EGCG inhibits atrial fibrosis and reduces the occurrence and maintenance of atrial fibrillation and its possible mechanisms

doi:10.21203/rs.3.rs-2331153/v1

Purpose The aim of this study was to investigate the effect and mechanism of epigallocatechin-3-gallate (EGCG) on atrial fibrillation (AF) in rats.

Methods A rat AF model was established by angiotensin-II (Ang-II) induction, to verify the relationship between atrial fibrosis and the AF. The expression levels of TGF-β/Smad3 pathway molecules and lysyl oxidase (LOX) in AF were detected. Subsequently, EGCG was used to intervene Ang-Ⅱ-induced atrial fibrosis, to explore the role of EGCG in the treatment of AF and its inhibitory mechanism on fibrosis. It was further verified that EGCG inhibited the production of collagen and the expression of LOX through the TGF-β/Smad3 pathway at the cellular level.

Results The results showed that the induction rate and maintenance time of AF in rats increased with the increase of the degree of atrial fibrosis. Meanwhile, the expressions of Col I, Col III, molecules related to TGF-β/Smad3 pathway, and LOX increasedsignificantly in the atrial tissues of rats in the Ang-II induced group. EGCG could reduce the occurrence and maintenance time of AF by inhibiting the degree of Ang-induced rat atrial fibrosis. Cell experiments confirmed that EGCG could reduce the synthesis of collagen and the expression of LOX in cardiac fibroblast induced by Ang-II. The possible mechanism is to down-regulate the expression of genes and proteins related to TGF-βSmad3 pathway.

Conclusion EGCG could downregulate the expression levels of collagen and LOX by inhibiting the TGF-β/Smad3 signaling pathway, alleviating Ang-II-induced atrial fibrosis, which in turn inhibited the occurrence and curtailed the duration of AF.

Atrial fibrillation

Epigallocatechin-3-gallate

Lysyl oxidase

Atrial fibrosis

Angiotensin II

Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice in which the normally ordered pacing mechanisms of the atria are replaced by diffuse, chaotic electrical activity patterns[1, 2]. AF has the characteristics of insidious onset, many complications, and high disability. With the rapid advancement of social aging, the number of patients with AF will increase exponentially. Thrombosis and/or thromboembolism can occur due to the stasis of blood in the atria. More than 60% of patients with AF have significantly reduced quality of life or exercise tolerance, in addition, AF can increase the risk of stroke by 5 times and increase the risk of death by 1.5-2 times[3]. AF has caused a substantial social burden and there is an urgent need to significantly increase the investment in social medical resources[4, 5].

Currently, the treatment of AF is mainly in two areas: heart rate (HR) control and rhythm control[6]. HR control improves subjective symptoms through the long-term use of ventricular rate-lowering drugs in patients, but it does not cure AF, and potentially has serious complications. Currently, it is mainly limited to patients with failed rhythm control[7]. Rhythm control mainly adopts cardioversion methods such as antiarrhythmic drugs, electrical defibrillation, and surgical ablation, among which radiofrequency ablation is the most effective method. Even so, the sinus maintenance rate at 6 months after surgery is only about 50–75%, which does not meet the expectations of doctors and patients[8]. The latest literature reports that after cardioversion treatment for persistent AF, atrial electrical remodeling and nerve remodeling can be reversed entirely, while structural remodeling characterized by atrial fibrosis continues to be maintained, so AF is still prone to relapse at the later stage[9].

Drinking tea may reduce the risks of heart disease and reduce cardiovascular mortality[10–12]. Oral administration of tea catechins attenuates chronic ventricular remodeling after myocardial ischemia and improves left ventricular dysfunction in autoimmune myocarditis[13]. Epigallocatechin-3-gallate (EGCG), the most critical phenolic in tea, has been shown to inhibit oxidation, reduce inflammation and resist atherosclerotic plaque formation, reduce collagen deposition, and has been shown to promote cardiovascular health[14]. EGCG prevents cardiac hypertrophy in rats and exhibits positive inotropic effects on isolated rat and guinea pig atria[15–17]. EGCG has also been shown to be effective in preventing an increase in left ventricular size and an improvement in systolic function in animals with coarctation of the aorta[18]. Previous studies[19] have shown that EGCG is a specific inhibitor of transforming growth factor-β (TGF-β) type I and II receptors. In a clinical study[19] of oral ECGG therapy for specific pulmonary fibrosis, the content of extractable type I collagen (Col I) and the expression level of p-Smad3 in lung tissue of patients receiving EGCG treatment was also significantly reduced, suggesting that EGCG may inhibit the expression of lysyl oxidase (LOX), which can oxidize lysine and hydroxylysine in collagen fibers to form cross-links, through the TGF-β signaling pathway, reducing the cross-linking of collagen fibers and collagen deposition. At the same time, a study have also confirmed that EGCG can directly regulate left atrial electrophysiological characteristics and Ca²⁺ homeostasis and restrain isoproterenol-induced atrial arrhythmias by inhibiting Ca²⁺/calmodulin or cyclic guanosine monophosphate-dependent protein kinase[20].

Therefore, this study mainly explores the role and mechanism of EGCG in the treatment of AF. Firstly, we verified the relationship between the degree of atrial fibrosis and the occurrence and maintenance of AF. Secondly, we intervened in the atrial fibrosis process in rats using EGCG to determine whether it could reduce the occurrence of AF and shorten the duration of AF. Finally, we explored the possible mechanism of EGCG, which reduced the content of p-Smad3 by inhibiting TGF-β receptor activity, inhibited the expression of the downstream target gene LOX, and reduced the cross-linking and deposition of collagen fibers.

Experimental animals

In this experiment, male Sprague Dawley (SD) rats, aged 5-6 weeks, weighing 180-190 g, were selected for specific pathogen-free experiments, all of which were ordered from Chengdu Dashuo Laboratory Animal Co., Ltd. (China). SD rats were reared in a standard environment for 1 week to adapt to the new environment, followed by subsequent experiments. The feeding and experimental procedures of all SD rats strictly adhere to the regulations on the protection of experimental animals of Sichuan University, and all meet the ethical requirements of experimental animals.

Establish a rat model of atrial fibrosis

Angiotensin II (Ang II, 200ng/kg/min, Rebosci, Shanghai, China) was used to induce atrial fibrosis with osmotic minipumps (model 2004; Alzet) for 4 weeks as previously described[21]. A total of 30 rats were randomly divided into 3 groups. Namely, blank group, kept in the original environment for 4 weeks (n=11); N.S. group, filled with Osmotic minipumps with normal saline (n=9); Ang-Ⅱ group (n=10).

Ingestion of EGCG in a rat fibrosis model

From the second day after implantation of the osmotic minipumps filled with Ang-II/N.S., the intragastrical treatment of EGCG (Solarbio, Beijing, China) was performed for 4 weeks with different doses related to the weight of the rat, daily. The experiment was divided into 8 groups as follows: Group A: Blank group, namely the previous N.S. group; Group B: Sham group, namely the previous Ang-Ⅱ group; Group C: N.S.+EGCG (25mg/kg/d) group, fill the osmotic minipumps with normal saline; Group D: N.S.+EGCG (50mg/kg/d) group; Group E: N.S.+EGCG (100 mg/kg/d) group; Group F: Ang-Ⅱ+EGCG (25mg/kg/d) group, fill the osmotic minipumps with Ang II; Group G: Ang-Ⅱ+EGCG (50mg/kg/d) group; Group H: Ang-Ⅱ+EGCG (100mg/kg/d) group.

Detection of electrophysiological indicators in rats and induction of AF

Electrophysiological studies were performed on all rats after 4 weeks of feeding as required. Rats were anesthetized by inhalation of isoflurane (3-5%, Reward, Shenzhen, China). Basic electrophysiological parameters, including HR, PR interval, QRS width, and QT interval, were recorded by limb lead electrocardiograms (ECG) with the system (BF-4205, Chengdu, China). After that, a temporary 5F cardiac pacing electrode (St. Jude Medical, America) was inserted through the right jugular vein to the entrance of the right atrium, at which point the atrial ECG showed a bi-directional P wave (Fig. 1A, B, C). After the electrocardiogram of the rat was stable and the heart rates tend to be stable, the detection of electrophysiological indexes and subsequent induction of AF will be started.

First, the pacing threshold was detected by the bipolar programmed stimulator (YC-3, Chengdu, China) with S₁S₁mode (using the advanced increment/decrement mode, initial stimulus voltage of 0.1 v, wave width 5 ms, pulse number 20, increasing the pacing voltage based on 0.1v, when the ECG showed 1:1 atrial capture, the pacing voltage was gradually reduced by 0.01v based on the pacing voltage at the moment until the atrial capture occurred again, and the voltage at the moment was the pacing threshold. In subsequent experiments, the pacing voltage was 4 times the pacing threshold. Sinus node recovery time (SNRT), conduction time (SACT), AV nodal effective refractory period (AVNERP), and atrial effective refractory period (AERP) were determined by the S1S1 and S1S2 stimulation procedures according to the previous description[22].

High-frequency stimulation was given for 30 consecutive seconds, with the frequency successively starting from 15Hz to 80Hz with an increase of 5Hz. The pulse width was 5ms, and the stimulation voltage was 4 times the pacing threshold. The stimulation of each frequency was repeated twice, and the interval of each stimulation was 5min. Attacks of AF are defined as atrial arrhythmias with a duration of >1s after high-frequency stimulation. The ECG shows that the sinus P wave is replaced by a wavy f wave, and that RR intervals are absolutely unequal. After electrophysiological studies, all animals were sacrificed, and hearts were removed.

Cell culture

Adult rat cardiac fibroblasts were prepared and cultured as previously described[23]. The purity of cardiac fibroblasts was greater than 95% as determined by positive staining for vimentin and fibroblast-specific protein-1 (FSP-1) (Bioss, Beijing, China). The cardiac fibroblasts of the second to third passage were used for all experiments, and the cells were starved for 6 h in Dulbecco’s modified Eagle’s medium (DMEM) (KeyGen, Jiangsu, China) supplemented with 0.5% fetal bovine serum (Gibco, America) before the corresponding intervention.

CCK-8 assay and scratch-wound assay

The CCK-8 assay (Dojindo, Shanghai, China) and scratch-wound were performed as previously described[24].

Exploration of the mechanism of action of EGCG

The second generation of cardiac fibroblasts in good growth condition were selected, diluted to 3×10⁵/ml, and inoculated into 6-well plates, each well was added with 1ml, then filled with medium to 2ml, and cultured in a 37℃ incubator with 5%CO₂ for 12h. After changing the fresh medium, related drugs were added to different groups to continue the culture for 24h. The groups are as follows: group A: blank control group; group B: EGCG group, fibroblasts were intervened with 10 uM EGCG; group C: Ang-Ⅱ+EGCG group, firstly culture fibroblasts with 10 uM EGCG for 30min, then add 10nM Ang-Ⅱ; group D: Ang-Ⅱ+BAPN+EGCG group, cultured fibroblasts with 10 uM EGCG and 1 mM BAPN for 30 min, then added 10 nM Ang-Ⅱ; group E: Ang-Ⅱ+TGF-β group, cultured fibroblasts with 5ng/ml TGF-β for 30min, then added 10nM Ang-Ⅱ; group F: Ang-Ⅱ group, fibroblasts were intervened with 10 nM Ang-Ⅱ; group G: Ang-Ⅱ+TGF-β+EGCG group, cultured fibroblasts with 10 uM EGCG and 5ng/ml TGF-β for 30min, then added 10nM Ang-Ⅱ; group H: Ang-Ⅱ+BAPN group, cultured fibroblasts with 1mM BAPN for 30min, then added 10nM Ang-Ⅱ.

Histological analysis

All atria were fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin. Sections of atria (4–5mm thick) were prepared and stained with Masson and picrosirius red (PSR) for collagen deposition. To determine the degree of cardiac fibrosis, the collagen area fraction was calculated as the percent of the surface area occupied by collagen. The quantification was performed with Image Pro-Plus 6.0 software (Media-cybernetics) by two independent investigators blinded to the experimental procedure.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA from tissues or cultured cells was extracted using Trizol reagent (Invitrogen). One microgram of total RNA was reversely transcripted using One-step RT Kit (Takara Biotechnology) and the resulting cDNA was used as a PCR template. The mRNA expression levels were determined using SYBR-Green Quantitative PCR Kit (Takara Biotechnology) by iCycler iQ system (Bio-Rad). All PCRs were performed in triplicate. Rat-specific primers (Table S1) for collagen-I, smad3 and LOX were synthesized by Invitrogen. Relative expression level of gene was calculated following the manufacturer’s instructions using the ΔΔCt method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control to normalize the Ct value of the target genes.

Western blot analysis

Rabbit anti-Col I, Col III antibody was purchased from Boster (Wuhan, China). Rabbit anti-Smad3, p-Smad3 and LOX antibodies were purchased from Sigma (America). Western blot analyses were performed as previously described[25], and GAPDH (Boster, Wuhan, China) was used as a loading control.

Statistical analysis

All experimental data were divided into count data and measurement data. All count data were described by median (interquartile range) and percentage, while measurement data were described by mean ± standard deviation (M±SD). After the data were summarized, statistical software GraphPad Prism 9.0 and SPSS 24.0 were used to analyze and make statistical analysis graphs. The differences between the experimental data of measurement data were statistically analyzed by Student's t-test and One-way ANOVA, while the count data were statistically analyzed using the chi-square (χ²) test. When P<0.05, the difference between two groups or between multiple groups is considered to be statistically significant. If a pairwise comparison between multiple groups is required, the adjusted P value will be used. When the adjusted P<0.05, pairwise comparisons in multiple groups can be identified as having statistically different.

Establishment of a rat model of atrial fibrillation and evaluation of the degree of atrial fibrosis

A total of 30 rats, after the implantation of micro-osmotic pumps, showed no infection, abscess, or death during the observation period. After 4W, all rats were anesthetized with the same isoflurane concentration to record the basic electrophysiological indicators, and the main indicators include HR (beats/min), PR interval (ms), QRS width (ms), and QT interval (ms). The results showed that there were no significant differences in each index among the blank group, N.S. group and Ang-Ⅱ group (Adjusted P>0.05) (Table S2).

Electrophysiological indicators detected by atrial pacing electrode in rats in each group

Atrial pacing was performed in all rats after recording basic electrophysiological indicators to detect the pacing threshold (voltage, V), SNRT (ms), SACT (ms), AVNERP (ms), AERP (ms) and other indicators, assessing the stability of cardiac electrical activity and atrial fibrillation in rats. The pacing threshold voltage of each group fluctuated between 0.40-0.70V, but there were no significant differences between the groups (P>0.05). There was no statistical difference in SNRT and SACT between the groups (P>0.05), indicating that cardiac electrical activity was relatively stable among rats in each group. The AVNERP and AERP of the Ang-Ⅱ group were significantly decreased compared to the Ang-Ⅱ group (P<0.05), suggesting that continuous pumping of Ang-Ⅱ may increase susceptibility of rats. There were no significant differences in AVNERP and AERP between the blank group and the N.S. group (P>0.05), indicating that the implantation of Osmotic minipumps alone does not affect the electrical activity of the rat heart (Table 1).

Induction and maintenance time of AF in rats

AF of the rat was defined as an atrial arrhythmia with a duration of more than 1s on the ECG, manifested as sawtooth f waves and absolute unequal RR intervals; maintenance time refers to the duration from the end of rapid high-frequency stimulation to the resumption of sinus rhythm (Fig. 1C, D, E). Persistent AF was defined as the persistence of AF features on the ECG for more than 5 minutes without returning to sinus rhythm.

During high-frequency stimulation, AF was induced in each group of rats. Compared with the N.S. group, the Ang-Ⅱ group had an increased number of AF occurrences and a significantly higher AF induction rate (Fig. 2A, 3B; N.S. group: 58/252, Ang-Ⅱ group: 171/280, P<0.05), the maintenance time of non-persistent AF also increased significantly (Fig. 2C; N.S. group: 73.3±8.8s, Ang-Ⅱ group: 195.2±9.4s, P<0.05), and the occurrence of persistent AF showed an increasing trend (Fig. 2D; N.S. group: 11/252, Ang II group: 37/280).

However, compared with the N.S. group and the Blank group, there were no statistically significant differences in the number of AF occurrences, the AF induction rate, the duration of non-sustained AF, and the number of persistent AF episodes (Fig. 2; P>0.05). It can be seen that Ang-Ⅱ could increase the occurrence and maintenance time of AF in rats. Similarly, osmotic minipumps did not affect on the induction and maintenance time of AF in rats.

Masson staining of the atrial tissue of rats in each group

Masson staining was performed on the paraffin sections of the rat atrial tissue in each group, and blue collagen fibers were observed between cells (Fig. 3). Image-Pro Plus 6.0 software was used to measure the blue collagen area and the tissue area of the entire visual field, and calculate CVF (collagen area/tissue area×100%). The results showed that the CVF of atrial tissue in the Ang-Ⅱ group was significantly higher than that in the N.S. group (Fig. 3; Ang-Ⅱ group: 9.74±0.77%, N.S. group: 7.47±0.46%, P<0.05), suggesting that Ang II could promote collagen production and atrial fibrosis. There was no significant difference in atrial fibrosis between the blank group and the N.S. group (Fig. 3; blank group: 7.48±0.24, P>0.05), indicating that the implantation of Osmotic minipumps did not affect the fibrotic process of atrial tissue.

Picrosirius red staining of the atrial tissue of rats in each group

The paraffin sections of the rat atrial tissue in each group were stained with picrosirius red, and Col I showed thick orange and bright red fibers under a polarized light microscope, while Col III showed thin green fibers (Fig. 4). Image-Pro Plus 6.0 software was used to measure the area of Col I and the area of Col III, and to calculate the collagen type ratio (area of Col I/area of Col III). The results showed that the Col I ratio in the Ang-Ⅱ group was significantly higher than that in the N.S. group (Fig. 4D; Ang-Ⅱ group: 3.09±1.92, N.S. group: 1.49±0.32, P<0.05), suggesting that Ang-Ⅱ can promote the qualitative degree of atrial fibrosis and increase the cross-linking of fibers to increase insoluble collagen fibers. At the same time, there was no statistical difference in the degree of atrial fibrosis between the blank group and the N.S. group (Fig. 4D; blank group: 1.57±0.42, P>0.05), indicating that the implantation of Osmotic minipumps did not affect the fibrotic process of the atrial tissue.

Expression of fibrosis-associated molecules in the atrial tissue of rats in each group

The above results show that Ang-Ⅱ could induce atrial fibrosis and increase the induction and maintenance time of AF. At the same time, Ang-II increased the expression of Col I and Col III, and the content of Col I was also significantly higher. On this basis, we further verified the gene and protein expression levels of Ang-II-induced atrial fibrosis pathway-related molecules by qRT-PCR and Western blot to explore the possible mechanism.

In the qRT-PCR experiment, we detected the mRNA expression levels of Col I, Smad3, and the downstream effector molecule LOX. The results showed that the expressions of Col I mRNA, Smad3 mRNA, and LOX mRNA in the Ang-Ⅱ group were significantly higher than those in the N.S. group, and the differences were statistically significant. The expression levels of the above genes in the blank group and the N.S. group had the same trend, and the difference was not statistically significant (Fig. 5B; P>0.05).

In the Western blot experiment, we detected the protein expression levels of Col I, Col III, TGF-β1, Smad3, p-Smad3, and LOX. Statistical analysis of relative gray value showed that compared with the N.S. group, the expressions of Col I, Col III, TGF-β1 and LOX and the phosphorylation levels of Smad3 increased significantly in the Ang-Ⅱ group (Fig. 5A; P<0.05), indicating that Ang-II induced the expression of Col I, Col Ⅲ, TGF-β1, Smad3, p-Smad3, and LOX during atrial fibrosis, contributing to increased collagen fiber production and cross-linking of collagen fibers. The expression of each index tended to be the same in the blank group compared with the N.S. group, and the difference was not statistically significant (Fig. 5A; P>0.05), indicating that the implantation of osmotic minipumps had no significant effect on the expression of the above genes and proteins in the rat atria.

EGCG inhibits atrial fibrosis and reduces the occurrence and maintenance of AF by down-regulating LOX expression through TGF-β/Smad3 pathway

The rat AF model verifies that EGCG can reduce the occurrence and maintenance of AF

This part of the experiment was divided into 8 groups with a total of 79 rats. Group A and B were set as the N.S. group and the Ang-Ⅱ group in the upper part of the experiment. The drug treatment groups were divided into 6 groups with a total of 60 rats, and the oral dose of EGCG was 25 mg/kg/d, 50 mg/kg/d, 100 mg/kg/d, respectively. No incision infection or abscess occurred in all rats after the implantation of osmotic minipumps, and no death occurred during the observation period. The oral dose of EGCG increased with increasing rat body weight, and the required dose of EGCG was recalculated by measuring the body weight of the rats once a week. Diet and exercise were normal in all rats, and no rats showed depression or poor mobility.

Basic electrophysiological indicators of limb lead ECG of rats in each group

After 4W of different doses of EGCG intervention, all rats were anesthetized with the same isoflurane concentration for basic recording electrophysiological indicators, and the results showed that there was no statistical significance among groups (Table S3; P>0.05).

Atrial pacing was performed in all rats after recording basic electrophysiological indicators to detect the pacing threshold (voltage, V), SNRT (ms), SACT (ms), AVNERP (ms), AERP (ms). The results showed that the pacing threshold voltage of each group fluctuated between 0.40V-0.73V, SNRT and SACT tended to be consistent among the groups, but there were no statistically significant differences (Table 2; Adjusted P>0.05) , indicating that the intervention of EGCG did not have a significant effect on the stability of cardiac electrical activity. Both AVNERP and AERP increased in group E compared with group A (P<0.05), indicating that the 100 mg/kg/d EGCG intervention in rats reduced atrial susceptibility with age in normal rats. AVNERP and AERP also increased significantly in groups G and H compared with group B (P<0.05), indicating that the intake of EGCG could inhibit Ang-II-induced atrial susceptibility in rats, and the 100 mg/kg/d intake group had the most obvious effect. There were no significant differences in AVNERP and AERP between group C and group A, between group D and group A, and between group F and group B (P>0.05), indicating that low dose of EGCG intake had no significant effect on age and susceptibility induced by Ang-II in rats (Table 2).

Induction and maintenance time of AF in each group of rats

After measuring the corresponding electrophysiological indicators by atrial pacing, burst stimulation induced AF. Compared with group A, the frequency of AF occurrence and the induction rate of AF in groups D and E decreased, and the difference was statistically significant (group A: 58/252, group D: 38/280, group E: 9/280; Fig. 6A, B; P< 0.05), the maintenance time of non-sustained AF in groups C, D, and E was significantly reduced (group A: 73.3±8.8s, group C: 56.7±7.3s, group D: 47.9 ±5.9s, group E: 17.1±1.8s; Fig. 6C; P<0.05) and the number of persistent AF also decreased gradually (group A: 11/252, group C: 6/280, group D: 4/280, Group E: 0/280, Fig. 6D), indicating that the intake of a certain dose of EGCG could inhibit the induction rate of AF and reduce the maintenance time of AF with age, and 100 mg/kg/d EGCG could significantly inhibit the occurrence of persistent AF.

Compared with group B, the occurrence of AF and the AF induction rate in groups G and H also reduced significantly, and the differences were statistically significant (group B: 171/280, group G: 64/280, group H: 21/280; Fig. 6A, B; P<0.05), the maintenance time of non-sustained AF reduced significantly (group B: 195±9.4s, group G: 69.9±7.1s, group H: 25.7±3.2s; Fig. 6C; P <0.05), the occurrence of persistent AF reduced significantly (group B: 37/280, group G: 3/280, group H: 1/280; Fig. 6D; P<0.05), indicating that the intake of 50 mg/kg/d and 100 mg/kg/d of EGCG could inhibit the induction rate of Ang II-induced AF, shorten the maintenance time of AF, and reduce the occurrence of persistent AF, but low-dose EGCG may not offset the changes of rat atrial structure caused by Ang-Ⅱ.

EGCG inhibited the elevation of collagen expression and the alteration of collagen typeinduced by Ang-II in rat atrial tissue

To clarify the pathological changes of the rat atrial tissue induced by different doses of EGCG, we performed Masson and PSR staining in each group of rats, and the results are shown in Fig. 7A and B. Results of the statistical analysis of Masson staining showed that compared with group A, the CVF of the atrial tissue of rats in group E decreased significantly (group E: 6.13±0.42%, group A: 7.47±0.46%; Fig. 7A; P<0.05), suggesting that 100 mg/kg/d EGCG could inhibit the increase of collagen fibers in the atrial tissue of rats with age, thereby slowing down the process of atrial fibrosis of rats. Compared with group B, the CVF of group G and group H decreased significantly (group G: 8.45±0.13%, group H: 7.00±0.38%, group B: 9.74±0.77%; Fig. 7A; P< 0.05), indicating that 50 mg/kg/d and 100 mg/kg/d EGCG could alleviate the production of collagen fibers in rat atrial tissue caused by Ang-Ⅱ, and reduce the quantitative degree of atrial fibrosis. At the same time, there was no significant difference in atrial CVF between groups F and B, and groups C, D, and A (group F: 8.77±0.16%, group C: 7.54±0.59%, group D: 6.54±0.43 %; Fig. 7A; P>0.05), indicating that low-dose EGCG could not counteract the process of Ang II-induced atrial fibrosis.

Results of the statistical analysis of PSR staining showed that compared with group A, the ratio of collagen types in the atrial tissue of rats in groups D and E decreased, and the difference was statistically significant (group D: 0.70±0.08, group E: 0.54±0.04, group A: 1.49±0.08 0.32; Fig. 7B; P<0.05), suggesting that a certain dose of EGCG can inhibit the cross-linking of collagen fibers, reduce the production of insoluble collagen fibers, and then inhibit the qualitative degree of atrial fibrosis. Compared with group B, the ratio of atrial collagen types in group G and group H was significantly lower (group G: 2.49±0.27, group H: 0.96±0.02, group B: 3.09±0.19; Fig. 7B; P<0.05), indicating that the degree of atrial fibrosis induced by Ang II can be alleviated by taking a certain dose of EGCG. At the same time, there was no significant difference in the ratio of atrial collagen types between groups F and B and between groups C and A (group F: 2.79±0.01, group C: 1.12±0.08; Fig. 7B; P>0.05), indicating that low-dose EGCG could not counteract Ang-Ⅱ-induced atrial fibrosis.

Expression of fibrosis-related genes and proteins in atrial tissue of rats in each group

Basic electrophysiological studies and pathological staining results indicated that EGCG could inhibit Ang-Ⅱ-induced atrial fibrosis and then reduce the induction and maintenance time of AF and reduce the production of collagen fibers. On this basis, we further verified the genes and proteins of the pathway-related molecules of atrial fibrosis by qRT-PCR and Western blotting and explored the possible mechanism.

It was detected by qRT-PCR experiment that the expression levels of Col I mRNA, Smad3 mRNA, and LOX mRNA in atrial tissue decreased after EGCG intake intervention and that the expression of the above genes decreased significantly in group E compared with group A, and in groups G and H compared with group B, respectively (Fig. 8B; P<0.05). Moreover, the expression of Smad3 mRNA was significantly inhibited by the intake of EGCG, and the low dose of EGCG also reduced the expression of Smad3 mRNA.

In the Western blot experiment, we detected expressions of Col I, Col III, TGF-β1, Smad3, p-Smad3, and LOX proteins, and the results are shown in Fig. 8A. Statistical analysis of relative gray value showed that the expression of the above proteins was significantly decreased in group E compared with group A, and in groups F, G and H compared with group B, respectively, with statistically significant differences (Fig. 8A; P<0.05).

The results of these two assays showed that EGCG at 50 mg/kg/d and 100 mg/kg/d inhibited the expression of Col I and Col Ⅲ in atrial tissue and had an inhibitory effect on the proteins of the classical TGF-β/Smad3 pathway in the fibrosis process, reducing the expression of molecules related to this pathway. Meanwhile, to clarify that EGCG could inhibit the degree of fibrosis by reducing the cross-linking of collagen fibers, LOX, a key enzyme for collagen fiber cross-linking, was examined. The results showed that EGCG significantly inhibited the expression of LOX, suggesting that EGCG inhibited the quantifiable and qualitative degree of Ang II-induced atrial fibrosis probably by inhibiting collagen production and down-regulating LOX expression.

Extraction, culture and identification of primary rat cardiac fibroblasts phenotypes

Cardiac fibroblasts, an essential part of the heart, provide structural support for cardiomyocytes. When cultured in vitro, the cell morphology was irregular triangle or spindle under an inverted microscope, usually with 2-3 elliptical nuclei. The cells were arranged disordered and did not beat spontaneously. FSP-1 and Vimentin are phenotypic proteins expressed specifically by cardiac fibroblasts, and positive expression of specific antibodies can be seen by cellular immunofluorescence staining, namely the red fluorescence in Fig. 9, confirming that the primary cells we extracted were cardiac fibroblasts.

CCK-8 assay to detect the proliferation-toxicity of EGCG at different concentrations on fibroblasts

To determine the optimal concentration of EGCG, we set the concentration gradient of EGCG, which are 0uM, 1uM, 10uM, 100uM, and 1000uM, respectively. After the intervention with different concentrations of EGCG, the culture was continued for 12h, and then the CCK-8 kit was used to detect the proliferation-toxicity of EGCG on cardiac fibroblasts. Since the discoloration of the EGCG solution by oxidation in the air may affect the detection of absorbance, the remaining medium and drug were removed prior to the addition of CCK-8 reagent, and the complete medium was added again. The results showed that when the concentration of EGCG was 100uM, the cytotoxicity increased significantly (Fig. S1). Combined with previous animal experiments, a low concentration of EGCG may not be able to compensate for Ang-II-induced atrial fibrosis, so we determined that the optimal concentration of EGCG was 10uM for subsequent experiments.

Cell scratch test results

The scratch test can verify the effect of EGCG on the migration of cardiac fibroblasts at 24h and 48h. The results showed that at 24h and 48h, the migration rate of the blank group was significantly higher than that of the EGCG intervention group and the difference was statistically significant (Fig. S2; P<0.05), suggesting that EGCG reduced the crawling and healing abilities of cardiac fibroblasts.

Western blot and qRT-PCR detection of expression levels of TGF-β/Smad3 pathway molecules and LOX

Since TGF-β1, as one of the intervention measures in this group of experiments, was added separately as an agonist of the pathway in the corresponding group, it was not detected in the Western blot experiment. Cells were divided into 8 groups for intervention, namely: A: blank control group; B: EGCG group; C: Ang-Ⅱ+EGCG group; D: Ang-Ⅱ+BAPN+EGCG group; E: Ang-Ⅱ+TGF-β group; F: Ang-Ⅱ group; G: Ang-Ⅱ+TGF-β+EGCG group; H: Ang-Ⅱ+BAPN group.

The Western blot and qRT-PCR experiments were performed on cells from the above 8 groups after drug intervention. The results are shown in Fig. 10. The statistical analysis of the relative gray value shows that: compared with the F group, the expressions of Col I, Col III, Smad3, p-Smad3, LOX, Col I mRNA, Smad3 mRNA, and LOX mRNA in the E group were significantly increased (Fig. 10; P<0.05), suggesting that TGF-β1 could further enhance Ang-Ⅱ-induced collagen expression and the expression of TGF-β/Smad3 pathway corresponding molecules and LOX in cardiac fibroblasts; collagen，pathway-related molecules were significantly lower in groups C, D, and G, respectively, compared with group F, with statistically significant differences (Fig. 10; P<0.05), suggesting that EGCG could downregulate the expression of the above molecules in Ang II-stimulated cardiac fibroblasts and that EGCG significantly inhibited the promoting effect of TGF-β1. The expression of the above molecules was consistent in group C compared with group D and in group F compared with group H. The differences were not statistically significant (Fig. 10; P>0.05), suggesting that the BAPN, LOX inhibitor, did not inhibit the expression of collagen and LOX by Ang II and TGF-β1 on cardiac fibroblasts.

Therefore, EGCG could reduce the occurrence and maintenance time of AF and significantly reduce the occurrence of persistent AF by inhibiting the quantitative and qualitative degree of atrial fibrosis induced by Ang-Ⅱ in rats. The mechanism may be that EGCG reduces collagen fiber synthesis in rat atrial tissue through the TGF-β/Smad3 pathway, while decreasing the expression of LOX, a key enzyme for collagen fiber cross-linking, and thus inhibiting collagen fiber cross-linking, alleviating atrial fibrosis. Cell experiments also confirmed that EGCG could inhibit the migration and healing ability of cardiac fibroblasts and reduce the synthesis of Col Ⅰ and Col Ⅲ and the expression of LOX in cardiac fibroblasts. The possible mechanism is down-regulation of the expression of genes and proteins related TGF-β/Smad3 pathway.

Establishment of an animal model of AF

In this experiment, we successfully established a paroxysmal AF model in rats by constructing Ang-II continuous pumping to induce atrial fibrosis and inducing AF by rapid pacing with bipolar atrial pacing electrodes after a certain induction time. The experimental results showed that AF could be induced in all SD rats in the Ang-II induction group, and the total success rate of AF induction was up to 61% at different pacing frequencies.

Currently, common animal models of AF are mainly established using large animals because large animals are more conducive to macroscopic observation of the structural features of the central atrium of AF and facilitate the study of atrial arrhythmias[26]. However, the establishment of AF in large animals mostly requires open-heart surgery and implantation of rapid pacemakers, which is expensive to establish the model and keep the animals. The operation process is complicated, and the stability of AF establishment is also not high, while the individual differences among large animals are large, making the study results vary significantly within the group and the conviction of the study conclusions is reduced. In contrast, small animal models can effectively avoid the above disadvantages and, at the same time, provide a reliable way to study the genetic and molecular mechanisms of atrial arrhythmias, but small animal AF models are mostly paroxysmal, and the success rate in establishing stable long-term AF is low[26,27].

One of the main purposes of this study is to clarify the relationship between the pathway molecules involved in the quantification and qualitative degree of atrial fibrosis and the occurrence and maintenance of AF. Therefore, it is necessary to induce atrial fibrosis at first. We established atrial fibrosis induced by Ang-II to achieve structural reconstruction, and then AF was induced by rapid atrial pacing, which was consistent with the experimental purpose.

Elevated expression of TGF-β/Smad3 pathway molecules and LOX in the atrial fibrosis process in AF

Our experimental results showed that continuous pumping of Ang II by subcutaneously implanted osmotic minipumps containing Ang II resulted in decreased expression of Col I and Col Ⅲ, and significantly increased expression of protein and mRNA of molecules related to the TGF-β/Smad3 pathway and LOX, a critical enzyme for collagen fiber cross-linking, in the atrial tissue of SD rats. Electrophysiological assays showed a significant decrease in AVNERP and AERP in the Ang II-induced group, suggesting an increased susceptibility to AF, and AF-induced experiments showed a significant increase in the success rate and maintenance time of AF induction. The experimental results above indicate that the quantification and qualitative degree of Ang II-induced atrial fibrosis are consistent with the induction rate and maintenance time of AF, verifying that structural remodeling based on fibrosis is one of the key substrates for the development of AF, and the susceptibility and maintenance time of AF increase with increasing fibrosis.

Ang-II is one of the crucial molecules of pro-fibrosis and plays a vital role in the development and maintenance of AF[28]. It was shown[29] that continuous pumping of Ang II (200 ng/kg/min) for 4 weeks caused a significant increase in collagen content and increased fibrosis in rat atrial tissue; therefore, in this experiment, a dose of 200 ng/kg/min was used to induce atrial fibrosis, and the results were consistent with the above study. In the cellular model, with reference to the relevant literature[29] and to avoid the toxic and proliferative effects of high concentrations of Ang II on cardiac fibroblasts, we, therefore, chose 10 nM of Ang II in culture for 24 h to establish a cardiac fibroblast fibrosis model for subsequent drug treatment experiments. TGF-β/Smad3 is one of the classic pathways in cardiac fibrosis, and it has been reported[30-32] that Ang II can promote the expression of TGF-β1 mRNA and protein to activate the downstream Smad signaling pathway to increase the synthesis of Col I and Col Ⅲ. Our experimental results also showed elevated TGF-β1 and Smad3 expression and significant atrial fibrosis in the Ang II-induced group of rats.

The primary manifestation of atrial structural remodeling is fibrosis due to the deposition of collagen fibers in atrial tissue[33-37]. Studies[38] have shown that fibrosis consists of both quantitative (quantification) and qualitative (quality) changes in collagen, with quantification determined by the amount of Col I and Col III and quality by the cross-linking of collagen fibers. Qian et al.[39] reported that transmission electron microscopy of atrial tissue of AF patients showed that the longer the duration of AF, the easier it is to pounce on collagen fiber crosslinks in anatomical areas such as near the orifice of the pulmonary vein and the posterior wall of the left atrium. Conversely, collagen fibrillar crosslinks could not be captured by electron microscopy at shorter AF durations. Subsequently, further studies found that the higher the degree of fiber cross-linking, the longer the duration of AF; and the correlation between the degree of fiber cross-linking and AF was higher compared with the quantitative degree of Col I and Col III[40]. Our results also showed that Ang-II induction led to increased fibrous cross-linking in atrial tissue resulting in increased insoluble collagen content, suggesting that increased quantitative and qualitative degree of atrial tissue fibrosis may provide the material basis for the maintenance and recurrence of AF.

After LOX is secreted extracellularly by fibroblasts, it can cleave the N and C terminus of protocollagen, the collagen precursor molecule, to form helical monomers in the intercellular matrix, followed by spontaneous formation of Col I and Col Ⅲ collagen filaments under protein hydrolysis and insoluble Col I and Col Ⅲ are formed under the action of fibrous cross-linking[41-43]. Adam et al.[40] examined left atrial tissue from AF patients , found that LOX and fiber cross-linking increased significantly in the left atrium of the AF group compared with patients with sinus rhythm, and that BAPN, a specific small-molecule inhibitor of LOX, inhibited the expression of ligand protein in cardiac fibroblast, all of which are consistent with the results of the present experiment, in which LOX expression was significantly elevated in both Ang II-induced rat atrial tissue and cardiac fibroblasts. Therefore, LOX might be a target to inhibit the degree of fibrosis and thus the onset and maintenance of AF[41-46].

EGCG inhibits atrial fibrosis through TGF-β/Smad3 pathway by downregulating LOX expression to reduce the onset and maintenance of AF

We intervened in Ang II-induced atrial fibrosis and AF development by orally administering EGCG at graded concentrations. The results showed that the induction rate and maintenance time of AF were significantly reduced in rats orally administered 50 mg/kg/d and 100 mg/kg/d EGCG, and the quantitative and qualitative extent of atrial fibrosis were correspondingly significantly reduced, and the expression of Col I and Col Ⅲ was decreased. Preliminary investigation of the related mechanisms revealed that the expression of TGF-β1, Smad3, p-Smad3, and LOX was reduced in the EGCG-treated group, suggesting that EGCG may block the TGF-β/Smad3 signaling pathway to reduce the expression of collagen and down-regulate the content of LOX, which then affects the fibrosis process in the atria and reduces the occurrence and maintenance time of AF.

EGCG is the most dominant and essential polyphenol in green tea and has played a significant role in medical research and treatment due to its anti-bacterial and viral proliferation and parasitism, antioxidant capacity, anti-vascular sclerosis, inhibition of thrombosis, anti-inflammatory cell aggregation, and anti-tumor proliferation[14,47-49]. Recent studies have shown that EGCG also has sound anti-fibrotic effects, showing excellent anti-fibrotic effects in skin scar repair, liver fibrosis, kidney fibrosis, lung fibrosis, and ventricular fibrosis[50-56]. Wang et al.[52]studied the hepatoprotective effects of catechins using CCl₄-induced liver injury and showed that intraperitoneal injection of EGCG 600 mg/ kg/w significantly attenuated the extent of liver injury and fibrosis process, and the mechanism may be inhibition of TGF-β and downstream ERK and Smad signaling pathways, while Kochi et al.[57] believed that EGCG could inhibit nonalcoholic liver fibrosis by inhibiting inflammation and reducing oxidative stress. In a study of protective effects on the kidney, Chen et al.[58] treated a rat kidney injury model exposed to cadmium contamination by gavage with 300 mg/kg/d EGCG and showed that EGCG ameliorated cadmium-induced kidney injury, inhibited oxidative stress levels, normalized the antioxidant status of renal enzymes and E-calmodulin levels, and attenuated TGF-β1, p-Smad3 waveform protein and α-SMA overproduction. In an in vivo study of acute myocardial infarction-related cardiac fibrosis, treatment with EGCG (50 mg/kg) reduced the area of cardiac fibrosis by 64.5% and reduced end-diastolic and end-systolic dimensions of the left ventricular, improving cardiac function[56]. Thus, EGCG has shown wide therapeutic promise in the course of fibrotic lesions in various organs, but whether EGCG also plays a protective role against AF has not been reported. Therefore, in this study, EGCG was ingestion by gavage to relieve Ang-II-induced atrial fibrosis, and the results were consistent with the above studies, which confirmed that EGCG could reduce the occurrence and maintenance time of AF by inhibiting atrial fibrosis.

Harold et al.[19] reported the use of EGCG as an inhibitor of TGF-β receptor in the treatment of patients with pulmonary-specific fibrosis and showed significantly less collagen deposition and cross-linking in the patients' lung tissue and significantly lower expression of the downstream molecule p-Smad3, suggesting that EGCG may affect the expression of LOX through the TGF-β/Smad3 signaling pathway. Therefore, we also proposed the hypothesis that EGCG could inhibit the expression of atrial LOX to slow down the process of atrial fibrosis, inhibit the occurrence of AF, and shorten the maintenance time of AF (Fig. 11), and the experimental results also confirmed our conjecture. Therefore, EGCG may have great application prospects in the anti-fibrotic effect of AF.

Meanwhile, in adult rat cardiac fibroblasts, the upregulation of mRNA, protein and activity of LOX (accompanied by an increase in Col I, Col Ⅲ) by TGF-β1 involves multiple signaling pathways such as PI3K, Smad3, p38-MAPK, JNK and ERK1/2[59]. Upregulation of LOX through TGF-β/Smad2/3 signaling was shown to promote cardiac fibrosis and chronic heart failure. In this rat model, TGF-β/Smad2/3 promoted the expression of C-jun, a subunit of the AP-1 transcription factor (which induces LOX gene expression)[60]. The results of our cellular experiments also showed that the stimulation of Ang-II and TGF-β1 promoted collagen and LOX expression in cardiac fibroblasts, whereas EGCG significantly inhibited the activation of TGF-β1. Therefore, we inferred that EGCG may downregulate LOX expression by inhibiting the TGF-β/Smad3 pathway, reducing the synthesis of Col I and Col Ⅲ, thereby improving the quantification and qualitative extent of atrial fibrosis.

Of course, the present study still has many limitations: firstly, the failure to establish chronic persistent AF in an animal model may have some limitations to the wider application of EGCG; secondly, the drug concentration of EGCG in vivo was not tested, and the pharmacokinetic characteristics of EGCG and the most suitable therapeutic concentration could not be accurately elaborated; finally, the therapeutic effect of EGCG on AF may involve the involvement of multiple mechanisms, but the present study only explored the interaction between EGCG and atrial fibrosis which is closely related to atrial fibrillation. In future experiments, we will further improve the establishment of animal models to make them more consistent with actual clinical cases, and we may choose to increase the observation time of the persistent AF rats in this experiment or keep them alive to establish a long-term persistent AF animal model; secondly, when selecting the concentration of EGCG, we will design more concentration gradients to study the optimal concentration suitable for atrial antifibrosis, and conduct plasma and tissue drug concentration to clarify its pharmacokinetic properties; finally, we will further explore other mechanisms by which EGCG inhibits the onset and maintenance of AF in subsequent experiments.

Quantification and qualitative extent of atrial fibrosis in rat atrial tissues induced by Ang-II were significantly increased, consistent with the increased induction rate and maintenance time of AF. The TGF-β/Smad3 pathway plays a crucial role in the process of atrial fibrosis, and the expression levels of its related molecules are significantly elevated, while LOX expression is elevated in fibrotic atrial tissues and increases with the degree of fibrosis; EGCG may downregulate the expression levels of Col I, Col Ⅲ and LOX by inhibiting the TGF-β/Smad3 signaling pathway, alleviating Ang II-induced quantification and qualitative extent of atrial fibrosis in rats, which in turn inhibited the occurrence and curtailed the duration of AF.

Author Contributions

Tao Li: Conceptualization, Methodology, Investigation, Software, Validation, Data curation, Formal analysis, Writing – original draft, Visualization; Tong Qi: Conceptualization, Methodology, Validation, Data curation, Formal analysis, Writing – review & editing; Zhengjie Wang: Formal analysis, Resources, Writing – review & editing, Visualization; Ziqi Yang: Software, Resources, Writing – review & editing; Ziqi Yang: Conduct animal experiments; Yiren Sun: Conduct animal experiments; Jie Cai: Data Collection; Qiyue Xu: Writing – review & editing; Yuan Lu: Writing – review & editing; Xuemei Liu: Statistical analysis; Ke Lin: Conceptualization, Writing – review & editing, Supervision, Project administration, Funding acquisition, Yongjun Qian: Conceptualization, Writing – review & editing, Supervision, Project administration, Funding acquisition. All authors read and approved the final manuscript.

Acknowledgments

This work was funded by the Scientific Research Project of National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University (No. Z2018B19), and 1⋅3⋅5 project for disciplines of excellence–Clinical Research Incubation Project, West China Hospital, Sichuan University (No. 2019HXFH029), the Major Science and Technology Project of Sichuan Province, China (No. 2021YFS0121), and Technology innovation research and development project of Chengdu Science and Technology Bureau (No. 2019YF05-00183-SN), and the Science and Technology Project of Sichuan Province Health Commission, China (No. 21PJ035).

Supplementary Material

Supplementary data to this article can be found online.

Data Availability Statement

The data sets generated and analyzed in the course of this study are available from the corresponding authors upon reasonable request.

Ethics Approval

All applicable international national and/or institutional animal care and use guidelines have been followed. This article does not contain any studies conducted by any author with any human participants.

Consent to Participate

Not applicable.

Consent for Publication

Not applicable.

Conflict of Interest

The authors declare no conflict of interest.

Informed Consent

Not applicable.

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Table 1

Electrophysiological indexes of jugular vein - right atrium after pacing in each group

Indexes	Blank （n=11）	N.S. （n=9）	AngII （n=10）
pacing threshold（V）	0.53±0.07	0.51±0.09	0.61±0.07
SNRT（ms）	185.6±21.0	197.7±23.8	213.9±24.5
SACT（ms）	18.2±3.1	19.2±2.4	18.1±4.0
AVERP（ms）
140	86.8±4.6	90.0±3.9	83.4±3.8^#
130	88.6±3.6	88.6±3.7	83.3±3.2^#
120	87.1±4.2	89.8±3.8	83.1±3.3^#
AERP（ms）
140	78.3±4.8	79.7±4.3	73.6±2.6^#
130	78.4±4.7	79.4±3.6	73.3±1.9^#
120	78.0±4.1	79.9±4.1	73.1±3.0^#

^#, vs N.S. P<0.05

Table 2

Electrophysiological indexes of jugular vein - right atrium after pacing in each group

Indexes	A （n=9）	B （n=10）	C （n=10）	D （n=10）	E （n=10）	F （n=10）	G （n=10）	H （n=10）
pacing threshold（V）	0.51±0.09	0.61±0.07	0.54±0.07	0.55±0.08	0.53±0.08	0.59±0.06	0.60±0.06	0.54±0.05
SNRT（ms）	197.7±23.8	198.9±20.4	184.2±16	180.1±11.9	179.9±17.2	200.1±21.3	181.1±13.6	184.3±16.4
SACT（ms）	19.2±2.4	18.1±4	15.6±3.6	16.7±3.1	15.7±3.1	17.8±3.4	16±2.7	17.6±2.6
AVERP（ms）
140	90±3.9	83.4±3.8^*	92.8±4.7	93.6±4.2	96.4±3.5^*	84.5±3.2	89.3±2.8^#	95.9±3.9^#
130	88.6±3.7	83.3±3.2^*	92.3±4.8	93.1±3.3	95.9±2.7^*	84±3	87.4±4.7	95.8±4.7^#
120	89.8±3.8	83.1±3.3^*	92.8±4.5	93.8±4.3	96.5±3.6^*	84.9±3.3	88.9±3.1^#	95.3±3.5^#
AERP（ms）
140	79.7±4.3	73.6±2.6^*	82.5±4.8	84.4±4.3	85.3±3.1^*	75.8±4.3	80±4.1^#	83.5±4.2^#
130	79.4±3.6	73.3±1.9^*	81.9±4.9	84.4±3.0^*	85.0±2.2^*	75.9±3.8	80.3±3.8^#	83.6±5.0^#
120	79.9±4.1	73.1±3.0^*	82.6±4.9	84.0±4.8	86.5±3.6^*	76.9±4.0	79.3±2.8^#	84.0±4.0^#

*, vs group A, P<0.05；#, vs group B, P<0.05.

EGCG inhibits atrial fibrosis and reduces the occurrence and maintenance of atrial fibrillation and its possible mechanisms

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Experimental animals

Establish a rat model of atrial fibrosis

Ingestion of EGCG in a rat fibrosis model

Detection of electrophysiological indicators in rats and induction of AF

Cell culture

CCK-8 assay and scratch-wound assay

Exploration of the mechanism of action of EGCG

Histological analysis

Quantitative real-time polymerase chain reaction (qRT-PCR)

Western blot analysis

Statistical analysis

Results

Establishment of a rat model of atrial fibrillation and evaluation of the degree of atrial fibrosis

EGCG inhibits atrial fibrosis and reduces the occurrence and maintenance of AF by down-regulating LOX expression through TGF-β/Smad3 pathway

Discussion

Establishment of an animal model of AF

Elevated expression of TGF-β/Smad3 pathway molecules and LOX in the atrial fibrosis process in AF

EGCG inhibits atrial fibrosis through TGF-β/Smad3 pathway by downregulating LOX expression to reduce the onset and maintenance of AF

Conclusion

Declarations

Author Contributions

Acknowledgments

Supplementary Material

Data Availability Statement

Ethics Approval

Consent to Participate

Consent for Publication

Conflict of Interest

Informed Consent

References

Tables

Supplementary Files

Status:

Journal Publication

Version 1