Hydroxysafflor yellow A inhibits lipopolysaccharide-induced vascular smooth muscle cell-derived foam cell formation through the NLPR3/IL-1β/PCSK9 signaling pathway via activation of autophagy

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To the Editor: Atherosclerosis (AS), the pathological basis of most cardiovascular diseases (CVDs) and the leading cause of death and disability worldwide, is a complex disease involving multiple mechanisms. Previous studies have indicated that phenotypic switching of vascular smooth muscle cells (VSMCs) leads to a lack of VSMC marker cell forms, such as macrophage-like cells, which directly facilitate AS.[1] Macrophage-like cells derived from VSMCs accelerate AS by inhibiting lipid clearance, promoting cell death, and exacerbating inflammation.[2] Therefore, preventing the formation of VSMC-derived foam cells may represent a novel treatment strategy for AS and associated CVDs. Proprotein convertase subtilisin/kexin type 9 (PCSK9), the third most prevalent genetic cause of autosomal dominant familial hypercholesterolemia, aggravates AS and CVDs. The low-density lipoprotein (LDL) receptor (LDLR) facilitates the uptake of LDL by cells, which is the main pathway for the clearance of circulating cholesterol, resulting in a protective effect against hypercholesterolemia and AS. PCSK9 promotes LDLR degradation, thereby preventing LDLR recirculation to the cell surface and leading to an increase in circulatory LDL-cholesterol (LDL-C) levels.[3] Downregulation of PCSK9 expression ameliorates AS by inhibiting the formation of VSMC-derived foam cells. Autophagy, a self-regulatory process that destroys and degrades proteins and organelles in lysosomes, plays an essential role in AS. Activation of autophagy can inhibit AS and the formation of VSMC-derived foam cells. Deficient autophagy promotes AS by aggravating macrophage inflammation mediated by NOD-like receptor protein 3 (NLRP3) inflammasome activation and interleukin (IL)-1β secretion, thereby promoting PCSK9 expression.[4] Hydroxysafflor yellow A (HSYA), the main active compound derived from safflower, is widely used to treat CVDs. HSYA has various beneficial effects, including antioxidant and anti-inflammatory properties. Moreover, HSYA exerts a protective role in CVDs by activating autophagy and inhibiting NLRP3 inflammasome activation. In our previous study, we found that HSYA can inhibit NLRP3 inflammasome activation through the toll-like receptor 4/nuclear factor κB (TLR4/NF-κB) pathway.[5] However, the specific role and underlying molecular mechanisms of HSYA in preventing the formation of VSMC-derived foam cells have yet to be determined. Therefore, in this study, we investigated the activity of HSYA in VSMCs treated with lipopolysaccharide (LPS) and 3-methyladenine (3-MA, an autophagy inhibitor). Cell viability was assessed by cell counting kit-8 (CCK-8) assay, and VSMC-derived foam cells were evaluated using Oil Red O staining. Western blotting, real-time quantitative polymerase chain reaction (qPCR), and immunofluorescence assays were used to determine the expression of PCSK9, LDLR, and the assembly and activation of the NLRP3 inflammasome. Cellular autophagy was assayed using monodansylcadaverine (MDC). Statistical differences were analyzed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA) or SPSS software (version 26.0; IBM Corp, Endicott, New York, USA). Cell activity measurements determined that the optimal concentration and treatment time of HSYA were 30 µmol/L, and 24 h, respectively [Supplementary Figure 1A, https://links.lww.com/CM9/C629]. Further Oil Red O staining showed that HSYA significantly downregulated VSMC-derived foam cell formation induced by LPS [Supplementary Figure 1B, https://links.lww.com/CM9/C629]. Enhanced autophagy facilitates excessive lipid clearance, and reduces lipid accumulation and foam cell formation by VSMCs. To assess the effects of HSYA on autophagy during foam cell formation by VSMCs, the autophagy inhibitor 3-MA was used. Oli Red O staining indicated that HSYA significantly lowered LPS- and 3-MA-induced lipid accumulation and foam cell formation [Figure 1A and Supplementary Figure 2A–C, https://links.lww.com/CM9/C629]. The MDC assay results indicated that both LPS and 3-MA inhibited autophagy, whereas HSYA had the opposite effect by increasing the intensity of autophagy-fluorescence spots [Figure 1B]. To further investigate the effects of HSYA on autophagy, the protein levels of key molecules involved in the autophagy signaling pathway, including microtuble-associated protein light chain 3 (LC3), sequestosome 1 (p62), and Beclin 1, were analyzed by Western blotting. HSYA significantly inhibited the upregulation of LAMP1 and p62 protein and the downregulation of Beclin 1 and LC3 induced by LPS or 3-MA [Figure 1C and Supplementary Figure 2D, https://links.lww.com/CM9/C629].Figure 1: HSYA inhibits LPS-induced VSMC-derived foam cell formation through the NLRP3/IL-1β/PCSK9 signaling pathway via autophagy activation. (A) Oil Red O staining of the VSMCs in five groups (n = 6). Bar = 50 µm. (B) MDC assayof autophagosomes (blue) in VSMCs. Bar = 100 µm. Data are presented as the mean ± SEM (n = 6). (C) Western blotting assay of LAMP1, Beclin 1, p62, and LC3(A/B) (n = 6). (D) Western blotting analysis of the protein levels of NLRP3, ASC, and Caspase-1 (n = 6), ns: non-specific band. Analysis of PCSK9 (red) (E) and LDLR (green) (F) in VSMCs by immunofluorescence staining. n = 6, Bar = 200 µm. 3-MA: 3-Methyladenine; ASC: Apoptotic-associated speck-like adaptor protein with a caspase recruitment domain; Caspase-1: Cysteinyl aspartate specific proteinase 1; HSYA: Hydroxysafflor yellow A; IL-1β: Interleukin-1β; LC3: microtuble-associated protein light chain 3; LPS: Lipopolysaccharide; LDLR: Low-density lipoprotein receptor; MDC: Monodansylcadaverine; NLRP3: NOD-like receptor protein 3; p62: sequestosome 1; PCSK9: Proprotein convertase subtilisin/kexin type 9; VSMC: Vascular smooth muscle cell.Enhanced autophagy is known to significantly inhibit NLRP3 inflammasome activation. To assess the effects of HSYA on NLRP3 inflammasome activation in VSMCs, the expression of NLRP3 inflammasome-related proteins (NLRP3, apoptotic-associated speck-like adaptor protein with a caspase recruitment domain [ASC], and cysteinyl aspartate specific proteinase 1 [Caspase-1]) was assessed by Western blotting. HSYA greatly inhibited the upregulation of NLRP3, ACS, and Caspase-1 expression induced by LPS or 3-MA [Figure 1D and Supplementary Figure 2E, https://links.lww.com/CM9/C629]. NLRP3 inflammasome activation leads to the production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNFα), IL-6, and IL-1β. The qPCR results showed that HSYA profoundly inhibited the increased gene expression of these pro-inflammatory cytokines induced by LPS and 3-MA [Supplementary Figure 2F, https://links.lww.com/CM9/C629]. The NLRP3 inflammasome can regulate PCSK9 secretion via IL-1β, ASC, and Caspase-1. LDLR and PCSK9 exert essential effects on cholesterol uptake and metabolism in VSMC foaming. Therefore, the expression of LDLR and PCSK9 was determined using Western blotting, and the results demonstrated that LPS and 3-MA upregulated PCSK9 expression and downregulated LDLR expression that was significantly reversed by HSYA treatment [Figure 1E]. VSMCs, which are crucial cell types involved in the progression of AS, undergo phenotypic switching into foam cells during AS development. Lipid accumulation in VSMCs leads to their transformation into foam cells and aggravates AS. HSYA has shown promise in the treatment of AS by modulating the function of various cell types involved in the disease, including macrophages, vascular endothelial cells, VSMCs, and platelets. Inflammation plays a critical role in the formation of VSMC-derived foam cells. Thus, we used LPS stimulation to induce the transformation of VSMCs into foam cells, thereby confirming that HSYA significantly inhibited LPS-induced VSMC-derived foam cell formation [Supplementary Figure 1, https://links.lww.com/CM9/C629]. Autophagy plays a crucial role in lipid metabolism and the pathogenesis of AS. Autophagy has been shown to

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