The Effect Of Exogenous Phosphocreatine On The Formation And Stability Of Atherosclerotic Plaque

Atherosclerosis (AS), which is related to oxidative stress and inflammation, is the common pathological basis of many cardiac-cerebral vascular disease (CCVD).Phosphocreatine (PCr), a high energetic phosphate compound, could supply phosphate group to ATP and transport energy as one of the most direct and main energetic materials inside the body. Nowadays PCr has been applied in heart surgery to provide energy and protect the heart from dysbolism and oxygen free radical caused by cardiac ischemia. Many researches have demonstrated that PCr aslo has good therapeutic effect on heart failure, myocardial infraction, cerebral ischemia, etc. because of its anti-oxidation and membrane stabilizing effects. Besides, a study in vitro indicated that PCr could inhibit the apoptosis of ovarian carcinoma cells (SKOV3 cells) by blocking the cell cycle.A patent published in 2012 in the U.S. gave out an interesting hint that PCr could resolve the AS plaque outside the body. So whether PCr has the effect on the prevention and treatment of atherosclerosis and what the potential mechanisms became an meaningful question. In this study, we simulated AS model both in vivo and in vitro, gave the treatment of PCr i.v. and found that PCr could inhibit atherosclerotic lesion formation and enhance plaque stability, which provide a new choice of clinic medicine in cardiac-cerebral vascular disease and atherosclerosis.Objective1、Observe the effect of exogenous PCr on the formation and stability of atherosclerotic plaque.2、Discuss the potential mechanisms and the effects of PCr on endothelial cells.Methods1、Animal models and groups7-8 weeks male ApoE-/- mice, feeding with high-fat food (0.25% cholesterol and 15% cocoa butter) for 12 weeks. Four weeks after the hight-fat diet, a constrictive silastic collar (length,3mm; inner diameter,0.3mm) was placed around the right common carotid artery near the bifurcation under sterile conditions to establish the advanced carotid atherosclerosis lesion model. Sutured the wounds and gave high-fat food for another 8 weeks. At the end of the 8th week, all the ApoE-/-mice were allocated randomly into five groups:negative control group(0.9% saline 0.1 ml/10g/d); phosphocreatine groups (low dosage:150mg/kg/d; middle dosage: 300mg/kg/d; high dosage:600mg/kg/d; positive control group:simvastatin 50mg/kg/d. After 8 weeks’ high-fat food feeding, mice were injected intravenously into NS and phosphocreatine everyday respectively for four weeks, and simvastatin was added to food. Thereafter, mice were euthanized and tissues were harvested for further experiments.2、Body weight and the serum lipid profileBefore sacrifice, mice were weighted and blood samples were collected from the inferior vena cava of each mouse. Separated the serum and the levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured in the center laborary of Qilu hospital.3、Histopathology and immunohistochemistry stainingAfter blood collection, aorta and carotid artery were separated. Area of atherosclerotic lesions in general aorta was messured by en face oil red O staining and the ratio of positive staining area to the enface area was quantified by Image-Pro Plus 6.0.Frozen sections of aortic root and carotid artery (5μm thickness) were performed for the routine staining:hematoxylin and eosin, oil red O and Sirius red, respectively. Immunohistochemistry was used for the detection of targeted protein, alpha smooth muscle Actin. DHE staining was used to observe the content of ROS.4^ Molecular biology testAortas were collected and a serious cytokines were tested. The contents of MMP-2 and MMP-9 were tested by Western Blot and IL-6, IL-10, TNFa, MCP-1 were measured by BD-CBA kit. The mRNA levels of IL-6, IL-10, TNFa, MCP-1, TIMP-1, MMP-2 and MMP-9 were detected by qRT-PCR. Zymography was used to mesure the activity of pro-MMP-9, MMP-9, pro-MMP-2 and MMP-2.5> Investigate the effects of PCr on endothelia cellsFor EA.hy926 cells, dosage of PCr (20mM) was determined by CCK-8 test. Cells were pretreated by PCr and then stimulated by oxLDL (100μg/ml). The expression of NOX2 and MAPK family members were tested by Western blot, mRNA levels of inflammation cytokines were measured by qRT-PCR, and DCFH-DA and DHE staining coupled with Fluorescence Microscopy and Flow cytometry were performed to measure the generation of ROS in cells.Results1、In comparison to the NS group, PCr could down regulate the total cholesterol(Cho), triglyceride(TG) and low density lipoprotein(LDL-C), but has no significant influence on high density lipoprotein(HDL-C).2、In comparison to the NS group, PCr could significantly decrease the plaque area and the expression of ROS in aorta, aortic arch and aorta root respectively. Plaque were more stabilize in PCr groups.3、In comparison to the NS group, PCr middle dosage group (300mg/kg/d) and high dosage group (600mg/kg/d) decreased the expression of MMP-9, IL-6 and TNF-α (P<0.05) but has no significant effect on MMP-2 and IL-10. The protein level of MCP-1 was reduced only in high dosage group.As for the levels of mRNA, middle dosage group and high dosage group could significantly down regulate IL-6 and TNF- αlevels and rise TIMP-1 and IL-10 mRNA levels. High dosage group could decrease the mRNA level of MCP-1 significantly. (P <0.05)The activities of pro-MMP-9 and MMP-9 were reduced in all three PCr groups, while pro-MMP-2 and MMP-2 seemed not been affected by PCr treatment.4, In EA.hy926 cells, PCr(20mM) could decrease the generation of ROS after stimulated by oxLDL, reduce the expression of NOX2 and mRNA levels of IL-6, and inhibit the phosphorylation of JNK to inactivate the MAPKs signal pathways which could reduce the oxidative stress and inflammation and as a result alleviate the injury of endothelial cells.ConclusionIntravenous injection of PCr play a role in anti-atherosclerosis by inhibiting atherosclerotic lesion formation and enhancing plaque stability, and the mechanism might be related to the regulation of serum lipid, antioxidation and anti-inflammation, which provide a new choice of clinic medicine in cardiac-cerebral vascular disease and atherosclerosis.