Erxian Decoction Ameliorates Myocardial Damage in Ovariectomized Rats by Regulating the Gut Microbiota and TMAO-Mediated NLRP3 Inflammatory Pathway

TL;DR

Erxian Decoction improves heart health in menopause-like rats by changing gut bacteria and reducing inflammation linked to heart disease.

Contribution

Erxian Decoction's novel cardiovascular benefits via gut microbiota and TMAO-mediated NLRP3 pathway modulation in ovariectomized rats are demonstrated.

Findings

Erxian Decoction improved heart function and reduced inflammation in ovariectomized rats.

The decoction altered gut microbiota composition and lowered TMAO and its precursor metabolites.

Erxian Decoction reduced blood pressure and lipid levels in the rats.

Abstract

Menopausal women face an increased risk of cardiovascular diseases (CVDs), and exploring effective therapeutic strategies from traditional Chinese medicine is of great clinical significance. Erxian decoction (EXD), a classic formula for alleviating menopausal symptoms, has potential cardiovascular protective effects, but its underlying mechanisms remain unclear. In this study, the effects of EXD on gut microbiota, TMAO levels, and inflammatory levels were evaluated in ovariectomized (OVX) rats, a well-established model of menopausal CVDs. EXD elevated serum E2 levels and improved both the cardiac systolic and diastolic functions in OVX rats. EXD decreased Firmicutes and Ruminococcaceae, increased Bacteroidota, Muribaculaceae and Escherichia-Shigella. EXD significantly decreased serum levels of TMAO and its precursor metabolites. Additionally, it attenuated myocardial expression of ROS, TXNIP, NLRP3, ASC, Caspase 1, IL-18, and IL-1β. EXD could reduce the levels of SBP and DBP, and reduce the serum levels of TC, TG and LDL-C. Spearman correlation analysis revealed positive associations between TMAO and blood pressure, as well as inflammatory factors and dyslipidemia. Collectively, EXD may ameliorate myocardial damage in OVX rats through modulation of the gut microbiota and suppression of the TMAO-mediated NLRP3 inflammasome pathway, demonstrating its potential therapeutic implications for cardiovascular disorders.

Figures

A](#F1) depicts the blood pressure changes in the rats. In comparison to the Sham group, the DBP and SBP exhibited a significant increase in the OVX group. The DBP and SBP were markedly reduced in the treatment groups receiving 0.18 mg/kg estradiol valerate and EXD at doses of 7.5 and 9 g/kg. In comparison to the Sham group ([Fig. 1B](#F1)), the OVX group had significantly decreased E2 levels and increased serum levels of IL-6. E, EXD-7.5 and EXD-9 groups had increased the E2 levels and decreased the IL-6 levels. Exogenous E2 was indeed employed as the positive control in this experiment, and

D](#F4) and [4E](#F4), respectively, display the bar chart and heatmap of the top 15 phylum-level classifications. *Firmicutes* and *Bacteroidota* emerged as the predominant phyla. A significant decrease in *Bacteroidota* abundance was observed in the OVX group, whereas the EXD group exhibited a marked increase. Conversely, *Firmicutes* abundance increased in the OVX group but decreased in the EXD group. Additionally, the EXD group demonstrated a markedly reduced abundance of *Desulfobacterota* compared to the OVX group ([Fig. 4F](#F4)). At the genus level ([Fig. 4G](#F4) and [4H](#F4)), in co

C](#F5)). The results demonstrated that TMAO and its precursor metabolite creatinine were positively correlated with blood pressure (SBP and DBP), inflammatory factors (IL-1β and IL-18), and serum lipid (TC, TG, and LDL-C). This study focused on TMAO-mediated inflammation. As TMAO increases ROS levels in myocardial tissue to activate the NLRP3 inflammasome [[25](#ref25)], the expression of the TMAO-mediated inflammatory pathway in the myocardium was subsequently examined.

A](#F7) and [7B](#F7)), the OVX group had significantly increased relative TXNIP, NLRP3, ASC, and Caspase 1 expression levels. The E and EXD groups had significantly decreased relative TXNIP, NLRP3, ASC, and Caspase 1 expression levels. In this study, EXD decreased the relative expression levels of IL-1β and IL-18 in the myocardium and serum ([Figs. 7A-C](#F7)), which are the downstream inflammatory factors that follow NLRP3 inflammasome activation.

Full text

Introduction

Epidemiological evidence indicates elevated cardiovascular disease (CVD) incidence among postmenopausal women or those undergoing early oophorectomy [1]. Furthermore, observed gender disparities in CVD suggest heightened susceptibility to cardiac dysfunction in postmenopausal women due to diminished estrogen (E2) levels [2]. However, long-term hormone replacement therapy remains controversial owing to significant adverse effects, including vaginal bleeding, thrombosis, and hearing impairment [3, 4]. Currently, therapeutic options for CVD prevention and management in postmenopausal or early-oophorectomy women are notably limited. Traditional Chinese medicine (TCM) demonstrates efficacy in alleviating CVD-related symptoms and enhancing patients' quality of life [5]. Additionally, various TCM-derived active compounds, herbal formulations, and medicinal prescriptions exhibit E2-like effects [6, 7], suggesting their potential therapeutic value for postmenopausal CVD.

Trimethylamine N-oxide (TMAO) demonstrates causal involvement in CVD development [8], with elevated TMAO levels correlating with increased risk of major adverse cardiovascular events [9]. Animal studies further indicate that TMAO exacerbates left ventricular dysfunction and myocardial fibrosis in murine models [10]. Moreover, TMAO activates platelet hyperreactivity and potentiates thrombotic susceptibility [11]. Additionally, TMAO impairs myocardial contractility and intracellular calcium handling through compromised energy metabolism and mitochondrial function, attributable to its disruption of pyruvate and fatty acid oxidation [12]. TMAO biosynthesis is fundamentally dependent on gut microbiota metabolism [13], where microbial processing of dietary choline yields TMA as an intermediate metabolite, which is subsequently converted to TMAO in the liver. Gut microbiota dysbiosis consequently modulates TMAO levels [14], thereby promoting CVD progression.

The contribution of inflammatory processes in CVD development and progression is an ongoing research topic [15]. Elevated serum inflammatory cytokine concentrations are commonly observed after menopause [16], which suggests that the inflammation state might be associated with E2 depletion at menopause. TMAO increases reactive oxygen species (ROS) production and promotes assembly activation of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome [17]. Subsequently, the inflammatory response mediated by NLRP3 inflammasome activation induces myocardial injury [18]. Therefore, TMAO-mediated inflammatory responses might be related to postmenopausal CVD progression.

Erxian decoction (EXD) is a classical traditional Chinese herbal formula for clinical treatment of menopausal symptoms [19]. Previously, our research found that EXD improved abnormal cardiac function, myocardial gene remodeling, lipid metabolism disorder, and inflammatory response in ovariectomized (OVX) rats[20]. More importantly, EXD regulated gut microbiota structure and function in OVX rats. However, whether EXD can regulate TMAO-mediated inflammatory response to achieve myocardial protection remains unclear. In this study, the protective effect of EXD on OVX rat myocardia was systematically investigated using 16S rRNA amplicon sequencing and metabolomics combined with molecular biology. The results demonstrated that EXD might be involved in regulating the TMAO-mediated NLRP3 inflammatory pathway.

Materials and Methods

Preparation of EXD and Quality Control

EXD comprises six herbs: Curculigo orchioides Gaertn. (Curculiginis Rhizoma, CR), Epimedium brevicornu Maxim. (Epimedii Folium, EF), Morinda officinalis How. (Morindae Officinalis Radix, MO), Angelica sinensis (Oliv.) Diels (Angelicae Sinensis Radix, AS), Phellodendron chinense Schneid. (Phellodendri Chinensis Cortex, PC), and Anemarrhena asphodeloides Bge. (Anemarrhenae Rhizoma, AR). The aforementioned plant names were all checked with www.worldfloraonline.org. CR, EF, MO, AS, PC, and AR were weighed at a ratio of 2:2:2:2:1:1, combined and concentrated to 0.75 g/ml and 0.9 g/ml. The method for decocting EXD and the main active components was detected by high-performance liquid chromatography (HPLC), as described in the published materials [21].

Establishment of the Rat Model and Groups

The animal involvement in this study was approved by the China Academy of Chinese Medical Sciences Institutional Ethics Committee (ethics approval number: IBTCMCACMS21-2104-05). Ten-week-old female Sprague-Dawley (SD) rats (n = 30, 180 ± 20 g) were provided by Si Pei Fu Biotechnology Co., Ltd. [SCXK (Beijing) 2019-0010, China]. This experiment involved a bilateral OVX rat model. Thirty rats underwent a one-week acclimation period. Subsequently, 24 rats were randomly selected for OVX surgery. During the surgery, rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital, and the ovaries were removed. The remaining six rats formed the Sham group, in which only small pieces of adipose tissue were removed from the abdominal cavity, while the ovaries were preserved. After two weeks, the 24 OVX rats were randomly divided into OVX, E, EXD low dose and EXD high dose groups (n = 6 rats per group). The E group received 0.18 mg/kg estradiol valerate (Bayer Healthcare Co., Ltd., China), the Sham and OVX groups both received same amount of purified water, and the EXD group received 7.5 g/kg EXD (EXD-7.5) and 9 g/kg EXD (EXD-9). After 12 weeks of administration, the blood pressure and echocardiogram of the rats were measured in the early morning, followed by sampling and subsequent experiments.

Tail-Cuff Method Detection of Blood Pressure

The systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured by a small animal noninvasive sphygmomanometer (BP-98A, Softron Biotechnology, China). Each rat was measured three times consecutively, and the average value was taken.

Echocardiography Detection of Cardiac Function

Echocardiography was performed using an ultrahigh resolution small animal ultrasound imaging system (VisualSonics, Vevo2100, Canada). The following parameters were measured and calculated: left ventricular end-systolic dimension (LVESD), left ventricular end-systolic volume (LVESV), left ventricular end-diastolic dimension (LVEDD), left ventricular end-diastolic volume (LVEDV), ejection fraction (EF%), and fraction shortening (FS%).

Assessment of Myocardial Histopathology

The rat myocardium was fixed in 4% paraformaldehyde, paraffin-embedded, sliced 7 μm thick and stained with hematoxylin-eosin (HE). The left ventricle myocardia (3 mm × 3 mm × 1 mm) were fixed in 4% glutaraldehyde, then ultrathin sections of myocardium (50-nm thickness) were prepared with an ultrathin microtome and stained with uranyl acetate-lead citrate. Pathological changes and ultrastructural alterations in the myocardium were observed using a 200 × optical microscope and a transmission electron microscope (TEM), respectively.

Detection of Serum E2, Inflammatory Factors, and Serum Lipids

The serum level of E2 (Beijing North Institute of Biotechnology Co., Ltd., China) was detected by radioimmunoassay. The levels of interleukin (IL)-6, IL-1β and IL-18 (all, Beijing North Institute of Biotechnology Co., Ltd.) were determined using enzyme-linked immunoassay (ELISA) kits. The levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) (InTec PRODUCTS, INC., China) were detected by kit instructions.

16S rRNA Amplicon Sequencing

Fresh feces samples were obtained from all rats before being transferred to sterile cryopreserved tubes. Samples were quickly frozen at −80°C after collection. The bacterial DNA in feces was extracted using the MagPure Soil DNA LQ Kit (China), the quality and quantity of DNA were verified with NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) and agarose gel. The DNA was used as the template for PCR amplification of bacterial 16S rRNA genes with the barcoded primers and Takara Ex Taq (Takara, Japan). The PCR products were purified with Agencourt AMPure XP beads (Beckman Coulter Co., USA) and quantified by Qubit dsDNA assay kit (YEASEN, China). Finally, Sequencing was performed on an Illumina NovaSeq6000 (Illumina Inc., OE Biotech Company, China).

Liquid Chromatography-Mass Spectrometry (LC-MS) Detection of Serum TMAO and Related Precursor Metabolites

Serum samples were processed and placed in LC bottles. The LC was performed on an AB ExionLC (AB Sciex, USA). The analysis was conducted using an ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm) (Waters, USA). The MS was performed on an AB Sciex Qtrap 6500+ System (AB Sciex). All metabolites were quantified using SCIEX OS-MQ software.

Dihydroethidium (DHE) Staining Detection of Myocardial ROS Levels

The frozen and sectioned myocardial tissue was incubated with DHE dye solution (Sigma-Aldrich, China) at 37°C in a dark incubator for 30 min. Then, the nuclei were stained with DAPI dye solution (Servicebio, China) and incubated at room temperature away from light for 10 min. Fluorescence microscopy revealed that the ROS were stained in a red color, while the nuclei were stained in a blue color. The positive rate of ROS (%) = red fluorescent cells/total number of cells × 100%.

qRT-PCR Analysis

The mRNA expression of FMO3 in the liver and TXNIP, NLRP3, caspase-1, ASC, IL-1β, and IL-18 in the myocardium was examined using qRT-PCR. Table 1 presents the primer sequences. Total tissue RNA was extracted by TRIzol (Takara). Then, oligo (dT) primer and M-MLV Reverse Transcriptase (Both, Takara) were used for reverse transcription. qRT-PCR was used SYBR Green system (YEASEN, China). Finally, the comparative threshold cycle (2^−ΔΔCt^) method was used to normalize relative mRNA expression levels to GAPDH.

Western Blot

The expression of FMO3 (ab126711, Abcam, USA) in liver and NLRP3 (ab263899, Abcam), TXNIP (ab188865, Abcam), ASC (67494-1-Ig, Proteintech, China), IL-1β (A11369, ABclonal, China), and IL-18 (A1115, ABclonal) in myocardium was analyzed with western blots. Total liver and myocardial proteins were extracted using cell and tissue lysis buffer, protease inhibitor cocktail, and phosphatase inhibitor cocktail (all, APPLYGEN, China). Subsequently, the quantification was performed using a bicinchoninic acid (BCA) protein assay kit (YEASEN). Proteins underwent separation through 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were subsequently transferred onto PVDF membranes. The membranes were blocked in 5% skim milk at room temperature for 1 h and incubated with the primary antibodies at 4°C overnight. Next, the membranes were exposed to the secondary antibodies for 1 h. Finally, the membranes were visualized with an enhanced chemiluminescence kit (Proteintech, China).

Statistical Analysis

The experimental results are expressed as the mean ± standard deviation. The statistical analysis and data statistical chart were drawn using GraphPad Prism 8. One-way analysis of variance (ANOVA) was used to compare the groups. For uniform variance, the least significant difference (LSD) test was employed, while the Kruskal-Wallis test was utilized for non-uniform variance. A statistically significant result was determined when the P value <0.05.

Results

Effects of EXD on Blood Pressure, E2, Inflammatory Factors, Serum Lipid and Cardiac Function

Fig. 1A depicts the blood pressure changes in the rats. In comparison to the Sham group, the DBP and SBP exhibited a significant increase in the OVX group. The DBP and SBP were markedly reduced in the treatment groups receiving 0.18 mg/kg estradiol valerate and EXD at doses of 7.5 and 9 g/kg. In comparison to the Sham group (Fig. 1B), the OVX group had significantly decreased E2 levels and increased serum levels of IL-6. E, EXD-7.5 and EXD-9 groups had increased the E2 levels and decreased the IL-6 levels. Exogenous E2 was indeed employed as the positive control in this experiment, and its selection was based on robust scientific rationale: OVX rats exhibit a rapid and profound decline in endogenous E2 levels, which is the core pathological feature mimicking postmenopausal women. The results demonstrated that EXD exerted phytoestrogen-like effects and improved the inflammatory response in OVX rats. In comparison to the Sham group (Fig. 1C), the OVX group had significantly decreased serum HDL-C levels and significantly increased TC, TG, and LDL-C levels. E, EXD-7.5 and EXD-9 groups had increased the HDL-C levels and decreased the TC, TG, and LDL-C levels. The results indicated that EXD improved dyslipidemia in OVX rats.

In comparison to the Sham group (Fig. 1D), the OVX group showed no significant differences in EF% and FS%, but exhibited significantly increased LVESD, LVESV, LVEDD, and LVEDV. In comparison to the OVX group, the EXD-7.5 group had significantly decreased LVEDD and LVEDV. The results demonstrated that EXD-7.5 could improve the diastolic function (LVEDD and LVEDV) in the OVX rats but had no significant effect on cardiac pumping function (EF% and FS%).

In summary, EXD-7.5 and EXD-9 groups could increase the level of estrogen, reduce the level of inflammatory factors and blood pressure, and improve dyslipidemia in OVX rats, and there is no statistical difference between EXD-7.5 and EXD-9 groups. EXD-7.5 could also improve the diastolic function (LVEDD and LVEDV) in the OVX rats; thus, the EXD-7.5 group was used for subsequent experiments.

HPLC Results of EXD

HPLC identified the six main EXD compounds: monotropein, mangiferin, berberine hydrochloride, ferulic acid, curculigoside, and epimedium glycoside (Fig. 2).

Effects of EXD on Myocardial Morphological Structure

Fig. 3 depicts the HE staining and TEM microscopy results. In comparison to the Sham group, the myocardial fibers in the OVX group were atrophic and thinned, and the space between cardiomyocytes was enlarged. The OVX group ultrastructure demonstrated disordered myocardial fiber arrangement, blurred sarcomere structure, and obviously broken and sparse myocardial mitochondria cristae. The E and EXD group had lighter atrophy and stromal hyperplasia of myocardial cells and had clearer mitochondrial crista structures than the OVX group. The results indicated that EXD improved the local pathological changes of myocardium in OVX rats.

Effects of EXD on Gut Microbiota

The cumulative species curve revealed that the 24 samples contained nearly 30,000 intestinal microorganism species, a finding that confirms our sequencing depth was sufficient to capture the vast majority of microbial taxa present in the samples and thus ensures the reliability of subsequent diversity analyses (Fig. 4A). For the alpha diversity assessment of the microbial community, we employed four complementary indices with distinct biological implications: the Chao1 index, which estimates the total number of species in a community (including rare and potentially undetected taxa), was used to characterize microbial richness; in contrast, the Shannon index, which integrates both species richness and the evenness of their relative abundance distribution, and the Simpson index, which emphasizes the dominance of the most abundant species in the community (with higher values indicating a more dominated, less diverse community structure), were utilized to evaluate microbial diversity. The results demonstrated that the chao1 and shannon indices in OVX rats exhibited an upward trend, and the simpson index demonstrated a downward trend, which EXD reversed (Fig. 4B). The Principal Coordinate Analysis (PCoA) results demonstrated that the projection distance of the gut microbiota composition in the Sham group and OVX group was far, whereas that of the EXD group was between the Sham group and OVX group, implying that EXD may exert a modulatory effect on OVX-induced gut microbial dysbiosis (Fig. 4C).

Fig. 4D and 4E, respectively, display the bar chart and heatmap of the top 15 phylum-level classifications. Firmicutes and Bacteroidota emerged as the predominant phyla. A significant decrease in Bacteroidota abundance was observed in the OVX group, whereas the EXD group exhibited a marked increase. Conversely, Firmicutes abundance increased in the OVX group but decreased in the EXD group. Additionally, the EXD group demonstrated a markedly reduced abundance of Desulfobacterota compared to the OVX group (Fig. 4F). At the genus level (Fig. 4G and 4H), in comparison to the Sham group, the OVX group had decreased Muribaculaceae and Escherichia-Shigella abundance and increased Roseburia, Oscillibacter, and Ruminococcaceae abundance. The EXD group had increased Muribaculaceae and Escherichia-Shigella abundance and decreased Roseburia, Oscillibacter, and Ruminococcaceae abundance. Furthermore, the EXD group also had increased Parabacteroides abundance and decreased [Eubacterium]_hallii_group and UCG-003 abundance (Fig. 4I). These results indicate that EXD may partially reverse or mitigate the OVX-induced alterations in gut microbiota composition in rats, aligning with the PCoA-derived observation of EXD’s modulatory effect on microbial dysbiosis.

Firmicutes contain many cholinergic bacteria, which can significantly increase the body’s TMA and TMAO levels [22, 23]. The Ruminococcaceae of Firmicutes was significantly associated with plasma TMAO [24]. In this study, the OVX rats had significantly increased Firmicutes and Ruminococcaceae related to TMAO production, and EXD reduced the increasing trend of the TMAO production-related bacteria. It was speculated that EXD has the potential to reduce TMAO levels. Furthermore, the expression and activity of flavin-containing monooxygenase 3 (FMO3) in the liver could also affect TMAO levels. Therefore, the serum levels of TMAO and its associated metabolites and FMO3 expression in the liver were examined.

Effects of EXD on TMAO-Related Metabolites

TMA enters the circulation and becomes TMAO in the liver. The TMA precursor metabolites are L-carnitine, choline, and betaine. In comparison to the Sham group (Fig. 5A), the OVX group had significantly increased serum TMAO, creatinine, and L-carnitine, and the serum TMA and choline demonstrated an increasing trend. In comparison to the OVX group, the EXD group had significantly decreased creatinine, L-carnitine, and TMAO levels.

FMO3 is a key rate-limiting enzyme involved in TMA conversion to TMAO in the liver. In comparison to the Sham group (Fig. 5B), the OVX group had significantly increased FMO3 expression. The EXD and E groups had significantly decreased FMO3 levels. The results demonstrated that EXD decreased the serum level of TMAO in the OVX rats, which might be related to the decreased level of TMAO-related precursor metabolites and FMO3 levels in the liver.

Correlation Analysis of TMAO and Blood Pressure, Serum Lipid, and Inflammatory Factors

TMAO promotes inflammation and affects blood pressure and serum lipids. The correlation between TMAO and blood pressure, blood lipid, and inflammatory factors was analyzed (Fig. 5C). The results demonstrated that TMAO and its precursor metabolite creatinine were positively correlated with blood pressure (SBP and DBP), inflammatory factors (IL-1β and IL-18), and serum lipid (TC, TG, and LDL-C). This study focused on TMAO-mediated inflammation. As TMAO increases ROS levels in myocardial tissue to activate the NLRP3 inflammasome [25], the expression of the TMAO-mediated inflammatory pathway in the myocardium was subsequently examined.

Effects of EXD on ROS Level in the Myocardium

In comparison to the Sham group (Fig. 6), the OVX group had significantly increased positive production of ROS. In comparison to the OVX group, the EXD and E groups had significantly decreased ROS positive production. These results indicated that EXD reduced ROS production in the myocardium.

Effects of EXD on the TXNIP–NLRP3 Pathway in the Myocardium

In comparison to the Sham group (Fig. 7A and 7B), the OVX group had significantly increased relative TXNIP, NLRP3, ASC, and Caspase 1 expression levels. The E and EXD groups had significantly decreased relative TXNIP, NLRP3, ASC, and Caspase 1 expression levels. In this study, EXD decreased the relative expression levels of IL-1β and IL-18 in the myocardium and serum (Figs. 7A-C), which are the downstream inflammatory factors that follow NLRP3 inflammasome activation.

Discussion

Premenopausal women have lower CVD rates than men of the same age, but have higher CVD rates after menopause [26]. This trend is mainly attributed to the role of E2, which dilates the coronary artery and improves the myocardial blood and oxygen supply. Furthermore, E2 protects myocardial contractility and improves diastolic function in both humans and animals [27]. In this experiment, EXD supplemented the serum E2 level in the OVX rats and improved the cardiac systolic and diastolic functions. Moreover, EXD improved the myocardial atrophy and interstitial hyperplasia in the OVX rats and protected mitochondrial structural integrity. Thus, EXD was protective against cardiac dysfunction and damaged myocardium in OVX rats.

Gut microbiota can directly or indirectly regulate host physiological processes through alterations in their composition, functional profiles, or associated metabolites, thereby influencing the occurrence and progression of CVD [28, 29]. While OVX rats exhibit perturbations in gut microbiota structure and function, the specific mechanisms by which such microbial dysbiosis contributes to CVD pathogenesis remain unclear. In the present study, PCoA results indicated that EXD may mitigate OVX-induced gut microbial imbalance. Notably, previous studies have highlighted that an imbalance between Firmicutes and Bacteroidota is closely associated with the initiation and development of CVD, and related research reported that myocardial hypertrophy occurrence might be related to the increased Ruminococcaceae [30]. Consistent with these findings, our data showed that EXD treatment reduced the relative abundances of Firmicutes and Ruminococcaceae while increasing that of Bacteroidota, suggesting a potential regulatory role of EXD in CVD progression via modulating these key microbial taxa. Additionally, accumulating evidence has linked the abundances of Escherichia-Shigella and Muribaculaceae to anti-inflammatory effects [31]; our results demonstrated that EXD increased the relative abundances of these two taxa, which was accompanied by reduced inflammation in OVX rats. Collectively, these observations suggest that EXD may regulate the abundance of specific gut microbial taxa, thereby modulating inflammatory responses and ultimately influencing CVD development in OVX rats.

Gut microbiota-derived metabolites are increasingly recognized to participate in the development and progression of CVD, with TMAO and short-chain fatty acids being well-characterized examples [32]. The association between elevated TMAO levels and an increased risk of adverse cardiovascular events has been extensively validated in previous studies [33]. Notably, accumulating evidence indicates that Firmicutes and Ruminococcaceae are closely linked to TMAO biosynthesis; consistent with this, our data show that EXD treatment reduced the relative abundances of these microbial taxa in OVX rats, suggesting a potential mechanism by which EXD may modulate TMAO-related CVD pathways. Biologically, choline, betaine, and L-carnitine are dietary precursors that undergo microbial metabolism in the gut to produce can produce TMA, which is subsequently transported to the liver and oxidized by FMO3 to form TMAO [9]. In line with this, our results demonstrated that EXD treatment significantly reduced serum TMAO levels in OVX rats. We speculate that this reduction may be associated with EXD-mediated modulation of TMAO biosynthesis, potential decreases in the serum levels of TMAO precursor metabolites (choline and L-carnitine) and downregulated hepatic FMO3 expression, which will require further validation in future studies. Collectively, these findings suggest that OVX-induced gut microbial dysbiosis may promote TMA metabolism, thereby accelerating TMAO production, circulation, and in vivo accumulation. However, fully elucidating the precise regulatory mechanisms underlying these observations will require more experimental evidence from targeted functional validation studies in the future.

Dyslipidemia and hypertension are CVD risk factors [34]. TMAO-related metabolites are significantly associated with serum lipids, where TMAO and TG were significantly positively correlated [35]. According to the Spearman correlation analysis, TMAO showed a positive correlation with TC, TG, and LDL-C. Several animal experiments determined that increased TMAO levels could promote an increase in plasma osmolality, which ultimately leads to an increase in blood pressure [36]. A subsequent systematic review reported that high TMAO concentrations were associated with a higher prevalence of hypertension compared with low TMAO concentrations [37]. Our present study demonstrated that TMAO was positively correlated with SBP and DBP. Therefore, it was surmised that the abnormal blood lipids and blood pressure might be related to the increased TMAO in the OVX rats, where EXD reduced TMAO accumulation and improved blood lipid and blood pressure. Additionally, TMAO is positively correlated with inflammatory factors, and the TMAO-mediated inflammatory response has attracted more extensive attention, which requires further in-depth research.

NLRP3 inflammasome-mediated inflammatory response is a well-established driver of various CVD [38, 39]. Mechanistically, TMAO has been shown to increase intracellular ROS levels [25], where ROS enables TXNIP protein to separate from TRX protein, where it binds and activates the NLRP3 inflammasome, thus triggering the activation of Caspase 1 [40] and production of IL-1β and IL-18 [41]. IL-1β and IL-18 could promote atherosclerosis and its complications [42]. Consistent with this mechanistic pathway, the present study demonstrated that EXD treatment reduced intracellular ROS accumulation and TXNIP expression, while inhibiting NLRP3 inflammasome activation in the myocardium of OVX rats. Consequently, EXD downregulated the expression of IL-1β and IL-18 in both serum and myocardial tissue, suggesting that EXD exerts a protective effect on the myocardium by targeting the ROS-TXNIP-NLRP3 inflammasome pathway.

In conclusion, the present study demonstrates that EXD ameliorates myocardial damage in OVX rats by gut microbiota and inhibiting the TMAO-mediated NLRP3 inflammatory pathway. Specifically, EXD modulates the relative abundances of TMAO biosynthesis-associated microbial taxa (Firmicutes and Ruminococcaceae). EXD inhibits NLRP3 inflammasome activation, attenuates myocardial inflammatory responses, and ultimately mitigates myocardial damage in OVX rats. However, evidence supporting causality within this pathway remains correlative, as definitive functional validation experiments were not performed in the current study. Future work employing in vitro fecal microbiota assays, FMT, and pharmacological NLRP3 inhibition will be critical to confirm the direct causal link underlying EXD’s cardioprotective effects.

Supplemental Materials

Supplementary data for this paper are available on-line only at http://jmb.or.kr.