induce p65 to transfer intranuclearly and induce TNF-? after LEE treatment LEE group, p65 to the nucleus of the transfer of the situation has been significantly inhibited. This indicates that LEE can inhibit NF-?B activation.(10) Western blot was used to detect the effect of LEE on the phosphorylation of p38, ERK and JNK in MAPK signaling pathway. The results showed that LEE could inhibit the phosphorylation of ERK and JNK.(11) Real-time quantitative PCR and western blot were used to detect the levels of CX3CR1 and CX3CL1 in the aortic wall of mice. The results showed that icariin could decrease the levels of CX3CR1 and CX3CL1 in the aortic wall of ApoE knockout mice(12) gene chip analysis showed that the treatment of icariin could make the expression of CX3CR1 in LPS-induced macrophage RAW264.7.(13) Real-time quantitative PCR and western blot analysis of CX3CR1 expression in RAW264.7 cells showed that Icariin treatment could significantly inhibit LPS-induced CX3CR1.(14) Trans well migration experiments showed that icariin could inhibit CX3CL1-induced macrophage migration in a dose-dependent manner.Conclusions(1) LEE and icariin can significantly inhibit the progression of AS;(2) LEE and Icariin could significantly inhibit the infiltration of monocytes and macrophages in AS plaques.(3) LEE and icariin can affect the levels of adhesion molecules and chemokines in serum(4) LEE can inhibit TNF-a-induced endothelial cell dysfunction(5) LEE can inhibit the activation of NF?B and the nuclear transfer of p65 subunit in TNF-a-induced endothelial cells;(6) LEE can inhibit the phosphorylation of ERX and JNK in MAPK signaling pathway.(7) Icariin could decrease the levels of CX3CR1 and CX3CL1 in the aortic wall of ApoE knockout mice.(8) Icariin could decrease the expression of CX3CR1 in LPS-induced macrophages.(9) Icariin can inhibit the migration of macrophages induced by CX3CL1.LEE inhibits the activation of NFr?B by phosphorylation of ERK and JNK in MAPK signaling pathway in endothelial cells, and decreases the expression of ICAM-1 and MCP-1, Nuclear cells to endothelial cells adhesion and migration, so that the AS plaque mononuclear macrophages infiltration, and play its anti-atherosclerotic effect.Icariin reduces the expression of CX3CR1 in macrophages and reduces the migration of macrophages under the action of chemokine CX3CL1, thereby infiltrating mononuclear macrophages in AS plaques and exerting its anti-atherogenic Hardening effect.