Linggui Qihua Decoction Inhibits Atrial Fibrosis by Regulating TGF-β1/Smad2/3 Signal Pathway

Myocardial fibrosis is a critical factor in the development of heart failure with preserved ejection fraction (HFpEF). Linggui Qihua decoction (LGQHD) is an experienced formula, which has been proven to be effective on HFpEF in clinical and in experiments. Objective. This study aimed to observe the effect of LGQHD on HFpEF and its underlying mechanism. Methods. Spontaneously hypertensive rats (SHR) were induced with high-glucose and high-fat to establish HFpEF models and were treated with LGQHD for 8 weeks. The heart structure was detected by echocardiography, and the histopathological changes of the myocardium were observed by hematoxylin-eosin (HE) and Masson staining. Reverse transcription PCR (RT-PCR) and western blot were used to detect mRNA and protein expression of the target gene in rat myocardium. Results. In this study, LGQHD improved cardiac morphology and atrial fibrosis in HfpEF rats, decreased tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expression, up-regulated matrix metalloproteinase-9 (MMP-9) mRNA expression, and inhibited the expression of angiotensin II (Ang II), angiotensin II type 1 receptor (AT1), transforming growth factor β1 (TGF-β1), Smad2/3 mRNA, and protein in myocardial tissue of HFpEF rats. Conclusion. LGQHD can suppress atrial fibrosis in HFpEF by modulating the TGF-β1/Smad2/3 pathway.


Introduction
Heart failure with preserved ejection fraction (HFpEF) is a special heart failure that has clinical symptoms and signs, abnormal cardiac structure and function, and elevated natriuretic peptides, but with normal ejection fraction (left ventricular ejection fraction, LVEF ≥50%) [1]. It was reported the overall morbidity of HFpEF in the general population is 1.1-5.5% [2], accounting for approximately 50% of all patients with heart failure [3]. Its morbidity and hospitalization are increasing annually [4,5] and are expected to soon surpass that of heart failure with reduced ejection fraction (HFrEF) [6]. Te current treatment for HFpEF includes angiotensin-converting enzyme inhibitor (ACE-I)/angiotensin-receptor blocker (ARB), beta-blockers, mineralocorticoid receptor antagonists (MRAs), and sacubitril/valsartan or sodium-glucose cotransporter 2 (SGLT2) inhibitors [1]. Tese treatments can improve certain specifc phenotypes and reduce hospitalization rates in HFpEF. However, none of the large randomized controlled trials (RCTs) conducted in HFpEF have achieved their primary endpoints [1]. Te SGLT2 inhibitor trials were conducted in HF patients with LVEF >40% [7]. Terefore, it is vital to explore the pathogenesis of HFpEF and develop potential medical therapies with improved therapeutic efcacy.
Hypertension and left ventricular hypertrophy stimulate interstitial cardiac fbrosis, which has long been considered to be a cause of passive muscle stifening and reduced chamber compliance in HFpEF [8,9]. Myocardial fbrosis also results from diabetic heart disease via multiple signaling cascades and alterations in extracellular matrix proteins, such as the formation of insoluble advanced glycation endproducts [10]. Transforming growth factor β (TGFβ)/Smads signaling pathway is most closely related to the formation of myocardial fbrosis. TGF-β is the most important fbrous growth factor in the process of myocardial fbrosis. It activates the Smad2/3 signaling pathway mainly through binding TGF-β receptors to facilitate the myocardial fbrosis process [11]. As a key mediator of myocardial fbrosis, angiotensin II (Ang II) can up-regulate the expression of TGF-β1 by binding with angiotensin type 1 receptor (AT1R), which induces myocardial cell hypertrophy and enhances its secretion of profbrotic growth factor [12]. On the other hand, left atrial (LA) enlargement is common in HFpEF and correlates with the severity and duration of left ventricular (LV) diastolic dysfunction. In the setting of hypertensive HFpEF, LA remodeling occurs early and exhibits maladaptive alterations in LA compliance and left atrioventricular coupling which compromises overall cardiac performance and may exacerbate increases in LA and pulmonary pressures [13]. Terefore, exploring the potential roles of the TGF-β/Smads signaling pathway in the pathogenesis of HFpEF may provide a new method for the treatment of HFpEF.
Linggui Qihua decoction (LGQHD) is a patented and efective decoction for the clinical treatment of HFpEF which consists of Poria cocos Schw Wolf (Poria coco, 茯苓), Cinnamomum cassia Presl (Cassia twig, 桂枝), Atractylodes macrocephala Koidz (Atractylodes macrocephala, 白术), Paeonia lactifora Pall., and P. veitchii Lynch (Radices paeoniae rubra, 赤芍). Te whole formula is efective in warming yang for resolving fuid retention and promoting blood circulation and diuresis. Our previous studies showed that LGQHD can improve cardiac diastolic function, inhibit atrial fbrosis, as well as reduce infammatory factors, and improve glucose and lipid metabolism and endothelial function in HFpEF model rats [14]. However, the mechanism underneath it is unclear. To explore the possible mechanism of LGQHD-treated HFpEF, we observe cardiac structure by echocardiography, the pathological changes of cardiomyocytes by Masson staining, and the activation of TGF-β/Smads signaling pathway in myocardial tissue by real-time polymerase chain reaction (RT-PCR) and Western blot analysis. Moreover, the potential mechanism of LGQHD for HFpEF was elucidated for the frst time.

Animals.
Tirty males and thirty females specifcpathogen-free (SPF) grade spontaneously hypertensive rats (SHR) and fve males and fve females with the same genetic background SPF Wistar-Kyoto rats (WKY) (age, 14 Weeks; weight, 180-260 g) were provided by the Vital Laboratory Animal Technology Company, Beijing, China (experimental animal license number: SCXK (Beijing) 2016-0006). Tese rats were housed in the barrier-class animal room of Xiyuan hospital of the Chinese Academy of Chinese Medical Sciences in a controlled environment (constant room temperature 23 ± 2°C, humidity 60% ± 10%, and 12 h light-dark cycle). Te rats were fed on time, and the experiment was conducted after 1 week of adaptation. High-fat and highsugar feed (10% lard, 10% sucrose, 2.0% cholesterol, 0.5% bile salt, and 77.5% basal feed) was manufactured by Beijing Co-operative Feeds Ltd. (feed certifcate: Beijing Feed Certifcate (2014) 06054).

Drugs Preparation and Chemical Composition Analysis.
LGQHD is composed of Poria cocos Schw Wolf (Poria coco, 茯苓) 20 g, Cinnamomum cassia Presl (Cassia twig, 桂枝) 15 g, Atractylodes macrocephala Koidz (Atractylodes macrocephala, 白术) 10 g, Paeonia lactifora Pall., and P. veitchii Lynch (Radices paeoniae rubra, 赤芍) 10 g. Tey are, respectively, derived from the dried mycorrhizal nucleus of Poria coco, the dried shoots of Cassia twig, the dried rhizome of Atractylodes macrocephala, and the dried roots of Radices paeoniae rubra. All herbs are purchased by Hebei Bai Cao Kang Pharmaceutical Co., Ltd. Te preparation process consists of the traditional process of water extraction and volatile oil of the tablets to make β-CD inclusions in a ratio of about 1 : 4, and the aqueous solution is concentrated into an infusion. It was prepared as an extract at a concentration of 2.28 g of raw drug/g, which was provided by the Department of Pharmaceutics at Xiyuan hospital, China Academy of Chinese Medicine. Sacubitril Valsartan Sodium Tablets (trade name: Entresto) were manufactured by Novartis Pharma Schweiz AG with the (approval number H20170344, Switzerland) with the specifcation of 50 mg/ tablet.
Te Traditional Chinese Medicine Preparation Department at Xiyuan hospital, Chinese Academy of Traditional Chinese Medicine, provides LGQHD Infusion. Precisely 90 ml of LGQHD extraction is taken, 5.5 ml of the 70% methanol solution is added, kept at room temperature for 30 minutes, and then it is fltered through a 0.22 μm flter membrane. Te LGQHD extract test solution was then made and kept in the refrigerator at a temperature of −20°C. Te proper amount of LGQHD extraction solution is taken, it is liquefed with 70% methanol, and the sample for analysis is injected. Chromatographic circumstances mobile phase: gradient elution of water (A)-acetonitrile (B) as described in Table 1. Te column temperature was 40°C, the fow rate was 0.4 ml/min, and the injection volume was 2 μl. Te ESI source, positive and negative ion scan, and scan range m/z: 50-1200 Da were the mass spectrometry conditions. Data were acquired using Masslynx V4.1 software, and the resulting data were processed using Progenesis QI software and calibrated and peak aligned by peak detection and calibration processing algorithms, respectively. Meanwhile, the chemical formulae of the possible target compounds were imported into the software, and the following main components of the LGQHD were identifed by qualitative analysis of the components through accurate molecular weight comparison.

LGQHD Extract Preparation.
According to the weight, the compatibility ratio of the four medicines of Poria coco, Cassia twig, Atractylodes macrocephala, and Radices paeoniae rubra in LGQHD is 4 : 3 : 2 : 2. Te drug was decocted three times with water and fltered through the process each time, and the fltrate to be combined fnally. Te extract was then further fltered to remove solid impurities and fnally condensed into granules. According to the table of equivalent dose ratios converted by body surface area between humans and animals [15], based on a standard adult body mass of 70 kg, the dose for rats � standard adult clinical dose mg/kg × 70 kg × 0.018/200. Te LGQH-H group was administered 9.92 g/kg/d, which is 2 times the clinically equivalent dosage. Te LGQH-L group was administered 4.96 g/kg/d, which is the clinical equivalent dosage.

Animal Model and
Groups. Fifty 14-week-old SHR rats were given a high-fat diet after 16 weeks of continuous feeding, and after 2 weeks of a high-fat diet, STZ (25 mg/ kg body weight) was injected intraperitoneally, and the high-fat diet was continued to induce the establishment of the HFpEF model in rats [16][17][18]. Surviving model rats were grouped and the survival rate was approximately 80%.
Te rats were randomly divided into SHR model group (SHR group) and HFpEF model group (HFpEF group). Te rats were split into the SHR model group, the HFpEF model group, the positive drug control group (Entresto group), the LGQHD high-dose group, and the LGQHD small-dose group at random (LGQH-L group). In which the same volume of pure water was gavaged to the SHR group and the HFpEF group. Shakubatra valsartan sodium tablets, 18 mg/ kg body mass, were gavaged to the Entresto group; 8.10 g of the raw drug were given to the LGQH-H group; and 4.05 g was given to the LGQH-L group. Each group contained 10 animals, and the medications under test were diluted and given in equal amounts for 8 weeks. In addition, the normal control group (normal group) of 10 WKY rats with the same genetic background was administered the same amount of pure water. Except for the SHR and WKY groups, the rats were fed with a normal diet, while the other groups continued to be fed with a high-fat and high-sugar diet. Four rats were sacrifced during the experiment.
Te animal model and experiment processes were performed strictly in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Tis experiment was approved by the Committee on Ethics of Animal Experiments of Xiyuan Hospital of China Academy of Chinese Medical Sciences (No. 2018XLC004-2).

Echocardiography Parameters Measurements and Cardiac
Histopathology. After the treatment, the rats were anesthetized intraperitoneally with 2% sodium pentobarbital aqueous solution (0.2 mL/100 g body weight) for 12 h on the 2nd day of an empty stomach, and after anesthesia, the rats were evaluated by echocardiography with a high-frequency line array ultrasound diagnostic instrument (GE VividE95 cardiac ultrasound diagnostic instrument, manufactured by General Electric Company, USA) for cardiac function and echocardiographic morphological changes. Cardiac ultrasound morphological indices include left atrium diameter index (LADI), such as the left atrium diameter/Left (DI/L) and the left atrium diameter/Right(DI/R), left atrium volume index (LAVI); right atrium diameter index (RADI), such as, namely, right atrium diameter/Left (DI/L), right atrium inner diameter/Right (DI/R), interventricular septal thickness (IVST), and left atrial ejection fraction (LAEF). Te operation of ultrasound examination was completed by two experienced ultrasound specialists who were blind to the experiment.
Myocardial tissues of 5 rats were randomly selected from each group and fxed in 10% central formaldehyde solution, embedded in immersion wax, dewaxed in sections, debenzene in ethanol step by step, and then stained sequentially with hematoxylin-eosin (HE) stain/Masson stain, acid alcohol separation, 1% eosin stain, and fnally debenzened in ethanol step by step and sealed in neutral gum. Te cardiac structure was photographed by a gross sample imaging Evidence-Based Complementary and Alternative Medicine system (Beijing Ruizhiaoheng Vision Technology Co., LTD.), and the morphology of myocardial tissue was observed under an optical microscope (Olympus (Japan) Co., LTD.).

Quantitative Real-Time Reverse Transcription PCR (RT-PCR).
We use TRNzol total RNA extraction reagent for sample RNA extraction, then use the PrimeScript ™ RT reagent Kit with gDNA Eraser was used for cDNA reverse transcription, and then RT-PCR samples were detected. Te 96-PCR plate was placed on the RT-PCR instrument for PCR reaction, and the melting curve of PCR products was established. Te experimental operation was carried out according to the product instructions. RT-PCR was used to detect 3 Wells in each sample, and the data were analyzed by 2 −ΔΔCT method. Primer sequences are shown in Table 1.

Western Blot Analysis.
Precooled RIPA lysate was used for protein extraction, and a protease inhibitor was added along with a phosphatase inhibitor. Te bicinchoninic acid method (Bicinchoninic acid, BCA) was used for protein concentration detection, along with protein concentration conversion and protein concentration adjustment, and electrophoretic stopping time was determined by prestaining protein marker. Te primary antibody was incubated with 3% BSA-TBST dilution, incubated for 10 minutes at room temperature, and placed at 4°C overnight. TBST washes the flm. ECL is added to the flm and then the reaction flm is exposed, developed, and fxed. Te membrane was washed with stripping bufer, the internal reference was incubated, beta-actin murine monoclonal antibody was, respectively, added and the antibodies were diluted with 3% BSA-TBST, 1 : 5000 dilution for AT1 and AngII, 1 : 4000 dilution for TGF-β1, Smads2/3 and P-Smada2/3, 1.1 dilution for MMP-9 and TIMP-1: 1000, 1 : 4000 dilution for type Icollage, 1 : 2000 dilution for type III collagen, and 1 : 2000 dilution for α-SMA protein and incubated at room temperature. Te membrane was washed again, incubated with secondary antibody, goat antimouse IgG (H + L) HRP, diluted antibody with 3% BSA-TBST at a ratio of 1 : 10000, shaken gently at room temperature for 40 minutes, and the membrane was washed. ECL was added to the membrane and reacted, the flm was exposed for development and fxation, and a specifc positive band could be detected. Te gray value of Western blot protein expression was transformed from JPEG to TIF by the software image J. Te integrated optical density (IOD) values of the reading strips were calculated using Total Lab Quant V11.5, manufactured in Newcastle upon Tyne, UK.
2.9. Statistical Analysis. SPSS 13.0 statistical software was used for statistical analysis. All data were expressed as the mean-± standard deviation (mean ± SD). One-way ANOVA was used to compare the measured data for each group when the data met the normal distribution and homogeneity of variance, and the LSD test was used for further multiple comparisons between groups. If the homogeneity of variance was not satisfed, the F value was corrected by the Welch method, and the Dunnett T3 method was used for further multiple comparisons. Te difference was considered statistically signifcant at P < 0.05.

Chemical Composition Analysis of the Main Constitutes in
LGQHD Extracts. Te LC-MS method was adopted to compare the compounds between the standard and LGQHD using positive and negative ion detection modes. Te ion chromatograms of the main compounds and LGQHD are shown in Figures 1 and 2. Te main compounds detected in positive ion mode were conifer aldehyde, coumarin, atractylenolide I, and atractylenolide II; the main compounds detected in negative ion mode were sucrose, gallic acid, oxypaeonifora, dihydromelilotoside/dihydrocinnacasside, galloylpaeoniforin, paeoniforin, poricoic acid A, dehydropachymic acid, and pachymic acid.

LGQHD Improved Cardiac Ultrasound Morphology in HFpEF Rats.
Echocardiography is an important means to detect the morphological changes of rat heart, and the results showed that compared with the control group (WKY group), the LAVI, IVST, LADI/R, and RADI/L of the model group (HFpEF group) were increased (P < 0.01, P < 0.05), indicating that the rat heart morphology changed after modeling. LAVI was decreased in the small and large dose groups of LGQHD and Entresto group (P < 0.05); LADI/R and RADI/R were decreased in the high-dose group of LGQHD (LGQH-H) (P < 0.05, P < 0.01), and LADI/R was decreased in the low-dose group of LGQHD (LGQH-L) (P < 0.01); the results showed that LGQHD improved cardiac morphological changes. Te results are shown in Figure 3 and Table 2.

LGQHD Ameliorated Atrial Fibrosis in HFpEF Rat
Models. To verify the protective efect of LGQHD in the process of HFpEF atrial fbrosis, a high-glucose and high-fat induced HFpEF model in SHR rats was established, and the pathological changes of the myocardium were observed by Masson staining. Tis staining method detects the extent of collagen fbers deposition, and because it employs the combination of three dyes, can also distinguish muscle fbers (red), from collagen (blue) and nuclei (black), simultaneously. Tis method is suitable for the assessment of the extent and distribution of fbrosis in a quantifable manner and can be successfully applied for the detection/diagnosis of fbrotic diseases [19]. Te medical image analysis software Image J was applied to analyze the collagen fber expression of each group and calculate the collagen volume fraction (CVF � collagen area/total area). Te myocardial fbers of WKY rats were neatly arranged with normal cell morphology and a slightly proliferation of collagen fbers. In SHR rats, myocardial interstitial fbers were deposited, the myocardial cell arrangement gap was increased, and some collagen fbers were proliferated. In the model group, myocardial cells were arranged in a disorderly manner, and some myocardial cells were replaced by collagen fbers, collagen fbers were signifcantly increased, and the cell tissue gap was enlarged. After treatment in the Entresto group, LGQH-H group, and LGQH-L group, myocardial fber collagen deposition was reduced and collagen fber proliferation was decreased, especially in the LGQH-H group. Te Image J results showed that compared with the control group, the myocardial tissue collagen area was larger and CVF was greater in the model group; after drug treatment, the myocardial tissue collagen fbers were reduced to diferent degrees in all groups. Te results are shown in Figure 4.

LGQHD Regulates the Expression of Myocardial Fibrosis-Related Genes in Atrial Tissue of HFpEF Rats.
TGF-β/Smads signaling pathway plays an important role in the development of myocardial fbrosis. We detected AngII, AT1, TGF-β1, Smad2, Smad3, MMP-9, and TIMP-1 mRNA in the atrial tissue of HFpEF rats by RT-PCR level. Compared with the control group, the expression of MMP-9 mRNA was down-regulated (P < 0.05), while AngII, AT1, TGF-β1, Smad2, Smad3, and TIMP-1 mRNA expression were up-regulated in model group (P < 0.01). After drug treatment in each administration group, the expression of AT1, TGF-β1, TIMP-1, Smad2, and Smad3 mRNA were down-regulated in the LGQH-H group, LGQH-L group and Entresto group (P < 0.01), while the mRNA expression of MMP-9 was up-regulated (P < 0.05). Te LGQH-H group and Entresto group could also down-regulate the expression level of AngII mRNA (P < 0.01), while the LGQH-L group had no obvious improvement on AngII mRNA. As shown in Figure 5.

LGQHD Regulates the Expression of Myocardial Fibrosis-Related Proteins in Atrial Tissue of HFpEF Rats.
Western blot analysis the expression levels of AngII, AT1, TGF-β1, Smad2, Smad3, MMP-9, TIMP-1 proteins, Type I collagen, Type III collagen, and α-SMA protein in TGFβ/Smads signaling pathway in atrial tissue of HFpEF rats. Compared with the control group, the expression of TGF-β1, PSmad2/3, AT1 receptor, and Type III collagen protein were up-regulated in the model group (P < 0.01 or P < 0.05). Type I collagen and α-SMA protein expression were diferentially upregulated. After 8 weeks of drug treatment, the LGQH-H group and Entresto group could down-regulate MMP-9 and PSmad2/3 protein expression (P < 0.05, P < 0.01), AT1 receptor and Smad2/ 3 protein expression were down-regulated in LGQH-H group (P < 0.01). It also down-regulated Type I collagen protein expression (P < 0.05). Te expression of PSmad2/ 3 protein was down-regulated in the LGQH-L group (P < 0.05).
LGQH-H and LGQH-L groups also downregulated Ang II, TGF-β1, TIMP-1, Type III collagen, and α-SMA protein expression, but the diference was not statistically signifcant. As shown in Figure 6.

Discussion
Myocardial fbrosis is a critical factor in the development of HFpEF, which refers to the proliferation of fbroblasts, excessive collagen synthesis, and deposition of extracellular matrix (ECM) in the structure of myocardial tissue [20]. In the myocardium, the extracellular matrix consists primarily of fbrillar collagen, and cardiac fbroblasts regulate the balance of extracellular matrix synthesis and degradation. Myocardial injury induces the diferentiation of cardiac fbroblasts into myofbroblasts, and the expression of α-SMA indicates successful transformation into a phenotype that has a strong ability to synthesize extracellular matrix proteins [21]. Cardiac myofbroblasts contribute to the structural and functional changes in the heart by increased deposition of ECM components, predominantly collagen types I and III, within the interstitium by regulating autocrine/ paracrine factors [22]. It was shown that decreased myocardial compliance and increased stifness caused by myocardial fbrosis may be a potential mechanisms for diastolic dysfunction in HFpEF [23][24][25][26].

Evidence-Based Complementary and Alternative Medicine
Te expression balance between matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinase (TIMPs) plays a critical role in the maintenance of extracellular matrix [27]. MMP-9 and TIMP-1 are the most abundant MMPs and TIMPs in the myocardium, respectively. MMP-9 disrupts the collagen network architecture mainly by degrading type I and IV collagen and increasing type III collagen in the extracellular matrix, which causes the atrial wall to thin and then the atria to enlarge under pressure. Te level of MMP-9 is positively correlated with the severity of myocardial fbrosis [28]. TIMP-1 can specifcally inhibit the activity of MMP-9 and alleviate the degradation of extracellular matrix collagen, thus improving myocardial fbrosis. Under physiological conditions, MMP-9 and TIMP-1 maintain a balance at a ratio of 1 : 1. Te balance is disturbed in pathological conditions, resulting in increased or decreased collagen degradation [29]. Tus, it accelerates or slows down the deposition of collagen fbers and aggravates or ameliorates atrial fbrosis.
In this study, the expression of MMP-9 mRNA was signifcantly down-regulated while the expression of TIMP-1 mRNA was signifcantly up-regulated in the myocardium of SHR rats after continuous high-sugar and high-fat diet and intraperitoneal injection of STZ for modeling. Masson staining showed that myocardial cells were disorganized, cellular tissue gap was increased. And, some of the myocardial cells were replaced by collagen fbers, so the collagen fbers were signifcantly increased. Furthermore, we observed signifcant upregulation of α-SMA protein and types I and III collagen expression, suggesting that cardiac fbroblasts had differentiated into myofbroblasts, resulting in increased collagen synthesis. It suggests that myocardial fbrosis occurred in the atrial tissue of rats after modeling. After 8 weeks of administration, the TIMP-1 mRNA and type I collagen expression were markedly down-regulated, both α-SMA protein and type III collagen expression were downregulated to diferent degrees, while the MMP-9 mRNA expression was signifcantly up-regulated in cardiac tissue of HFpEF rats. Te decrease of collagen deposition and collagen fber proliferation in myocardial fbers of rats under Masson staining indicated that LGQHD could efectively improve myocardial fbrosis. Te protein levels of MMP-9 and TIMP-1 were inconsistent with the mRNA expression levels, which may be due to the fact that mRNA was easily degraded, exists in tissues for a short time and had stable properties after being translated into protein. Or, the regulation of MMP-9 and TIMP-1 were regulated by gene polymorphisms, and further analysis of susceptibility genes and susceptibility biomarkers was needed in the future.
Myocardial fbrosis is a complex pathological process involving multiple cytokine and molecular pathways, in which multiple pathways lead to diferent tissue molecular patterns of fbrosis, which in turn translate into diverse clinical phenotypes [30]. Te major pathways are TGFβ/Smads, p38 MAPK, Wnt/β-Catenin, Gprotein-coupled receptor kinase (GRK), and Hippo [31], among which the TGF-β/Smads signaling pathway is most closely associated with myocardial fbrosis formation and is the key to the prevention and treatment of myocardial fbrosis [32]. TGF-β is part of the superfamily of growth factors. TGF-β1 is a key regulator in myocardial fbrosis, which can afect cell growth, apoptosis, and diferentiation, increase ECM production, inhibit the production of MMPs, and reduce collagen degradation [33]. Smad proteins are TGF-β downstream signal molecule that mediates the signal transduction of TGF-β. At present, the TGF-β1/Smads signaling pathway is the most signifcant pathway in the process of myocardial fbrosis. TGF-β1 exacerbates myocardial fbrosis by binding to receptors, activating Smad2 and 3, and further translocating to the nucleus, up-regulating the expression of genes associated with ECM synthesis, and promoting ECM deposition [34]. Ang II is a well-known fbrogenic factor and has been associated with fbrosis in various organs, including myocardial fbrosis. Te combination of Ang II and AT1 can induce TGF-β1 expression, cause cardiomyocyte hypertrophy and fbroblast proliferation, and consequently afect atrial fbrosis [35]. Conversely, it can also improve myocardial fbrosis by inhibiting the expression of TGF-β1 [36].
In this study, we found that the expression of AT1, TGF-β1, and Smad2/3 in myocardial tissues of rats in the small and large dose groups of LGQHD was signifcantly reduced, suggesting that the improvement of myocardial fbrosis by LGQHD may be related to the inhibition of TGF-β1/Smad2/ 3 signaling pathway. Tis is manifested that LGQHD can reduce the expression of Ang II and AT1 receptors, downregulate the expression of TGF-β1 in cardiomyocytes, inhibit Smad2/3 expression, reduce Smad2/3 hyperphosphorylation, and negatively regulate the biological efect of TGF-β1 on myocardial fbrosis, and thus ameliorating myocardial fbrosis.
LGQHD is composed of four components Poria coco, Cassia twig, Atractylodes macrocephala, and Radices paeoniae rubra. Poria coco as the JUN, and the efect is to       Figure 6: Efect of LGQHD on protein expression in myocardial tissue of HFpEF rats. ( * : Compared with WKY group, HFpEF P < 0.05. * * : Compared with WKY group, HFpEF P < 0.01. # : Compared with HFpEF group, each drug treatment group P < 0.05. ## : Compared with HFpEF group, each drug treatment group P < 0.01. AT1: angiotensin II type 1 receptor. AngII: angiotensin II. TGF-β1: transforming growth factor-β. MMP-9: matrix metalloproteinases. TIMP-1: tissue inhibitors of matrix metalloproteinase). and delay the progression of heart failure [37]. Cassia twig can promote yang to transform qi, eliminate blood stasis and dissolve symptoms, and has a strong efect of diuresis and dehumidifcation. It can also increase urine output in mice, and the diuretic efect is longer than furosemide [38]. Te combination of Poria coco and Cassia twig has been studied to inhibit myocardial hypertrophy and ventricular remodeling, improve myocardial fbrosis, as well as control the infammatory response and inhibit neuroendocrine hyperactivation in rats with heart failure [39]. Radices paeoniae rubra has been shown to intervene in early ventricular remodeling after acute myocardial infarction, inhibit serum infammation levels, protect ischemic cardiomyocytes, and promote recovery of cardiac function [40]. Its improvement of ventricular remodeling may be related to its ability to inhibit the TGF-β/Smad signaling pathway to reduce fbrosis [41][42][43][44]. Atractylodes macrocephala has the efects of immune regulation, anti-infammatory, gastrointestinal mucosa protection, and blood sugar reduction [45]. Previous studies have found that the combination of Linggui Zhugan Decoction with the function of promoting blood circulation and removing stasis can signifcantly improve the clinical symptoms and cardiac function of HFpEF patients [46]. Tis was further confrmed in the research group's previous study on the efect of LGQHD on HFpEF rat models [14].

Conclusions
In this study, we identifed the protective role of LGQHD in HFpEF.
LGQHD can inhibit atrial fbrosis by suppressing the activation of TGF-β1/Smad2/3 signaling in HFpEF rats. Our fndings provide a solid foundation for future investigation of the role of atrial fbrosis in HFpEF progression and the traditional Chinese medicine LGQHD is a promising candidate for the development of novel HFpEF treatments.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon reasonable request.