Fifty-eight patients with non-valvular AF treated at the first clinical medical college of China Three Gorges University and Tongji Hospital between January 2023 and December 2024 were included in the observation group. The inclusion criteria were as follows: (1) electrocardiograph (ECG) showing absence of P waves, f-waves at 350–600 bpm, and irregularly irregular QRS complexes, as defined by current guidelines [18]; and (2) documented AF duration $>$3 months. Exclusion criteria were as follows: (1) history of valvular heart disease; (2) rheumatic heart disease or dilated cardiomyopathy; (3) hyperthyroidism; (4) malignancy; and (5) any surgery within the preceding six months. The control group comprised 58 hospitalized patients without a history of AF, matched for age and other baseline characteristics. No significant differences were observed in age, hypertension, sex, coronary artery disease, smoking history, fasting blood glucose, serum creatinine, total cholesterol, triglycerides, high-density lipoprotein, and low-density lipoprotein (p $>$ 0.05). The observation group had a significantly lower ejection fraction than the control group (p 0.05). Additionally, both the left atrial diameter and the left ventricular end-diastolic diameter were significantly larger in the observation group than in the control group (p 0.05). All patients were thoroughly informed about the study’s objectives and procedures and provided written informed consent. This study received approval from the Medical Ethics Committee of Tongji Hospital.

Sixteen specific pathogen-free (SPF) Sprague-Dawley (SD) rats, aged 6 to 8 weeks and weighing between 200 and 220 grams, were supplied by the Animal Experiment Center of Three Gorges University. An AF animal model was established in eight SD rats through daily intravenous injection of a mixture of acetylcholine and calcium chloride (ACH-CaCl₂, 1 mg$\cdot$kg^-1 day^-1) via the tail vein for four consecutive weeks (AF group). The remaining eight rats were raised under normal conditions and served as negative controls (the normal group). After discontinuation of injections, surface ECGs were recorded in conscious rats. Successful induction of the AF model was indicated by the absence of the P wave, supplanted by small f-waves with a frequency of 350–600 beats/min on the ECG. All rats were anesthetized using 3% sodium pentobarbital, administered intraperitoneally at 30 mg/kg. After euthanasia by air embolism, atrial tissues were collected for subsequent analysis. The reporting of animal experiments followed the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. All animal experiments received authorization from the institutional ethics committees of China Three Gorges University (2024050L).

Five milliliters of fasting venous blood were collected from each patient and processed using density gradient centrifugation. At room temperature, the blood was combined with an equal volume of pre-warmed phosphate-buffered saline (PBS) and lymphocyte separation medium in a 50 mL centrifuge tube, followed by centrifugation at 1700 rpm for 15 minutes. After centrifugation, the sample was separated into four distinct layers. The mononuclear cell layer was meticulously transferred to a 15 mL tube and subsequently washed with PBS, using a volume 2–5 times greater than that of the cell layer, and centrifuged again (1000 rpm, 10 min, room temperature). After discarding the supernatant, the cells were resuspended in PBS to achieve a PBMC concentration of 2 $\times$ 10⁶/mL.

Blood samples were collected in standard tubes, allowed to clot at room temperature for 30 minutes and then centrifuged at 3000 rpm for 20 minutes. The resulting supernatant serum was then separated and stored at –80 °C until further analysis. The levels of SPP1 (Shanghai Jianglai Biotechnology Co., Ltd., Cat. No.: JL10368, Shanghai, China), collagen I (Shanghai Jianglai Biotechnology Co., Ltd., Cat. No.: JL47278, Shanghai, China), IL-10 (Xinboseng Biotechnology Co., Ltd., Cat. No.: EHC009.96, Shanghai, China), IL-17A (Xinboseng Biotechnology Co., Ltd., Cat. No.: EHC170.96, Shanghai, China), and IL-23 (Xinboseng Biotechnology Co., Ltd., Cat. No.: EHC171.96, Wuhan, China) were measured following the manufacturer’s ELISA kit instructions.

The levels of SPP1 (Shanghai Jianglai Biotechnology Co., Ltd., Cat. No.: JL14517, Shanghai, China), collagen I (Wuhan Jilide Biotechnology Co., Ltd., Cat. No.: J23744, Wuhan, China), IL-10 (Wuhan Jilide Biotechnology Co., Ltd., Cat. No.: J22449, Wuhan, China), IL-17A (Wuhan Jilide Biotechnology Co., Ltd., Cat. No.: J23469, Wuhan, China), and IL-23 (Wuhan Jilide Biotechnology Co., Ltd., Cat. No.: J22374, Wuhan, China) in the left atrium of rats was detected using ELISA following the manufacturer’s guidelines.

The left atrial tissue samples were embedded in paraffin and sectioned at 5 µm thickness. Masson’s trichrome staining was employed to assess the extent of fibrosis in the left atrium. These sections were examined under a microscope, and the measurements were taken using Image Pro-Plus software (Media Cybernetics Inc., Silver Spring, MD, USA).

HDL-C, LDL-C, TC, TG and FBG were measured by the clinical laboratory department of Tongji Hospital.

Statistical analyses were conducted utilizing SPSS version 22.0 (IBM Corp., Armonk, NY, USA). Continuous variables are expressed as mean $\pm$ standard deviation. Homogeneity-of-variance testing was performed first. If variances were equal (p $>$ 0.05), the two-sample t-test was used for inter-group comparisons; if variances were unequal (p 0.05), Welch’s t-test was applied. Categorical data are represented as n (%), with the chi-square test applied. Pearson’s correlation analysis was performed to assess correlations, with a significance threshold set at p 0.05.

Baseline characteristics showed no significant differences between the observation and control groups in terms of age, sex, hypertension, coronary artery disease, smoking status, fasting glucose, serum creatinine, or lipid profile parameters (p $>$ 0.05). In contrast, the observation group exhibited a significantly lower ejection fraction and larger left atrial and left ventricular end-diastolic diameters than the control group (p 0.05) (Table 1).

The proportions of CD4⁺CD25⁺CD127^– Treg cells and CD3⁺CD8^–IL17⁺ Th17 cells in PBMCs were determined. Compared to the control group, the observation group exhibited a significant reduction in the proportion of CD4⁺CD25⁺CD127^– Treg cells (p 0.001). Conversely, there was a significant increase in the proportion of CD3⁺CD8^–IL17⁺ Th17 cells (p 0.001), and the Treg/Th17 ratio was significantly decreased (p 0.001) (Table 2).

In the observation group, RORC mRNA was significantly up-regulated, whereas Foxp3 mRNA was down-regulated (p 0.05) (Fig. 1).

Fig. 1.

The Foxp3 and RORC mRNA expressions. (A) In the observation group, Foxp3 mRNA was lower (n = 6, p 0.05); (B) RORC mRNA was higher (n = 6, p 0.05). Foxp3, forkhead box protein P3; RORC, retinoic acid-related orphan nuclear receptor C; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Serum levels of SPP1, collagen I, IL-10, IL-17A, and IL-23 were measured using ELISA. Compared to the control group, the observation group exhibited a significant increase in SPP1 and collagen I concentrations (p 0.001). Pro-inflammatory cytokines associated with Th17 cells (IL-17A and IL-23) were significantly increased, whereas the anti-inflammatory cytokines linked to Treg cells (IL-10) were significantly reduced (p 0.001) (Table 3).

After tail-vein injection, all eight rats exhibited typical AF waveforms: f waves replaced P waves and R-R intervals were irregular. The duration of AF in all rats exceeded 10 seconds (18.92 $\pm$ 4.76 s, n = 8). Fig. 2 shows a typical ECG of AF. Left atrial tissue concentrations of SPP1, IL-10, IL-17A, and IL-23 were quantified using ELISA. Compared to the normal group, the atrial fibrillation group showed significantly elevated levels of SPP1 (p 0.001). Pro-inflammatory cytokines associated with Th17 cells (IL-17A and IL-23) were significantly increased, whereas the anti-inflammatory cytokines linked to Treg cells (IL-10) were significantly reduced (p 0.001) (Table 4).

Fig. 2.

A typical ECG of AF. AF, atrial fibrillation; ECG, electrocardiograph.

Atrial fibrosis was assessed using Masson’s trichrome staining. Our findings indicate that left atrial fibrosis was significantly more extensive in the AF group than that of the control group (Fig. 3).

Fig. 3.

Atrial fibrosis was assessed by Masson staining. (A) Representative images of Masson staining of the left atrium (magnification, $\times$200); (B) Quantitative analysis of atrial fibrosis (%) calculated from Masson staining (n = 8, p 0.001).

Correlation analysis revealed a statistically significant inverse relationship between SPP1 and the Treg/Th17 ratio (n = 58, r = –0.655, p 0.001) (Fig. 4).

Fig. 4.

A significant negative correlation between SPP1 and Treg/Th17 (n = 58, r = –0.655, p 0.001). SPP1, secreted phosphoprotein 1; Treg, regulatory T cells; Th17, T helper 17.