Tanshinone IIA alleviates atherosclerosis in LDLR−/− mice by regulating efferocytosis of macrophages
Jiarou Wang, Yifan Zhang, Xiaoteng Feng, Min Du, Sijin Li, Xindi Chang, Ping Liu
Abstract
Background: Tanshinone IIA (TIIA) is the major lipid-soluble active ingredient of the traditional Chinese medicine Salvia miltiorrhiza , which slows down atherosclerosis (AS). However, it remains unclear whether TIIA has the potential to enhance macrophage efferocytosis and thereby improve atherosclerosis. Objective: The focus of this examination was to determine if TIIA could reduce lipid accumulation and treat AS by enhancing efferocytosis. Methods: Firstly, we conducted in vivo experiments using LDLR knockout (LDLR −/− ) mice for a period of 24 weeks, using histopathological staining, immunofluorescence and Western blot experiments to validate from the efficacy and mechanism parts, respectively; in addition, we utilized cells to validate our study again in vitro . The specific experimental design scheme is as follows: In vivo , Western diet-fed LDLR −/− mice for 12 weeks were constructed as an AS model, and normal diet-fed LDLR −/− mice were taken as a blank control group. The TIIA group and positive control group (atorvastatin, ATO) were intervened for 12 weeks by intraperitoneal injection (15 mg/kg/d) and gavage (1.3 mg/kg/d), respectively. In vitro , RAW264.7 cells were cultured with ox-LDL (50 ug/mL) or ox-LDL (50 ug/mL) + TIIA (20 uM/L or 40 uM/L). Pathological changes in aortic plaques and foam cell formation in RAW264.7 cells were evaluated using Masson and Oil Red O staining, respectively. Biochemical methods were used to detect lipid levels in mice. The immunofluorescence assay was performed to detect apoptotic cells and efferocytosis-related signal expression at the plaques. RT-qPCR and Western blot were carried out to observe the trend change of efferocytosis-related molecules in both mouse aorta and RAW264.7 cells. We also used the neutral red assay to assess RAW264.7 cells’ phagocytic capacity. Results: Compared with the model group, TIIA decreased serum TC, TG, and LDL-C levels ( p < 0.01), reduced the relative lumen area of murine aortic lipid-rich plaques ( p < 0.01), enhanced the stability of murine aortic plaques ( p < 0.01), reduced ox-LDL-induced lipid build-up in RAW264.7 cells ( p < 0.01), and upregulated efferocytosis-related molecules expression and enhance the efferocytosis rate of ox-LDL-induced RAW264.7 cells. Conclusion: TIIA might reduce lipid accumulation by enhancing the efferocytosis of macrophages and thus treat AS.