Validation experiment designed to validate causal mechanisms targeting P2RY12 in ApoE-deficient mice. Primary outcome: autophagy activity in VSMCs in vivo
This in vivo experiment validated the autophagy mechanism by treating ApoE-deficient mice with P2RY12 inhibitors in combination with the autophagy inhibitor chloroquine. The study aimed to demonstrate that P2RY12 receptor inhibition promotes autophagy in VSMCs through the PI3K-AKT-MTOR pathway in advanced atherosclerosis. Mice were assessed for autophagy markers in vascular tissue and atherosclerotic plaque characteristics.
Animals: ApoE-/- mice, 8-10 weeks, n=12 per group. Groups: (1) Vehicle (saline). (2) Ticagrelor (100 mg/kg/day, oral). (3) Chloroquine (CQ, 60 mg/kg/day, i.p., autophagy inhibitor). (4) Ticagrelor + CQ. Diet: High-fat diet (60% kcal fat) for 12 weeks. Treatment starts at week 4 (after plaque initiation). Euthanasia at week 12. Plaque analysis: Aortic root sections (5 μm), Oil Red O for lipid area, α-SMA for VSMC content. Quantify foam cell area (α-SMA+ cells with lipid droplets). Autophagy in vivo: (1) Isolate aortic arch, dissect medial layer, Western blot for LC3-II/I, p62, p-MTOR, p-S6K. (2) Immunofluorescence on aortic sections: LC3 puncta (antibody staining), colocalize with α-SMA. Count LC3+ puncta per VSMC (n=50 cells per mouse).
...Quantitative predictions: (1) Vehicle (HFD): Plaque area 450,000 μm², LC3-II/I ~0.4 (low autophagy in diseased VSMCs), p62 elevated 2-fold vs. normal chow. (2) Ticagrelor: Plaque reduced 35%, LC3-II/I increases to 0.7-0.9 (restored autophagy), p62 decreases 50%, TEM autophagosomes 4-6 per cell (vs. 1-2 in vehicle). (3) Chloroquine alone: Worsens plaque (increase 20-30%), LC3-II accumulates (0.8-1.0, but flux blocked, not functional autophagy), p62 further elevated, autophagosomes rare but autolysosomes visible on TEM.
...Primary: Ticagrelor reduces plaque area by >25% vs. vehicle (p<0.05, one-way ANOVA), and this effect is significantly attenuated by CQ cotreatment (ticagrelor+CQ differs from ticagrelor alone, p<0.05; reduces benefit by >50%). Secondary: (1) Ticagrelor increases LC3-II/I in aortic tissue by >50% (p<0.01). (2) CQ alone does not reduce plaque (p>0.1 vs. vehicle or increases plaque, p<0.05). (3) p62 levels: inversely correlate with treatment benefit (ticagrelor reduces, CQ increases, R² >0.6).
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