Yang Hai-chen, Geng Jia-yi, Li Hao-yu, Xu Ning, Liu Chen-yang, Kang Pin-fang?, Zhang Heng?

1. Department of Cardiology, The First Affiliated Hospital of Bengbu Medical College, Bengbu 233000, China

2. Research Center of Cardiovascular and Cerebrovascular Diseases, Bengbu Medical College, Bengbu 233000, China

3. Class 4, Preventive Medicine, Grade 2020, School of Public Health, Bengbu Medical College, Bengbu 233000, China

4. Public Service Management of Clinical Medical College of Anhui Medical University, Bengbu 233000, China

Keywords:TGF-β2 Galectin-2 Atrial fbirillation

ABSTRACT Objective: To investigate the expression of serum transforming growth factor β2(TGFβ2) and galectin-2 (Galectin-2) in patients with atrial fibrillation and its clinical significance.Methods: A total of 150 inpatients in the Department of Cardiology of the First Affiliated Hospital of Bengbu Medical College from January 2019 to June 2019 were selected,including 84 patients in the paroxysmal atrial fibrillation group, 66 patients in the persistent atrial fibrillation group, and 120 patients with sinus rhythm excluding atrial fibrillation who were hospitalized during the same period were selected as the control group. Blood routine,biochemical routine and cardiac ultrasonic morphological examination were improved after admission. In addition, the serum levels of TGF-β2 and Galectin-2 in each group were detected by Elisa and Western Blot, and the differences and clinical significance of TGF-β2 and Galectin-2 in different groups were statistically analyzed. The correlation between TGFβ2, Galectin-2 and left atrial end-diastolic diameter (LAD) was further determined by Pearson correlation analysis. By means of binary logistics regression analysis, the factors affecting atrial fibrillation were obtained. Results: Compared with the control group, age and LAD in atrial fibrillation group were significantly increased (P＜0.05). The serum concentrations of TGF-β2 and galectin-2 in persistent af group were significantly increased compared with those in paroxysmal af group and control group (P＜0.05), while those in control group were significantly decreased compared with paroxysmal AF group (P＜0.05). Pearson correlation analysis showed that TGF-β2 and Galectin-2 concentrations were significantly positively correlated with LAD size in the experimental group (P＜0.05). Binary logistic regression analysis showed that TGF-β2, galectin-2 and LAD were risk factors for atrial fibrillation(P＜0.05). Conclusion: TGF-β2 and Galectin-2 are closely related to the occurrence and development of atrial fibrillation. Both TGF-β2 and Galectin-2 were positively correlated with LAD. The three have a synergistic effect on the occurrence and progression of atrial inflammatory fibrosis in patients with ATRIAL fibrillation, jointly promoting mechanical and electrical remodeling of atrial, and can be used as risk factors to evaluate the occurrence and progression of atrial fibrillation.

1. Introduction

Atrial fibrillation (AF) is one of the most common clinical arrhythmias and a major global health burden. Regardless of whether patients have corresponding symptoms, the occurrence of AF will greatly increase the risk of thromboembolism, among which ischemic stroke events are the most significant, further increasing the all-cause mortality [1]. As the pathophysiological basis of AF, inflammation and fibrosis mediate related pathological processes, such as oxidative stress and apoptosis, and cause conduction disorders of cardiac myocytes, exacerbating the progress of atrial remodeling [2]. Chronic inflammatory response and atrial fibrosis caused by atrial remodeling result in the release of related inflammatory and fibrogenic products and regulatory factors into the blood and become detectable serum markers.

The pathological mechanism of AF is widely studied at home and abroad, but there are few studies on the role of TGF-β2 and Galectin-2 in the occurrence and development of AF. For transforming growth factor β(TGF-β), it belongs to the TGF-β superfamily, which has a certain regulatory effect on cell growth and differentiation, and is a typical fibrogenic factor [3]. He pointed out that the increased frequency of some alleles related to TGFβ2 transcription would adversely affect the transcription and translation expression of TGFβ2 gene, thus affecting the proliferation and differentiation of cardiac myocytes, and causing fibrosis of atrial myocytes under the action of immune inflammatory mechanism[4]. Galectins are a member of a superfamily of lectins with special amino acid sequences and specific affinity for sugar conjugants containing β-galactoside residues, which are involved in various biological processes such as cell adhesion, inflammatory cell migration, differentiation and apoptosis [5-7]. Fifteen members of the galectin family of proteins have been found in mammals,among which, Galectin-2 can be obtained by different inflammatory immune cells to induce cell migration, activation and release of inflammatory factors to produce immunomodulatory effects [8].Relevant literature indicates that chronic inflammatory responses caused by dysregulation of galactose agglutinin-related pathways also play a non-negligible role in the occurrence and development of cardiovascular diseases [9]. Yildirim [10] showed that Galectin-2 binds to CD14 on the surface of monocytes and monocyte derived macrophages, induces downstream by Toll-like receptor 4 (TLR4),and converts macrophages into M1 pro-inflammatory subtypes,thus triggering a series of pro-inflammatory reactions. In addition,Galectin-2 acts as a ligand of lymphotoxin α(LTA), which together activate pro-infalmmatory cytokine mediated immune infalmmation.The aim of this study was to analyze the relationship between TGFβ2 and Galectin-2 and AF by detecting the concentrations of TGFβ2 and Galectin-2 in serum, and to explore the roles of TGFβ2 and Galectin-2 in the occurrence and development of AF.

2. Objects and methods

2.1 Subjects

In this study, 150 inpatients in the Department of Cardiology of the First Affiliated Hospital of Bengbu Medical College from January 2019 to June 2019 were enrolled, including 84 patients in the paroxysmal AF (PAF) group and 66 patients in the persistent AF (PeAF) group. Inclusion criteria refer to the American College of Cardiology (ACC) guidelines for AF patient Management[11] :PAF mostly terminates within 48h, lasts no more than 7 days, and can terminate spontaneously. PeAF lasts more than 7 days or longer and is terminated by medication or direct current cardioversion.Exclusion criteria: acute coronary syndrome, myocarditis,rheumatic heart disease, pulmonary heart disease, valvular heart disease, hypertrophic cardiomyopathy, dilated cardiomyopathy,hepatic and renal insufficiency, pulmonary embolism and trauma,hyperthyroidism, acute and chronic infections, and autoimmune systemic diseases.

2.2 Detection Indicators

The clinical data of the subjects were compared and analyzed,such as gender, age, history of stroke, history of diabetes, history of hypertension, history of smoking, blood routine, biochemical routine and other relevant indicators. Fasting peripheral venous blood was collected at 6:00 am after admission and before treatment for both patients with AF and the control group, and then the levels of serum TGF-β2 and Galectin-2 Elisa kit (Shanghai Yuduo) were detected.Blood routine, biochemical indexes, left atrial diameter (LAD), left ventricular ejection fraction (LVEF) were completed by laboratory and cardiac color doppler ultrasound room.

2.3 Statistical Treatment

After obtaining the research data, IBM SPSS 22.0 was used for analysis. For the data of normal distribution measurement data, it is expressed by means of mean ± standard deviation, and by means of median ± quartile spacing, the continuous variable that goes against the normal distribution is described. In order to better analyze the continuous variable, one-way analysis of variance is adopted now.Among them, the counting data were expressed in percentage form,and the χ2 test was passed for comparison between groups. Pearson correlation analysis was used to analyze the correlation between indicators. Logistic multifactor regression analysis was used to determine the risk factors of AF. When 0.05, there is significant statistical significance.

3. Results

3.1 Comparison of clinical baseline data among the three groups

Between the three study groups, gender, history of hypertension,diabetes and stroke, smoking history, red blood cell (RBC) count,white blood cell (WBC) count, alanine aminotransferase (ALT),aspartate aminotransferase (AST), creatinine (CR), low density lipoprotein (LDL), left ventricular ejection fraction (LVEF) and left ventricular end-diastolic diameter (LVDD) had no statistical significance (P＞ 0.05); In addition, there was a significant difference in age between the AF group and the control group (P＜ 0.05), the left atrial diameter (LAD) in the control group was significantly different between the PAF group and the PeAF group (P＜ 0.05), and the LAD in PeAF group was significantly higher than that in PAF group (P＜ 0.05), as shown in Table 1.

Table 1 Comparison of baseline data of the three groups of subjects

3.2 Comparison of serum concentrations of TGF-β2 and Galectin-2 among three groups

The serum concentrations of TGF-β2 and Galectin-2 in the three groups were compared, and the conclusions were as follows:compared with the PAF group and the control group, the levels of TGF-β2 and Galectin-2 in the PeAF group were significantly higher (P＜ 0.05). The control group was significantly lower than the PAF group (P＜ 0.05). See Figure 1 and Table 2 for details.

3.3 Expression comparison of TGF-β2 and Galectin-2 in peripheral blood mononuclear cells (PBMC)

Western Blot results showed that the expression of TGF-β2 and Galectin-2 in PBMC of patients with AF was significantly higher than that of the control group (P＜ 0.05), the levels of TGF-β2 and Galectin-2 in patients with PeAF were significantly higher than those in patients with PAF (P＜0.05).

Table 2 Comparison of serum concentrations of TGF-β2 and Galectin-2 in each group of patients (±s)

Table 2 Comparison of serum concentrations of TGF-β2 and Galectin-2 in each group of patients (±s)

Note: Compared with control group,*P＜0.05;Compared with the PAF group, #P＜0.05.

Indicators Control(n=120) PAF(n=84) PeAF(n=66) F P TGFβ2(pg/ml) 498.42±150.46 717.51±117.87* 763.55±99.19*# 115.976 ＜0.05 Galectin-2(ng/ml) 44.92±12.41 88.73±21.10* 126.02±30.95*# 308.433 ＜0.05

Figure 1 Comparison of serum concentrations of TGF-β2 and Galectin-2 among the three groups

Figure 2 Expression of TGF-β2 and Galectin-2 in PBMC of three groups

Table 3 Comparison of expression levels of TGF-β2 and Galectin-2 in PBMCs of each group(±s)

Table 3 Comparison of expression levels of TGF-β2 and Galectin-2 in PBMCs of each group(±s)

Note: Compared with control group,*P＜0.05;Compared with the PAF group, #P＜0.05.

Indicators Control(n=120) PAF(n=84) PeAF(n=66) F P TGF-β2/GAPDH 0.853±0.051 0.945±0.013* 1.050±0.017*# 28.32 ＜0.05 Galectin-2/GAPDH 0.499±0.066 0.705±0.047* 0.888±0.032*# 44.85 ＜0.05

3.4 Correlation analysis of TGF-β2, Galectin-2 and LAD

Pearson correlation analysis showed that TGF-β2 was significantly positively correlated with LAD in patients with AF (PAF and PeAF)(r= 0.529, P＜ 0.05, R2=0.3221), Galectin-2 was positively correlated with LAD (r= 0.718, P＜ 0.05, R2=0.5494), Galectin-2 was positively correlated with TGF-β2 concentration (r= 0.669, P＜ 0.05,R2=0.4458), indicating the same role in AF. See Figure 3 and Table 4 for details.

Figure 3 Correlation analysis between TGF-β2 and LAD, Galectin-2 and LAD, Galectin-2 and TGF-β2 in the experimental group

Table 4 Correlation analysis of TGF-β2, Galectin-2 and LAD

3.5 Logistic multifactor regression analysis of risk factors of AF

150 patients with AF and 120 controls were analyzed by Logistic multivariate regression analysis, with AF as the dependent variable,TGF-β2, Galectin-2, and LAD as covariables. The results showed that TGF-β2, Galectin-2 serological levels and LAD were risk factors for AF, as shown in Table 5.

Table 5 Results of logistic multifactor regression analysis

4. Discussion

Chronic inflammation and fibrosis of the myocardium are characteristic structural changes of many arrhythmias including AF. Myocardial inflammation and fibrosis can change the normal connection between myocardial fiber bundles, destroy the normal coupling between myocardial cells, so that the heart loses normal contraction and diastolic function, causing abnormal conduction of cardiac signals. It can be concluded that mechanical and electrical remodeling induced by atrial myocyte fibrosis is an important basis for the occurrence and development of AF, among which extracellular matrix (ECM) secreted by cardiac fibroblasts caused by chronic inflammation plays a key role in myocardial fibrosis [12].So far, there are few effective treatment strategies for myocardial fibrosis, mainly because the migration, proliferation and ECM protein deposition of fibroblasts caused by the chronic inflammatory response of myocardial cells is a very difficult process to reverse[13]. Inflammation and fibrosis are thus an interwoven process.However, under pathological conditions, these processes produce a vicious cycle and synergistically promote the development of atrial remodeling. Although the mechanismic link between inflammation and fibrosis is not fully understood, clinical evaluation of underlying inflammatory conditions and targeted interventions are promising treatment strategies for atrial fibrillation. This study confirmed the close relationship between TGF-β2 and Galectin-2 and the occurrence and development of atrial fibrillation. Both TGF-β2 and Galectin-2 were positively correlated with LAD, suggesting that TGF-β2 and Galectin-2 had synergistic effects on the occurrence and progression of atrial inflammatory fibrosis in patients with AF,which jointly promoted mechanical and electrical remodeling of atrial, and could be used as risk factors to evaluate the occurrence and progression of AF.

For TGF-β, it belongs to the TGF-β superfamily and has a certain regulatory effect on the process of cell growth and differentiation. It is a typical fibrogenic factor, mainly including TGF-β1, TGF-β2,TGF-β3, activin, growth and differentiation factors (GDFs) and bone morphogenetic protein (BMP)[14]. A large number of studies have shown that TGF-β can achieve fibrogenic effects mainly through matrix protein expression and inducing cell differentiation into myofibroblasts [15-17]. In addition, endothelial cells can become mesenchymal cells through a process called endothelium-stromal transformation, which can not only migrate but also secrete collagen,playing an important role in myocardial fibrosis. Pardali found that TGF-β2 plays a significant mediating role in this process,eventually becoming fibrocytes and participating in atrial remodeling in atrial fibrillation [18]. In addition, He pointed out that in patients with atrial fibrillation, when the frequency of G allele of RS6658835 locus of TGFβ2 gene increased, the transcription and translation expression of TGFβ2 gene would be adversely affected, thus affecting the proliferation and differentiation process of myocardial cells, and also under the action of immune inflammation mechanism.Leading to fibrosis of atrial myocytes [4]. The results showed that the serological level of TGFβ2 in the experimental group was significantly higher than that in the control group (P＜ 0.05), and TGFβ2 concentration in patients with PeAF was significantly different from that in patients with PAF (P＜ 0.05); In addition,Pearson correlation analysis showed that TGFβ2 was significantly positively correlated with LAD (r=0.529, P＜ 0.05, R2= 0.3221).

Galectin is a class of β- galactoside binding proteins, which are widely distributed in cells and tissues and play an important role in mediating inflammatory response, promoting cell apoptosis and participating in cell migration and adhesion. Relevant literature also pointed out that chronic inflammatory response caused by dysregulation of galactose agglutinin-related pathways also plays a non-negligible role in the occurrence and development of cardiovascular diseases [19]. Wang showed that plasma Galectin-3 concentration levels were significantly correlated with duration of atrial fibrillation, and could provide a basis for risk stratification of atrial fibrillation[20]. However, no clear study has shown the expression of Galectin-2 in patients with atrial fibrillation. The results of this study suggest that Galectin-2 can mediate relevant inflammatory response in patients with atrial fibrillation, and the level of Galectin-2 is closely related to the duration of atrial fibrillation, which can make atrial fibrillation continue. In addition,LAD in patients with persistent atrial fibrillation was significantly higher than that in patients with paroxysmal atrial fibrillation.Pearson correlation analysis showed that Galectin-2 was significantly positively correlated with LAD, suggesting that Galectin-2 and Galectin-2 play a synergistic role in the occurrence and progression of atrial fibrillation. Both Galectin-2 and LAD can be used as risk factors for atrial fibrillation, suggesting that chronic inflammation mediated by Galectin-2 combined with changes in left atrial structure and function may lead to further deterioration of left atrial function in patients with atrial fibrillation. This is also consistent with Menichelli findings on LAD as a risk factor for atrial fibrillation [21].Therefore, the elevated expression of Galectin-2 in AF suggests that it may cause chronic inflammation in left atrium through relevant inflammatory pathways, leading to the occurrence of inflammatory cascade reaction, which has significant adverse effects on left atrium function and increases the risk of recurrent malignant events in AF.The results of this study showed that the serum expression levels of Galectin-2 and TGF-β2 in patients with PAF and PeAF were higher than those in the control group (P＜ 0.05), there was also a statistical difference between the PAF and PeAF group (P＜ 0.05). These results indicated that Galectin-2 and TGF-β2 were closely related to AF, and the inflammatory response and atrial myocyte fibrosis were more intense during the formation and maintenance of AF. Pearson correlation analysis showed that Galectin-2 and TGF-β2 were significantly positively correlated with LAD in AF group (P＜ 0.05),Galectin-2 was positively correlated with TGF-β2 concentration(r=0.669, P＜ 0.05, R2=0.445 8), indicating that the two have the same role in AF, and the two may have mutual influence on the related pathways mediating the inflammatory fibrosis process in patients with AF, which provides a theoretical basis for the early prediction of AF and in-depth understanding of the related mechanisms of the occurrence and development of AF. Logistic multivariate regression analysis showed that Galectin-2, TGF-β2 serological levels and LAD were risk factors for AF, providing a theoretical basis for early prediction of AF and in-depth understanding of the pathogenesis of atrial fibrillation. With only a small sample size, large studies must be conducted to complete validation.

5. Author contribution

Yang Haichen is responsible for the data analysis and writing of this paper. Geng Jiayi, Li Haoyu are responsible for the experimental operation of this paper. Xu Ning, Liu Chenyang are responsible for the experimental data processing and drawing of this paper. Kang Pinfang, Zhang Heng are responsible for the implementation of the project, and guide the writing and modification of the paper. The author of this article declares that there is no conflict of interest in relation to this manuscript.