Objective: To investigate the process where quercetin suppresses atherosclerosis by upregulating MST1-mediated autophagy in Organic264. the manifestation of MST1, which increase was clogged by quercetin, which offered a potential system where quercetin may shield foam cells against age-related harmful effects. Summary: Quercetin can inhibit the forming of foam cells induced by ox-LDL and hold off senescence. The system could be linked to the rules of CD2 MST1-mediated autophagy of Natural264.7 cells. < 0.05; ** < 0.01. 2.2. Quercetin Delayed Senescence and Reduced the Accumulation CRAC intermediate 2 of Lipid in RAW264.7 Cells Oil red O and SA–gal staining were used to detect the effects of QUE on lipid accumulation and senescence in RAW264.7 cells. As shown in Figure 2A,B, there was a large amount of red staining lipid accumulation in the M group (ox-LDL-treated) compared with the Con group (untreated), indicating that 100 g/mL ox-LDL successfully induced the foam cell model. Furthermore, the addition of QUE to ox-LDL-induced foam cells (M + Q group) significantly decreased the lipid accumulation. The results of the SA-beta-gal staining assay also demonstrated that the number of positive staining cells in the M + Q group was significantly lower than that in the M group (Figure 2C,D), which confirmed these findings. We further studied the effect of QUE on the expression of P16 and P21. The results of immunofluorescence revealed more protein aggregation of P16 and P21 in the M group; however, after using QUE, the protein aggregation of P16 and P21 decreased (Figure 3A,C).The results of Western blot showed that the expression of each of these markers of senescence was increased dramatically in the M group, and that the expression in the M + Q group was significantly lower than that in the M group (Figure 3D,F). Therefore, the results suggested that QUE can effectively delay the senescence of ox-LDL-induced RAW264. 7 cells and significantly reduce intracellular lipid accumulation. Open in a separate window Figure 2 Quercetin can delay senescence of RAW264.7 cells and reduce the accumulation of intracellular lipid. (A) Oil red O staining. (B) Intracellular lipid deposition. (C) SA–gal staining. (D) Percentage of SA–gal positive stained cells. Con, control; M, model; Q, quercetin; M + Q, model + quercetin. Data are presented as means SD, * < 0.05; ** < 0.01. Open in a separate window Figure 3 Expression of P21 and P16 in macrophage cells detected by immunofluorescence and Western blot. (A) Immunofluorescence. (B,C) Results of P21 and P16 immunofluorescence. (D,E) Results of P21 and P16 Western blot. (F) Western blot. Con, control; M, model; Q, quercetin; M + CRAC intermediate 2 Q, model + quercetin. Data are presented as means SD, * < 0.05; ** < 0.01; *** < 0.001. 2.3. Inhibition of Autophagy Promoted the Lipid Accumulation and Senescence of RAW264.7 Cells Therefore, we used 3-MA (3-methyladenine) to study the role of autophagy deficiency in ox-LDL-treated RAW264.7 cells. The results demonstrated that inhibition of autophagy aggravated the lipid accumulation in ox-LDL-treated RAW264.7 cells (Figure 4A,B). Consistently, SA--gal staining showed more positive staining cells (Figure 4C,D), and the expression of P16 and P21 protein increased significantly (Figure 5ACF). These total results suggested that inhibition of autophagy promoted lipid accumulation and senescence in RAW264.7 cells. Open up in another window Shape 4 3-MA advertised senescence of Natural264.7 cells and aggravated accumulation of intracellular lipid. (A) Essential oil reddish colored O staining. (B) Intracellular CRAC intermediate 2 lipid deposition. (C) SA--gal staining. (D) Percentage of SA--gal-positive stained cells. Con, control; M, model; 3-MA, 3-methyladenine; M + 3-MA, model + 3-methyladenine. Data are shown as means SD, * < 0.05. Open up in another window Shape 5 Manifestation of P21 and P16 in macrophage cells recognized by immunofluorescence and Traditional western blot. (A) Immunofluorescence. (B,C) Outcomes of P21 and P16 immunofluorescence. (D,E) Outcomes of P21 and CRAC intermediate 2 P16 Traditional western blot. (F) Traditional western blot. Con, control; M, model; 3-MA, 3-methyladenine; M + 3-MA, model.