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.