Honey-fried licorice decoction ameliorates atrial fibrillation susceptibility by inhibiting the NOX2-ROS-TGF-β1 pathway

Abstract

Objective: Honey-fried licorice decoction (HFLD), a well-established traditional Chinese medicine, is widely used to treat atrial fibrillation (AF) in China. However, the specific cardioprotective mechanisms of HFLD in treating AF remain unclear. This study aimed to determine the efficacy of HFLD and validate the efficacy and mechanisms of action of HFLD in reducing AF susceptibility.

Methods: Serum oxidative stress biomarker levels of healthy controls and patients with paroxysmal AF were detected using enzyme-linked immunosorbent assay kits. The HFLD components were identified using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Wistar rats were intraperitoneally injected with isoprenaline (5 mg/kg) for 2 weeks to construct an AF rat model. The effect of HFLD on AF was assessed with the transesophageal atrial pacing technique and histopathological analysis. The expression levels of NOX2-ROS-TGF-β1 signaling pathway related proteins were detected using Western blot and dihydroethidium staining.

Results: Our clinical trial verified that the expression of MDA increased, whereas SOD, CAT and GSH/GSSG ratio decreased in the serum of patients with paroxysmal AF compared with that in individuals with a normal sinus rhythm. Notably, HFLD treatment could reverse these imbalances. In rat experiments, HFLD was found to reduce oxidative stress and extracellular matrix deposition, thereby effectively reducing AF's induction rate and duration. Western blot analysis indicated that HFLD downregulated the expression of NOX2 and its regulatory proteins, leading to the inhibition of the downstream TGF-β1-SMAD3 signaling pathway.

Conclusion: HFLD may reduce AF susceptibility by inhibiting the NOX2-ROS-TGF-β1 pathway, potentially providing new perspectives on AF treatment.

Keywords: NOX2-ROS-TGF-β1 pathway; atrial fibrillation; fibrosis; honey-fried licorice decoction; oxidative stress.

FIGURE 1

Schematic experimental procedure for HFLD treatment of the rat model.

FIGURE 2

HFLD treated PAF by alleviating oxidative stress and inflammation. (A–F) The levels of MDA, SOD, CAT, GSH/GSSG ratio, TNF-α and IL-6 in serum samples from patients in the NSR and PAF groups. NSR, n = 33; PAF, n = 31. Compared using unpaired Student’s t-test. ^* P < 0.05, ^** P < 0.01 compared with the NSR group. (G–M) The changes in serum MDA, SOD, CAT, GSH/GSSG ratio, TNF-α, IL-6 and TCM syndrome scores in patients with PAF between the pre-treatment and post 28 days HFLD treatment (n = 6). Compared using paired Student’s t-test. ^* P < 0.05, ^** P < 0.01 compared with pre-treatment.

FIGURE 3

Base-peak intensity chromatograms of HFLD using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. (A) Negative-ion mode. (B) Positive-ion mode. The peak number corresponds to the compound number in Supplementary Table S3.

FIGURE 4

ISO caused myocardial damage in rats. (A) Representative ECG tracings on day 14. (B) RR, PR, P wave, QRS, and QTc duration quantification (n = 8). (C) P wave, QRS, and T wave amplitude quantification (n = 8). (D) Representative M-mode images of echocardiography. (E–H) Statistical analyses of the ejection fraction, fractional shortening, cardiac output, and stroke volume (n = 8). (I–K) Serum levels of CK-MB, LDH, and NT-proBNP (n = 8). ^* P < 0.05, ^** P < 0.01 compared with the vehicle group.

FIGURE 5

HFLD decreased the susceptibility to AF in rats 4 weeks after ISO injection. (A) Representative ECG recordings after atrial-burst pacing. (B) Total induction rates of AF in each group. (C) Total AF durations in each group. ^* P < 0.05 compared with the vehicle group. ^# P < 0.05 compared with the ISO group.

FIGURE 6

HFLD prevented ISO-induced myocardial damage in rats. (A) Representative WGA staining (×20 and ×100 magnification; scale bar: 100 μm and 20 μm). (B) Quantification data of WGA staining (n = 3). (C) HW: TL ratio in each group (n = 8). (D–G) Levels of serum NT-proBNP, MMP-9, TNF-α, and IL-6 (n = 4).^* P < 0.05, ^** P < 0.01 compared with the vehicle group. ^# P < 0.05, ^## P < 0.01 compared with the ISO group.

FIGURE 7

HFLD suppressed oxidative stress by blocking the activation of NOX2 signaling in rats. (A) Representative dihydroethidium staining performed to analyze ROS production in the atrium (×40 magnification; scale bars: 50 μm). (B) MDA contents of rat atrial tissues (n = 4). (C) SOD activities in atrial tissues (n = 4). (D) The activities of CAT in rat atrial tissues (n = 4). (E) The relative concentrations of GSH, GSSG and GSH/GSSG ratio in rat atrial tissues (n = 4). (F) Western blot analysis of NOX2 and its subunits protein expression (n = 3). ^* P < 0.05, ^** P < 0.01 compared with the vehicle group. ^# P < 0.05, ^## P < 0.01 compared with the ISO group.

FIGURE 8

HFLD reduced atrial fibrosis by regulating the TGF-β1–SMAD3 pathway in rats. (A) Masson’s trichrome staining and immunochemistry revealed collagen I and collagen III expression in atrial tissues (×5 and ×25 magnification; scale bars: 400 μm and 80 μm). (B–D) Quantitative data of fibrosis and collagen I and collagen III expressions (n = 3). (E) Western blot analysis of TGF-β1, p-SMAD3, SMAD3, and α-SMA protein expression (n = 3). ^* P < 0.05, ^** P < 0.01 compared with the vehicle group. ^# P < 0.05, ^## P < 0.01 compared with the ISO group.