Anti‐inflammatory mechanisms and research progress of colchicine in atherosclerotic therapy

Abstract Inflammatory responses play a vital role in the onset and development of atherosclerosis, and throughout the entire process of the chronic disease. The inflammatory responses in atherosclerosis are mainly mediated by the NLRP3 inflammasome and its downstream inflammatory factors. As a powerful anti‐inflammatory medicine, colchicine has a history of more than 200 years in clinical application and is the first‐choice treatment for immune diseases such as gout and familial Mediterranean fever. In atherosclerosis, colchicine can inhibit the assembly and activation of NLRP3 inflammasome via various mechanisms to effectively reduce the expression of inflammatory factors, thereby reducing the inflammation. Recent clinical trials show that a low dose of colchicine (0.5 mg per day) has a certain protective effect in stable angina patients or those with acute myocardial infarction after PCI. This article summarizes and discusses the mechanisms of colchicine in the treatment of atherosclerosis and the latest research progress.

as atherosclerosis research progressed, the role of inflammation in the progression of coronary atherosclerosis became more prominent.

| Lipoproteins deposited subcutaneously in blood vessels activate inflammatory responses
There are many hypotheses about the mechanism of atherosclerosis, and one widely accepted among them is the endothelial injury theory. The hypothesis suggests that a disorder haemodynamics or hypoxia-affected local vasculature could lead to vascular endothelial damage, and apolipoproteins carrying cholesterol can continuously deposit under vascular endothelium with blood circulation. 6 The lipoproteins are very easily oxidized under the intima of blood vessels and mainly composed of oxidized low-density lipoprotein (OxLDL) and cholesterol crystals. 7 OxLDL can induce leukocyte recruitment and activation to promote inflammation. OxLDL could also activate macrophages through the CD36-TLR4-TLR6 complex to promote NLRP3 inflammasome-related inflammatory responses. 8

| Expression of NLRP3 inflammasome
In general, NLRP3 inflammasomes are expressed in myeloid cells, such as monocytes, neutrophils and eosinophils. 12 The expression of NLRP3 inflammasomes is activated by PAMP and DAMP. Rosenfeld ME and Campbell LA reported that infections of different pathogens, including bacteria and viruses, can promote and aggravate the inflammatory response by activating the NLRP3 inflammasome, thereby increasing the risk of cardiovascular disease. 13 Both PAMP and DAMP activate the downstream NF-κB signal transduction pathway through the pattern recognition receptor (PRR) to promote the NLRP3 inflammasome expression. In atherosclerosis, OxLDL can directly activate the downstream NF-κB signal transduction pathway through DAMP to increase the NLRP3 inflammasome of the expression, ASC (apoptosis-associated speck-like protein containing a CARD), pro-IL-1 β and pro-IL-18 ( Figure 1).

| Assembly and activation of NLRP3 inflammasome
At present, it is believed that the activation of the NLRP3 inflammasome is extremely dependent on the intracellular K + concentration: only when it is less than 70 mM can the NLRP3 inflammasome be assembled and activated. When P2X7 receptors are activated by extracellular ATP, they open K + channels and a massive moment of K + flows out. The decrease in intracellular K + concentration provides a basic prerequisite for the assembly and activation of the NLRP3 inflammasome. 14,15 The proteins induced by NF-κB signal transduction pathway, such as NLRP2 and ASC, could then assemble with pro-caspase in the cytoplasm to form the NLRP3 inflammasome. 16 Among them, the micro-tubule plays a critical role in the assembly of NLRP3 and ASC. 17 In addition, cholesterol crystals (CCs) deposited under the damaged endothelium can be swallowed into lysosomes in macrophages through endocytosis, while OxLDL can also be swallowed by macrophages through receptor-mediated endocytosis. OxLDL can be converted into cholesterol crystals in the lysosome to form intracellular CCs. Either intracellular or extracellular CCs can result in lysosomal membrane instability and rupture. 9 At this time, a large amount of cathepsin B in lysosomes will be released into the cytoplasm, thus inducing the activation of the NLRP3 inflammasome. 16,18 Other common upstream NLRP3 inflammasome activation mechanisms include mitochondrial damage, release of cardiolipin and mitochondrial DNA, and release of reactive oxygen species [19][20][21] (Figure 1). can enter the liver through blood circulation and stimulate hepatocytes to synthesize acute-phase reactants, such as fibrinogen and plasminogen activator inhibitor. It can also induce the liver to synthesize a marker of inflammatory state: C-reactive protein. 27,28 As early as 1997, Paul M. et al. used the detection of plasma C-reactive protein levels to predict the risk of patients' future myocardial infarction and stroke. 29 It is currently believed that hsCRP level of greater than or equal to 2 mg/L can be considered as an inflammatory response ( Figure 2).

| ANTI -INFL AMMATORY MECHANIS MS OF COLCHI CINE IN ATHEROSCLEROS IS
Colchicine, as a powerful anti-inflammatory drug, has been used for rheumatic immune diseases for many years, as well as gout, familial Mediterranean fever and osteoarthritis. Compared with nonsteroidal anti-inflammatory drugs and antibiotics, colchicine has a different anti-inflammatory effect.
F I G U R E 1 Inflammasome assembly and activation mechanism. OxLDL can activate the NF-κB signal transduction pathway through PRR, which not only increases the expression of NLRP3, pro-Caspase and ASC, but also upregulates the pro-IL-1β and pro-IL-18 levels. Extracellular ATP can combine with P2X2R to activate P2X2R, increase K + efflux and decrease intracellular K + concentration, which provides a basis for the assembly and activation of NLRP3 inflammasomes. At the same time, OxLDL can be swallowed by macrophages through membrane receptors and can be converted into cholesterol crystals in the lysosome. The CCs (formed intracellularly or derived extracellularly) can cause the lysosome to rupture, resulting in Cathepsin B being released into the cytoplasm and inflammasome activation. Microtubules can transport ASC so that it can combine with NLRP3 and assemble NLRP3 inflammasomes into complexes. After the inflammasome is activated, pro-Caspase can be converted into Caspase by shearing, and the activated Caspase can cleave pro-IL-1β and pro-IL-18 into IL-1β and IL-18, and secrete them outside the cell, causing an outbreak of inflammation. However, colchicine can inhibit the activation of NLRP3 inflammasomes and reduce the release of IL-1β through a variety of ways to inhibit inflammation, mainly in three ways as follows: ① restriction of P2X7 receptor and reduction of K + outflow; ② damping of microtubule synthesis, and inhibition of the assembly of NLRP3 inflammasome and NLRP3 inflammasome complex; ③ inhibition of NLRP3 inflammasome activation and IL-1 β release

| Inhibition of neutrophil chemotaxis, adhesion and recruitment
In the early stage of inflammation, neutrophils, as the forerunner of immune responses, can be affected by chemokines and then reach the inflammatory site first through blood circulation, and adhere to vascular endothelial cells though E-selectin and L-selectin. Through deformation, neutrophils can go through the intercellular space to reach the site of inflammation. Phelps P et al. discovered that in vitro, 0.1 nM colchicine can inhibit the chemotaxis of neutrophils and inhibit the release of chemokine S100A8 and S100A9 in neutrophils. 30 What's more, Cronstein B et al. found that colchicine can also inhibit neutrophil adhesion and recruitment by inhibiting microtubule synthesis and promoting microtubule depolymerization. 31,32 Colchicine at 300 nM can directly cause the exfoliation of the adhesion molecule L-selectin on the surface of endothelial cells and hinder the recruitment of neutrophils. 32 Colchicine can also inhibit the synthesis of superoxide in neutrophils by inhibiting microtubules, thus reducing inflammation. 2,33 In vitro 3 nM colchicine can change the distribution of E-selectin on the surface of endothelial cells and inhibit the adhesion of neutrophils. It is because colchicine can inhibit the chemotaxis, adhesion and recruitment of neutrophils that its clinical dosage needs to be precise. A high dose will often cause myelosuppression and neutropenia, therefore resulting in infection.

| Inhibition of NLRP3 inflammasome activation and IL-1 β release
Colchicine can inhibit not only neutrophils, but also NLRP3 inflammasome activation in many ways, thus exert a powerful antiinflammatory effect. Martinon F. et al. proved that colchicine can inhibit the activation of the NLRP3 inflammasome in cultured monocytes. 34 Current studies have shown that the mechanism of colchicine on the NLRP3 inflammasome can be summarized into the following three types.

| Colchicine can restrict P2X7 receptor
Marques-da-Silva C et al. evaluated the function of heterologous P2X2 and P2X7 receptors after ATP treatment by electrophysiology F I G U R E 2 NLRP3 inflammasome, IL-1 β, IL-6, C-reactive protein inflammatory response axis. The activation of NLRP3 inflammasomes in macrophages can activate caspase-1 and release a large amount of IL-1β, which can induce inflammatory factor storms through selfactivation. In addition, IL-1β can activate endothelial cells, smooth muscle cells and macrophages to release a number of IL-6. IL-6 can circulate through the blood to the liver and induce hepatocytes to produce CRP. The marker of CRP as an indicator of clinical inflammation can be detected in patient blood samples. With CRP ≥2 mg/L, we can consider there to be an inflammatory response in the patient's body. Colchicine can inhibit the activation of NLRP3 inflammasomes, thereby causing the downstream levels of IL-1β, IL-6 and CRP to decrease and dye uptake, and verified it with colchicine in vitro. The results showed that colchicine could effectively inhibit the pore formation induced by P2X7. 35,36 As mentioned above, ATP can activate P2X7 receptors to open K + channels and reduce intracellular K + concentration, thus promoting the activation of the NLRP3 inflammasome.
When P2X7 receptors are inhibited, K + outflow is blocked and a high concentration of K + prevents NLRP3 inflammasome assembly and activation.

| Colchicine can damp microtubule synthesis
By immunofluorescence staining and other methods, Takuma

| Colchicine can effectively inhibit Caspase-1
In the study of small intestinal injury induced by NSAID in mice,

| RE S E ARCH PROG RE SS OF COLCHI CINE IN THE TRE ATMENT OF ATHEROSCLEROS IS
At present, lipid-regulating therapy is still an unshakable cornerstone in the treatment of atherosclerosis. However, inflammation runs through the onset and development of atherosclerosis. Right now, anti-inflammatory therapy is still under exploration and has not been formally put into clinical practice. Current studies have shown that colchicine, as an anti-inflammatory drug, is likely to become a first-line treatment for atherosclerosis and other cardiovascular inflammatory diseases in the future.

| Progress in Basic Research of Colchicine in the treatment of Atherosclerosis
As a classic anti-inflammatory drug, colchicine has been widely stud-

| Progress in Clinical Research of Colchicine in the treatment of Coronary Atherosclerotic Disease (CAD)
In the past ten years, several clinical studies were carried out to observe the therapeutic role of anti-inflammatory drugs on cardiovascular diseases. Distinguished among them is the CANTOs trial CAD patients (colchicine n = 5774; placebo n = 5820) through databases and conducted a systematic review and meta-analysis of randomized controlled trials, showing that in terms of secondary cardiovascular prevention, compared with the standard drug therapy alone, adding low-dose colchicine can reduce the incidence of major cardiovascular events. 44 However, in 2020, the COPS trial conducted a randomized, double-blind, placebo-controlled study on 795 ACS patients.
Patients in the colchicine group took 1 mg/day of colchicine in the first month after admission to the hospital with a diagnosis of ACS.
The dose of colchicine taken for the next 11 was months 0.5 mg/day.
After 1 year of follow-up study, the results of the study showed that low-dose colchicine (0.5 mg per day) not only failed to have a significant effect on cardiovascular results, but also was associated with a higher rate of mortality. 45 (Table 1).

| CON CLUS I ON AND PROS PEC TS
First of all, the development of atherosclerosis is inseparable from inflammation. The inflammatory response is mainly induced by OxLDL and cholesterol crystals, which cause the assembly and activation of NLRP3 inflammasomes in macrophages, and increase the expression of downstream inflammatory factors, through the NLRP3 inflammasome, IL-1β, IL-6 and C-reactive protein inflammatory response axis. As an anti-inflammatory drug, colchicine mainly inhibits the inflammatory response in atherosclerosis in three ways, as follows: ① damping P2X7-induced K + channel opening and reduce K + efflux; ② restraining microtubule synthesis and reducing the assembly of NLRP3 inflammasomes and ASC and the formation of NLRP3 inflammatory complex; ③ inhibiting caspase-1 conversion pro-IL-1β into the active form of IL-1β. Eventually, it will reduce the levels of IL-1β, thereby inhibiting the inflammatory response in atherosclerosis. While colchicine is anti-inflammatory, it also comes with many side effects, such as diarrhoea and abdominal pain. When the dose of colchicine is not well controlled, it will not only cause reductions in platelets and neutrophils, and increase the risk of infection in patients, 46 but even also induce aplastic anaemia in severe cases. These side effects and adverse reactions put certain limitations on colchicine in the anti-inflammatory treatment of cardiovascular diseases, but we believe that as long as the dosage of colchicine is well controlled, its safety properly monitored, and a new mode of administration developed, colchicine will certainly have a very important place in the future treatment of cardiovascular inflammatory diseases.

ACK N OWLED G EM ENTS
We want to thank Dr. Yu Qi and Dr. Jinxuan Zhao for scientific advices and Dr. Jiaxin Hu for technical assistance.

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no data sets were generated or analysed during the current study.