EFFECTS OF COLCHICINE ADJUVANT THERAPY ON DISEASE CONTROL, SERUM NALP3, SICAM-1,

Background: To investigate the impact of colchicine adjuvant therapy on disease control and serum levels of nucleotide-binding oligomerization domain-like receptor (NALP) 3, soluble intercellular adhesion molecule (sICAM)-1, matrix metalloproteinase (MMP)-9


Introduction
Coronary heart disease (CHD), as a cardiovascular disease, is the most common type of organ damage caused by atherosclerosis, predominantly affecting the middle-aged and elderly population.It has a high incidence, disability rate, and mortality rate (1).Given the close association between the occurrence of cardiovascular diseases and high uric acid levels, and because high uric acid is a direct cause of acute gout attacks (2), CHD patients often experience acute gout attacks, significantly worsening their condition.Therefore, early control and active treatment have a positive impact on improving the patient's condition and promoting recovery.
Currently, the clinical treatment of CHD patients with acute gout attacks mainly focuses on antiplatelet therapy, lipid regulation, control of risk factors, and uric acid reduction.However, in recent years, increasing evidence suggests that inflammation plays a significant role in the formation and progression of atherosclerosis (3).It is thus speculated that antiinflammatory treatment will become a new direction in the treatment of CHD.Previous research by Ridker PM and others has also shown that anti-inflammatory treatment can improve the prognosis of CHD patients (4).
Colchicine is a lipophilic alkaloid extracted from autumn crocus and has been approved for use in the treatment of acute gout and familial Mediterranean fever, showing significant efficacy (5).As an antiinflammatory drug, it has been found to have broad prospects in the secondary prevention of CHD, promoting plaque stability, reducing acute plaque events, and major adverse cardiovascular events (6,7).
Given these considerations, this study will use colchicine as adjuvant therapy for CHD patients with acute gout attacks to investigate its effects on disease control, serum levels of nucleotide-binding oligomerization domain-like receptor protein (NALP) 3, soluble intercellular adhesion molecules (sICAM)-1, matrix metalloproteinase (MMP)-9, MMP-13, and immune function cells.

Materials and Methods
This study was approved by the ethics committee of Cangzhou Central Hospital (approval number: 2022-051-02(z)).Signed written informed consents were obtained from the patients and/or guardians.

Basic Characteristics
A total of 92 patients with CHD complicated by acute gout attacks, who were admitted to our hospital from October 2021 to January 2023, were selected for this study.They were divided into an observation group and a control group, with 46 patients in each group.Control group: Male/Female = 39/7, age Results: The total effective rate in the observation group was significantly higher than that in the control group (93.48% vs 79.07%) (P<0.05).After treatment, the levels of NALP3, MMP-9, and MMP-13 in both groups decreased, with the observation group being lower than the control group (P<0.05).After treatment, LVPWT and LVEDD levels in the observation group were lower than those in the control group, and LVEF was higher (P<0.05).After treatment, the levels of ET-1 and sICAM-1 in the observation group were lower than those in the control group, and VEGF levels were higher (P<0.05).After treatment, the proportions of CD3+ lymphocytes, CD3+CD4+ lymphocytes, and CD3+CD4+/CD3+CD8+ ratio were significantly higher in the observation group than in the control group (P<0.05).There was no significant difference in the incidence of adverse reactions between the two groups (P>0.05).The occurrence of major adverse cardiovascular events in the observation group was lower than that in the control group (2.17% vs 13.04%).Conclusions: Colchicine adjuvant therapy improves the efficacy of CHD patients with acute gout attacks, helps improve cardiac function and vascular endothelial function, reduces serum levels of NALP3, sICAM-1, MMP-9, and MMP-13, enhances patient immunity, and controls disease progression.
Klju~ne re~i: kolhicin, koronarna bolest srca, akutni napad gihta, receptorski protein 3 nalik domeni oligomerizacije vezivanja nukleotida, rastvorljivi me|u}elijski adhezioni molekul-1, matriks metaloproteinaza-9, matriks metalo proteinaza- .There were no significant differences in baseline data between the two groups (P > 0.05).This study was approved by the hospital ethics committee.Inclusion criteria: All patients met the diagnostic criteria for CHD (8) and acute gout attack (9).Patients and their families were informed about this study.Exclusion criteria: Patients with allergies to the medications used in this study; patients with psychiatric disorders; patients with other types of cardiovascular diseases; patients with significant liver or kidney dysfunction; patients with autoimmune diseases.

Observation Parameters
Clinical Efficacy: At the end of the treatment, the clinical efficacy was evaluated based on reference to previous literature (10) and categorized into three groups: significant efficacy, efficacy, and inefficacy.Significant efficacy: Patients exhibited a significant improvement in clinical symptoms, good recovery of joint function, nearly normal electrocardiogram (ECG) results, and a return to nearly normal laboratory indicator levels.Efficacy: Patients experienced improvements in clinical symptoms and joint function, with ECG and laboratory indicators showing improvement.Inefficacy: Patients did not show improvements in clinical symptoms, joint function, ECG results, or laboratory indicator levels, and may have even worsened.The total effective rate was calculated as the sum of the significant efficacy rate and the efficacy rate.
Inflammatory Factors: Prior to treatment and at the end of treatment, fasting venous blood samples (3 mL) were collected from the patients.After centrifugation (3000 r/min, 10 min, 10 cm radius), the upper clear liquid was collected and stored at -40 °C for further analysis.Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of NALP3, MMP-9, and MMP-13.The reagents and kits were purchased from Shanghai XunYa Biotechnology Co., Ltd., and the measurements were performed strictly following the manufacturer's instructions.
Cardiac Function: Before treatment and at the end of treatment, a color Doppler ultrasound machine (Instrument: GE, LOC-ZQ9, USA) was used to measure the left ventricular posterior wall thickness (LVPWT), left ventricular end-diastolic diameter (LVEDD), and left ventricular ejection fraction (LVEF) of the patients.
Endothelial Function: Before treatment and at the end of treatment, fasting venous blood samples (3 mL) were collected from the patients.After centrifugation (3000 r/min, 10 min, 10 cm radius), the upper clear liquid was collected and stored at -40 °C for further analysis.Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of endothelin-1 (ET-1), soluble intercellular adhesion molecule-1 (sICAM-1), and vascular endothelial growth factor (VEGF).The reagents and kits were purchased from Shanghai Enzyme Research Biotechnology Co., Ltd., and the measurements were performed strictly following the manufacturer's instructions.
Detection of Immune Function Cells: Before and after treatment, 3 mL of peripheral blood was collected from patients and placed in EDTA anticoagulant tubes.From each blood sample, 100 mL was taken and 5 mL of CD45-Percp, CD3-FITC, CD8-PE, and CD4-APC monoclonal antibodies were added separately.After incubation in the dark for 30 minutes, 1 mL of lysing reagent was added to lyse red blood cells.After thorough mixing and centrifugation, the supernatant was removed.The cells were washed twice with PBS and then analyzed using the BD FACS Canto flow cytometer to detect changes in the proportions of immune function cells, including CD3+ lymphocytes, CD3+CD4+ lymphocytes, and CD3+ CD8+ lymphocytes, in peripheral blood before and after treatment.The immune function index (CD3+ CD4+/CD3+CD8+ ratio) was also calculated.
Adverse Reactions: Adverse reactions occurring during the treatment period in both groups were recorded.These primarily included nausea, vomiting, headache, gastrointestinal bleeding, digestive tract bleeding, diarrhea, etc.If a patient experienced multiple adverse reactions, only the most severe one was recorded.
Major Cardiovascular Events: Three months after the end of treatment, both groups were followed up to record the occurrence of major cardiovascular events, including cardiovascular death, myocardial infarction, and stroke.

Statistical Analysis
Data processing was performed using Statistic Package for Social Science (SPSS) 22.0 software (IBM, Armonk, NY, USA).For cardiac function, vascular endothelial function, and inflammatory factor levels, which all followed a normal distribution and are represented as (mean ± standard deviation), differences between groups were analyzed using the twosample independent t-test.For comparisons within each group before and after treatment, paired t-tests were used.Categorical data such as adverse reactions, gender, and efficacy were presented as (n (%)), and chi-square tests were employed.A significance level of P < 0.05 was considered statistically significant.

Comparison of Total Effective Rates between the Two Groups
The total effective rate in the observation group was significantly higher than that in the control group (93.48% vs. 79.07%)(P < 0.05).Detail information was shown in Table I.

Comparison of Inflammatory Factor Levels between the Two Groups
Before treatment, there were no significant differences in NALP3, MMP-9, and MMP-13 levels between the two groups (P > 0.05).After treatment, the levels of NALP3, MMP-9, and MMP-13 decreased in both groups, with the observation group showing lower levels compared to the control group (P < 0.05).Detail information was shown in Table II.

Comparison of Cardiac Function between the Two Groups
Before treatment, there were no significant differences in LVPWT, LVEDD, and LVEF levels between the two groups (P > 0.05).After treatment, the LVPWT and LVEDD levels in the observation group were lower than those in the control group, while the LVEF was higher in the observation group compared to the control group (P < 0.05).Detail information was shown in Table III.

Comparison of Endothelial Function between the Two Groups
After treatment, the levels of ET-1 and sICAM-1 decreased, while the level of VEGF increased in both groups.Additionally, the observation group had lower levels of ET-1 and sICAM-1 compared to the control group, and higher levels of VEGF (P < 0.05).Detail information was shown in Table IV.

Comparison of Immune Function Cells between the Two Groups
Using flow cytometry, changes in immune cell function were detected in both groups before and after treatment.Before treatment, there were no sig-  nificant differences in the proportions of CD3+ lymphocytes, CD3+CD4+ lymphocytes, CD3+CD8+ lymphocytes, and the immune function index (CD3+CD4+/CD3+CD8+ ratio) in peripheral blood between the two groups (P > 0.05).However, after treatment, the proportions of CD3+ lymphocytes, CD3+CD4+ lymphocytes, and the CD3+CD4+/ CD3+CD8+ ratio increased significantly in both groups (P < 0.05).Moreover, the observation group showed significantly higher proportions of CD3+ lymphocytes, CD3+CD4+ lymphocytes, and a higher CD3+CD4+/CD3+CD8+ ratio compared to the control group (P < 0.05).The detailed information was presented in Figure 1 and Table V.

Comparison of Adverse Reactions between the Two Groups
There were no significant differences in the occurrence rates of adverse reactions between the two groups (P > 0.05).The detailed information was shown in Table VI.

Comparison of the Incidence of Major Cardiovascular Events between the Two Groups
In the observation group, there was only 1 case of myocardial infarction, resulting in a major cardiovascular event incidence rate of 2.17%.In the control group, there were 3 cases of stroke, 2 cases of myocardial infarction, and 1 case of cardiovascular death, resulting in a major cardiovascular event incidence rate of 13.04%.Upon comparison, the major cardiovascular event incidence rate in the observation group was lower than that in the control group (c 2 =3.866,P=0.049).

Discussion
Research has shown that high uric acid levels are the end product of purine metabolism and the underlying cause of acute gout attacks, which is related to CHD (11).In recent years, the rapid aging of the population and improved living standards in China have led to an increasing incidence of CHD.This has resulted in a substantial rise in the number of patients with acute gout attacks complicating CHD.This not only poses a significant threat to the lives of patients but also increases the economic and societal burdens.Therefore, adopting safe and effective treatment measures is of great importance for controlling disease progression and improving prognosis.There is a growing interest in anti-inflammatory treatment for CHD patients with acute gout attacks, as recent studies have highlighted the role of inflammation in the development of atherosclerosis (12).Colchicine, as a cost-effective anti-inflammatory drug, has a wide range of anti-inflammatory effects.Hence, this study applied colchicine as an adjunctive therapy for CHD patients with acute gout attacks, aiming to explore its effects on disease control and its impact on relevant inflammatory factors and vascular endothelial function factors.
In the current study, the observation group showed a higher overall clinical effective rate compared to the control group, indicating that colchicine adjuvant therapy is beneficial for improving the clinical efficacy in patients with CHD and acute gout attacks.It plays a positive role in controlling the further development of the disease.This effect may be related to the anti-inflammatory, immunosuppressive, anti-fibrotic, and cardiovascular protective properties of colchicine.Additionally, a study conducted by Wang Pengfei et al. ( 13) also demonstrated that colchicine had good efficacy in treating patients with angina pectoris and acute gout, leading to improvements in gout and serum markers.This finding aligns with the results of the current study.
CHD is a chronic vascular inflammatory condition characterized by the accumulation of subendothelial lipoproteins, triggering abnormal immune responses and resulting in the formation of inflammatory plaques (14).Furthermore, research has indicated a close association between inflammatory responses and both CHD and acute gout attacks (15)(16).Serum NALP3 plays a critical role in the inflammatory response, as it activates caspase-1, which in turn regulates the maturation of interleukins (IL)-1b and IL-18 in white blood cells.This activation  (20).Moreover, colchicine can stimulate M2 macrophages to increase the expression of transforming growth factor-beta (TGF-b), which limits the proliferation and activity of smooth muscle cells and fibroblasts, thereby promoting the resolution and healing of plaque inflammation (21).This leads to a reduction in inflammatory responses and a decrease in serum NALP3, MMP-9, and MMP-13 levels.
Vascular endothelial dysfunction plays a role in the development of CHD combined with acute gout attacks.ET-1, as a vasoconstrictive peptide, can induce vascular constriction.Elevated levels of ET-1 can led to insufficient myocardial blood supply.sICAM-1 can increase plaque vulnerability and is often used as an important biochemical indicator to assess the severity of CHD (22).VEGF promotes endothelial cell regeneration and angiogenesis.When endothelial function is impaired, it can lead to coronary artery narrowing and myocardial ischemia and hypoxia, resulting in reduced VEGF levels.This study demonstrates that after treatment, the observation group had lower levels of ET-1 and sICAM-1 compared to the control group, while VEGF levels were higher than in the control group.This suggests that adjunctive treatment with colchicine is beneficial for improving endothelial dysfunction in patients with CHD and concurrent acute gout attacks.This may be due to colchicine's effective uric acid-lowering properties, which reduce urate crystal deposition, thereby minimizing damage to the vascular endothelium and preventing thrombus formation.Furthermore, colchicine can inhibit the interaction between white blood cells and platelets, reduce endothelial cell selectin expression, and exert anti-platelet aggregation effects, thus protecting endothelial function (23).In this study, after treatment, the observation group had lower levels of LVPWT and LVEDD while LVEF was higher compared to the control group, indicating that adjunctive colchicine treatment in patients with CHD and concurrent acute gout attacks is associated with improved heart function.This improvement may be attributed to colchicine's anti-inflammatory properties and its role in improving endothelial function.The study also found that the observation group had a lower rate of major cardiovascular events compared to the control group, which is consistent with previous research (20).This suggests that adjunctive colchicine therapy has a positive impact on reducing the occurrence of major cardiovascular events.Additionally, research by Fiolet et al. ( 24) also demonstrated that colchicine treatment can reduce the risk of cardiovascular events in stable CHD patients.
Furthermore, the study showed that the immune cell subsets (CD3+ lymphocytes, CD3+CD4+ lymphocytes ratio) and immune index (CD3+CD4+/ CD3+CD8+ ratio) in the observation group were significantly higher than in the control group, indicating that colchicine adjunctive therapy can enhance the immune response recovery in patients with CHD and concurrent acute gout attacks.Finally, the study found no significant difference in the incidence of adverse reactions between the two groups, reaffirming the safety of adjunctive colchicine therapy.However this paper still has some shortcomings.Firstly, the adequacy of the sample size was not calculated for this study, however we collected a more than adequate number of patients.Secondly this is a single centre study, which may limit the application of the results of this study.In the future we will conduct a multi-centre study.
In conclusion, adjunctive treatment with colchicine in patients with CHD and concurrent acute gout attacks has shown superior therapeutic efficacy.It helps reduce inflammation, improve endothelial function and cardiac function, promotes immune recovery, and reduces the occurrence of major cardiovascular events.

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Wang et al.: Colchicine effects on coronary disease and acute gout markers Table I Comparison of Total Effective Rates between the Two Groups (n (%), n=46).
*Compared to before treatment, P<0.05; #compared to the control group, P<0.05.

Table III
Comparison of Cardiac Function between the Two Groups ( ⎯x±s, n=46).Compared to before treatment, P<0.05; # compared to the control group, P<0.05.

Table V
Comparison of Immune Function Cells between the Two Groups ( ⎯x±s, n=46).
Figure 1 Flow Cytometry Analysis of Changes in Immune Cell Subsets in Peripheral Blood of Patients in Two Groups Before and After Treatment: Left: CD3+ Lymphocyte Subpopulation; Middle: CD3+CD4+ Lymphocyte Subpopulation; Right: CD3+CD8+ Lymphocyte Subpopulation.

Table VI
Comparison of Adverse Reactions between the Two Groups (n (%), n=46).