Xiong's Shiwei Wendan decoction (熊氏十味温胆汤) attenuates plaque lesions and balances gut microbiota dysbiosis in ApoE-/- mice with high-fat diet

Abstract

OBJECTIVE:

To evaluate the anti-atherosclerotic potential and gut microbiota (GM) modulation effects of Xiong's Shiwei Wendan decoction (熊氏十味温胆汤, XSWD).

METHODS:

For in vitro study, Tsuchiya human peripheral blood mononuclear cell-1 (THP-1) derived foam cells were used to examine the possible anti-atherosclerotic effect of XSWD and XSWD-medicated serum. Atherosclerosis-prone apolipoprotein E-deficient (ApoE-/-) mice were utilized for in vivo analysis. After an 8-week high-fat diet (HFD) adminstration, 25 male ApoE-/- mice were randomly divided into the model group, different doses of XSWD groups (1.25, 2.5, 5 mg/mL), and atorvastatin group (2.6 mg/kg). Following a continuous 8-week intervention, all mice underwent examination for AS lesion formation and assessment of its serum lipid profile. To investigate the effect on the gut microbiome, 16S rRNA gene sequencing targeting the V3-V4 hypervariable region was performed on the colonic content of mice.

RESULTS:

XSWD administration attenuated lipid deposition in THP-1 cells, significantly reduced aortic plaque lesions, improved the lipid profile, and normalized GM composition in HFD-fed ApoE-/- mice.

CONCLUSION:

This study investigated the potential anti-atherosclerotic and gut microbio-ta-restoring effects of XSWD in ApoE-/- mice, with findings suggesting that XSWD may be a promising preventive measure against atherosclerosis through its ability to reduce lipid accumula-tion in foam cells, improve lipid profile, and restore gut microbiota composition.

1. INTRODUCTION

Atherosclerosis (AS) is a chronic pathological process characterized by inflammation and lipid deposition within arterial walls. It is intricately to link risk factors including hypertension, hyperlipidemia, hyperglycemia, and hyperuricemia.¹ Nonetheless, the mechanism of underlying the onset and progression of AS remains incompletely understood. Clinically approved treatments for AS encompass cholesterol-lowering drugs, notably statins and fibrates. Although statins effectively prevent the development and progression of atherosclerotic diseases, their clinical utility is constrained by adverse effects, such as statin-induced renal disease, myopathy, and hepatobiliary dysfunction.² Thus, the introduction of novel clinically relevant anti-atherosclerosis drugs and modalities holds utmost significance.

AS is classified as "thoracic obstruction" in Traditional Chinese Medicine (TCM). The deficiency of Heart Qi impairs venous blood circulation, causing phlegm dampness obstruction and sluggish blood circulation, ultimately inducing Qi stagnation and blood stasis. The cumulative effects of phlegm retention, Qi stagnation, blood stasis, cold coagulation, and thermojunction culminate in heat stagnation, which then develops into heart vessel blockage stasis and pain. Notably, increasing evidence has shown that TCM has a significant effect on atherosclerotic diseases. For instance, Qingxue Xiaozhi formula (清血消脂方) promotes lipid efflux and inhibits inflammation associated with AS. Additionally, Ruanmailing (软脉灵) affects macrophage foam cell formation to ameliorate AS,³ with low toxicity to patients. Huanglian (Rhizoma Coptidis)-Wuzhuyu (Fructus Evodiae Rutaecarpae) herb pair is effective in improves cellular lipid metabolism, reducing steatosis.⁴ The encouraging therapeutic effects of TCM against AS have enticed wide attention.

Xiong's Shiwei Wendan decoction (熊氏十味温胆汤, XSWD), formulated by Professor Xiong Jibai, a master of TCM, is extensively documented as a treatment for thoracic obstruction with a pathogenesis attributed to the "Yang Micro-yin String" theory.⁵ XSWD is composed of Xiyangshen (Radix Panacis Quinquefolii) 10 g, Danshen (Radix Salviae Miltionrrhizae) 10 g, Chenpi (Pericarpium Citri Reticulatae) 10 g, Banxia (Rhizoma Pinelliae) 10 g, Fuling (Poria) 10 g, Zhishi (Fructus Aurantii Immaturus), Zhuru (Caulis Bambusae in Taeniam) 10 g, Suanzaoren (Semen Ziziphi Spinosae) 20 g, Yuanzhi (Radix Palygalae) 10 g, and Zhigancao (processed Radix Glycyrrhizae) 10 g (supplementary Table 1). Xiyangshen (Radix Panacis Quinquefolii) replenishes heart Qi and benefits heart blood, danshen nourishes blood, calms nerves, cools the blood, and removes blood stasis. Banxia (Rhizoma Pinelliae), Chenpi (Pericarpium Citri Reticulatae), Fuling (Poria), Zhishi (Fructus Aurantii Immaturus), Zhuru (Caulis Bambusae in Taeniam), and Gancao (Radix Glycyrrhizae) [Wendan decoction (温胆汤, WDD)] harmonize the stomach, remove phlegm, nourish the heart, and calm the mind. Suanzaoren possesses properties that nourish the heart and tranquillizes the mind, thereby safeguarding against Yin depletion induced by other herbs. Yuanzhi (Radix Palygalae) can tranquillize the heart and calm the mind, dispel phlegm, and reduce swelling. Gancao (Radix Glycyrrhizae) harmonizes medicines, benefits Qi, and eases the spleen and stomach. In our previous study, XSWD has shown beneficial effects on reducing symptoms in AS Patients.⁶ However, its exact mechanism of mitigating AS remains unknown.^7,8

Accumulating evidence has elucidated that gut microbiota (GM) impacts the progression of AS by regulating metabolism, immunity, and the mucosal barrier.^9-11 Given the in-depth understanding of the relationship between GM imbalance and AS, research focusing on the regulation of GM as a novel treatment modality for AS has garnered significant attention.

TCM formulations have been associated with alterations in gut microbial composition in patients with metabolic disorders. WDD, which serves as the foundation of XSWD, has been confirmed to alter gut microbial composition, plasma lipids, and glucose metabolism in patients with olanzapine-induced metabolic syndrome.¹² Of note, Jiawei Wendan decoction (加味温胆汤, JWD), a homologous formula of XSWD, modifies specific herbs from WDD and exhibits multiple biological effects, including the positive regulation of insulin resistance, improvement of body metabolism, and inhibiton of macrophage-derived foam cell formation.¹³ In addition, several studies indicate that ingredients of XSWD, such as Chenpi (Pericarpium Citri Reticulatae) and water-insoluble polysaccharides from Fuling (Poria), alter GM species structure, GM-derived metabolites production, and improve lipid profiles.^14,15 The existing literature indicates that XSWD serves as a promising candidate for against AS, potentially hindering its progression through modifications in GM structure and regulation of lipid metabolism.

This study aimed to explore the capacity of XSWD to inhibit AS in vivo by elucidating its role in regulating lipid metabolism and identifying alterations in the GM composition in mice, consequently, the detailed mechanisms underlying alleviation of AS by XSWD and its correlation with GM were elucidated.

2. MATERIAL AND METHODS

2.1. Drug source and preparation process

Atorvastatin calcium tablets (Lepu Pharmaceutical Technology Co., Xiangcheng, China) and XSWD were utilized in our study. Herbs required for the preparation of XSWD were sourced from Yangtianhe TCM Library Co. (Changsha, China). All medicinal plants used in the decoction were authenticated by experts from the College of Pharmacy of Hunan University of TCM. The preparation of XSWD adhered to the standardized protocol outlined in the Chinese Pharmacopoeia Commission (2015) to ensure the consistency in the herbal composition consistency of XSWD decoction.¹⁶ The herbs were soaked in water for 30 min, subsequently decocted for 25 min, and then concentrated to a paste (5 g/mL) to obtain the final XSWD product, which was stored at 4 ℃ for further use.

Quality control assessment and presence of major compounds (fingerprints shown in supplementary Figure 1) in XSWD were determined by High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) technique from Agilent Technologies (Santa Clara, CA, USA). Each standard solution and herbal compound test solution underwent filtration using a 0.22 μm membrane filter before HPLC-MS analysis. A Hypersil Octadecylsilyl column (4.6 mm × 150 mm diameter, 5 μm) from Welch Materials (Shanghai, China) was used as stationary phase; while 0.1% phosphoric acid A: and acetonitrile (B) were used as mobile phases. The gradient elution program was as follows: 0-5 min, 10% phase B; 5-45 min, 10-90% phase B; 45-49 min, 90% phase B; 49-60 min, 100% phase B. The injection volume was 10 μL and temperature was maintained at 30 ℃. The flow rate was set as 1 mL/min, with detection wavelengths configured at 203, 237, and 330 nm. The drug dosage for in vitro and in vivo studies were chosen based on our previous reports.¹⁷

2.2. Preparation of XSWD-medicated serum

The protocol for animal experiments was approved by the Ethics Committee of the Hunan University of Chinese Medicine (No. LL2020052704) in accordance with the Chinese National Laboratory Guidelines for experimental animals. Sixty rats of were purchased from Experimental Animal Center of Hunan university of TCM (Changsha, China), weighing (240 ± 20) g. The rats were housed in a cage under a specific-pathogen-free (SPF) environment, maintained at a controlled ambient temperature ranging from 21 to 25 ℃, with humidity level of 40%-50%, and a 12 h light/dark cycle with free access to food and water. After an adaptive feeding for acclimatization, 6-week-old male Sprague-Dawley rats were administered with 1.25 mg/mL (low dose-XSWD), 2.5 mg/mL (medium dose-XSWD), 5 mg/mL (high dose-XSWD) of XSWD decoction, as well as an equivalent volume of saline for the control group. After the 7-day period, blood was collected from the abdominal aorta, and serum was obtained by low-speed centrifugation (500 rpm) and subsequently stored at －80 ℃.

2.3. Cell culture

Tsuchiya Human Peripheral blood Mononuclear cell-1 (THP-1) were obtained from National Collection of Authenticated Cell Cultures (Shanghai, China), and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Cellmax, Beijing, China), supplemented with 10% fetal bovine serum (Cellmax, Beijing, China), and maintained in a CO₂ incubator with 5% carbon dioxide at 37 ℃. Once the cell density reached 80%, the THP-1 monocytes were cultured in 24-well plates and treated with 100 ng/mL Phorbol Myristate Acetate (PMA) from Sigma (St. Louis, MO, USA) for 24 h, inducing their differentiation into macrophages. Foam cells were generated by pre-incubating the cells with 25 μg/mL oxidized low-density lipoprotein (ox-LDL) from Yiyuan Biotech (Guangzhou, China) for 24 h. For Oil Red O (ORO) staining, the macrophages were divided into five groups: control (normal macrophages), model (ox-LDL-induced foam cells), and three experimental groups, namely low-dose XSWD-treated ox-LDL-induced cells (L-XSWD; 1.25 mg/mL), medium-dose XSWD-treated ox-LDL-induced cells (M-XSWD; 2.5 mg/mL), and high-dose XSWD-treated ox-LDL-induced cells (H-XSWD; 5 mg/mL).

2.4. Cell counting kit-8 (CCK-8) assay

THP-1 cells in logarithmic growth phase were differentiated into macrophages with PMA and subsequently cultured in 96-well plates at a density of approximately 2 × 10⁴ cells/well. After 24 h of incubation, the cells were exposed to 10% XSWD-containing serum. The cells were divided into following groups: normal control (cells without any treatment), blank serum (RPMI 1640 + saline serum), L-XSWD (RPMI 1640 + serum containing L-XSWD), M-XSWD (RPMI 1640 + serum containing L-XSWD), and H-XSWD (RPMI 1640 + serum containing L-XSWD). Following 24 h, the existing medium was discarded, and 110 μL of fresh medium containing 10 μL of CCK-8 detection solution was added to each well. After a 2-h incubation period at 37 ℃, the absorbance was recorded at a wavelength of 450 nm by a microplate reader from Bio-tek (Winooski, VT, USA). The cell viability (%) was calculated as follows: Cell viability (%) = [(intervetion wells) － (cell-free wells) / [(normal control wells) － (cell-free wells)]*100%.

2.5. Oil red O staining

After discarding the medium, THP-1 macrophages were rinsed with phosphate buffered saline (PBS; Solarbio, Beijing, China) and fixed with 4% paraformaldehyde (PFA; Biosharp, Hefei, China) at room temperature, followed by decolorization with 60% isopropanol (Sinopharm, Beijing, China) for 2 min and staining with the ORO filtration solution (GBCBIO, Guangzhou, China) for 30 min at 37 ℃. Subsequently, the cells were re-stained with hematoxylin (Biosharp, Hefei, China) for 3 min. Next，1% Acid Alcohol (Beyotime, Shanghai, China) was added to differentiate cells and then light ammonia (Solarbio, Beijing, China) was added immediately. Finally, the lipid deposition was observed under the 912x microscope from Thermo Fisher (Invitrogen EVOS M5000; Waltham, MA, USA), and images were analyzed by ImageJ software (vers 2.1.0, NIH, Bethesda, MD, USA).

2.6. Animals

The Ethics Committee of Hunan University of Chinese Medicine approved all animal experiments conducted in this study (No. LL2020052704) in accordance with the Guide for Care and Use of Laboratory Animals. All male ApoE^-/- mice (5 weeks old) were purchased from Viton Lever Laboratory Animal Technology Company (Beijing, China). The mice were housed in a split-cage (5 mice per cage) environment under SPF conditions with an ambient temperature of (21-25) ℃, a humidity level 40%-50%, and a 12 h light/dark cycle. Daily food and water supplements were provided, and the bedding was changed every four days. The body weights of the mice were recorded weekly. Following a week of acclimation feeding, mice were randomized into six weight-based groups by random number table method: control (n = 5), model (n = 5), atorvastatin (n = 5), low-dose XSWD (L-XSWD, n = 5), medium-dose XSWD (M-XSWD, n = 5), and high-dose XSWD (H-XSWD, n = 5). The control group was continuously fed with normal chow (NC). Other groups were fed with high-fat diet (HFD, 1% cholesterol + 10% pork fat + 10% dried yolk + 0.2% porcine bile salt + 78.8% basal diet) for 8 weeks to establish AS model. After establishing model, all groups were fed with NC daily during the subsequent 8 weeks of drugs or vehicle (sterile saline) administration. The atorvastatin group (positive control) was given 2.6 mg·kg^-1·d^-1 of atorvastatin calcium tablets, while L-XSWD group, M-XSWD group, and H-XSWD group received 9, 18 and 36 g·kg^-1·d^-1 of XSWD, respectively.¹⁷

2.7. Samples collection

Colonic contents of ApoE^-/- mice were collected in the final week and stored in sterile Eppendorf tubes. After the final gavage, mice were anesthetized via intraperitoneal injection of 0.3% sodium pentobarbital (Sigma, St. Louis, MO, USA)) at a dose of 15 μL/g, and blood samples were collected from the orbital venous plexus. Subsequently, the right auricle was clipped and the left ventricle was perfused with PBS and 4% PFA to remove impurities. Following excision of excess organs and adipose tissue, the vessel branches were clipped, and the entire aorta was subsequently stripped with precision. Subsequently, the aorta was preserved in 4% PFA. Finally, the liver was excised and weighed. All samples were stored at －80 ℃for examination.

2.8. Histochemical stain

Following a 24-h soak in 4% PFA, the aortas of mice were excised and washed twice with PBS. Subsequently, aortic peripheral vascular tissue was dissected, fixed in longitudinal sections, and stained with ORO. Frozen sections (4-6 μm thickness) for aortic root staining were prepared using Frozen Slicer (Leica, Wetzlar, Germany), and sections were stained by ORO, hematoxylin, and eosin reagents according to routine procedures. To detect hepatic steatosis, the liver was saturated with 4% PFA, and sections were produced as described above, followed by staining with hematoxylin and eosin. Lipid accumulation in the artery was photographed using camera (NIKON D5200; Tokyo, Japan), and those in the aortic root was photographed under a microscope. The quantitative analysis was done by ImageJ software (vers 2.1.0, NIH, Bethesda, MD, USA).

2.9. Lipid profile assessment

Plasma lipid profile was measured by high-density lipoprotein (HDL), low-density lipoprotein (LDL), total cholesterol (TC), and triglyceride (TG) assessment kit (Jiancheng Bio, Nanjing, China), using an Enzyme Immunoassay Analyzer (Bio-tek, Winooski, VT, USA).

2.10. 16S rRNA sequencing of colonic microbiota

Samples of colon contents were subjected to DNA extraction. Tks Gflex DNA Polymerase (Takara, Dalian, China) was used to amplify the variable V3-V4 region of the microbial 16S rRNA and sequenced by Illumina Novaseq (San Diego, CA, USA). Vsearch (version 2.4.2, Oslo, Norway) was employed to cluster valid tags into operational taxonomic units (OTUs) based on a 97% similarity threshold, and the representative sequences were subjected to species taxonomy analysis. The species taxonomy analysis was conducted using the Naive Bayesian classification algorithm in the ribosomal database project classifier. QIIME (vers 1.8.0, Shenzhen, China) was used to assess the Alpha diversity, Beta diversity, and inter-group variability of GM.

2.11. Statistical analysis

The data are represented as mean ± standard deviation. Kruskal-Wallis test, Welch’s analysis of variance (ANOVA) and one-way ANOVA were performed using SPSS 25.0 (IBM Corp., Armonk, NY, USA). Species abundances among different subgroups was analyzed by Kruskal-Wallis test. The compositional similarity of GM was determined using UniFrac Adonis and Bray-Curtis analyses. Statistical significance among groups was defined when P < 0.05.

3. RESUITS

3.1. XSWD inhibits foam cell formation

Foam cells play a pivotal role in initiation and development of atherosclerosis. Given the established role of TCM-medicated serum in obesity mitigation,¹⁸ we initially investigated whether serum medicated with XSWD exhibits the ability to inhibit lipid deposition. We performed cytotoxicity experiments to choose an appropriate XSWD-containing serum intervention concentration. The CCK-8 assay revealed that serum derived from XSWD-treated rats at a 10% concentration exhibited no significant impact on cell viability, demonstrating no discernible difference from the non-XSWD-containing serum group (Figure 1A). Then, we investigated the mechanisms of how XSWD modulates lipid deposition in foam cells under in vitro conditions. ORO staining results showed that XSWD (M-XSWD and H-XSWD) evidently reduced intracellular lipid deposition in ox-LDL-induced foam cells (Figures 1B, 1C), indicating the XSWD-mediated lipid accumulation reduction in macrophage-derived foam cells. These results are consistent with the potential results of our previous clinical study.⁶

Figure 1. Effect of XSWD-medicated serum and XSWD on THP-1 macrophages differentiation.

A: CCK-8 assay for THP-1 macrophages cultured in the presence of 10% XSWD-containing serum; B: ORO staining of THP-1 macrophages (× 912, scale bars = 10 μm); B1: cells of the control group; B2: cells of the model group; B3: cells of the L-XSWD group; B4: cells of the M-XSWD group; B5: cells of the H-XSWD group; C: area of Oil Red O measured from the images shown in (B) using ImageJ software. Control: normal control (incubated by complete medium); Model: model group (25 μg/mL ox-LDL + complete medium); L-XSWD: low-dose XSWD group (25 μg/mL ox-LDL + 1.25 mg/mL XSWD); M-XSWD: medium-dose XSWD group (25 μg/mL ox-LDL + 2.5 mg/mL XSWD); H-XSWD: high-dose XSWD group (25 μg/mL ox-LDL + 5 mg/mL XSWD). XSWD: Xiong's Shiwei Wendan decoction; CCK-8: cell counting kit-8; THP-1: Tsuchiya human peripheral blood mononuclear cell-1; ORO: oil red O; ANOVA: analysis of variance. Statistical significance was assessed using one-way ANOVA. All data were expressed as the mean ± standard deviation (n = 3). ^aP < 0.0001, compared with the control group; ^bP < 0.01 and ^cP < 0.001, compared with the model group.

3.2. XSWD attenuates atherosclerotic lesions in AS mice

To elucidate the protective effect of XSWD against AS in vivo, ApoE-/- mice prone to atherosclerosis were selected and fed HFD to establish an AS model. The mice were then treated with different administraion for next 8 weeks. Apart from a marked weight gain observed in the HFD-fed mice, the establishment of AS model was confirmed by the detection of plaque lesions and lipid deposition in the aortic root and artery of HFD-fed mice, both in en face and aortic root staining. Compared to the NC-fed group (Figures 2A, 2B), the establishment of AS model was unequivocally confirmed. Further, the same tendency was observed for lipid accumulation in above mentioned mice (Figure 2C). Interestingly, H-XSWD treatment reduced the lipid area in AS mice, whereas no similar differences were observed in atorvastatin, L-XSWD, and M-XSWD groups. The results suggest that H-XSWD inhibited aortic atherosclerosis and reduced the lipid deposition in AS mice.

Figure 2. Effects of XSWD on AS lesions in ApoE^-/- mice.

A: en face analysis of the aorta; A1: aorta of the control group; A2: aorta of the model group; A3: aorta of the atorvastatin group; A4: aorta of the L-XSWD group; A5: aorta of the M-XSWD group; A6: aorta of the H-XSWD group. B: ORO staining and analysis of aortic root plaque lesions (× 40, scale bars = 300 μm); B1: aortic root plaque lesions of the control group; B2: aortic root plaque lesions of the model group; B3: aortic root plaque lesions of the atorvastatin group; B4: aortic root plaque lesions of the L-XSWD group; B5: aortic root plaque lesions of the M-XSWD group; B6: aortic root plaque lesions of the H-XSWD group. C: lipid area of ORO in aortic root plaque. Control: normal control (fed by normal chow); Model: model group (fed by HFD); Atorvastatin: atorvastatin group (2.6 mg/kg atorvastatin + HFD); L-XSWD: low-dose XSWD group (9 g/kg XSWD + HFD); M-XSWD: medium-dose XSWD group (18 g/kg XSWD + HFD); H-XSWD: high-dose XSWD group (36 g/kg XSWD + HFD). XSWD: Xiong’s Shiwei Wendan decoction; ORO: oil red O; HFD: high-fat diet; ANOVA: analysis of variance. The statistical significance of lipid area was assessed by Welch’s ANOVA. All data were expressed as the mean ± standard deviation (n = 5). ^aP < 0.001, compared with the control group; ^bP < 0.05, compared with the model group.

3.3. XSWD improves serum lipids in AS mice

To investigate whether XSWD mitigates lipid deposition in atherosclerotic plaques by modulating lipid metabolism, we conducted a comprehensive analysis of serum lipids and assessed the body and liver weights of the mice. The results showed that compared to HDF-fed mice, H-XSWD significantly decrease the body weight and liver index of mice (supplementary Figures 2A-2C). The serum lipid analysis showed that the high-fat diet in ApoE^-/- mice significantly impacted the lipid metabolism (Figures 3A-3D). Nevertheless, XSWD treatment improved the serum lipid profile, which was in line with the lipid-lowering effect of atorvastatin. Above results indicated that XSWD positively regulated the serum lipid profile, while L-XSWD and H-XSWD have potential hepatoprotective effects.

Figure 3. Effect of XSWD on serum lipid profile in ApoE^-/- mice.

A: serum TG level. B: serum TC level. C: serum LDL level. D: serum HDL level. Control: normal control (fed by normal chow); Model: model group (fed by HFD); Atorvastatin: atorvastatin group (2.6 mg/kg atorvastatin + HFD); L-XSWD: low-dose XSWD group (9 g/kg XSWD + HFD); M-XSWD: medium-dose XSWD group (18 g/kg XSWD + HFD); H-XSWD: high-dose XSWD group (36 g/kg XSWD + HFD). XSWD: Xiong’s Shiwei Wendan decoction; TG: triglycerides; TC: total cholesterol; LDL: low-density lipoprotein; HDL: high-density lipoprotein; HFD: high-fat diet; ANOVA: analysis of variance. Statistical significance of all data was assessed using one-way ANOVA. All data were expressed as the mean ± standard deviation (n = 5). ^aP < 0.01 and ^bP < 0.05, compared with the model group.

3.4. XSWD transforms the composition of GM in AS mice

Gut microbiota plays a crucial physiological role in the human body and communicates through gut-systemic axis. GM is modifiable by dietary intake and pharmacological interventions. Modulation of gut microbiome has demonstrated its ability to influence the progression of AS progression by reducing inflammation and regulating lipid metabolism.¹⁹ To determine whether the protective effect of XSWD against AS was associated with altered microbiota structure, we sequenced the bacterial 16S rRNA V3-V4 region in colonic samples. The coverage index (supplementary Figure 3A) represented the sequencing depth of the analyzed samples, indicating the maximum coverage of the microorganisms present in samples.

In our study, HFD and drug administration resulted in tremendous alterations in the GM composition of mice, especially Bacteroidetes and Firmicutes. As illustrated in Figures 4A, 4B, we counted the top 15 colonic gut microorganisms at phylum and genus level. Bacteroidetes and Firmicutes were the prominent microorganisms at the phylum level, which have shown association with host inflammatory responses.²⁰ Additionally, the relative abundance of Firmicutes in the model group decreased while that of Bacteroidetes increased compared to the untreated group. (Figure 4A). Upon adding atorvastatin and XSWD, the relative abundance of Bacteroidetes decreased, and those of Firmicutes increased, except L-XSWD group. At the genus level (Figure 4B), XSWD-treated groups showed a clear increase in Lachnospiraceae_NK4A136_group (4.81%, 10.90%, 9.76% in L-XSWD group, M-XSWD group, and H-XSWD group, respectively), and Alistipes (5.44%, 8.32%, 4.87% in L-XSWD group, M-XSWD group, and H-XSWD group, respectively). In addition, XSWD did not restore Bacteroides levels, XSWD upregulated Bacteroides (3.06%, 3.50%, 3.73%, 4.54%, 3.54% in control group, model group, L-XSWD group, M-XSWD group, and H-XSWD group, respectively) instead. Our data show that the species abundance of GM is fractionally altered in AS mice after XSWD and atorvastatin administration.

Figure 4. GM composition in AS mice after XSWD administration.

A: top 15 colonic gut microorganisms at phylum level; B: top 15 colonic gut microorganisms at genus level; C: Chao1 index of Alpha diversity; D: Shannon index of Alpha diversity; E: un-weighted unifrac PcoA plot. Control: normal control (fed by normal chow); Model: model group (fed by HFD); Atorvastatin: atorvastatin group (2.6 mg/kg atorvastatin + HFD); L-XSWD: low-dose XSWD group (9 g/kg XSWD + HFD); M-XSWD: medium-dose XSWD group (18 g/kg XSWD + HFD); H-XSWD: high-dose XSWD group (36 g/kg XSWD + HFD). GM: gut microbiota; AS: atherosclerosis; XSWD: Xiong’s Shiwei Wendan decoction; PcoA: principal coordinates analysis; HFD: high-fat diet. The statistical significance of Chao1 index and Shannon index were assessed by kruskal-wallis. All data were expressed as the mean ± standard deviation (n = 5).

Microbial diversity served as an indicator of alterations in gut microbial population and structure (Figures 4C, 4D). HFD disrupted microbial diversity and abundance in AS mice, evidenced by a decreased in the Chao1 index (P = 0.015) and Shannon index (P = 0.061). Although Shannon index is not statistically significant, its trend is consistent with Chao1 index. Atorvastatin and XSWD, however, mitigated the negative effects of HFD, restoring the composition of microorganism. Subsequently, we used principal coordinate analysis (PCoA), principal component analysis (PCA), and UPGMA to represent the beta diversity of each group (Figure 4E, supplementary Figures 3B, 3C). PcoA is displayed by weighted unifrac distances, and the similarity of microbiota is positively related to the distance in PcoA, PCA and UPGMA. Composition of GM was differed between control group and model group, indicating HFD-induced alteration of GM in ApoE^-/- mice. But the heterogeneity was abolished upon atorvastatin and XSWD administration. Surprisingly, compared to all XSWD groups, atorvastatin group had less species homology with control group, inferring that XSWD has superior efficacy in positively adjusting gut microbiome in AS mice.

3.5. XSWD administration results in divergent microbial community and functional alterations

To elucidate the specific microbial colony that exerts a pivotal role following XSWD administration, we used linear discriminant analysis effect size (LefSe) to concurrently compare multiple groups and identify species exhibiting significantly distinct abundances in experimental group. The linear discriminant analysis (LDA) histogram (supplementary Figure 4A) showed 41 taxa in five groups with LDA scores > 2.5. Rumino-coccaceae_UCG-014, Prevotellaceae_NK-3B31_group, Eubacterium_ventriosum_group, Ruminococcus_1, Adlercreutzia were enriched in control group, the model group showed obvious enrichment in Allopreveotella, Muribaculum, Acrinobacteria, Methylobacterium, Actinomycetos-pora; nevertheless, Prevotellaceae_ UCG_001, Proteobacteria, Zymobacter, Glucomobacter, Acetobacter were the dominating strains in L-XSWD group, while Blautia was the remarkable strain in M-XSWD, and Ruminiclostridum_6, Marvinorvantia, Pantoea were the featured strains in H-XSWD, respectively. The cladogram (supplementary Figure 4B) shows that Tenericutes were notable in control group, Actinobacteria was influential in model group, while Proteobacteria was prominent in different-doses XSWD group.

4. DISCUSSION

XSWD exhibits heart nourishing, phlegm dissipating, and blood stasis removing effect. Our previous study demonstrated the therapeutic effect of XSWD in AS patients.⁶ However, its specific effect to AS is not yet reported in vivo. This study revealed that XSWD inhibited lipid accumulation, improved lipid metabolism, and reduced foam cell formation. Moreover, high-throughput sequencing of 16S rRNA’s showed that XSWD altered GM composition and diversity. To the best of our knowledge, this is the first study to report the in vivo inhibitory potential of XSWD on AS and its association with GM alteration.

Atherosclerosis is characterized by the accumulation of lipids in the walls of arteries, with dyslipidemia-induced catabolic stress leading to the transformation of macrophages into foam cells. The excessive accumulation of foam cells serves as a primary driver of atherosclerosis progression. Additionally, the Traditional Chinese Medicine formula XSWD, specifically its component WDD, has been shown to modulate macrophage foam cell formation through the regulation of reverse cholesterol transport.²¹ Salvianolic acid B and Tanshinone ⅡA (the contents of XSWD) reduced lipid deposition via upregulating transporter protein expression in macrophages.^22,23 It indicates a possible relationship between XSWD and foam cell formation. This study demonstrates the protective effects of XSWD against foam cell formation induced by ox-LDL, with high doses significantly reducing intracellular lipid deposition. Furthermore, both XSWD and atorvastatin were found to decrease arterial plaque lesions and improved serum lipid profile.

Increasing evidence suggests that dysbiosis of GM is a contributing factor in the development of obesity, metabolic syndrome, and atherosclerotic diseases.^24,25 The modulation of GM has been extensively researched as a potential therapeutic target for these conditions, particularly in relation to lipid management and reduction of inflammatory responses.²⁶ Interestingly, certain herbal and herb monomer have been shown to impact disease phenotypes by altering the composition of the gut microbiota.^{27- 29} Our hypothesis posits that XSWD influences GM composition to enhance lipid metabolism in AS mice. We used high-throughput 16S rRNA sequencing technology to investigate whether XSWD improved lipid metabolism in AS mice by affecting GM composition. As anticipated, the composition of GM was altered in mice subjected to HFD-induced and XSWD-treated mice. Firmicutes and Bacteroidetes were identified as the predominant bacterial taxa in gut. Both of which are involved in the synthesis of short-chain fatty acids (SCFAs). Previous research has suggested an inverse relationship between the Firmicutes/ Bacteroidetes ratio and the severity of AS,^30,31 although this association is not universally supported in the context of lipid metabolism and obesity.^32,33 Our findings revealed a decreased prevalence of Firmicutes and Bacteroidetes in the colonic microbiota of AS mice. Following treatment with atorvastatin and XSWD, the abundance of Firmicutes and Bacteroidetes returned to levels similar to those of the control group, partially restoring the GM diversity in AS mice. Indeed, there is variability in Firmicutes/Bacteroidetes ratios among individuals, influenced by a range of factors such as host genomics and environmental variables like lifestyle, hygiene, and diet.³⁴ Short-term dietary changes have been shown to impact gut microbiota composition in inbred mice,^35,36 potentially influenced by the feeding regimen utilized. It is possible that the introduction of NC in the final stage of the study may have disrupted the gut microbiota. Furthermore, SCFAs, as secondary metabolites produced by gut microbiota, its concentration may play a more significant role in regulating metabolism than the overall abundance of specific bacterial species.³² Further experimentation is required to confirm these findings. Our study found that both atorvastatin and XSWD were able to reverse the changes in species richness and diversity induced by a high-fat diet. However, there was no significant difference in their ability to increase richness and diversity in the gut microbiota of AS mice. Analysis of beta diversity revealed that XSWD groups were more similar to the control group, indicating that XSWD may be more effective than atorvastatin in positively modulating the gut microbiota.

Certain genera such as Lactobacillus and Prevotellacea, have been shown to regulate metabolism (glucose metabolism, lipid metabolism) in vivo.^37-39 To pinpoint the genera contributing to the protective role of XSWD, we used LefSE analysis to delve into the composition differences among all groups. Prevotellaceae_UCG_001 showed remarkable differences (LDA > 4) at the genus level. These results implied that Prevotellaceae_UCG_001 may have a crucial role in AS inhibition by XSWD. Prevotellaceae_UCG_001 is a member of Prevotellaceae family, which is a major fiber degrading (indigestible polysaccharide) bacterium and generates SCFAs during decomposition.⁴⁰ SCFAs are essential metabolites produced by GM, and their beneficial effects have been demonstrated in metabolic syndrome and cardiovascular pathologies.^41,42 SCFAs also promote intestinal barrier integrity as well as anti-inflammatory responses in immune cell.⁴³ Furthermore, SCFAs have been shown to potentially impede the progression of pathological processes by inhibiting histone deacetylases.⁴⁴ Additionally, SCFAs have demonstrated vascular benefits through their impact on cholesterol synthesis and metastasis.⁴⁵ Specifically, Butyric acid, a partial SCFA, has been found to decrease monocyte adhesion to endothelial cells and inhibit foam cell formation.⁴⁶ Despite limited research on the role of Blautia in cardiovascular disease, studies have indicated its potential to improve lipid metabolism in adipocytes.⁴⁷ Collectively, these findings suggest a possible association between the inhibition of atherosclerosis by XSWD and GM. However, further metabolomic characterization is still needed.

In conclusion, our study specifically, Prevotellaceae UCG 001 and Blautia exhibited altered expression levels following XSWD intervention, warranting additional exploration through metabolomics and transcriptomics analyses of atherosclerotic plaques to elucidate their potential roles in atherosclerosis. Moreover, the implementation of fecal microbiota transplantation could serve as a valuable method to validate the robustness of our findings. In summary, herein we demonstrated that XSWD treatment effectively mitigated atherosclerotic lesions and altered the composition and population of gut microbiota in HFD-induced AS mice. Prevotellaceae UCG 001 and Blautia showed differential expression after XSWD administration. however, their role in atherosclerosis needs further investigation by metabolomics and transcriptomics studies of atherosclerotic plaques. Moreover, the implementation of fecal microbiota transplantation could serve as a valuable method to validate the reliability of our findings.

5. SUPPORTING INFORMATION

Supporting data to this article can be found online at http://journaltcm.com.

Funding Statement

Supported by National Natural Science Foundation of China: the Anti-Atherosclerotic Mechanism of Xiong’s Shiwei Wendan decoction based on the Regulation of Reverse Cholesterol Transport via the MicroRNA-33A Adenosine Triphosphate-binding Cassette Subfamily A Member 1/ Adenosine Triphosphate-binding Cassette Subfamily A Member 1 Pathway (No. 81603600); the Natural Science Foundation of Hunan Province of China: the Anti-Atherosclerotic Mechanism of Xiong's Shiwei Wendan Decoction based on the Activation of Lipophagy Mediated by the AMP-activated Protein Kinase/Mechanistic Target of Rapamycin Pathway (No. 2021JJ30510) ; the Health Research Project of Hunan Provincial Health Commission: the An-ti-Atherosclerotic Mechanism of Xiong's Shiwei Wendan Decoction based on Lipophagy Mediated by the MicroRNA-499a-3p/ Autophagy-related 5 Pathway (No. D202303018265)