Effect of moxibustion at Danzhong(CV17) at different time scales on platelet activation in ApoE -/- mice overexpressed with GPVI lentivirus

Abstract

Objective: To observe the effect of moxibustion at Danzhong (CV17) at different time scales on the levels of molecular markers of platelet activation in ApoE-/- mice with atherosclerosis by tail vein injection of GP6 overexpression lentivirus, so as to explore whether moxibustion can improve atherosclerosis by reducing the level of platelet activation. Methods: A total of 63 ApoE-/- mice aged 8 weeks were randomly divided into model, moxibustion and clopidogrel groups, with 21 mice in each group. Another 21 8-week-old C57BL/6 mice with the same genetic background were used as the control group and fed with normal diet. The mice in the moxibustion group were treated with moxibustion at Danzhong (CV 17) for 20min/ day, the mice in the medication group were treated with clopidogrel solution 14mg/kg by gavage once a day, and the mice in the model group were treated with sham moxibustion. The intervention lasted for 5 days/week. The blank group received no additional intervention. We collected samples from five mice after 4, 8, and 12 weeks of intervention. One week before sampling, ApoE-/- mice were injected with 100μl GP6 lentivirus at a titer of 1.27×109 V.G./ml at 4, 8 and 12 weeks, and C57BL/6 mice were injected with 100μl EGFP fluorescent expression plasmid at 4,8 and 12 weeks. After 48h of injection, the intervention was continued for 5 days, after which the mice were sacrificed. The heart and thoracic aorta were taken from the sacrificed animals, and were stained by HE staining and Oil red "O" staining. Then, the pathological tissue were used for quantitative analysis of aortic plaque. The fluorescence transfection of bone marrow cells was observed under a fluorescence microscope to indirectly evaluate the success of lentivirus transfection in vivo. The platelet-rich blood were detected by flow cytometry for observing the expression levels of platelet activation molecular markers CD63, CD62p and CD154. Results: After 4 weeks of moxibustion intervention, the levels of CD63 and CD154 were down-regulated, and the levels of CD63 and CD154 in the moxibustion group were significantly lower than those in the clopidogrel group (P < 0.0001), and the level of CD63 in the moxibustion group was lower than that in the control group (P > 0.05). After 8 weeks of moxibustion intervention, the levels of CD63, CD62P and CD154 were down-regulated, and the levels of CD63 and CD62P were significantly lower than those in the clopidogrel group, and were close to the levels in the control group (P > 0.05). The levels of CD63, CD62P and CD154 in the 12-week moxibustion group were higher than those in the clopidogrel group, but there was no statistically significant difference (P > 0.05), suggesting that after over-expression of GPVI injection in vivo and continuous intervention for 12 weeks, the down-regulation effect of moxibustion on platelets was less than that of clopidogrel group. Conclusion: Moxibustion therapy has a certain inhibitory effect on platelet activation, which can effectively slow down the progress of atherosclerosis by reducing the platelet activation rate. The intervention effect has the characteristics of a time scale. Conclusion: Moxibustion therapy has a definite inhibitory effect on platelet activation, which can effectively slow down the progression of arteriosclerosis by reducing the platelet activation level, and the intervention has time-scale characteristics. The effect of moxibustion for the treatment of atherosclerosis by inhibiting platelet activation is more obvious in the early stage of the disease.