Discussion and Conclusions
At present, atherosclerosis and subsequent acute myocardial infarction are the most frequent causes of death worldwide (Hansson et al. , 2011; Grines, 2015). Therefore, the development of drugs that target lipid accumulation and the subsequent inflammatory cascade observed during the progression of this disease is urgently required. Over the last two decades, statins have emerged as effective drugs for reducing atherosclerotic lipoprotein levels and preventing major cardiovascular events. However, various adverse reactions, including rhabdomyolysis, hepatotoxicity, peripheral neuropathy, impaired myocardial contractility, and autoimmune diseases, have been reported to occur after the use of statins. Therefore, it is necessary to re-evaluate the existing statin treatment guidelines (Bellosta et al. , 2018). In recent years, clinical and laboratory studies have focused on the use of ATO as a heart stent coating. These studies found that ATO-eluting stents can significantly reduce the area and thickness of the neointima (Shen et al. , 2013). Another study found that placing ATO-eluting stents in the coronary arteries of pigs effectively inhibited local inflammation (Zhao et al. , 2018).These findings suggest that the anti-inflammatory effects of ATO may represent an effective protective strategy for atherosclerosis. In this study, for the first time, we indicated that ATO could suppress the progression and instability of atherosclerotic plaques by reducing CD36-mediated ox-LDL internalisation, inhibiting inflammatory responses and pyroptosis in macrophages and plaques. Accordingly, our results have revealed a pivotal role of ATO in suppressing the development of atherosclerosis, suggesting the use of ATO as a potential pharmacological atheroprotective strategy.
In our previous studies, we confirmed that ATO promoted autophagy in macrophages without affecting their viability in vitro(Fang et al., 2021) . In addition, we confirmed that an appropriate ATO dose would not cause liver and kidney damage or toxic changes in various organs; therefore, our chosen dosage was not significantly toxic and might be clinically useful. In the present study, we first subjected ApoE-/- model mice with atherosclerosis to treatment with ATO. The results showed that ATO significantly reduced atherosclerosis development and altered the plaque composition by reducing macrophage accumulation and increasing collagen content. This evidence highlights the potent effects of ATO against atherosclerosis.
The TC, TG, and LDL levels significantly affect the incidence of major vascular events and the development of atherosclerosis in humans (Landray et al. , 2014). Lowering TC and LDL levels can prevent atherosclerosis progression (Sarwar et al. , 2007). Additionally, many studies demonstrated that elevated TG levels represent an independent risk factor for coronary heart disease. Recently, CD36 has been reported to serve as an effective pharmacological target as it promotes lipid accumulation in the vascular wall and the internalisation of ox-LDL in macrophages (Febbraio et al. , 2000; Koelwyn et al. , 2018). Moreover, a study of medullary chimeras showed that atherosclerotic lesion formation was significantly reduced in mice receiving CD36-null macrophages, while the reintroduction of CD36 in macrophages doubled the area of ​​atherosclerotic lesions (Febbraio et al. , 2004). These studies suggest that lipid metabolism and CD36 expression are important therapeutic targets for atherosclerosis. Our results indicated that ATO markedly reduced the TC, TG, and LDL levels in ApoE-/- mice. In addition, ATO inhibited CD36 expression at the transcriptional and translational levels in vivo and in vitro ; thus, ATO reduced foam cell formation. These results suggest ATO inhibits atherosclerotic plaque progression by regulating blood lipid homeostasis and CD36 expression. However, in addition to CD36, previous studies showed that other members of the scavenger receptor family, including ABCA1, ABCG1, and SR-B1, can prevent foam cell formation and the development of atherosclerosis by promoting cholesterol efflux from macrophages (Navab et al. , 2011; Hazen et al. , 2012; Huang et al. , 2019; Ouimet et al. , 2019). Whether ATO can act on other scavenger receptor family members remains to be explored.
Our study demonstrates that ATO treatment suppresses inflammatory factor expression and reduces inflammatory responses. Previous studies have indicated that pro-inflammatory cytokines are involved in all stages of atherosclerosis, and vulnerable plaques are rich in inflammatory cells. Moreover, IL-6 signal transduction occurs through multiple mechanisms, including the release of other pro-inflammatory cytokines, which stimulates acute phase protein secretion and prethrombotic mediator release. Another study demonstrates that TNFα blockers have a beneficial effect on preventing the progression of subclinical atherosclerosis and arterial stiffness (Tam et al. , 2014; Tie et al. , 2015). Here, we show that ATO reduces IL-6 and TNF-α expression and increases IL-10 expression significantly in vivo and in vitro , which further emphasizes the anti-inflammatory and atheroprotective nature of ATO.
TLR4 is a classic pattern recognition receptor for activating macrophages, and its role in the occurrence and development of atherosclerotic lesions has been extensively studied (Li et al. , 2017). Previous studies showed that TLR4 and CD36 have a synergistic effect in mediating ox-LDL-induced inflammation. Activated TLR4 triggers the activation of downstream signalling molecules, leading to the nuclear translocation of p65 and the transcription of genes encoding various pro-inflammatory cytokines, including TNF, IL-6, and IL-12 p40 (Monaco et al. , 2004). Here, we provided the first evidence that ATO inhibited inflammatory responses in macrophages by modulating the TLR4/NF-κB signalling pathway. In this study, we demonstrated that ATO significantly reduced TLR4 expressionin vivo and in vitro . In vitro , ATO inhibited the nuclear translocation of p65 by inhibiting IκB-α degradation, similar to the action of BAY-11, an IκB-α degradation inhibitor. Our results suggest that the suppression of TLR4/NF-κB signalling pathway activation may be the specific mechanism by which ATO inhibits atherosclerotic inflammation in macrophages.
Duewell et al. transplanted NLRP3-/- and IL-1α/β-/- bone marrow cells into LDL-deficient mice and found that the knockdown of these inflammatory components reduced the formation of atherosclerotic lesions (Christ et al. , 2018). Similarly, caspase-1 deficiency significantly reduces macrophage infiltration and the formation of atherosclerotic lesions in ApoE-/-mice (Usui et al. , 2012). Other studies showed that NF-κB activation triggered IL-1β and NLRP3 transcription and ox-LDL could induce IL-1β release (Latz et al. , 2018; Zhang et al. , 2018). Based on these premises, we proposed whether ATO played an atheroprotective role by inhibiting the pyroptosis caused by NLRP3 inflammasome activation. Firstly, we observed that macrophages in ApoE-/- model mice aortic tissue showed characteristics of pyroprosis. In addition, we demonstrated that ATO inhibited NLRP3 expression at the transcriptional and translational levels in vivo and in vitro . Expression of mature caspase-1 and IL-1β, which were downstream effector molecules of NLRP3, were also reduced by ATO treatment in vivo and in vitro . Taken together, these results indicate that ATO inhibits IL-1β secretion and caspase-1 activation by NLRP3 inflammasomes. Hence, we demonstrate the underlying protective effects of ATO against atherosclerosis and provide mechanistic insights into its atheroprotective effects.
However, this study had a few limitations. Firstly, whether ATO inhibits NLRP3 transcription by inhibiting the TLR4/NF-κB signalling pathway lacks direct evidence and is worthy of further investigation. Secondly, in this study, we focused on the role of ATO in regulating lipid metabolism and inflammatory responses in atherosclerosis. However, other mechanisms, such as those underlying macrophage activation or other processes related to the atherosclerosis progression, need further investigation. Thirdly, although our chosen dosage is not significantly toxic and may be clinically useful in this experimental period, it remains unknown whether the side effects of ATO are cumulative over time. Herein, whether long-term treatment of ATO has side effects needs further observation. Lastly, there are important differences between animals and humans, and thus, additional work is needed to fully explore whether the results obtained herein are also applicable in humans.
In summary, we confirmed that ATO had protective effects against the progression of atherosclerosis and instability of atherosclerotic plaques in ApoE-/- model mice. Thus, the use of ATO to inhibit macrophage lipid endocytosis and inflammatory responses may represent a potential atheroprotective strategy.