Cardiovascular diseases (CVDs) continue to be the leading cause of death worldwide, significantly impacting public health and contributing to excessive healthcare costs. The global incidence of CVDs increased from 271 million (12.1 million death) in 1990 to 523 million (18.6 million death) in 2019, nearly doubling during this period (Roth et al., 2020; Vaduganathan et al., 2022). Atherosclerosis is a major contributor to CVDs globally (Herrington et al., 2016). It is regarded as a multifactorial metabolic condition, with key risk factors comprising hypertension, elevated lipid levels, smoking, diabetes, obesity, and genetic susceptibility (Falk, 2006). The mechanisms underlying atherosclerosis involve multiple factors, such as high cholesterol, inflammation, endothelial dysfunction, immune response, smooth muscle cell dysfunction, and plaque rupture (Libby et al., 2019). In addition, recent studies have increasingly emphasized the role of intestinal microbes in the onset and progression of atherosclerosis (Björkegren and Lusis, 2022). Although pathogenesis of atherosclerosis is complex, multiple treatments are available, including statins that regulate lipid metabolism, antiplatelet drugs, medications that improve endothelial function, and procedures such as stent implantation, interventional surgery, and endarterectomy (Pu et al., 2023; Sahebkar et al., 2023; Wang X. et al., 2025; Wang C. et al., 2025). At present, the use of antiplatelet agents combined with cholesterol-lowering medications, including aspirin and atorvastatin, remains the conventional therapy for atherosclerosis (Castellano et al., 2022; Davidson et al., 2022). Typical treatments in Western medicine for atherosclerosis have shown significant effects in combating atherosclerosis and reducing lipid levels. However, adverse effects, including gastrointestinal discomfort, increased bleeding risk, and potential liver and kidney damage, were observed during treatment, which could restrict their broader clinical use (Thompson, 2016; Ji et al., 2020).

Amomum tsao-ko Crevost et Lemarie (T-K) is processed by sun-drying, stir-frying until it turns to charred golden-yellow, and dehulling. The kernel obtained through this process is then termed as Caoguo Ren in Chinese. This herb has a strong, pungent aroma and is considered warm in nature, primarily acting on the spleen and stomach meridians. Moreover, this herb exerts therapeutic effects, including drying dampness, warming the middle jiao, eliminating phlegm, and intercepting malaria. Clinically, it is used to treat epigastric distension and pain, nausea, vomiting, phlegm-associated cough, and malaria, while also alleviating alcohol toxicity and managing halitosis. Studies have reported that its extract possesses pharmacological activities, including the regulation of gastrointestinal function and possessing antibacterial, anti-inflammatory, hypoglycemic, and lipid-regulating effects. In our study, we aimed to evaluate the efficacy of oral administration of T-K in alleviating atherosclerosis in ApoE knockout (ApoE^−/−) mice. Given the strong connection between gut microbiota and liver function, we further confirmed how T-K exerts its therapeutic effects by reducing hepatic lipid deposition and inflammatory responses. Using 16S rRNA sequencing and histological techniques, we deeply explored the therapeutic mechanisms of T-K for atherosclerosis in the ApoE^−/− mice model.

Extensive research has demonstrated that lipid accumulation, oxidative stress, and inflammation responses are key pathogenic mechanisms underlying atherosclerosis (Liu N. et al., 2022; Liu H. et al., 2022). This study systematically evaluated the effects of T-K in ApoE^−/− mice with atherosclerosis. T-K significantly reduced lipid deposition in the aortic sinus and increased collagen content within plaques, suggesting improved plaque stability. It also alleviated inflammation in both the liver and aorta. By lowering serum TC, TG, and LDL-C levels and increasing HDL-C, T-K exerted hepatoprotective effects and improved colonic morphology. In addition, T-K enhanced gut microbiota diversity and reshaped microbial community structure. LEfSe analysis identified specific bacterial taxa enriched in the T-K group, potentially associated with its regulatory effects on the gut microbiota. Compared to HFHC diet-fed mice, the T-K treatment led to distinct shifts in gut microbial composition, suggesting that T-K may optimize gut microecology through targeted microbiota modulation, thereby offering new insights into atherosclerosis management. These microbial changes may also contribute to the regulation of host metabolism, immune function, and inflammatory responses. Moreover, correlation heatmap analysis revealed significant associations between specific bacterial taxa and hepatic or serum biomarkers, including SOD, GSH, ALT, MDA, HDL-C, LDL-C, and TG. Collectively, these findings support the therapeutic potential of T-K in the multifactorial management of atherosclerosis, in line with previous reports (Yoshida et al., 2018; Lavillegrand et al., 2024).

Through analysis of gut microbiota abundance and composition, the significantly different species in T-K group include the phyla Bacteroidota, Candidatus Melainabacteria, and genera Parabacteroides, Bacteroides, Christensenella, Oscillibacter, and Alistipes. Previous studies have shown that the genus Bacteroides is less abundant in individuals with coronary artery disease (CAD) compared to those with risk factors but without CAD or healthy controls. In particular, reduced abundance of Bacteroides vulgatus and Bacteroides dorei is associated with elevated fecal LPS levels in CAD patients, whereas supplementation with these strains alleviated inflammation and atherosclerotic lesion formation in mice. These findings are consistent with the gut microbiota alterations observed in our experimental mice (Yoshida et al., 2018). It is associated with parameters such as liver IL-6 level, which suggests that gut microbiota might play an indirect role in the pathological process of atherosclerosis by promoting inflammatory responses. After drug treatment in hyperlipidemic mice, levels of Candidatus Melainabacteria in microbiota markedly elevated, further confirming the crucial importance of intestinal microbiota to host health (Lu et al., 2022). Emerging evidence suggesting that a Ganoderma meroterpene derivative alleviates obesity-related atherosclerosis via enrichment of intestinal Parabacteroides. At the same time, supplementation with viable Parabacteroides in ApoE^−/− mice fed a high-fat diet can reduce the atherosclerotic plaques (Qiao et al., 2022). Evidence suggests that CAD is associated with reductions in Bacteroidetes and Alistipes populations. In particular, the abundance of Alistipes is negatively correlated with liver IL-17 and liver IL-6 levels, while positively correlated with serum HDL-C, indicating that dysbiosis of gut microbiota promotes endothelial inflammation and progression of atherosclerosis (Choroszy et al., 2022). In our study, treatment with T-K extract led to an increased abundance of Alistipes in atherosclerotic mice. Similarly, Wang et al. reported that Ginkgo biloba extract supplementation in Western diet-fed Ldlr^−/− mice elevated Alistipes, a shift associated with improved intestinal barrier function and alleviated atherosclerotic lesions (Wang Q. et al., 2022; Wang Y. et al., 2022). These consistent observations suggest that modulation of Alistipes may represent a common microbial response to botanical interventions in atherosclerosis, although its functional implications warrant further clarification (Liang et al., 2024). In our study, the relative abundance of Faecalibaculum was markedly elevated in atherosclerotic mice but significantly reduced following the treatment with T-K extract. This finding is in line with recent reports indicating that the abundance of Faecalibaculum is associated with bile acid dysregulation and activation of the intestinal FXR-FGF19 axis, thereby contributing to lipid metabolism disorders and atherosclerosis progression (Xu et al., 2023). Conversely, suppression of Faecalibaculum by T-K extract may attenuate bile acid-driven dysmetabolism, reduce pro-inflammatory signaling, and ultimately alleviate atherosclerotic lesions (Liang et al., 2024). Nevertheless, the associations of Christensenella and Oscillibacter with atherosclerosis remain incompletely characterized in current research.

Analysis of the functional prediction results using PICRUSt2 indicated that T-K may ameliorate atherosclerosis by enhancing lipid metabolism pathways, restoring the balance of PPAR and insulin signaling, and suppressing the excessive activation of apoptosis and ferroptosis, thereby exerting coordinated effects on multiple pathological processes. However, it should be noted that the KEGG functional annotation and pathway analysis in this study were primarily based on predictive results, which entails certain uncertainties, particularly as the enrichment significance at Levels 2 and 3 was limited after multiple-testing correction. Therefore, future studies should incorporate transcriptomic, proteomic, or metabolomic data for multi-omics integration to strengthen the reliability of the conclusions.

Recent studies have highlighted the significant role of the gut-liver axis in modulating systemic and hepatic inflammation. Gut microbiota dysbiosis increases intestinal permeability, allowing bacterial products such as lipopolysaccharides (LPS) to translocate into the portal circulation, which subsequently activates hepatic immune responses and promotes liver inflammation and oxidative stress (Anand and Mande, 2022). Moreover, short-chain fatty acids (SCFAs), produced by beneficial gut microbes, exert anti-inflammatory effects and contribute to intestinal barrier integrity (Verhaar et al., 2020). These findings support our observation that T-K improves liver inflammation in parallel with microbiota remodeling. Although our current study is based on correlation analysis, the consistency with previous mechanistic studies suggests a plausible link between microbial modulation and hepatic improvement. In addition, microbiota-derived metabolites such as TMAO and bile acids have been implicated in both cholesterol metabolism and vascular inflammation, indicating that the observed improvement in atherosclerosis may also be partially microbiota-mediated (Mao et al., 2024; Buchynskyi et al., 2025). Notably, the study by Zhu et al. (2020) directly demonstrates that Alisma orientale beverage reduces atherosclerotic plaque formation by modulating gut microbiota and lowering TMAO levels in ApoE^−/− mice. Together, these findings underscore the importance of gut-derived metabolites such as TMAO in linking intestinal dysbiosis to vascular pathology and suggest that targeting TMAO may be a promising strategy in future anti-atherosclerotic therapies (Liu et al., 2023).

Although this study provides new insights into the atheroprotective effects of T-K via modulation of the gut–liver axis, several limitations should be taken into consideration. The absence of a control + T-K group (healthy mice receiving T-K without disease induction) limits the evaluation of baseline effects of T-K on gut microbiota and host metabolism under physiological conditions. Although no overt toxicity was observed during the 8-week intervention, the long-term safety profile of T-K remains unclear; comprehensive toxicological assessments, including chronic toxicity and organ-specific evaluations, will be important in future studies. Histological analysis was restricted to Oil Red O staining, without cellular characterization of lesions such as smooth muscle cells, macrophages, and leukocytes. These parameters are essential for better understanding the impact of T-K on plaque composition. The relatively small number of animals included in histological evaluation may also reduce statistical power. Moreover, while PLS-DA and LEfSe analyses revealed significant alterations in gut microbiota composition, the absence of microbiota-depleted (pseudo-germ-free) controls prevents definitive conclusions about causality. These limitations highlight the need for more comprehensive experimental designs in future research to fully elucidate the therapeutic potential and underlying mechanisms of T-K.

Till date, the pharmacological research progress of T-K has been systematically summarized by Lu et al. (2022). At least 209 compounds have been isolated and identified from T-K, yet most of these components remain functionally uncharacterized and require further investigation. In the present study, the phytochemical constituents of T-K were identified and are listed in Supplementary Table 2. We have also added a brief summary of several key bioactive constituents that may contribute to the observed pharmacological effects of T-K. For instance, hyperoside (quercetin-3-O-galactoside) has been reported to inhibit LPS-induced NF-κB activation in macrophages, thereby reducing the production of TNF-α, IL-6, and nitric oxide (Kim et al., 2011). It also improves lipid profiles and alleviates hepatic steatosis in high-fat diet-induced models via activation of the PPARγ and FXR/LXRα pathways (Wang et al., 2021). Vanillin and its oxidative metabolite vanillic acid exhibit strong antioxidant and anti-inflammatory activities by scavenging reactive oxygen species and suppressing proinflammatory cytokines, in part through NF-κB inhibition (Kafali et al., 2024). Chlorogenic acid is known to modulate endothelial function, inhibit platelet aggregation, and regulate lipid metabolism, providing cardiovascular protection (Fuentes et al., 2014). In addition, quercetin, a well-studied flavonoid, has been shown to reduce atherosclerotic plaque area in ApoE^−/− mice by attenuating oxidative stress and upregulating HO-1 and eNOS expression (Shen et al., 2013). Although some of these components have been functionally validated in in vitro or animal studies, clinical evidence remains scarce. This represents an important future direction, as clinical validation will be essential for translating these findings into therapeutic applications (Zhang et al., 2022). Furthermore, the precise mechanisms through which T-K alleviates atherosclerosis remain incompletely understood. Therefore, future studies should focus on the isolation and characterization of individual bioactive compounds, in vivo functional validation, and ultimately clinical trials. Such investigations are expected to provide deeper insight into the therapeutic potential of T-K.

In summary, this study indicates that T-K may exert anti-atherosclerotic effects by regulating blood lipid levels, alleviating inflammation, and reshaping the composition of gut microbiota. However, our current research findings are primarily correlational rather than causal, and it is very important to further clarify the mechanisms. In future work, approaches such as fecal microbiota transplantation, antibiotic-induced microbiota depletion, and multi-omics integration will be essential to determine whether the gut microbiota directly mediates the therapeutic effects of T-K. In particular, metabolomics focusing on gut microbiota-derived metabolites, including SCFAs, bile acids and TMAO, will provide mechanistic insight into the microbiota-host interaction. In addition, isolation and functional characterization of individual bioactive compounds will help differentiate the contribution of phytochemical constituents and microbiota-derived mechanisms. By addressing these questions, the therapeutic potential of T-K can be fully elucidated, providing a stronger foundation for its possible use in the prevention and treatment of atherosclerosis and related metabolic disorders.