The Relationship between the Serum NLRP1 Level and Coronary Lesions in Patients with Coronary Artery Disease

Background The pathogenesis of coronary artery disease is complex, and inflammation is one of the regulatory factors. The nucleotide-binding oligomerization domain (NOD)-like receptor protein 1 (NLRP1) plays an important role in the cellular inflammatory response, cell apoptosis, cell death, and autoimmune diseases. Whether the level of NLRP1 is related to the severity of coronary artery stenosis in patients with coronary artery disease (CAD) has not been reported. Objective To test the serum level of NLRP1 in unstable angina (UA) patients and investigate the effect of NLRP1 on coronary stenosis severity of the coronary artery disease (CAD). Methods 307 patients hospitalized in the Department of Cardiology of the Affiliated Hospital of Xuzhou Medical University for coronary angiography from January 1, 2021, to December 31, 2022 were included. We detect the level of NLRP1 in the serum of the included patients. Patients were divided into UA group and control group according to coronary angiography results and other clinical data. We use logistic regression to screen the influencing factors of UA. Then, subgroups were divided according to the Gensini score and the number of coronary artery lesions, and the difference of serum NLRP1 level between the groups was compared. Spearman correlation analysis was used to explore the correlation between the serum NLRP1 level and Gensini score. We analyze the diagnostic value of NLRP1 for UA by drawing ROC curve. Results The median level of serum NLRP1 in patients with UA (n = 257) was 49.71 pg/ml, IQR 30.15, 80.21, and that in patients without UA (n = 50) was 24.75 pg/ml, IQR 13.49, 41.95. Serum NLRP1 levels were significantly different among different subgroups. The patient's Gensini score was correlated with the patient's serum NLRP1 level. Conclusion The serum NLRP1 level is increased in patients with UA, which is increased with the increasing severity of coronary lesions.


Introduction
Cardiovascular disease is one of the main causes of global incidence rate and mortality. According to the statistics of the World Health Organization, atherosclerotic cardiovascular disease (ASCVD) has become one of the main diseases threatening human health, with high morbidity, rapid progress, and high mortality as the main characteristics [1]. Infammation is involved in the occurrence and development of atherosclerosis [2,3]. With the in-depth and comprehensive study of apoptotic bodies and autophagosomes, infammatory bodies are increasingly favored by researchers. Te nucleotide-binding oligomerization domain (NOD)-like receptor protein 1 (NLRP1, also known as NALP1, DEFCAP, NAC, and CARD7) can activate caspase-1 and promote the infammatory cytokine interleukin-1β (IL-1β) and interleukin-18 (IL-18) mature and aggravate infammatory reaction [4]. Te NLRP1 infammatory body is the frst infammatory body found in human THP-1 monocytes. It is composed of NLRP1, caspase-1, caspase-5, junction protein ASC, and CARDI-NAL. It forms the frst line of defense of the host by activating infammatory cytokines [5]. Tere is evidence that coronary atherosclerotic heart disease is the result of multiple signal transduction pathways [6]. It was found that the knockout of NLRP1 gene alleviates the occurrence and development of myocardial hypertrophy and infammation in mice [7,8]. However, whether NLRP1 will play a similar role in the occurrence and development of coronary atherosclerosis and whether the level of NLRP1 is positively related to the degree of coronary stenosis in patients with coronary artery disease have not been reported yet. In this study, we determined the infuence of the NLRP1 level on the degree of coronary stenosis and prognosis of patients with coronary artery disease by detecting the level of NLRP1 in the serum of patients with coronary artery disease, in order to explore new targets and new strategies for the prevention and treatment of coronary atherosclerotic artery disease.

Study Population.
Tis study continuously included 307 patients who underwent coronary angiography in the Cardiology Department of Xuzhou Medical University Afliated Hospital from January 1, 2021, to December 31, 2022, due to unexplained chest pain ( Figure 1). All patients should complete relevant examinations such as blood routine, liver and kidney functions, coagulation functions, electrocardiogram, chest CT, and cardiac ultrasound before surgery. Te preoperative evaluation of the patient was based on the above auxiliary examinations, and surgical contraindications such as severe liver and kidney dysfunction, electrolyte disorders, coagulation dysfunction, and iodine contrast agent allergy were ruled out. Te patients with lung infection, malignant tumor, hematologic diseases, liver and kidney insufciency, autoimmune disease, heart valve disease, myocardiopathy, data loss, and failure to do relevant examinations were not included in this study. According to the results of coronary angiography and clinical data, CAD is diagnosed when there is at least one major epicardial vessels stenosis diameter ≥50%. Te 257 patients were diagnosed with UA through clinical manifestation, angiography, and other clinical examinations [9]. Te control group consisted of 50 patients with unexplained chest pain. Tey received coronary angiography, but the results did not meet the diagnostic criteria for CAD. All patients agreed to undergo angiography and plasma collection. Blood samples were collected during cardiac catheterization before angiography. According to the Gensini scoring system specifed by the American Heart Association and the severity of stenosis and corresponding coefcients of each coronary artery were calculated [10]. Te subjects were divided into three groups according to Gensini score, namely, low-risk group (Gensini score < 30 points, n � 148), medium-risk group (30 ≤ Gensini score < 60 points, n � 66), and high-risk group (Gensini score ≥ 60 points, n � 43). According to the results of coronary angiography, the number of branches of the three main arteries [(eft anterior descending artery (LAD), left circumfex artery (LCX), and right coronary artery (RCA)) involved in the lesion were divided into control group (n � 50), single vessel lesion group (n � 88), and multivessel lesion group (n � 169). Since the left main coronary artery (LMCA) is the main branch of LAD and LCX, LMCA lesions are considered as two branch lesions.
Te severity of coronary artery disease was evaluated by Gensini score and the number of coronary artery lesions. Te study was conducted in accordance with the Helsinki Declaration and approved by the Ethics Committee of the Afliated Hospital of Xuzhou Medical University (No: XYFYLW2017-002) [11].

Methods.
Patients' general data (gender, age, BMI, smoking history, alcohol history, hypertension history, and diabetes history), laboratory data (platelets, neutrophils, lymphocytes, monocytes, C-reactive protein, aspartate aminotransferase, alanine aminotransferase, total bilirubin, fbrinogen, glutamine transpeptidase, urea, creatinine, uric acid, cholesterol, triglyceride, high-density lipoprotein, lowdensity lipoprotein, and lipoprotein-a), statin use, aspirin use, and Imaging data, etc., were analyzed to compare the difference of clinical data among diferent groups of patients. All the subjects collected venous blood on an empty stomach within 24 hours after admission and tested it in the Biochemical Department of the Afliated Hospital of Xuzhou Medical University. Te Enzyme-linked Immunosorbent Assay (ELISA) was used to detect the serum NLRP1 level in enrolled patients. Patients who were tested for serum NLRP1 levels experienced discomfort symptoms such as chest pain within 24 hours before blood sampling. Te ELISA kit was purchased from CUSABIO BIOTECH CO., Ltd, and the serum NLRP1 level was detected in strict accordance with the user manual [12]. To analyze the diference of serum NLRP1 level between UA patients and non-UA patients, the relationship between serum NLRP1 level and Gensini score, the relationship between serum NLRP1 level and the number of coronary artery lesions, and the correlation between serum NLRP1 level and Gensini score were considered.

Statistical Methods.
First, Shapiro-Wilk test was used to test the normality of the measurement data. If it obeys the normal distribution, it is represented by X ± SD. If the normal distribution is not obeyed, the median (interquartile interval, IQR) shall be used. Classifcation variables are expressed in frequency (percentage). Te variables between control group and UA group, each Gensini scoring group, and each coronary artery disease branch group were compared with ANOVA, nonparametric test, and chi-square test, correcting the results of intergroup comparisons using the FDR method. Logistic regression analysis is used to screen for risk factors of UA onset. Spearman correlation analysis was used to explore the correlation between the serum NLRP1 level and Gensini score. We draw the ROC curve to explore the predictive value of NLRP1 for UA.

Multivariate Logistic Analysis of Unstable Angina.
NLRP1, age, diabetes, TG, LDL-C, FIB, and NE were used for multivariate binary logistic analysis. Multivariate logistic regression analysis was conducted based on whether UA occurred: yes � 1, no � 0. Te results showed that NLRP1 was an independent risk factor for UA (P < 0.05), and its level was positively correlated with the occurrence of UA, as shown in Table 2.

Serum NLRP1 Level and the Severity of Coronary
Atherosclerosis. According to the results of coronary angiography, a total of 257 patients (median Gensini score 24.0, IQR 14.3, 46.0) can be diagnosed as UA. Te mean number of coronary artery lesions was 1.66 ± 0.48. In all grouping methods, the serum NLRP1 level (median 24.75 pg/ml, IQR 13.49, 41.95) of patients with non-UA was always the lowest and had statistically signifcant diference with other groups (P < 0.05).    Figure 2).

Serum NLRP1 Expression Level and Branch Number of Coronary Lesions.
In the study of grouping based on the number of coronary artery lesions (

Correlation Analysis of Serum NLRP1 Level and Gensini
Score. Spearman correlation analysis of the serum NLRP1 level and Gensini score in patients with coronary artery disease showed that there was a correlation between serum the NLRP1 level and Gensini score (ρ � 0.580, P < 0.05). Figure 4 shows a scatter plot based on NLRP1 and Gensini score.

Discussion
Tis experiment was based on the hospital to explore the correlation between the serum NLRP1 level and the severity of coronary atherosclerosis in patients undergoing coronary angiography. To investigate the relationship between the severity of coronary atherosclerosis and serum NLRP1 levels    in patients with coronary artery disease, we selected unstable angina, which is a common type of coronary artery disease. According to our results, the serum NLRP1 level in patients with UA was signifcantly higher than that in patients without UA. Trough binary logistic regression analysis, we found that NLRP1 is an independent risk factor for the occurrence of unstable angina. According to the patients' coronary arteriosclerosis Gensini score, there were diferences in serum NLRP1 levels among the control group, lowrisk group, medium-risk group, and high-risk group. Te serum NLRP1 level of patients showed an obvious upward trend with the increase of Gensini score. Similarly, according to the number of coronary diseased branches, the serum NLRP1 level increased with the increase of the number of coronary diseased branches. Te serum NLRP1 level in patients with multivessel disease was signifcantly higher than that in control group and patients with single vessel disease. Te level of serum NLRP1 is positively correlated with the severity of coronary atherosclerosis. Because the serum NLRP1 level may change due to the patients sufering from certain infammatory diseases, autoimmune diseases, blood diseases, malignant tumors, liver and kidney dysfunction, and other diseases, this experiment has not been included in this population. However, the experimental results are still signifcant to a considerable extent, which further indicates that the experimental results are reliable. Coronary atherosclerotic heart disease is a relatively common type of cardiovascular disease, which refers to the heart disease that coronary atherosclerotic lesions or vasospasm cause coronary artery lumen stenosis or occlusion, and then cause myocardial ischemia, hypoxia, or necrosis, as referred to as coronary artery disease. Age, smoking, body mass index, dyslipidemia, hypertension, diabetes, and hyperhomocysteinemia (HCY) are important risk factors afecting the occurrence and development of coronary artery disease. Te pathogenesis of coronary artery disease is complex, which is the result of many factors. Among them, infammation is one of the regulatory factors of myocardial infarction pathology [13]. After myocardial infarction and necrosis death of myocardial cells, cardiac repair/remodeling can cause infammatory reaction, leading to some important structural changes in the infarcted area and distal end. Te degree and duration of infammation can change the subsequent development process. In addition to activating infammation and destroying myocardial tissue, migrated neutrophils and classically activated macrophages also alternately activate macrophages and myocardial myosin for fbrosis repair and reducing infammatory response [14,15].
Infammatory corpuscles belong to the pattern recognition receptors (PRRs) family, called NOD-like receptors (NLRs). Tey are multiprotein complexes and members of  the cytoplasmic protein family. Tey promote the production of caspase-1 dependent bioactive IL-1β through the formation of infammatory corpuscles, thereby promoting the secretion of IL-1β and the downstream infammatory response [4]. It has been confrmed that NLRP1 is associated with a variety of autoimmune diseases, such as enteritis, Addison's disease, vitiligo, type I diabetes, and rheumatoid arthritis [16,17]. NLRP1 also plays an important role in nervous system diseases and is involved in the pathogenesis of acute angle closure glaucoma [18,19]. NLRP1 is also an innate immune sensor, which produces IL-18 under metabolic stress and inhibits obesity and metabolic syndrome [20]. Studies have confrmed that apoptosis plays an important role in the occurrence and development of myocardial infarction [21,22]. NLRP1 is the frst member of the CED-4 family of apoptotic proteins found, which is essential for the initiation of programmed cell death in developing nematodes [23]. Related studies have confrmed that overexpression of NLRP1 in mammalian cells can lead to apoptosis [24]. NLRP1 plays an important role in cellular infammation, apoptosis, cell death, and autoimmune diseases. NLRP1 is closely related to apoptosis and infammation signal pathway. Some studies have shown that inhibiting NLRP1 can reduce the infammatory pathway and disease progression of some nervous system diseases [25][26][27]. In stroke patients, the expression of NLRP1 will increase and will activate the infammatory reaction and eventually lead to neuronal death [28]. As for the pathogenesis of coronary atherosclerosis, the current mainstream is the endothelial injury response theory, which involves a variety of infammatory cells, secretes a variety of growth factors and proinfammatory mediators, and ultimately leads to coronary stenosis. Once there is a contradiction between coronary blood supply and myocardial blood demand, coronary blood fow cannot meet the needs of myocardial metabolism, which can cause myocardial ischemia and hypoxia. Temporary ischemia and hypoxia cause angina pectoris, while continuous severe myocardial ischemia can cause myocardial necrosis, namely, myocardial infarction. Te process of coronary atherosclerosis and coronary artery disease is closely related to infammation, but the relationship between NLRP1 and coronary atherosclerosis needs further exploration. Whether NLRP1 can directly or indirectly protect or damage vascular endothelial cells at the physiological and pathological levels is still unknown. At present, there are few studies on NLRP1 and coronary arteriosclerosis, and there is no systematic and comprehensive elaboration on its mechanism. Te complex relationship network between NLRP1 and coronary atherosclerosis needs to be further explored, and the correlation between serum NLRP1 and coronary atherosclerosis needs to be further clarifed. In previous research, our research team found that the knockout of NLRP1 gene will inhibit MAPKs and NF-κ B signal pathway, thereby reducing the occurrence and development of myocardial hypertrophy and infammation in mice [7,8]. Compared to NLRP1, research on NLRP3 is relatively extensive. NLRP3 has been considered to be related to autoimmune diseases, tumors and, of course, coronary atherosclerosis. Te triggering and activation mechanisms of NLRP3 activation are usually consistent among diferent species. However, the situation of NLRP1 in humans and mice may not be the same, as they exhibit signifcant diferences, which makes NLRP1 more important in clinical research [29,30]. But up to now, there is no clinical study involving the relationship between coronary atherosclerosis and NLRP1.
Te advantage of this study is that the number of coronary artery lesions and Gensini score is used to represent the severity of coronary atherosclerosis. Te Gensini score combined with the number of coronary artery lesions to evaluate and express the severity of coronary atherosclerosis is more reliable and objective than a single indicator. Te results suggest that NLRP1 is closely related to the severity of coronary artery disease in patients with UA. It can lay a clinical foundation for the later study of the role and molecular mechanism of NLRP1 on cardiac remodeling and heart failure in patients with CAD and has strong feasibility in theory. Te test method is easy to learn, not complicated, and can be popularized. Our study laid the foundation for exploring the relationship between NLRP1 and coronary atherosclerosis and provided objective evidence support. It has important clinical signifcance to provide a new perspective and direction for the pathogenesis and treatment of atherosclerosis and its related diseases. Te results can provide ideas for exploring new targets and strategies for the prevention and treatment of coronary atherosclerotic heart disease.
Based on the baseline characteristics of this study, there is no diference in multiple cardiovascular risk factors between patients with non-UA and patients with UA [31], which may be due to the failure to control the drug use of patients in the experimental design. Most patients have begun to receive drug treatment before hospitalization or coronary angiography, which afects some of the patients' laboratory indicators. However, whether the use of drugs has an impact on the serum NLRP1 level of patients needs further research to confrm. Terefore, this is the limitation of this experiment. Secondly, our assessment of the severity of coronary atherosclerosis is based on coronary angiography, which is unethical for normal people. Terefore, we included patients with chest pain and suspected coronary artery disease. Tis may signifcantly reduce the sample size of the control group.

Conclusion
Te level of serum NLRP1 in patients with UA was signifcantly higher than that in patients without UA. NLRP1 is closely related to coronary atherosclerosis and it can serve as an efective indicator for predicting UA.

Data Availability
Te datasets used to support the fndings of this study available from the corresponding author upon reasonable request.

Disclosure
Jing Zong and Yixiao Wang are listed as co-frst authors.
International Journal of Clinical Practice