ATO reduces pyroptosis in the aorta of ApoE-/-mice and macrophages
Previous studies showed that apoptosis caused by NLRP3 inflammasomes promotes the development of atherosclerosis. Caspase-1 activation, which converts IL-1β into its mature form, is a critical step (Zhang et al. , 2018). First, we investigated whether the expression of pyroptosis-related proteins in the aorta was altered by ATO. Notably, the NLRP3 levels in the aorta were significantly reduced, caspase-1 activation was inhibited, and the abundance of the mature form of IL1β (c-IL-1β) was reduced (Figure 5A, D, B, E). Consistently, the NLRP3 and IL-1β mRNA expression levels in the aorta were significantly reduced in ATO-treated mice fed a high-fat diet (Figure 5F). Furthermore, we performed CD68/IL-1β and CD68/TUNEL double staining of aortic sinuses isolated from ApoE-/- mice. The IL-1β levels in, and proportion of TUNEL-positive cells among, macrophages (CD68+) were significantly reduced after ATO treatment in mice fed a high-fat diet (Figure 5K, L, Figure S1H, I).To elucidate the relationship between ATO and pyroptosis, we used THP-1 cells for the in vitro experiments. We treated these cells with ox-LDL (50 μg mL-1) to mimic the atherosclerotic environment in vitro . The results showed that ox-LDL significantly increased the expression levels of NLRP3, C-cas1, and C-IL-1β (Figure 5C, G, H,I,J,M), while ATO treatment (2.5 or 5.0 μM) reversed this increase. Consistently, THP-1 cells treated with ATO (2.5 or 5.0 μM) showed similar changes in IL-1β levels in the cell supernatant (Figure 5N). Taken together, these data indicate that ATO inhibits caspase-1 activation, thereby reducing the secretion of mature IL-1β and inhibiting the pyrolysis of macrophages in aortic lesions of ApoE-/- mice(Figure 5O).