EMPA stabilizes the established AS in ApoE -/- mice
To evaluate the therapeutic role of EMPA in AS in mice, we employed ApoE −/− mice to generate an in vivo model of AS. The aortic arch, thorax and abdomen of the four groups were processed for H&E staining. According to the staining results shown in Fig. 1, the atherosclerotic plaque lesion size of ApoE -/- mice in the EMPA group was significantly smaller than that in the control group. The higher dose of EMPA had a more profound affect than the lower dose. These findings indicated that EMPA stabilizes established atherosclerotic plaques in ApoE -/- mice.
EMPA inhibits oxLDL-induced formation of foam cells and decreases the synthesis of proinflammatory cytokines by promoting autophagy in the RAW 246.7 cells
The formation of macrophage-derived foam cells is a critical step in the development of AS [22]. To identify the effects of EMPA on lipid accumulation, RAW264.7 cells were treated with oxLDL (80 µg/mL) for 24 h. In RAW 264.7 cells, the presence of Oil Red O staining increased in the oxLDL group, but the amount of lipid accumulation in the EMPA group was significantly reduced compared to the oxLDL group (Fig. 2A), suggesting that EMPA inhibits the formation of oxLDL-induced macrophage-derived foam cells. To further clarify the mechanism by which EMPA affects the progression of AS, we analyzed the effect of EMPA on autophagosomes using a GFP-LC3 puncta assay. oxLDL treatment (80 µg/ml) for 24 h followed by incubation with EMPA (50 µM) increased the GFP-LC3 green fluorescent puncta in the cytoplasm of RAW 264.7 cells (Fig. 2B, C). Western blotting was also used to detect the levels of autophagy-related proteins in RAW 264.7 cells. After treatment of RAW 264.7 cells with EMPA for 24 hours, the expression levels of Beclin1 was increased, LC3B-II/I ratio was also augmented, and the expression of P62 decreased (Fig. 2D-G). These results suggested that EMPA may induce autophagy in RAW 246.7 cells. We next investigated the effect of 3MA as shown in Fig. 2F-I. Compared to the EMPA group, Oil Red O staining was significantly increased in the 3MA group, and western blot analysis demonstrated that the expression level of inflammatory factors in the 3MA group was also significantly increased. These results demonstrated that EMPA inhibits the formation of macrophage foam cells and the expression of inflammatory factors via autophagy induction in RAW 264.7 cells, thus playing an antiatherosclerotic role.
EMPA Hinders the OxLDL-induced Inflammatory Response in HUVECs by Inducing Autophagy in HUVECs
Endothelial dysfunction is the key cause of AS. Endothelial dysfunction can cause lipid accumulation, inflammation and AS. Therefore, the reduction in proinflammatory cytokine synthesis reflects the effect of EMPA on AS [23]. As depicted in Fig. 3A and C, the expression of proinflammatory cytokines (TNF-α and IL-6) in the EMPA group was significantly reduced compared to that in the oxLDL group.
To further explore the mechanism by which EMPA affects inflammatory factors, we used western blot analysis and green fluorescent puncta experiments. There were more green fluorescent puncta in the EMPA group compared to the oxLDL group. Compared to the oxLDL group, western blot analysis showed that the protein expression of LC3B was significantly increased in the EMPA group, while the protein expression of P62 was decreased in the EMPA group (Fig. 3A, B, D, E). These findings showed that EMPA promotes autophagy in HUVECs. To explore whether EMPA affects the expression of proinflammatory cytokines in HUVECs through autophagy induction, we treated HUVECs with 3MA. Western blot analysis demonstrated that the expression of proinflammatory cytokines was increased after treatment with 3MA (Fig. 3A, C), suggesting that EMPA inhibits the oxLDL-induced inflammatory response in HUVECs by inducing autophagy.
EMPA inhibits the formation of HASMC-derived foam cells induced by OxLDL by promoting autophagy and inhibiting the proliferation and migration of HASMCs
Smooth muscle cells are an important source of foam cells. To explore the effect of EMPA on lipid accumulation in HASMCs, Oil Red O staining experiments were performed. Compared to the blank control group, Oil Red O staining was significantly increased in the oxLDL group, and treatment of HASMCs with EMPA significantly reduced the lipid accumulation compared to the oxLDL group (Fig. 4A). These results suggested that EMPA reduces the oxLDL-induced lipid accumulation, indicating that EMPA may have an inhibitory effect on the lipid accumulation of HASMCs.
To further clarify whether EMPA affects the autophagy of HASMCs and whether it inhibits the foaming of HASMCs by inducing autophagy, we transfected GFP-LC3 into cells and observed the number of autophagosomes. The protein expression of autophagy-related proteins was also determined by western blot analysis. Similar results were obtained, indicating that EMPA promoted autophagy in HASMCs (Fig. 4B-E). Moreover, the autophagy inhibitor, 3MA, weakened the effect of EMPA in inhibiting the foaming of HASMCs (Fig. 4F). The above results indicated that EMPA inhibits oxLDL-induced HASMC foam cell formation by promoting autophagy.
Because the proliferation and migration of HASMCs accelerate the process of AS [24], we tested the effect of EMPA on the proliferation and migration of HASMCs. The CCK-8 cell viability assay indicated that treatment of HASMCs with EMPA for 24 h resulted in inhibition of cell proliferation (Fig. 5A). Similarly, the EdU experiment demonstrated that the proportion of proliferating cells was significantly reduced after HASMCs were treated with EMPA (Fig. 5B,C). Subsequently, the effect of EMPA on cell cycle progression was investigated. The addition of EMPA (50 and 100 µM) blocked HASMCs in the G0/G1 phase of the cell cycle as indicated by an increase in the proportion of cells in the G0/G1 phase and a decrease in the proportion of cells in the S phase, indicating that EMPA inhibits HASMC DNA synthesis (Fig. 5D, E). Using scratch assays, we further tested the effect of EMPA on the migration of HASMCs. The results showed that the healing rate of cell scratches was slower than that of the control group after EMPA treatment (Fig. 5F, G). The above results indicated that EMPA inhibits the foaming of HASMCs via autophagy induction as well as the proliferation and migration of HASMCs.