The clinical trial was approved by the Ethics Committee of Jiangsu Province Academy of Traditional Chinese Medicine (2022-LWKY-025) and has been registered at the Chinese Clinical Trial Registry (ChiCTR2400085614). Informed consent was obtained from all participants. According to the guidelines (Hindricks et al., 2021), PAF is defined as AF that resolves spontaneously or is terminated by intervention within 7 days of onset. Thirty-one adult patients diagnosed with PAF based on 12-lead or 24-h ambulatory electrocardiograph (ECG) reports at our institution between July 2023 and October 2023 were included in this study. Exclusion criteria included: patients with history of coronary artery disease, valvular heart disease, chronic renal failure, hyperthyroidism, cerebrovascular accident and ongoing systemic inflammation (such as in the case of infection, cancer, rheumatoid arthritis or chronic obstructive pulmonary disease) (Li et al., 2017). In addition, six of the patients with PAF were treated with HFLD for 4 weeks. The HFLD granules were provided by Jiangyin Tianjiang Pharmaceutical (China). TCM syndrome scores (see detailed evaluation method in Supplementary Table S1) and sera were examined at baseline and at the end of treatment. Thirty-three healthy volunteers without a history of arrhythmia were included in the normal sinus rhythm (NSR) group. The collected sera were stored at −80°C until use for expression analysis.

According to Shang-Han-Lun, HFLD is composed of Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle (also named honey-fried licorice, Zhigancao in Chinese, 12 g), Zingiberis Rhizoma Recens (Shengjiang in Chinese, 9 g), Ginseng Radix et Rhizoma (Renshen in Chinese, 6 g), Rhemanniae Radix (Dihuang in Chinese, 48 g), Cinnamoni Ramulus (Guizhi in Chinese, 9 g), Asini Corii Colla (Ejiao in Chinese, 6 g), Ophiopogonis Radix (Maidong in Chinese, 10 g), Cannabis Fructus (Maren in Chinese, 10 g), and Jujubae Fructus (Dazao in Chinese, 25 g). All herbs were purchased from the Dispensary of TCM of the Jiangsu Provincial Hospital on Integration of Chinese and Western Medicine.

HFLD was mixed and immersed in 1,400 mL 8% glutinous rice wine and 1,600 mL distilled water for 0.5 h. The compounds were extracted for 1 h with reflux. The extracted solution was filtered through gauze, and the procedure was repeated. Both extracts were combined, added to melted Ejiao, and concentrated to 3.1 g/mL for rats experiments. The remainder solution was concentrated and then made into a lyophilized powder for ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS).

For UPLC, an Acquity HSS T3 chromatographic column (Waters; 2.1 × 150 mm, 1.8 μm) was used. UPLC was performed in conjunction with a SCIEX Exion LC instrument and an X500B Q-TOF mass spectrometer (AB SCIEX, United States). The injection volume was 2 μL, and the flow rate was 0.3 mL/min. The mobile phase was divided into 0.01% aqueous formic acid solution (phase A) and acetonitrile (phase B). Separation was achieved using the following gradient program: 3%–8% B at 0–5 min, 8%–30% B at 5–11 min, 30%–80% B at 11–20 min, 80%–95% B at 20–21 min, 95% B at 21–27 min, and 3% B at 27.5–34 min. The column and autosampler temperature were maintained at 35°C and 4°C, respectively. Standard substances were used to identify the components of the HFLD.

Sixty adult male Wistar rats weighing 250 ± 20 g were purchased from the Experimental Animal Business Department of the Shanghai Institute of Planned Parenthood Research (China). All animals were reared in a room under 12 h light/dark cycles with a temperature of 22°C–24°C and a relative humidity of 40%–60% and had free access to food and water. All animal experiments were approved by the Committee of Experimental Animals of Jiangsu Province Academy of Traditional Chinese Medicine (AEWC-20221015-242) and complied with the National Institutes of Health’s Guidelines for the Care and Use of Laboratory Animals.

As shown in Figure 1, the rats were randomly divided into two groups after 1 week of adaptive feeding. The rats in the experimental group (n = 52) were intraperitoneally injected with isoprenaline hydrochloride (ISO; 5 mg kg^–1·d^–1; TargetMol; United States; T1056) for 2 weeks (Wei et al., 2020), whereas those in the vehicle group (n = 8) underwent equal treatments of normal saline. On day 14, The ECGs were monitored using PowerLab monitoring systems and LabChart 8 software (ADInstruments, Australia) to identify drug-sensitive rats. Later, 40 rats in the experimental group were randomly assigned to five groups and gavaged for 4 weeks with saline (ISO group, n = 8), a low dose of HFLD (6.08 g kg^–1·d^–1; HFLDL group, n = 8), moderate dose of HFLD (12.15 g kg^–1·d^–1; human-equivalent dosage; HFLDM group, n = 8), high dose of HFLD (24.30 g kg^–1·d^–1; HFLDH group, n = 8), or Met (10 mg kg^–1·d^–1 Met tartrate tablets; Met group, n = 8) (Fowler et al., 2018). The same volume of saline was administered to the vehicle group for 4 weeks. The rats were euthanized after atrial stimulation, and their sera and hearts were collected for further research.

The rats were initially anesthetized with 4.5% isoflurane, after which, the concentration was maintained at 1.5%–2%. Echocardiography was conducted using a VINNO 6 LAB ultrasound system (VINNO, China). Two-dimensional guide M-mode echocardiographic data were recorded. Each echocardiographic variable was determined from at least three cardiac cycles.

Before injection, at the time of the last ISO injection, and on the day after the last gavage, the rats were anesthetized and then implanted with subcutaneous electrodes to monitor ECGs.

For atrial stimulation, a 6-French quadripolar catheter was guided through the esophagus and positioned at the site with the lowest threshold for atrial capture. A 50 Hz burst pacing was applied at 3 V above the diastolic threshold for 3 s to assess the susceptibility to AF (Luo et al., 2022; Halațiu et al., 2022). Five successive stimulation cycles were applied to each rat. The onset of AF was indicated by the disappearance of P waves, an irregular R–R interval, and the appearance of tiny f waves, whereas its termination was marked by the recovery of P waves and sinus rhythm, and the disappearance of tiny f waves. We recorded total AF inductions and total AF durations.

The serum levels of creatine kinase-MB (CK-MB; SCIBEN, AE4147), lactate dehydrogenase (LDH; SCIBEN, AE4093), N-terminal pro-brain natriuretic peptide (NT-proBNP; SCIBEN, AE4148), matrix metalloproteinase-9 (MMP-9; SCIBEN, AE4152), interleukin-6 (IL-6; SCIBEN, AE4149 and AE0048), and tumor necrosis factor-alpha (TNF-α; SCIBEN, AE4087 and AE0120) were assessed using commercial ELISA kits according to the manufacturer’s protocols.

Human sera and rat-myocardial tissue homogenates were prepared, and the malondialdehyde (MDA; Beyotime, S0131), superoxide dismutase (SOD; Beyotime, S0101), catalase (CAT; Servicebio, G4307) and glutathione/glutathione disulfide (GSH/GSSG; Servicebio, G4304) contents were measured according to the manufacturer’s instructions. Protein concentrations of tissue homogenates were determined using the BCA Protein Assay Kit (Beyotime, P0012).

Histopathological analyses of atrial tissues were performed to observe the effect of HFLD on the structural remodeling in rats. Heart sections were dehydrated, embedded in paraffin, cut into 4-μm-thick sections using a microtome, and mounted on slides. After dewaxing, Masson trichrome staining (Servicebio, G1006) were performed to reveal fibrotic areas. Briefly, the sections were dehydrated by being treated with gradient ethanol one by one and then washed with tap water. Subsequently, they were immersed in potassium dichromate overnight, and then dyed in a haematoxylin solution for 1 min. Further, the sections were incubated with ponceau and acid fuchsin dye for 6 min, phosphomolybdic acid aqueous solution for 1 min, and aniline blue dye for 30 s. Finally, the slides were treated with 1% acetic acid solution, dehydrated, mounted with mounting solution, and observed under ECLIPSE E100 microscope (Nikon, Japan).

For immunohistochemical staining, the heart sections were incubated with antibodies against collagen I (Servicebio, GB124197-100) and collagen III (Servicebio, GB115674-100) overnight at 4°C. After incubation, the sections were washed gently in phosphate-buffered saline (PBS) and incubated with horseradish peroxide secondary antibodies for 50 min. The sections were then subjected to DAB colorization followed by hematoxylin restaining. Finally, a microscope was utilized to observe the tissue sections.

The cross-sectional areas of the cardiomyocytes were observed via fluorescein-conjugated wheat germ agglutini (WGA) staining. Briefly, paraffin sections were subjected to EDTA antigen repair solution (Servicebio, G1206) at 100°C for 20 min and returned to room temperature naturally, then washed with PBS. The sections were stained with sufficient staining solution (Sigma Aldrich, L4895) and incubated at 37°C in the dark for 1 h. PBS was rinsed three times again. Nuclei were stained with DAPI. After sealing the slide with cover glass, the images were observed and collected by ECLIPSE Ci fluorescence microscopy (Nikon, Japan) in the dark room. The positive areas were quantified using ImageJ software (National Institutes of Health, United States).

DHE staining was used to assay the production of ROS O₂ ^•− in situ (Ren et al., 2019). Briefly, atrial tissue slides were incubated with DHE (Sigma Aldrich, D7008) at 37°C for 30 min, then washed, fixed, mounted, and subjected to fluorescence microscopy.

Atrial tissues were dissected and homogenized in RIPA buffer (Beyotime, P0013) containing protease and phosphatase inhibitors (Thermo Fisher Scientific, A32959). Proteins were separated via 8%–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane. The membranes were blocked with Tris-buffered saline with Tween (TBST) containing 5% bovine serum albumin for 1 h and then incubated with a primary antibody at 4°C overnight. On the following day, the membranes were washed thrice with TBST, incubated with the secondary antibody for 1.5 h, and washed again. Protein bands were then visualized using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, United States; 34580) and an automated 5200 chemiluminescent imaging system (Tanon, China). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, and the protein bands were analyzed using ImageJ. The antibodies used for Western blot analysis are listed in Supplementary Table S2.

The data are expressed as the mean ± standard error of the mean (SEM) and analyzed using GraphPad Prism software (version 9.0). Comparisons of categorical variables between two groups were performed using the chi-square test, differences in continuous variables between two groups were compared using Student’s t-test. Differences among the six groups were analyzed using one-way analysis of variance (ANOVA) after confirming the assumptions of normality (Shapiro-Wilk test) and homogeneity of variance (Brown-Forsythe test). Following a significant one-way ANOVA, Tukey’s post hoc test was performed to assess pairwise differences among all six groups. Adjusted P-values are reported to account for multiple comparisons. P < 0.05 was considered to reflect a statistically significant difference.

The clinical manifestations observed in the NSR and PAF groups were remarkably similar (Table 1), indicating that these samples were statistically qualified for a comparative study.

While evaluating the expression levels of oxidative stress markers and key inflammatory cytokines, it was observed that patients with PAF exhibited higher MDA, TNF-α and IL-6 contents (Figures 2A,E,F), but lower SOD, CAT activities and GSH/GSSG ratio (Figures 2B–D), than individuals with NSR. However, following a 4-week HFLD treatment, these indicators showed improvements (Figures 2G–L). In addition, TCM syndrome scores decreased in patients with PAF between the pre-treatment and post-28 days HFLD treatment (Figure 2M). Importantly, no recurrence of AF was observed during the whole HFLD-treatment period and the follow-up period. These results indicated that oxidative stress was involved in the progression of PAF and that HFLD could prevent AF recurrence and alleviate clinical symptoms related to PAF by inhibiting oxidative stress.

The base-peak ion chromatograms of both electrospray ionization modes (positive and negative) were obtained using UPLC-QTOF/MS (Figure 3A). A total of 139 components were identified based on the molecular formula, MS/MS-fragmentation behavior, and comparison with reference standards, including flavonoids, flavonoid glycosides, saponins, sugars, organic acids, coumarins, amino acids, and iridoid glycosides. The identities and MS/MS data of these compounds were obtained, and most ingredients originated from honey-fried licorice and Ginseng Radix et Rhizoma (Supplementary Table S3).

Rats in the experimental group were intraperitoneally injected with ISO (5 mg/kg) for 2 weeks, and rats in the vehicle group were injected with the same volume of normal saline (Figure 1).

There were notable changes in ECG morphology in rats of the ISO group (Figure 4A). Specifically, the RR interval was shortened and QTc prolonged in the ISO group compared to those in the vehicle group (Figure 4B). Meanwhile, the QRS amplitude decreased and the ST segment was significantly elevated (Figure 4C).

Cardiac structure and function were assessed using echocardiography (Figure 4D). Consistent with previous results (Li et al., 2021), echocardiographic examinations indicated a compensatory increase in ejection fraction (EF, Figure 4E) and fractional shortening (FS, Figure 4F). Additionally, ISO treatment significantly reduced cardiac output (Figure 4G) and stroke volumes (Figure 4H). Furthermore, ISO triggered increased interventricular septum and left-ventricular posterior wall thickness, along with a reduction in the left-ventricular internal diameter (Supplementary Table S4).

In addition, elevated levels of myocardial injury markers, including CK-MB, LDH, and NT-proBNP, were observed in rats following ISO treatment (Figures 4I–K). These findings suggest that ISO application significantly induced cardiac hypertrophy and established cardiac injury in rats, which could effectively increase their susceptibility to AF in rats (Ma et al., 2020).

Rats were then assigned into six groups and treated with normal saline, HFLD with different concentrations, or Met, respectively. One hour after the last treatment, programmed electrical stimulation was performed to detect the susceptibility to AF (Figure 5A). Normal saline did not cause AF in rats in the vehicle group, as the induction rate was 0 (0/8); one rat in the ISO group died during stimulation, so the total induction rate in the ISO group was 57.14% (4/7). HFLD or Met treatment effectively decreased the induction rate (Figure 5B). Additionally, the total AF duration significantly increased under ISO injection, whereas it was reduced after the HFLD and Met treatments (Figure 5C).

Post-treatment evaluation via ECG and echocardiography revealed no significant alterations in most cardiac parameters. However, a decline in EF and FS was observed, suggesting mild systolic dysfunction. HFLD and Met interventions partially mitigated these deteriorations, suggesting a potentially beneficial role in preserving cardiac performance (Supplementary Tables S5, S6).

Moreover, our results showed that compared with the vehicle group, ISO induced an increased cardiomyocyte size, and HFLD treatment attenuated the ISO-induced hypertrophy in cardiomyocytes in a concentration-dependent manner (Figures 6A,B). Moreover, the heart weight (HW): tibia length (TL) ratio increased in the ISO group, which was significantly prevented in HFLD-fed rats (Figure 6C). Met also alleviated these trends to some extent but the efficacy was not as good as that of HFLD. These results indicate that HFLD significantly suppressed cardiac hypertrophy in ISO-induced cardiac injury.

The vasoactive peptide, NT-proBNP, is a crucial biomarker of AF (Bai et al., 2023). MMP-9 and its associated tissue inhibitor of metalloproteinases are key molecules for tissue remodeling (Kaireviciute et al., 2010). Therefore, we measured serum NT-proBNP, MMP-9, TNF-α, and IL-6 levels. We observed that the ISO group displayed markedly higher levels of these biomarkers than the vehicle group, and HFLD treatment significantly mitigated this cytokine release caused by myocardial damage (Figures 6D–G). Notably, HFLD_H was able to attenuate these biomarkers to a level similar to that in the vehicle group. Based on these results, HFLD was shown to be protective against ISO-induced myocardial injury in rats.

ISO also causes oxidative stress (Liao et al., 2022). Therefore, we detected the expression of oxidative stress markers in atrial tissues. Compared with the vehicle group, significantly higher DHE fluorescence was observed in the ISO group, indicating that ISO successfully led to ROS production. Meanwhile, HFLD treatment attenuated the expression of ROS (Figure 7A). Furthermore, the activities of SOD, CAT and GSH/GSSG ratio were remarkably decreased, and MDA concentrations increased in atrial tissues after ISO treatment. These alterations were partially reversed by HFLD (Figures 7B–E), indicating that HFLD contributed to free-radical scavenging.

The expression levels of NOX2 and its subunits were further examined with Western blot analysis (Figure 7F). ISO treatment resulted in significantly higher protein expression of NOX2, p67^phox, p-p47^phox, p40^phox, p22^phox, and Rac1 than vehicle treatment. However, these ISO-induced changes were significantly inhibited by HFLD treatment. These results suggest that HFLD demonstrated cardioprotective effects by alleviating oxidative stress and inhibiting the NOX2 signaling pathway.

Oxidative stress is known to be involved in cardiac fibrosis (Liu Z-Y. et al., 2023). To further study the mechanism through which HFLD blocks myocardial injury, we first detected atrial fibrosis in rats via Masson’s trichrome staining and immunohistochemistry and the relevant results are shown in Figure 8A. Compared to the vehicle group, ISO induced obvious atrial fibrosis, and HFLD reduced the ISO-induced atrial fibrosis. The semi-analysis further supported our observations, showing that the atrial fibrosis areas in the ISO group were markedly larger than those in the vehicle group (Figures 8B–D). In addition, HFLD therapy decreased the positive areas in a dose-dependent manner, and positive drug Met attenuated the ISO-induced atrial fibrosis similarly to HFLD_L.

We further detected TGF-β1, p-SMAD3, and alpha-smooth muscle actin (α-SMA) expression via Western blot analysis (Figure 8E). Our results validated that ISO significantly increased the expression of TGF-β1, p-SMAD3, and α-SMA compared to the respective expressions in the vehicle group, and treatment with HFLD suppressed these expression. In contrast, these protein expression levels in the HFLD_H group decreased more significantly compared with those in other groups. These results suggest that HFLD attenuated atrial fibrosis by regulating the TGF-β1–SMAD3 pathway.