Recent studies have shown that most patients with cardiovascular diseases are normally accompanied by elevated plasma TMAO levels, which not only reflects the atherosclerotic plaque burden but also determines the incidence of cardiovascular events such as stroke and ischemic stroke and the prognosis of the disease to a certain extent [25] .As a study analyzing plasma TMAO levels in patients with ischemic stroke showed that patients with higher plasma TMAO at admission tended to have larger infarct volume and more severe stroke degree [26].In addition, A study including 4007 subjects strongly demonstrated that plasma TMAO levels possessed higher predictive value than traditional factors of atherosclerotic disease, and the correlation was much stronger than triglycerides, lipoproteins, fasting blood glucose, and triglycerides[27, 28]. Increased TMAO levels were associated with an increased risk of death within 5 years even after adjusting for traditional risk factor levels [29]. Based on this, most experts have reached a consensus that TMAO is regarded as a novel biomarker of cardiovascular diseases and adverse events independent of traditional risk factors [30]. Based on the close relationship between TMAO and cardiovascular diseases, a research upsurge about the mechanism of TMAO and AS diseases has been gradually launched in the medical field. TMAO has been confirmed that it can accelerate the progression of atherosclerotic plaque by reducing the expression of hepatic bile acid transporter genes Cyp7a1 and Cyp27a1, which leads to the dysfunction of cholesterol clearance in atherosclerotic plaque, thus interferes with lipid metabolism and inducing inflammation, which is often manifested as dysfunction of macrophages, smooth muscle cells and vascular endothelial cells and the release of inflammatory factors[7].Some recent studies have shown that the elevating TMAO level is often accompanied by the high expression of inflammatory factors ,the elevation of NLRP3 level, and the activation of the NF-κB pathway in aortic plaque ,endothelial cells and smooth muscle cells, which provides strong evidence for this study [31, 32].
Plaque formation and destability are the inevitable results of long-term chronic inflammatory stimulation of blood vessels, with the process of foam cell expression and release of inflammatory factors. A previous study has identified the activation of NLRP3 inflammasome as a key factor in enlarging lipid cores and accelerating plaque rupture. In this study, we confirmed that TMAO accelerated the inflammatory process of macrophages induced by ox-LDL, which was related to the activation of the NLRP3 inflammasome by TMAO. Part of studies have confirmed the relationship between excessive ox-LDL or cholesterol crystals in plaque and activation of NLRP3 inflammasome in macrophages. As an intracellular pattern recognition receptor, NLRP3 can be recognized by cholesterol crystals and subsequently recruits inactive pro caspase-1 via ASC bridging role, then activated by active caspase-1 and mediates the release of IL-1 family cytokines, thus regulating innate immune and inflammatory responses. Inflammatory factors such as IL-1 β stimulated macrophage smooth muscle cells to secrete matrix metalloproteinases (MMPs) and Tumor necrosis factor-α(TNF-α)and CD40-L synergistically degrade collagen caps to accelerate the unstable progression of AS plaque[33]. On the contrary, in some animal experiments of NLRP3, ASC, and IL-1 β gene knockout, the lymphocyte infiltration, lipid content and the level of inflammatory markers such as IL-18 in plaque were still lower than those in Wild mice and showed higher collagen content although were fed with high glucose and high fat. Without NLRP3, the aortic plaque of mice almost no longer appeared [34, 35]. These results confirmed the key role of NLRP3 inflammasome in initiating plaque inflammation and accelerating the progression of pathological plaque rupture. In the present study, we first treated macrophages with concentration gradient and time gradient and determined 400umol/L as the follow-up experimental condition. Secondly, added TMAO can further accelerate the NLRP3 inflammasome activation and IL-18, IL-1 β secretion in ox-LDL-induced macrophages. Then, treating with MCC950 can significantly inhibit the promoted effect of TMAO on the release of IL-18 and IL-1 β in ox-LDL -induced macrophages, which suggested that TMAO can accelerate the macrophage’s inflammatory process by activating NLRP3 inflammasome under the action of ox-LDL. The results of this study provided a new perspective for the potential mechanism of TMAO accelerating the progression of AS and affirmed the key role of NLRP3 in this process, then made up for the blank of TMAO in the research field of foam cell NLRP3 inflammatory body.
Then we further investigated the potential mechanism of TMAO accelerating the activation of NLRP3 inflammasome in ox-LDL-induced macrophages. Previous studies have shown that NF-κB, as a key regulatory factor in inflammatory-related gene transcription, plays an important role in activating the NLRP3 inflammasome [36]. It has been established that ox-LDL can promote the transcription level of NLRP3 by enhancing the phosphorylation of NF-κB and then improve the expression of NLRP3-related proteins and the activation of inflammasome [37–39]. Likewise, inhibition of NF-κB also significantly reduced the activation of NLRP3 inflammasome [40]. Based on this research, we examined whether NF-κB pathway mediated the process of NLRP3 inflammasome activation promoted by TMAO in the present paper. The expression of p-NF-κB and T-NF-κB in the ox-LDL group and TMAO + ox-LDL group were separately detected by WB to determine the phosphorylation degree of NF-κB under TMAO treatment. TMAO treatment promoted the phosphorylation of NF-κB in macrophages under ox-LDL, but did no effect on the total protein, suggesting that TMAO did not affect the total NF-κB level in the cytoplasm but enhanced the phosphorylation of NF-κB by promoting the release process of NF-κB from IKB and its entry into nucleus. Likewise, we further treated cells with JSH-23, a NF-κB specific phosphorylation inhibitor, down-regulated p-NF-κB levels to inhibit the promotive effect of TMAO on the activation of NLRP3 inflammasome under ox-LDL. These data suggested that TMAO enhanced ox-LDL-induced NLRP3 inflammasome activation in macrophages partly due to its enhanced NF-κB phosphorylation process.
Finally, our results confirmed the key role of PERK activation in mediating TMAO to promote ox-LDL-induced NF-κB phosphorylation and NLRP3 inflammasome activation in macrophages. PERK is one of the classical pathways in ERS, a form of endoplasmic reticulum dysfunction, caused by accumulating unfolded or misfolded proteins in the endoplasmic reticulum cavity. Although short-term ERS is considered an adaptive compensatory mechanism, persistent or excessive ERS often lead to inflammation, apoptosis, and cytopathic changes. ERS has been regarded as the common mechanism of cell dysfunction induced by many risk factors and involved in the occurrence and development of many metabolic diseases such as Atherosclerosis, diabetes, obesity, and neurodegeneration by IRE1, PERK, and ATF6 pathways [41] .As mentioned above, PERK/elf-2a is one of the classical pathways in unfolded protein response (UPR) and most studies have established that PERK/elf-2a branch can couple ER stress with apoptosis through CHOP, and participate in the process of apoptosis [42]. More importantly, some recent studies have shown that there seems to be a certain correlation between ERS and NF-κB pathway, which is mainly reflected in ATF6 activating NF-κB to trigger the transcription of inflammatory factors in nucleus and activating PERK to release NF-κB via p-elf-2a. As the most extensive branch of ERS, most studies focus on the activation of PERK, which reduces the transcription level of IkB by phosphorylating the downstream molecule elf-2a, resulting in the decrease of IkB synthesis, which leads to the release of a large number of NF-κB from its inhibitor IkB and enters the nucleus to be phosphorylated, thus regulating the transcription process of inflammatory factor genes[43]. In addition, PERK can be used as a direct receptor of TMAO to participate in the progression of metabolic diseases[44]. Therefore, we speculate whether PERK pathway involve in regulating the pro-inflammatory effect of TMAO in ox-LDL-induced macrophages. Consistent with the speculated, our results provided preliminary evidence that TMAO treatment significantly promoted the expression and mRNA level of endoplasmic reticulum stress marker Bip, and PERK in macrophages induced by ox-LDL, which suggesting that PERK/NF-κB may be involved in the process of TMAO accelerating the activation of NLRP3 inflammasome and cell inflammation in macrophages induced by ox-LDL in vitro. Pretreated with 4-PBA can inhibit ERS and then down-regulate TMAO-induced activation of NF-κB and NLRP3 inflammasome. Our results suggested that endoplasmic reticulum stress was involved in TMAO accelerating the activation of NLRP3 inflammasome and phosphorylation of NF-κB in ox-LDL-induced macrophages.
In conclusion, as shown in Fig. 5, TMAO promoted ox-LDL-induced activation of the NLRP3 inflammasome and release of IL-1β and IL-18 in THP-1 macrophages via ER stress and the NF-κB pathway. It is undeniable that our conclusions will be completer and more reliable if this study can fill the gap about lacking vivo study, which is what we seek in our future studies. Our findings suggest that the PERK/NF-κB axis plays an important role in the NLRP3 inflammasome activation and IL-18 、IL-1β release in ox-LDL-treated macrophages promoted by TMAO, which may be useful for targeting gut microbiota points to a new direction for the clinical prevention of atherosclerosis.