Effect Of Moxibustion On Platelet Activation And Purine Receptor Signal Transduction In Arteriosclerosis Mice With APOE

Objective:To investigate the effects of moxibustion in atherosclerosis model mice on platelet activation and coagulation-fibrinolysis system; And to illustrate the anti-arteriosclerosis mechanism of moxibustion through platelet activation signal transduction pathways which is mediated by P2urine receptor.Methods:Experiment one:Experiments were divided into blank group (C57BL/6 mice), model group, moxibustion danzhong (RN17) group, clopidogrel group (positive group). The model group, moxibustion group and clopidogrel group are selected 15 atherosclerosis ApoE-/- mice in each group, with high fat diet. C57BL/6 mice were selected as control group, and the normal diet was fed with. In the blank group and model group, mice were used to fix the head, tail and limbs of mice by fixing apparatus. These mice were grab and fixed for 20min everyday.After fixing the mouse head, tail and limbs in the group of moxibustion danzhong (RN17), the retainer plate is provided with small holes, exposed point in the middle of the chest of mutton, with special moxa ignited placed underneath the abdomen of mice, fire moxibustion through holes at the bottom of the fixation device is transmitted to the mice and mutton in the hole. The group of moxibustion danzhong (RN17) are fixed the mouse head, tail and limbs Device is fixed at the base plate is provided with small holes, which exposed point in the middle of the chest of mutton. Special ignited moxa is placed underneath the abdomen of mice, the fire through the small hole at the bottom of moxibustion fixation to RN17 for 20min. Positive drug group mice:Oral administration of clopidogrel solution 14mg/kg (times /day). After 6 days/week, the animals were sacrificed and the indexes were detected after 16 weeks.En-face longitudinal section, HE and Red oil ’O’ stain were used to observe the plague area in aorta arch and lipid droplets area ratio, so as to evaluate the anti-arteriosclerosis effect of moxibustion.Experiment two:The experiment group and method steps are same as experiment one. The APOE-/-mice were used in this experiment to observe the expression of platelet activation effectors:CD62p、CD63、 CD40L. Elisa method was used to test the level of t-PA,PAI-l;TXB2,6-Keto-PGFla (playing as significant role in coagulation and fibrinolysis system respectively) and the level of vWF(important factor in platelet activation) in plasma.Experiment three:The experiment group and method steps are same as experiment one. Western blot method was used to detect the PLC, PLC(Ser1105), PI3K, Akt, Akt(Thr308), Akt(Ser473) expression in APOE-/-mice. Elisa method was used to test the level of Ca2+and cAMP in platelet cytoplasm.Results:Experiment one:1. By contrast with model group, moxibustion could significantly reduce the weight of atherosclerosis mice. p< 0.05;2. By contrast with model group, moxibustion could significantly reduce the level of TG、 LDL-c in serum. p<0.05;3. By contrast with model group, moxibustion could enhance the level of HDL-c in serum, p>0.05; could reduce the level of TC, p>0.05;4. Plaque area in different group:model group9.56%, moxibustion group4.25%, the Clopidogrel group0.36%, model group> moxibustion group> Clopidogrel group;5. Lipid Droplet area in different group:model group1.66%, moxibustion group0.96%, Clopidogrel group0.36%, model group> moxibustion group> Clopidogrel group;6. By contrast with model group, moxibustion could reduce the lipid droplets area in the plaque of aortic arch. p>0.05;7. By contrast with model group, moxibustion could significantly alleviate the aortic arch pathological changes. p<0.058. By contrast with the Clopidogrel group(the positive group),moxibustion could significantly reduce the weight of the mice, p<0.05;9. En-face、HE and Red oil ’O’ stain were used to observe the disease degree of the AS:model group> Clopidogrel group>moxibustion group>control group。Experiment two:1.By contrast with model group, moxibustion could significantly inhibit the expression of CD62p. CD40L、CD63,p<0.05;2.By contrast with model group, moxibustion could enhance the level of t-PA in plasma.p>0.053. By contrast with model group, moxibustion could significantly reduce the level of PAI-1 in plasma. p<0.05;4. By contrast with model group, moxibustion could significantly reduce the level of vWF. p<0.05;5. By contrast with model group, moxibustion could significantly increase the level of 6-Keto-PGF1αin plasma. p<0.05;6. By contrast with model group, moxibustion could enhance the level of TXB2 in plasma. p>0.05;7. By contrast with the Clopidogrel group(the positive group),moxibustion could significantly reduce theexpression of CD62p、CD40L, p<0.05。Experiment three:1. By contrast with model group, moxibustion had no obvious effect on the expression of PLC and phosphorylation PLC(Ser1105). p>0.05;2. By contrast with model group, moxibustion had no obvious effect on the expression of Akt and phosphorylation Akt(Thr308). p>0.05;3. By contrast with model group, moxibustion could significantly inhibit the expression of PI3k and phosphorylation Akt(Ser473).p<0.05;4. By contrast with model group, moxibustion could effectively reduce the level of Ca2+ in platelet cytoplasm.p<0.05;5. By contrast with model group, moxibustion had the tendency to reduce platelet cAMP, but showing no obvious effect, p>0.05;6. By contrast with the Clopidogrel group(the positive group),moxibustion significantly enhance the level of expression of phosphorylation Akt(Ser473), p<0.05.Conclusion:1. Moxibustion could benignly regulate the lipid metabolism, inhibit the pathological progress of Atherosclerosis, so as to alleviate occurrence and development of atherosclerosis. The early intervention of Moxibustion has positive significance in both prevention and treatment against atherosclerosis.2. Moxibustion had the effect of inhibiting platelet activation.3. The regulation effect of Moxibustion on platelet activation shows more significant in the late period.4. Moxibustion had good adjustment function on coagulation-fibrinolysis system. By improving the activity of fibrinolysis system, moxibustion could raise the 6-Keto-PGF1α/ TXB2ratio, reduce the adhesion factor VWF, further play to the regulation of platelet activation and prevention of atherosclerosis.5. The effect of moxibustion inhibiting platelet activation might function through the mediation of P2Y12-PI3k-Akt urine receptor signal pathway, which is similar to the action pathway of P2Y12 receptor antagonist clopidogrel.6. Akt(Ser473) protein may be the effective target of moxibustion regulating platelet activation signal pathway.