Qige Huxin Formula Attenuates Isoprenaline-Induced Cardiac Fibrosis in Mice via Modulating Gut Microbiota and Protecting Intestinal Integrity

Background The composition and metabolic activities of gut microbiota are strongly interconnected with cardiac fibrosis (CF) and heart failure (HF). Qige Huxin formula (QHF), a traditional Chinese medicine (TCM) formulation originating from a classical Fangji Huangqi decoction, has been widely used to clinically treat HF for decades. However, it is still unclear whether QHF alleviates CF by modulating gut microbiota and intestinal integrity. Purpose This study aimed to investigate the cardioprotective effects of QHF in isoprenaline-induced CF through modulating gut microbiota and intestinal integrity. Methods Fifty mice were randomly divided into five groups after one week of acclimatization feeding: control group, model group, 2.56 g/kg/d group (low-dose QHF), 5.12 g/kg/d group (high-dose QHF), and meto group (15 mg/kg/d). The CF model was established by subcutaneously injecting the mice with isoprenaline (10 mg/kg/d for 14 days), followed by QHF treatment. The heart volume, cardiac weight index (CWI), serum myocardial enzymes, serum inflammatory cytokines, serum lipopolysaccharide, histopathology of the heart and colon tissues, ZO-1, and occludin of colon tissues were then measured. Fecal samples from mice were analyzed using 16S rRNA sequencing. Results QHF treatment significantly reduced heart volume, CWI, and serum CK and CK-MB levels, attenuated cardiac histopathological alterations, and alleviated CF. QHF treatment also downregulated TNF-α, IL-1β, and IL-6 in serum. Moreover, QHF treatment decreased the serum level of lipopolysaccharide and maintained intestinal integrity by upregulating ZO-1 and occludin. The 16S rRNA microbiota analysis revealed that QHF treatment increased the relative abundance of Marvinbryantia and Phascolarctobacterium. Conclusions QHF treatment exerts cardioprotective effects through modulating gut microbiota, protecting intestinal integrity, and alleviating inflammation. This study shows that gut microbiota may enhance heart-gut interaction.


Introduction
Cardiac fibrosis (CF) is characterized by fibroblast activation, fibroblast transformation into myofibroblast, and the deposition of the extracellular matrix (ECM) [1]. CF is an inevitable pathological process and an irreversible contributing factor of heart failure (HF) [2,3]. HF has the characteristics of high morbidity, mortality, and dismal prognosis, and seriously affects the health and life of patients [4]. However, the relevant mechanisms of CF remain unclear. ere are no effective treatments to halt or reverse the progression of fibrosis since the current medical therapies for CF are very limited. erefore, there is an urgent need to discover novel mechanisms of CF and identity new therapeutic targets for effective antifibrosis and delaying the progression of CF to HF.
Many studies have indicated that the composition and metabolic activities of gut microbiota are intimately interconnected with CF and HF [5][6][7]. HF decreases cardiac output, resulting in intestinal wall ischemia, intestinal mucosal edema, and destruction of intestinal integrity, eventually increasing intestinal permeability ("leaky gut") [8]. Previous studies have also indicated that gut microflora dysbiosis may damage intestinal integrity, increase intestinal mucosal permeability, and lead to the leakage of inflammatory metabolites into the blood, thus triggering an inflammatory response that results in CF and HF [9][10][11]. erefore, modulating gut microflora and protecting intestinal integrity can ameliorate CF and delay the progression of CF to HF.
Qige Huxin formula (QHF), a traditional Chinese medicine (TCM) formulation originating from a classical Fangji Huangqi decoction, has been widely used to clinically treat HF for decades. Pharmacological and phytochemical evidence has demonstrated that QHF contains various bioactive components and has multiple pharmacological functions, including antioxidation [12], antifibrosis [13], anti-inflammation [14], and anticardiac remodeling [15] through the synergic effects of multiple active ingredients. Recent studies have also indicated that astragaloside IV and puerarin, the main effective ingredients of QHF, can modulate gut microbiota and promote intestinal mucosal repair [16,17]. ese studies indicate that QHF can attenuate CF by modulating gut microbiota and protecting intestinal integrity. is study aimed to investigate whether QHF can ameliorate CF by modulating gut microbiota, intestinal integrity, and inflammation.

Animals and Drugs.
Fifty specific pathogen-free (SPF) male Kunming mice (weight, 22-26 g) were obtained from the Laboratory Animal Center of Chongqing Medical University (CQMU, Chongqing, China). e mice were then acclimated to the environment for one week. An air-conditioned environment with standardized conditions (relative humidity: 40%-60%, ambient temperature: 20°C-22°C, and light: 12 h light/dark cycle) was provided for the mice during the experiment. Ad libitum food and water were provided to each mouse during the experiments. Animal experiments were conducted following the guidelines for care and use of laboratory animals established by the International Council on Research Animal Care and the Chinese Animal Welfare Committee. Experimental animal protocols were approved by the Animal Experiments Ethical Review Committee of CQMU. QHF consists of Astragali Radix (Huangqi, 6 g), Puerariae Lobatae Radix (Gegen, 3 g), Atractylodis Macrocephalae Rhizoma (Baizhu, 4.5 g), Curcumae Longae Rhizoma (Jianghuang, 2.7 g), Stephaniae Tetrandrae Radix (Fangji, 0.72 g), and Fritillariae unbergii Bulbus (Zhebeimu, 3 g). Animal equivalent doses were converted from human doses based on body surface area to calculate the oral doses of drugs given to the mice [18]. e lavage doses of the low-dose and high-dose QHF groups were 2.56 g/kg/d and 5.12 g/kg/d, respectively. QHF was purchased from Guangdong Yifang Pharmaceutical Co., Ltd. (Guangdong, China) and identified by Professor Wenfu Cao. Metoprolol tartrate tablets (product batch number: 2012A48) were sourced from AstraZeneca Pharmaceuticals (China) Co., Ltd. (Taizhou, China).

CF Model Establishment and Grouping.
e mice were randomly divided into five groups (n � 10 per group) after one week of acclimatization feeding. e CF model was established as previously described [3,19]. Mice in the control group were subcutaneously administered 0.9% sterile saline solution at the back of the neck. Mice in other groups were subcutaneously injected with isoproterenol (ISO, 10 mg/kg/d) for 14 days. Mice in the control and model groups were orally fed 0.5% carboxymethyl cellulose (CMC) for 14 days. Mice in the meto group were orally fed metoprolol tartrate (15 mg/kg/ d) for 14 days, whereas mice in the low-dose QHF (QHF-L) and the high-dose QHF (QHF-H) groups were treated with QHF doses of 2.56 g/kg/d and 5.12 g/kg/d for 14 days, respectively. All experimental drugs were dissolved in a 0.5% aqueous CMC solution. Figure 1 illustrates the experimental timeline and schematic design of the study.

Plasma Biomarkers and Cardiac Weight Index.
e serum levels of myocardial enzymes, including creatine kinase (CK) and creatine kinase-MB (CK-MB), were measured using a Rayto Chemray 800 automatic biochemical analyzer. e serum level of lipopolysaccharide (LPS) was detected using an endotoxin detection kit (Nanjing Jiancheng Bioengineering Research Institute; Nanjing, China). Blood was collected, and then, the mice were euthanized by inhalation of 5% isoflurane. e mice's chests were opened and the hearts were fully exposed. e heart tissues were immediately removed and weighed. e Cardiac Weight Index (CWI) was calculated to evaluate cardiac function. CWI (mg/g) � heart weight (HW, mg)/ body weight (BW, g) × 100%.

Histopathology Assay and Quantification of Cardiomyocyte Sizes.
e hearts and colons of freshly excised mice were fixed in 4% paraformaldehyde at room temperature for 24 hours. e tissues were embedded in paraffin and cut into 5 μm sections using a microtome (Leica) after fixation. e heart tissue sections were dewaxed and stained with HE and Masson. e HE staining kit (Servicebio, G1003) and the Masson staining kit (Servicebio, G1006) were purchased from Wuhan Servicebio Technology Co., Ltd (Wuhan, China). An Olympus BX53 biological microscope (Olympus, Tokyo, Japan) was used to photograph at least five positive areas randomly selected from each section. e area of positive staining was quantified using Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MD, USA). e myocyte cross-sectional areas in HE-stained vertical sections were measured (n � 50 cells per animal) using Image-Pro Plus 6.0 to determine cardiac hypertrophy [20].

Measurement of Inflammatory Cytokines.
e serum levels of TNF-α, IL-1β, and IL-6 were analyzed using commercial kits following the manufacturer's instructions. ese commercial kits were purchased from Quanzhou Jiubang Biotechnology Co., Ltd. (Quanzhou, China).

Immunohistochemical Staining.
Immunohistochemistry staining of α-smooth muscle actin (α-SMA), zonula occludens-1 (ZO-1), and occludin were carried out following the standard protocol. Paraffin-embedded sections (5 μm) were dewaxed in xylene, and then rehydrated with gradient ethanol, followed by antigen retrieval in a microwave for 15 min using sodium citrate buffer (Servicebio, G1201, pH 6.0). e slices were protected from light and incubated in 3% H 2 O 2 (Servicebio, G0115) at ambient temperature for 25 minutes to deplete endogenous peroxidase. e sections were blocked with 3% bovine serum albumin (Servicebio, G5001) for 30 minutes, and then washed thrice using phosphate-buffered saline (PBS, PH 7.2) (five minutes each) at room temperature. Afterward, the sections were incubated with antibodies specific for α-SMA (Servicebio, GB111364, 1 : 1000), ZO-1 (Servicebio, GB111981, 1 : 400), and occludin (Servicebio, GB111401, 1 : 600) at 4°C overnight. e sections were washed thrice (five minutes each) using PBS (pH 7.4) after incubation with the primary antibody. e sections were then incubated with the appropriate secondary antibody at room temperature for 50 minutes. e sections were washed again thrice (5 minutes each) as mentioned above and stained with a freshly prepared DAB reagent (Servicebio, G1211). e sections were counterstained with hematoxylin (Servicebio, G1004), dehydrated in alcohol gradients and xylene, and then blocked with neutral gum. Five positive areas of each section were randomly selected under a light microscope (Olympus BX53) at a magnification of x400. e positive staining area, defined as brown-yellow, was quantified using Image-Pro Plus 6.0. Finally, sequencing libraries were prepared using a NEXTflex ™ rapid DNA-seq kit (Bioo Scientific, USA), and then, the sequencing was conducted on the Illumina MiSeq PE300 platform (Illumina, San Diego, CA). e raw image data files were converted into the original sequenced reads after base calling analysis. e final results were transformed into the FASTQ (abbreviated as fq) format using Bcl2Fastq [21]. FASTQ paired-end raw data were merged with FLASH [22] software. e low-quality (score <20) and the short (<50 bases) sequences were removed after the screening of raw sequences. e remaining qualifying sequences were then spliced. e UCLUST algorithm in QIIME2 [23] software was used to cluster and annotate the operational taxonomic units at a similarity threshold of 97%. Alpha and beta diversity analyses were conducted according to the clustering results. e naive classifier in QIIME2 was used for species taxonomic analysis of ASVs based on the SILVA 16S rRNA database. e sequencing data underwent a diversity analysis using the free online platform of Majorbio Cloud (https://www.cloud.majorbio.com).

Statistical
Analysis. Data were expressed as mean-± standard deviation (SD) and analyzed with SPSS 26.0 statistical software (SPSS Inc., Chicago, USA). One-way analysis of variance (ANOVA) was used to compare variance among multiple groups. All graphs were plotted in

Evidence-Based Complementary and Alternative Medicine
GraphPad Prism 9.20 (GraphPad Software, California, USA). P < 0.05 was considered statistically significant.

QHF Reduced CWI and Regulated Body Weight.
Cardiac morphology and CWI were observed and measured to determine how QHF protects against ISO-induced myocardial damage. e model group had a significantly larger heart volume than the control group. In contrast, the QHF group had a reduced heart volume compared with the model group (Figure 2(a)). Additionally, CWI significantly differed between the model group and other groups after 14 days (Figure 2(c)). Moreover, the body weight of each mouse was measured and recorded every three days throughout the experiment. Mice in the five groups had similar body weights at the beginning of the experiment (Figure 2(b)). However, the body weight in the model group showed a downward trend at the end of the experiment, while it showed an increasing trend in the other groups (Figure 2(b)). Nevertheless, the body weight was not significantly different among the experimental groups.

QHF Improved ISO-Induced Myocardial Damage.
e serum levels of myocardial injury markers CK and CK-MB were detected. e serum levels of CK and CK-MB significantly increased in the model group and obviously deceased in the QHF groups (Figures 2(d), 2(e)), indicating that QHF can protect against ISO-induced myocardial damage.

QHF Ameliorated ISO-Induced Histopathological
Changes. Myocardial histopathological changes, stained with hematoxylin-eosin (HE), were observed using a light microscope. Myocardial fibers in the model group were severely damaged, with focal necrosis, myocardial structural disorder, and inflammatory cell infiltration (Figure 3(a)). However, these histopathological changes were remarkably reduced by QHF treatment. e myocyte cross-sectional area is a measure of cardiac hypertrophy, which is closely related to CF [24]. Herein, the myocyte cross-sectional area was significantly elevated in the model group and was attenuated following QHF treatment (Figure 3(d)). ese results indicated that QHF can prevent ISO-induced CF.

QHF Alleviated ISO-Induced Cardiac Fibrosis.
Cardiac fibrosis resulting from myocardial interstitial collagen deposition among the ECM can be investigated by Masson staining. e area of collagen deposits in the interstitial of the myocardium was significantly different between the model and control groups (Figures 3(b), 3(e)). Compared with the model group, QHF significantly reduced the area of collagen deposition in ECM. Cardiac fibroblasts differentiate into myofibroblasts, which secrete large  Evidence-Based Complementary and Alternative Medicine amounts of collagens and express abundant α-SMA, thus promoting CF [25]. We further examined the expression level of α-SMA in myocardial tissue by immunohistochemistry. e expression level of α-SMA was significantly upregulated in the model group, while it was markedly reduced by QHF treatment (Figures 3(c), 3(f )). ese results demonstrated that QHF can improve collagen deposition and attenuate ISO-induced CF.  Evidence-Based Complementary and Alternative Medicine

QHF Decreased ISO-Induced Inflammation.
Inflammation is a protective response to various injuries; however, excessive inflammation may damage the failing heart, which contributes to CF and cardiac dysfunction [26]. e serum levels of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6 were higher in the model group than those in the control group (Figures 3(g)-3(i)). However, QHF treatment obviously decreased the levels of these inflammatory cytokines, indicating that QHF can decrease ISO-induced inflammation.

QHF Protected ISO-Induced Intestinal Integrity.
e intestinal barrier can effectively prevent harmful substances, such as bacteria and toxins from passing through intestinal mucosa into the body's tissues, organs, and blood circulation [10]. erefore, maintaining intestinal integrity is essential for cardiac health. HE staining revealed destruction of goblet cells and inflammatory infiltration of the colon in the model group (Figure 4(a)). However, QHF treatment (high dose) effectively repaired intestinal damage and reduced severe histologic inflammation. Tight junction proteins (TJPs), including ZO-1 and occludin, are the major elements in modulating the permeability in the intestine and maintaining intestinal integrity [27]. In this study, ZO-1 and occludin proteins were significantly downregulated in the colon tissues of the model group compared with those of the control group. However, QHF treatment (high dose) markedly upregulated ZO-1 and occludin (Figures 4(b)-4(e)).
Lipopolysaccharide (LPS) is a sensitive biomarker of barrier strength and intestinal permeability [28,29]. LPS crosses the intestinal barrier into the bloodstream after intestinal damage and disperses throughout the body. erefore, the serum level of LPS can indirectly reflect intestinal integrity. In this study, the serum level of LPS was significantly upregulated in the model group compared with the control group (Figure 4(f )). However, QHF treatment (high dose) decreased the serum level of LPS, demonstrating that QHF can reduce ISO-induced intestinal permeability and protect intestinal integrity.

QHF Improved ISO-Induced Gut Microbial Dysbiosis.
16S rRNA sequencing was used to assess whether QHF treatment (high dose) can regulate gut microbiota composition in mice. e principal components analysis (PCA) was performed to assess the composition of gut microbiota. e gut microbiota compositions of the three groups were distinctly separated, indicating that they had significantly different gut microbiota compositions (Figure 5(a)). e gut microbial community structure changes were analyzed using heatmap analysis. Compared with the control group, the gut microbiota of the model group was significantly different, whereas QHF treatment (high dose) reversed the microbe changes in ISO-induced mice ( Figure 5(b)). e phylumlevel community structure composition histogram revealed that the structure and abundance of the fecal microbial community were altered. Bacteroidetes and Firmicutes were the most dominant phyla (over 85%) in all groups ( Figure 5(c)). At the genus level, Prevotella had the highest relative abundance, followed by Bacteroides (Figure 5(d)). Compared with the model group, QHF treatment (high dose) increased the relative abundance of Lactobacillus and Bacteroides, while it decreased the relative abundance of Escherichia-Shigella ( Figure 5(d)). e differential bacteria composition was identified using the LEfSe and taxonomy cladogram. QHF treatment (high dose) obviously increased the relative abundance of family Marvinbryantia and Phascolarctobacterium at the genus levels ( Figures 5(e), 5(f )). ese results demonstrated that QHF can modulate gut microbiota composition, thus alleviating CF.

Discussions
Cardiac fibrosis (CF) is a continuous and dynamic pathologic process characterized by transforming resident fibroblasts into myofibroblasts and excess deposition of collagens in the myocardium [30]. e pathophysiology of CF can be reversed at the early stage [31]. However, it can lead to continuous accumulation of ECM if left untreated, eventually resulting in cardiac dysfunction and heart failure [32]. Nevertheless, the mechanisms of CF are unknown, and there is no efficacious treatment to halt the progression of CF [33]. A significant amount of research has focused on the therapeutic potential of TCM in the prevention and treatment of CF [34]. is study investigated whether QHF can achieve the antifibrosis effects in ISO-induced mice by regulating gut microbiota, intestinal integrity, and inflammation.
Our data indicated that QHF treatment could significantly alleviate ISO-induced myocardial injury and cardiac fibrosis by reducing serum levels of CK and CK-MB, heart volume, CWI, and cross-sectional area of cardiomyocytes. Moreover, HE staining demonstrated that QHF treatment corrected the structural disorder of cardiomyocytes and decreased inflammatory cell infiltration and myocardial necrosis. Masson staining displayed that QHF treatment inhibited myocardial collagen deposition. Immunohistochemistry staining also showed that QHF treatment markedly downregulated α-SMA in myocardial tissue.
Previous studies have indicated that inflammatory cytokines are significantly upregulated in myocardial fibrosis [35]. Inflammatory cytokines can promote the transformation of fibroblasts into myofibroblasts by increasing the expression of proteins involved in ECM, thus accelerating CF progression [26]. An inflammatory response is immediately triggered when the body or cells are stimulated, thus making inflammatory cells immediately release various proinflammatory mediators, including TNF-α, IL-1β, and IL-6. ese substances, as proinflammatory mediators, stimulate the proliferation and activation of the resident fibroblasts and their transformation into myofibroblasts [36], thus promoting the deposition of matrix proteins in the ECM. Previous studies have exhibited that decreasing proinflammatory factors can ameliorate myocardial fibrosis [37][38][39]. In our present study, the serum levels of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, were increased significantly in the model group. However, QHF treatment decreased the serum levels of inflammatory cytokines, illustrating that QHF treatment can protect against ISOinduced CF by inhibiting inflammation. e intestinal mucosal barrier consists of TJPs and intestinal epithelial cells. It is the first physiological barrier for the human body to resist harmful intestinal microorganisms and their metabolites. Among the most important TJPs, ZO-1 and occludin play a crucial role in regulating intestinal permeability [40]. Bacteria and their metabolites, especially LPS, enter blood through intestinal mucosa when intestinal permeability increases, leading to bacterial infection and inflammatory response, thus inducing heart injury [41]. In this study, QHF treatment upregulated ZO-1 and occludin in colonic mucosa, and suppressed the serum    Evidence-Based Complementary and Alternative Medicine LPS level. Furthermore, HE staining showed that QHF treatment increased the number of goblet cells in intestinal mucosa, reduced inflammatory cell infiltration, and effectively repaired the intestinal injury. ese findings showed that QHF exerted a protective effect on intestinal integrity.
A lot of evidence has demonstrated that the imbalance of intestinal flora promotes CF progression [42,43]. Herein, QHF treatment regulated the gut microbiota structure. QHF consumption significantly increased the relative abundance of Bacteroides and Mucispirillum in mice feces, while it decreased the relative abundance of Escherichia-Shigella. Several studies have shown that microbiota with low Bacteroides abundance or high Helicobacter abundance can increase the risk of cardiovascular diseases [44,45]. In this study, QHF treatment significantly increased the relative abundances of Marvinbryantia and Phascolarctobacterium at the genus level. Marvinbryantia, a genus of Firmicutes, can produce short-chain fatty acids (SCFAs) [46]. SCFAs are mainly produced by the fermentation of dietary fiber. SCFA reduction increases the risk of CF and HF [47]. Marvinbryantia is a beneficial intestinal bacterium that maintains the diversity and function of gut microbiota and can improve human health. For instance, it can protect the intestinal epithelium cells from destruction, and has anti-inflammatory properties [48]. Furthermore, QHF treatment improved the relative abundance of Phascolarctobacterium. A recent study found that Phascolarctobacterium is negatively correlated with the IL-6 level [49]. erefore, Marvinbryantia and Phascolarctobacterium promote the antifibrosis effects of QHF treatment in ISO-induced mice.

Conclusion
Collectively, QHF treatment exerts obvious cardioprotective effects in ISO-induced cardiac fibrosis mice by modulating gut microbiota, protecting intestinal integrity, and inhibiting inflammation.
is study provides a new therapeutic strategy for QHF to prevent and treat cardiac fibrosis.