Atherosclerosis is a cardiovascular disease that poses a major threat to human health. It is characterized by the accumulation of lipids on the blood vessel wall and the infiltrationof immune cells. Pathophysiological studies have identified arterial endothelial injury and lipid deposition as initiating factors of atherosclerosis. Foam cell formation is a core feature of atherosclerosis pathology 16, 17. Foam cells are mainly formed by macrophage phagocytosis of cholesterol and lipid peroxides, and these then form the main component of plaque lipid cores 2, 3,which in turn play a key role in the formation and development of atherosclerosis. Excessive accumulation of apoptotic cells and insufficient phagocytic clearance of macrophages have been suggested to be the main causes of the formation and development of unstable plaques in atherosclerosis. Reducing the entry of extracellular cholesterol into cells, reducing foam cell formation, and enhancing macrophage efferocytosis may delay the progression of atherosclerosis 18, 19. The current study explored the anti-atherosclerosis effects and mechanism of SIRT1 in primary mouse peritoneal macrophages.
SIRT1 inhibits atherosclerosis
Silent information regu1ator 2 (Sir2) is a newly discovered NAD + dependent deacylase, and there are currently at least seven mammalian Sir2 homologs (SIRT1–SIRT7). Among these, SIRT1 shows the highest homology with Sir2, and is widely present in various somatic and germ cells. SIRT1 is an important nuclear protein involved in the regulation of metabolism, inflammation, oxidative stress, cellular senescence, and apoptosis.Previous studies showed that the SIRT1 agonist, resveratrol, had anti-atherosclerotic effects 14. SIRT1 can also enhance macrophage phagocytosis, reduce macrophage ox-LDL uptake and reduce intracellular lipid deposition 20. However, the mechanism by which SIRT1 enhances macrophage phagocytosis to slow plaque progression is unclear.
In this study, we showed thatSIRT1 reduced macrophage lipid phagocytosis and inhibited apoptosis during the formation of foam cells, while enhancing macrophage efferocytosis to achieve phagocytosis of apoptotic cells, thus inhibiting foam macrophage generation and the formation and development of atherosclerotic plaques.
SIRT1 exerts anti-atherosclerosis effects by regulating macrophage polarization.
As innate immune and inflammatory cells, macrophages are involved in the whole process of atherosclerosis. Macrophages can differentiate into M1 or M2 macrophages, according to their activation mode and immune function. M1 macrophages express IL-6 and Dectin1, underexpress IL-10, secrete inflammatory cytokines including TNF-α and IL-1β, participate in antigen presentation, and express iNOS and ROS, which are involved in the inflammatory response and pathogen clearance. In contrast, M2 macrophages overexpress the inflammatory inhibitory factors IL-10 and TNF-β, scavenger receptor, mannose receptor, galactose receptor, and Arg-1, inhibit the inflammatory response, and promote tissue damage repair 21, 22. M1 and M2 macrophages both exist in atherosclerotic lesions. M1 and M2 macrophages can be transformed under certain conditions, and it was recently shown that regulating the transformation of M1 into M2 macrophages allowed macrophages to exert their immune defense function and clear apoptotic and necrotic cells, and thus actively maintain the stability of atherosclerotic plaques. Statins, peroxisome proliferator-activated receptors, and other drugs have been shown to induce M1 macrophages to differentiate into M2 macrophages, and to promote this transformation by regulating the functional differentiation of macrophages 23, 24. This transformation reduces M1-induced inflammatory damage to the vessel wall and increases the anti-inflammatory abilities of M2 macrophages to promote the repair of atherosclerotic inflammation 25, 26. This previous study also found that the angiotensin II-1 receptor accelerated the progression of renal atherosclerosis by regulating the proportion of M1/M2 macrophages and affecting macrophage efferocytosis27.
The current results showed that expression levels of the M1 macrophage phenotype markers IL-6, TNF-α, and iNOS were significantly increased by ox-LDL, while expression levels of the M2 markers IL-10, Dectin-1, and Arg-1 were decreased, macrophage apoptosis increased, and cytotoxicity decreased. The above results were reversed by SIRT1 overexpression. These results indicated thatSIRT1 regulated macrophage phenotypic transformation to antagonize the inflammatory response, thereby reducing apoptosis and enhancing macrophage efferocytosis.
SIRT1 exerts anti-atherosclerosis effect by macrophage polarization via TXNIP/NLRP3 inflammatory pathway.
NLRP3 is one of the most important inflammatory complexes discovered in recent years. Cholesterol crystallization has been shown to participate in the early stage of atherosclerosis by activating the NLRP3 inflammatory complex in macrophages. The NLRP3 inflammatory complex can be produced in large quantities by ROS-mediated TXNIP activation. The TRX system is a widely distributed NADPH-dependent disulfide reductase system consisting of TRX, TRX reductase, and reduced coenzyme II (NADPH II). TRX is the main cellular antioxidant stress molecule protecting tissues or cells from multiple stimuli. TXNIP is a TRX-binding protein that mediates oxidative stress, suppresses cell proliferation, and induces apoptosis by inhibiting the function of the TRX system. Previous studies showed that myocardial ischemia-reperfusion injury leads to a massive release of ROS, while TXNIP mediates the abnormal activation of the NLRP3 inflammatory complex and aggravates the inflammatory response of myocardial ischemia-reperfusion injury 28. In addition, resveratrol has been shown to promote SIRT1 expression to inhibit ROS production and reduce ischemic oxidative stress in the heart.
We investigated the role of the TXNIP/NLRP3pathway in the inhibitory effect of SIRT1 through macrophage polarization using theNLRP3 inhibitor MCC950. We showed that ox-LDL stimulation significantly increased ROS level, IL-1β level, TXNIP and NLRP3 protein expressions in macrophages, and that these effects were reversed by SIRT1 overexpression. Ox-LDL treatment shifted the macrophage phenotype from M2 to M1, attenuated macrophage efferocytosis, and increased macrophage apoptosis, whereas ox-LDL combined with SIRT1 or MCC950 pretreatment decreased the proportion of M1 macrophages and increased M2 macrophages, thus enhancing the increased macrophage efferocytosis and reducing macrophage apoptosis. SIRT1 thus exerted an anti-atherosclerosis effect by inhibiting the TXNIP/NLRP3 inflammatory pathway.
In summary, SIRT1 regulated macrophage polarization to enhance macrophage efferocytosis, inhibit macrophage lipid uptake, and reduce apoptosis. The underlying mechanisms of SIRT1 may involve inhibition of the TXNIP/NLRP3 inflammatory pathway, which could provide novel ideas and targets for the treatment of atherosclerosis.