Establishment method and application of phlegm-heat stasis type atrial fibrillation rat model

Abstract

The invention discloses an establishment method and application of a phlegm-heat stasis type atrial fibrillation rat model, relates to the technical field of medical research, and has the technical key points that an internal environment of phlegm-heat is established by adopting a method of combining high-fat diet with lipopolysaccharide intraperitoneal injection; an animal model of phlegm-heat stasis type atrial fibrillation is constructed in a mode of triggering an electrophysiological mechanism of atrial fibrillation by Ach-Cacl2, so that the internal mechanism of atrial fibrillation is reflected in multiple dimensions, a model evaluation system conforming to the multi-target action characteristic of traditional Chinese medicine is established, and individualized anti-arrhythmia treatment is promoted.

Description

Technical Field

[0001] The present invention relates to the field of medical research technology, and in particular to a method for establishing a rat model of phlegm-heat and blood stasis type atrial fibrillation and its application. Background Art

[0002] Atrial fibrillation is one of the most common arrhythmias in clinical practice. The 2021 Global Burden of Disease Study found that the prevalence of atrial fibrillation was 520,000 and the incidence was 550,000. The high incidence is accompanied by high disability and mortality rates. Atrial fibrillation can significantly increase the risk of stroke, coronary artery disease and heart failure, and increases with age. It will become a major disease burden in the future.

[0003] The pathophysiology of atrial fibrillation is complex, involving a vicious cycle of multi-dimensional remodeling, including electrical remodeling, structural remodeling, and autonomic remodeling. The underlying mechanisms remain unclear. Current treatments for atrial fibrillation primarily rely on antiarrhythmic drugs, radiofrequency ablation, and anticoagulation. These treatments are limited by numerous adverse reactions, low sinus conversion rates, and a high risk of recurrence. Furthermore, most drugs only target electrophysiological abnormalities, such as ion channels, while neglecting upstream mechanisms such as inflammation and oxidative stress.

[0004] Animal models of atrial fibrillation are crucial for exploring the pathophysiology of atrial fibrillation. Current small animal models of atrial fibrillation primarily rely on rapid atrial pacing, drug induction, and gene modification, offering advantages such as short cycles and high reproducibility. However, existing animal models fail to simulate the multidimensional pathological phenotypes of atrial fibrillation, hindering the development of novel therapies. Traditional animal models of atrial fibrillation only mimic the electrical and structural remodeling pathological phenotypes of Western medicine, while ignoring upstream inflammatory and oxidative stress mechanisms, failing to reflect the complex characteristics of TCM syndromes.

[0005] To this end, the present invention aims to provide a method for establishing a rat model of phlegm-heat and blood stasis type atrial fibrillation and its application to solve the above problems. Summary of the Invention

[0006] The purpose of the present invention is to solve the above problems and provide a method for establishing and applying a rat model of atrial fibrillation caused by phlegm-heat and blood stasis. The present invention adopts a high-fat diet combined with intraperitoneal injection of lipopolysaccharide to construct an internal environment of phlegm-heat, and constructs an animal model of atrial fibrillation caused by phlegm-heat and blood stasis by the electrophysiological mechanism of Ach-Cacl2 triggering atrial fibrillation. The invention aims to reflect the internal mechanism of atrial fibrillation in multiple dimensions, establish a model evaluation system that conforms to the multi-target action characteristics of traditional Chinese medicine, and promote individualized antiarrhythmic treatment.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows:

[0008] The present invention provides a method for establishing a rat model of phlegm-heat and blood stasis type atrial fibrillation and its application, the method comprising the following steps:

[0009] S1. Six-week-old male SPF SD rats were fed a hyperlipidemia model diet for 3 weeks;

[0010] S2. In the second week, rats were injected with Ach-Cacl2 (Ach 60 μg / ml, Cacl2 10 mg / ml) 0.1 ml / 100 g via tail vein for 14 consecutive days. In addition, 1 mg / ml lipopolysaccharide 0.1 ml / 100 g was injected intraperitoneally once a week to establish a rat model of phlegm-heat and blood stasis type atrial fibrillation.

[0011] S3. Perform cardiac function tests on rats with phlegm-heat and blood stasis type atrial fibrillation and record the data;

[0012] S4. Perform histopathological and molecular biological tests on the rat model of phlegm-heat and blood stasis type atrial fibrillation.

[0013] The hyperlipidemia model feed comprises basic feed+20% sucrose+15% lard+1.2% cholesterol+0.2% sodium cholate+others.

[0014] The phlegm-heat and blood stasis type atrial fibrillation rat model is cultured under the conditions of 12-hour light and dark alternation, temperature of 20-24° C., humidity of 50%-70%, and free food and water intake.

[0015] The data include atrial fibrillation induction rate, atrial fibrillation duration, electrocardiogram indicators, cardiac ultrasound indicators (left atrial size, aortic diameter, left ventricular end-systolic volume, left ventricular end-diastolic volume, left ventricular end-systolic minimum diameter, left ventricular end-diastolic maximum diameter, ejection fraction, shortening fraction), inflammatory factors, blood lipid levels, coagulation and blood rheology indicators, fibrosis indicators and other data.

[0016] The present invention also provides a use of a rat model of phlegm-heat and blood stasis type atrial fibrillation, wherein the rat model of phlegm-heat and blood stasis type atrial fibrillation is used to simulate atrial fibrillation fibrosis induced by chronic inflammatory response.

[0017] Compared with the existing technology, this solution has the following beneficial effects:

[0018] The present invention adopts a strategy of combining a high-fat diet with intraperitoneal injection of lipopolysaccharide to construct an experimental model simulating the internal environment of phlegm-heat, and constructs an animal model of phlegm-heat and blood stasis-type atrial fibrillation by using the electrophysiological mechanism of Ach-Cacl2-triggered atrial fibrillation. This model aims to fully reveal the intrinsic mechanism of atrial fibrillation and establish a model evaluation system that is consistent with the multi-target action characteristics of traditional Chinese medicine to promote the development of individualized treatment strategies for arrhythmias. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a flow chart for constructing an animal model of atrial fibrillation of phlegm-heat and blood stasis type according to an embodiment of the present invention;

[0020] Figure 2Schematic diagram of the comparison of the general conditions of rats in each group in the embodiment of the present invention, A: body weight; B: food intake; C: water intake; compared with group C, * p<0.05, ** p<0.01, **** p<0.0001;

[0021] Figure 3 Schematic diagram of electrocardiogram in an embodiment of the present invention, A: normal electrocardiogram; B: atrial fibrillation electrocardiogram;

[0022] Figure 4 Schematic diagram of the comparison of the electrocardiogram of rats in each group according to the embodiment of the present invention, A: heart rate; B: RR interval; C: QRS interval; D: QT interval; Compared with group C, **** p<0.0001; compared with group M, #### p<0.0001;

[0023] Figure 5 Schematic diagram of the comparison of cardiac function of rats in each group according to the embodiment of the present invention, A: relative size of left atrium; B: ejection fraction; C: shortening fraction; compared with group C, * p<0.5, **** p<0.0001; compared with group M, #### p<0.0001;

[0024] Figure 6 Schematic diagram of the pathological staining of the left atrium of each group of rats in the embodiment of the present invention (200X), A: HE staining and MASSOM staining of the left atrium of each group of rats; B: Masson staining fiber area quantification of each group of rats (n=6); compared with group C, * p<0.05, **** p<0.0001; compared with group M, #### p<0.0001;

[0025] Figure 7 Schematic diagram of the expression of fibrin in each group detected by Western Blot in the embodiment of the present invention, A: Western Blot diagram; B: relative protein expression of MMP9; C: relative protein expression of COI-1; D: relative protein expression of α-SAM; compared with group C, ** p<0.01, *** p<0.001, **** p<0.0001; compared with group M, # p<0.05, ## p<0.01;

[0026] Figure 8Schematic diagram of the changes in the expression of MMP-9, COI-1, and α-SAM RNA in each group detected by qRT-PCR in the embodiment of the present invention, A: relative mRNA expression of MMP9; B: relative mRNA expression of COI-1; C: relative mRNA expression of α-SAM; compared with group C, *** p<0.001, **** p<0.0001; compared with group M, # p<0.05, ## p<0.01;

[0027] Figure 9 Schematic diagram of the comparison of fiber indexes of rats in each group detected by ELISA method in the embodiment of the present invention, A: MMP-9; B: Cal-3; C: α-SAM; D: COI-1; Compared with group C, ** p<0.01, *** p<0.001, **** p<0.0001; compared with group M, #### p<0.0001;

[0028] Figure 10 Schematic diagram of the comparison of blood lipid levels in rats in each group according to the present invention, A: TC; B: TG; C: HDL-C; D: LDL-C; Compared with group C, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.001; compared with group M, ### p<0.001;

[0029] Figure 11 Schematic diagram of the comparison of fever syndrome indicators in rats of each group in the embodiment of the present invention, A: changes in rectal temperature; B: CRP level; C: IL-1β level; D: IL-6 level; E: IL-17 level; F: TNF-ɑ level; compared with group C, * p<0.05, *** p<0.001,* *** p<0.0001; compared with group M, ### p<0.001, #### p<0.0001;

[0030] Figure 12 3. It is a comparison diagram of tongue images of rats in each group before and after intervention in the embodiment of the present invention. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0032] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0033] Example:

[0034] 1. Materials and Methods

[0035] 1.1. Materials

[0036] 1.1.1. Animals

[0037] Forty 6-week-old, SPF-grade male Sprague-Dawley rats weighing 180–200 g were provided by Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (License SCXK(Beijing)2021-0006). This experiment was approved by the Animal Ethics Committee of Xiyuan Hospital, China Academy of Chinese Medical Sciences (Ethics Approval Number: 2024XLC033-3). The animals were housed in a barrier environment at the Animal Experimental Center of Xiyuan Hospital, China Academy of Chinese Medical Sciences, with a 12-h light-dark cycle, a temperature of (22 ± 2)°C, and a humidity of 50%–70%. The animals had free access to food and water.

[0038] 1.1.2. Animal feed

[0039] Hyperlipidemia model feed (purchased from Xiaoshu Youtai (Beijing) Biotechnology Co., Ltd.) has the following ratio: basic feed + 20% sucrose + 15% lard + 1.2% cholesterol + 0.2% sodium cholate + others.

[0040] Reagents and instruments

[0041] Acetylcholine chloride (MCE, HY-B0282), 500 mg / bottle; calcium chloride (Sigma, 449709), 10 g / bottle; lipopolysaccharide LPS (Sigma, L2880), 100 mg / bottle; 0.9% sodium chloride injection (Beijing Shuanghe Pharmaceutical Co., Ltd., batch number: 0906201w), 500 ml / bottle; PBS buffer (Solaibao, P1020), 500 ml / bottle; sodium pentobarbital powder (Sigma, 20170308); isoflurane (Lunan Beite Pharmaceutical Co., Ltd., batch number: 64191201), 100 ml / bottle.

[0042] Rat C-reactive protein (CRP) ELISA detection kit, rat interleukin 1β (IL-1β) ELISA detection kit, rat interleukin 6 (IL-6) ELISA detection kit, rat interleukin 17 (IL-17) ELISA detection kit, rat tumor necrosis factor α (TNF-α) ELISA detection kit, rat matrix metalloproteinase 9 (MMP-9) ELISA detection kit, rat galectin-3 ELISA detection kit, rat collagenase I (Collagenase I) ELISA detection kit, rat α-smooth muscle actin (α-SMA) ELISA detection kit, TG, CHOL, HDL-C, LDL-C detection kits (Beijing Beijian Xinchuangyuan Biotechnology Co., Ltd., batch numbers B2001, B2002, B2003, and B2004, respectively).

[0043] Wireless biological signal acquisition system (Shanghai Kexin Medical Biotechnology Co., Ltd.), portable small animal color Doppler ultrasound imager (VINNO6 LAB), Purang PUN-208A coagulation analyzer (Pulang Medical), Prism LBY-N6C fully automatic hemorheometer (Beijing Prism Instrument Co., Ltd.), Toshiba Japan fully automatic biochemical analyzer, precision electronic balance (Kunshan Youkeweit Electronic Technology Co., Ltd., ZF-C20001); colorimetric card (Foshan Qiatu Technology Co., Ltd.).

[0044] 1.2. Methods

[0045] 1.2.1. Grouping and model preparation

[0046] After one week of adaptive feeding, 40 SD rats were randomly divided into a blank group (C), a phlegm-heat and stasis-type atrial fibrillation group (M), an atrial fibrillation group (AF), and a phlegm-heat and stasis syndrome group (TRYZ), with 10 rats in each group. Except for groups C and AF, the other two groups were fed a hyperlipidemia model diet and given intraperitoneal injections of 1 mg / ml lipopolysaccharide (LPS) once weekly for 3 weeks. Starting from the second week, rats in groups AF and M received tail vein injections of 0.1 ml / 100 g of ACh-CaCl2 (ACh 60 μg / ml, CaCl2 10 mg / ml), while the other two groups received tail vein injections of 0.1 ml / 100 g of normal saline for 14 consecutive days.

[0047] 1.2.2. Electrocardiogram

[0048] Electrocardiograms were performed on days 15 and 22 of modeling. Rats were weighed and anesthetized with 2% isoflurane gas. The rats were connected to a wireless biosignal acquisition system to record their electrocardiograms. After the electrocardiogram stabilized, 0.1 ml / 100 g of Ach-CaCl was injected via the tail vein. The atrial fibrillation induction rate, atrial fibrillation duration, and electrocardiogram parameters were recorded.

[0049] 1.2.3. Cardiac ultrasound

[0050] On the 22nd day of modeling, the weighed rats were anesthetized with 2% isoflurane gas and then subjected to cardiac function testing. The left atrial size (LA), aortic diameter (AO), left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic minimum diameter (LVIDs), left ventricular end-diastolic maximum diameter (LVIDd), ejection fraction (EF), and shortening fraction (FS) data of the rats were recorded.

[0051] 1.2.4. Obtaining materials

[0052] After the model was established, the rats were fasted but not watered for 12 hours, anesthetized with 1% sodium pentobarbital, and placed flat on a dissecting rack for blood collection from the abdominal aorta. Part of the anticoagulated whole blood was subjected to hemorheology examination, and part of the anticoagulated whole blood was centrifuged at 3500 r / min for 10 minutes. The supernatant was taken as plasma to detect four coagulation parameters. The remaining whole blood was rested for 2 hours and then centrifuged at 3500 r / min for 10 minutes. The supernatant was taken as serum to detect inflammatory factors. Three rat hearts were taken from each group and divided into cryopreservation tubes and frozen in a -80°C refrigerator for Western Blot and qPCR examinations. The remaining rat hearts and left lateral lobe liver tissues were fixed in 4% paraformaldehyde for pathological examination.

[0053] 1.2.5. Histopathological examination

[0054] Fixed rat heart tissue was obtained and prepared into 4 μm paraffin sections. The left atrium was stained with hematoxylin and eosin and Masson staining, and photographed under a 10x20x optical microscope. Image software was used to calculate the area of ​​cardiac fibrosis.

[0055] 1.2.6. Molecular biology testing

[0056] Left atrial protein was extracted and analyzed for MMP9, COI-1, and α-SAM protein expression by Western Blot. RT-qPCR was used to detect changes in the gene expression of these markers, and Image software was used for protein and gene quantitative analysis. ELISA kits were used to assess serum fibrogenic factors, including MMP9, COI-1, α-SAM, and CAL-3.

[0057] 1.2.7. Evaluation of TCM Syndrome Types

[0058] Objective indicators were used to evaluate TCM syndrome types. Blood lipid levels were used to evaluate phlegm syndrome; anal temperature and inflammation levels were used to evaluate heat syndrome; and rat tongue images, four coagulation tests, and blood rheology changes were used to evaluate blood stasis.

[0059] Statistical analysis

[0060] SPSS 27.0 software was used for statistical analysis. If the data conformed to the normal distribution and the variance was homogeneous, the univariate method was used for inter-group comparison. The Dunnett's t test (one-sided) was used for pairwise comparison between groups. The paired T test was used for before-after comparison within the group. If the data did not conform to the normal distribution or the variance was unequal, a nonparametric test was used. Statistical significance was set at p < 0.05. Graphpad Prism 10.1.2 was used for plotting.

[0061] 2. Results

[0062] 2.1. General status

[0063] Compared with the blank group (C group), rats with phlegm-heat and stasis type atrial fibrillation (M group) and phlegm-heat and stasis syndrome (TRYZ group) showed mental depression, fatigue, huddling, dull hair, dry stool, and significant weight loss. The TRYZ group consumed less food and water, while the atrial fibrillation group (AF group) showed no significant changes. Figure 2 .

[0064] 2.2. Atrial fibrillation

[0065] 2.2.1. Atrial fibrillation induction

[0066] Atrial fibrillation was induced by tail vein injection of Ach-Cacl2. Atrial fibrillation was successfully induced in the M and AF groups. The induction rate of atrial fibrillation increased with the increase in modeling time, but it was not statistically significant. Compared with the C group, the induction rate of atrial fibrillation in the M and AF groups was significantly increased, and the duration of atrial fibrillation was prolonged, but there was no statistical difference between the M and AF groups.

[0067] Table 1 Atrial fibrillation induction in rats in each group

[0068]

[0069] Note: Compared with group C, *p<0.05.

[0070] 2.2.2. ECG status

[0071] Compared with group C, the heart rate in group M and group AF decreased significantly, and the PP interval prolonged, which was statistically significant (p<0.0001). There was no significant change in the QRS interval and QT interval, and there was no significant difference in electrocardiographic conditions between the group M and group AF.

[0072] 2.2.3. Cardiac function

[0073] Cardiac ultrasound was used to examine cardiac function in each group. Compared with group C, the left atrium of rats in group M was enlarged (p<0.05), and the ejection fraction (EF) and fractional shortening (FS) were significantly decreased, with statistically significant differences (p<0.0001). The left atrium of rats in group AF was not significantly enlarged, and the ejection fraction (EF) and fractional shortening (FS) were significantly decreased (p<0.0001). However, there was no significant difference between the AF and M groups.

[0074] Cardiac fibrosis

[0075] 2.3.1. Cardiac pathology

[0076] Left atrial tissue sections were stained with hematoxylin and eosin (HE) and Masson staining to compare the levels of myocardial fibrosis among the groups. HE staining revealed that compared with group C, atrial tissue in group M showed locally disorganized myofiber arrangement, partial myocyte swelling and deformation, interstitial edema, numerous inflammatory cell infiltration, and fibrous tissue hyperplasia. Compared with group M, myocardial fibers in groups AF and TRYZ were more uniformly arranged, with less pronounced inflammatory cell infiltration and mild fibrous tissue hyperplasia. Masson staining revealed that compared with group C, collagen and fibrin deposition was observed in groups M, AF, and TRYZ, with more pronounced deposition in group M. Fiber area analysis revealed that compared with group C, fiber area in groups M and AF was significantly increased (p < 0.05, p < 0.0001), while fiber area in groups AF and TRYZ was significantly decreased (p < 0.0001).

[0077] 2.3.2.Western Blot

[0078] Left atrial tissue proteins were extracted and analyzed by Western blot. Matrix metalloproteinase-9 (MMP-9), collagen I (Collagenase I), and ɑ-smooth muscle actin (ɑ-SAM) were compared among the rat groups. Compared with group C, the expression of MMP9, COI-1, and ɑ-SAM proteins in group M were significantly increased (p<0.01, p<0.001, and p<0.0001, respectively). Only ɑ-SAM protein expression increased in group AF (p<0.001). The expression of MMP9 and ɑ-SAM proteins in group TRYZ was significantly increased (p<0.01). Compared with group M, the expression of MMP9 and COI-1 proteins in group AF was significantly decreased, but there was no statistical difference in ɑ-SAM protein.

[0079] 2.3.3.qRT-PCR

[0080] Three samples were collected from each group. Quantitative PCR reactions were performed for the target genes MMP-9, COI-1, and ɑ-SAM, as well as for the internal control, with triplicate wells for each sample. Data were analyzed using the 2-ΔΔCT method. Compared with group C, the mRNA expression levels of MMP-9, COI-1, and ɑ-SAM in groups M, AF, and TRYZ were significantly increased, with the increase being most significant in group M (p < 0.001, p < 0.0001). Compared with group M, the mRNA expression levels of MMP-9 and ɑ-SAM in group AF were decreased, and the mRNA expression of COI-1 in group TRYZ was decreased (p < 0.05, p < 0.01).

[0081] 2.3.4.ELISA

[0082] ELISA was used to measure the levels of fibrogenic factors, including MMP-9, galectin-3 (CAL-3), α-SAM, and COI-I. Compared with group C, M and AF groups showed significantly increased levels of MMP-9, CAL-3, α-SAM, and COI-1 (p < 0.001, p < 0.0001), while TRYZ group showed increased levels of α-SAM and COI-I (p < 0.01, p < 0.0001). Compared with group M, all fibrogenic factors were significantly decreased in groups AF and TRYZ (p < 0.0001).

[0083] 2.4. TCM Syndrome Types

[0084] 2.4.1. Phlegm syndrome indicators

[0085] Phlegm and lipid metabolism disorders are similar, and phlegm syndrome is assessed by blood lipid levels. Compared with group C, total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels in group M and TRYZ groups were significantly increased, with statistical significance (p < 0.05, p < 0.01, p < 0.001, p < 0.0001), but there was no significant change in group AF. Compared with group M, group AF had lower TC and LDL-C levels (p < 0.001), but there was no significant change in group TRYZ and M. There were no significant differences in triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C) levels among the four groups.

[0086] 2.4.2. Heat syndrome indicators

[0087] In animal experiments, rectal temperature and inflammatory cytokine levels are often used to reflect heat syndrome. Compared with group C, rectal temperature in groups M and TRYZ was significantly elevated, while there was no significant change in group AF. There was no significant difference between groups M and TRYZ. Regarding inflammatory cytokines, compared with group C, levels of CRP, IL-1β, IL-6β, IL-17, and TNF-ɑ were significantly elevated in groups M and TRYZ (p < 0.0001), while levels of IL-1β and TNF-ɑ were slightly elevated in group AF (p < 0.05). Compared with group M, levels of CRP, IL-1β, IL-6β, IL-17, and TNF-ɑ were significantly decreased in groups AF and TRYZ (p < 0.001, p < 0.0001, respectively).

[0088] 2.4.3. Blood stasis syndrome indicators

[0089] The tongue image of rats can reflect the overall TCM syndrome type. The tongue images of rats in each group were photographed under a fixed light source, and color was corrected using a colorimetric card. The tongue surface of the rats was divided into a nine-square grid, and the RGB values ​​of the nine points were recorded and averaged. Before the intervention, there were no significant differences in the RGB values ​​of the tongue images among the rat groups. Compared with pre-intervention, the tongue color of the M and TRYZ groups after modeling became dark red, and the RGB values ​​were significantly decreased (p < 0.01, p < 0.001). Furthermore, compared with group C, the RGB values ​​of group M were significantly decreased after the intervention, while only the R value of group TRYZ decreased (p < 0.05, p < 0.01). There was a significant difference in the R value between group M and group AF (p < 0.01).

[0090] Hemorheology was assessed using an automatic hemorheometer. Compared with group C, low-shear whole blood viscosity, high-shear whole blood viscosity, plasma viscosity, erythrocyte sedimentation rate, and low-shear whole blood reduced viscosity were significantly increased in groups M and TRYZ, while hematocrit and erythrocyte aggregation index were decreased (p < 0.05, p < 0.01, and p < 0.001). Compared with group M, hematocrit was significantly increased in group AF (p < 0.01).

[0091] The chromogenic substrate method was used to detect four coagulation parameters. Compared with group C, the thrombin time (TT), activated partial thromboplastin time (APTT), and prothrombin time (PT) levels in groups M, AF, and TRYZ decreased to varying degrees, while the fibrinogen (FIB) level increased (p<0.05, p<0.01, p<0.001). The difference in group M was the most significant, but there was no significant difference between groups AF and M.

[0092] Table 2 Comparison of RGB values ​​of tongue images of rats in each group (N=6)

[0093]

[0094] Note: After intervention, compared with group C, * p<0.05, **p<0.01, compared with group M, ## p<0.01. Compared with before intervention, ΔΔ p<0.01, ΔΔΔ p<0.001.

[0095] Table 3 Comparison of blood rheology in rats of each group (N=6)

[0096]

[0097] Note: Compared with group C, * p<0.05, ** p<0.01, *** p<0.001; compared with group M, ## p<0.01.

[0098] Table 4 Comparison of coagulation parameters in rats of each group (N=6)

[0099]

[0100] Note: Compared with group C, * p<0.05, ** p<0.01, *** p<0.001.

[0101] 3. Discussion

[0102] 3.1. Construction of animal model of phlegm-heat and blood stasis syndrome

[0103] Our research team analyzed the syndrome patterns of 924 patients with atrial fibrillation at Xiyuan Hospital and found that the proportion of phlegm-heat internal disturbance syndrome and phlegm-stasis mutual obstruction syndrome continued to increase, and phlegm syndrome, heat syndrome and inflammatory indicators had a certain correlation. Therefore, constructing an animal model of atrial fibrillation with phlegm-heat and blood stasis syndrome has important clinical value for the traditional Chinese medicine differentiation and treatment of atrial fibrillation.

[0104] This study, drawing on previous literature, established a phlegm-heat and blood stasis syndrome model using a high-fat diet combined with lipopolysaccharide. The Medical Record states, "Phlegm can arise from external exposure to the six pathogenic qi, disrupting the ascending and descending mechanisms of the spleen, lungs, and stomach, leading to inadequate digestion and transport of food; from excessive consumption of fatty, sweet, and alcoholic beverages; and from spleen and stomach yang deficiency, resulting in dampness and turbidity." This indicates that phlegm is often associated with a chronic diet rich in fatty, sweet, and rich foods. Therefore, this study established a phlegm syndrome model using a high-fat diet. Elevated TC and LDL-C levels in rats confirmed the occurrence of phlegm and turbidity. Phlegm and turbidity obstruct qi flow, transforming into heat. Heat and toxicity burn the blood, resulting in a state of phlegm-heat and blood stasis. Stagnation and heat mimic inflammatory responses, altered blood physicochemical properties, and abnormal coagulation mechanisms. Therefore, low-dose lipopolysaccharide was used to intervene and simulate the process of chronic inflammatory response in vivo. The M and TRYZ groups had dry stools, elevated rectal temperatures, and dark red tongues, which were macroscopic manifestations of stasis and heat. The levels of inflammatory factors CRP, IL-1β, IL-6, IL-17, and TNF-ɑ increased, reflecting a state of excessive heat and toxicity. Whole blood viscosity, plasma viscosity, whole blood reduced viscosity, and fibrinogen increased, reflecting a state of blood stasis caused by phlegm-heat blocking the blood circulation. Therefore, the high-fat diet combined with lipopolysaccharide modeling method can reflect the cascade reaction of "phlegm turbidity obstruction → depression and heat → blood stasis blocking the collaterals" and reflect the biological manifestations of phlegm-heat and blood stasis syndrome.

[0105] 3.2. Effect of Phlegm-Heat and Stasis Syndrome on Atrial Fibrillation

[0106] Tail vein injection of Ach-CaCl2 is a common pharmacological method for inducing atrial fibrillation. This study, employing a strategy of etiological superposition and pathological triggering, successfully induced a rat model of phlegm-heat and blood stasis-induced atrial fibrillation. In this study, there were no significant differences in the inducibility or duration of atrial fibrillation between the M and AF groups, consistent with the characteristics of atrial fibrillation. Furthermore, the M group showed significantly increased levels of inflammation and myocardial fibrosis, a significant increase in left atrial volume, worse cardiac function, and more severe atrial remodeling, demonstrating significant value in the study of atrial remodeling induced by atrial fibrillation in rats.

[0107] Myocardial fibrosis is a key hallmark of structural remodeling, leading to uneven conduction within and between the atria, creating a matrix for local reentry and contributing to the progression and persistence of AF. Studies have shown that inflammation is a trigger for myocardial fibrosis in AF. Numerous inflammatory factors, including hs-CRP, TNF-α, IL-6, IL-17, and the NLRP3 inflammasome, can induce AF by promoting fibrosis. Among the Traditional Chinese Medicine (TCM) syndromes, "phlegm syndrome," "heat syndrome," and "stasis syndrome" can manifest as low-grade inflammatory responses. Studies have shown that "phlegm" in TCM is associated with cell and tissue deformation, inflammatory exudation, degeneration, and proliferation. Phlegm-heat syndrome is closely associated with disturbances in inflammatory cells such as eosinophils, mast cells, and T lymphocytes, as well as cytokines such as interleukin-1, interleukin-4, interleukin-5, and interleukin-10. The biological basis of blood stasis syndrome also involves pathological processes such as inflammatory responses. Establishing an animal model of phlegm-heat and blood stasis syndrome can accelerate the progression of inflammation on atrial fibrillation myocardial fibrosis, shorten the time of atrial fibrillation fibrosis modeling, and also contribute to the study of the mechanism of atrial fibrillation myocardial fibrosis.

[0108] 3.3. Significance of the animal model of phlegm-heat and blood stasis-type atrial fibrillation

[0109] Traditional animal models of atrial fibrillation only simulate the pathological phenotypes of electrical and structural remodeling in Western medicine, while ignoring the upstream inflammatory and oxidative stress mechanisms, and are unable to reflect the complex characteristics of TCM syndromes. However, this study revealed the interaction between metabolic disorders (phlegm), inflammatory activation (heat), and fibrosis (stasis) through the combined intervention of high-fat diet, lipopolysaccharide and electrophysiological induction, and accurately reproduced the pathological conduction chain of "phlegm-heat-stasis-fibrillation" in TCM, which is conducive to in-depth research on the mechanism of atrial fibrillation; on the other hand, the construction of an animal model of phlegm-heat and stasis-type atrial fibrillation verified the molecular basis of TCM syndromes, embodied the scientific nature of TCM, and was also conducive to the multi-dimensional research of TCM, reflecting the characteristics of the multi-target effects of TCM.

[0110] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the embodiments as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A method for establishing a rat model of phlegm-heat and blood stasis type atrial fibrillation, characterized by: The method comprises the following steps: S1. Six-week-old male SPF SD rats were fed a hyperlipidemia model diet for 3 weeks; S2. In the second week, rats were injected with Ach-Cacl2 0.1 ml / 100 g via tail vein for 14 consecutive days, and 1 mg / ml lipopolysaccharide 0.1 ml / 100 g was injected intraperitoneally once a week to establish a rat model of phlegm-heat and blood stasis type atrial fibrillation; S3. Perform cardiac function tests on rats with phlegm-heat and blood stasis type atrial fibrillation and record the data; S4. Perform histopathological and molecular biological tests on the rat model of phlegm-heat and blood stasis type atrial fibrillation.

2. The method for establishing a rat model of phlegm-heat and blood stasis type atrial fibrillation according to claim 1, wherein: The hyperlipidemia model feed contains 63.6% basal feed, 20% sucrose, 15% lard, 1.2% cholesterol and 0.2% sodium cholate; The Ach-Cacl2 contains Ach 60ug / ml and Cacl2 10mg / ml.

3. The method for establishing a rat model of phlegm-heat and blood stasis type atrial fibrillation according to claim 1, wherein: The phlegm-heat and blood stasis type atrial fibrillation rat model is cultured under the conditions of 12-hour light and dark alternation, temperature of 20-24° C., humidity of 50%-70%, and free food and water intake.

4. The method for establishing a rat model of phlegm-heat and blood stasis type atrial fibrillation according to claim 1, wherein: The data include atrial fibrillation induction rate, atrial fibrillation duration, electrocardiogram indicators, cardiac ultrasound indicators, inflammatory factors, blood lipid levels, coagulation and blood rheology indicators, fibrosis indicators and other data.

5. The method for establishing a rat model of phlegm-heat and blood stasis type atrial fibrillation according to claim 4, characterized in that: The cardiac ultrasound indicators include left atrial size, aortic diameter, left ventricular end-systolic volume, left ventricular end-diastolic volume, left ventricular end-systolic minimum diameter, left ventricular end-diastolic maximum diameter, ejection fraction and shortening fraction.

6. The use of the rat model of phlegm-heat and blood stasis type atrial fibrillation as claimed in claim 1, characterized in that: The phlegm-heat and blood stasis type atrial fibrillation rat model is used to simulate atrial fibrillation fibrosis caused by chronic inflammation.

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