Sepsis is a severe condition that poses a significant threat to global health, with persistently high incidence and mortality rates. The excessive inflammatory response triggered by sepsis can lead to multiple organ failure (Singer et al., 2016). Notably, the lungs are among the organs most susceptible to septic injury, ultimately resulting in sepsis-induced acute lung injury(SI-ALI). Approximately 45% of sepsis patients progress to acute respiratory distress syndrome (ARDS), characterized by disruption of the pulmonary endothelial barrier and diffuse lung injury (Li N, et al., 2023). Despite decades of research on SI-ALI, outstanding breakthroughs in clinical treatment remain lacking, highlighting an urgent need to develop novel pharmaceuticals and innovative therapeutic strategies.

Recent studies have increasingly emphasized the concept of “gut-derived sepsis”, underlining the critical role of the gut in driving multi-organ dysfunction during sepsis through complex organ crosstalk mechanisms (Li D, et al., 2024).Gut dysbiosis is a pivotal contributor to sepsis progression by promoting pathogenic bacterial proliferation, triggering excessive pro-inflammatory immune responses, and reducing the production of beneficial metabolites, thereby increasing host susceptibility (Charitos et al., 2025). This dysbiosis also profoundly disrupts host bile acid metabolic homeostasis. Dysregulation of bile acid metabolism plays a crucial role in sepsis-induced lung injury. Recent reviews emphasize the central role of bile acids and their receptors, especially FXR and TGR5, in coordinating metabolic homeostasis and inflammatory responses, and highlight that modulation of the bile acid-FXR signaling axis is a promising therapeutic strategy to suppress systemic inflammation and treat metabolic disorders (Fleishman and Kumar, 2024; Li et al., 2024).Mechanistically, bile acids exert broad regulatory effects on host metabolism, immunity, and inflammation through activation of nuclear receptors such as FXR and PXR as well as the G protein–coupled receptor TGR5 (Thomas, 2017; Li and Chiang, 2024). Sepsis itself exacerbates dysbiosis and bile acid imbalance (Haak and Wiersinga, 2017), forming a vicious cycle of dysbiosis-metabolic disorder-sepsis aggravation. The cohort observations and animal experiments have further confirmed that following the onset of sepsis, the composition of the gut microbiota and the profile of bile acids undergo rapid alterations. These changes are closely associated with elevated systemic inflammatory markers and adverse clinical outcomes. This suggests a potential causal pathway in which infection may induce dysbiosis, subsequently leading to disturbances in bile acid metabolism, and ultimately exacerbating the systemic inflammatory response (Li Z. et al., 2024; Shang et al., 2025). Therapeutic strategies targeting gut microbiota and bile acid metabolism modulation urgently require further exploration. In this context, traditional Chinese medicine (TCM), with its long-standing history, has gradually demonstrated unique advantages in the clinical treatment of sepsis. TCM has been observed to induce corresponding changes in gut microbiota while ameliorating organ damage caused by sepsis (Fan et al., 2023), exerting its effects through multiple mechanisms, encompassing the regulation of immune responses and anti-inflammatory effects. Although the significance of gut microbiota and their metabolites in sepsis and multiple organ dysfunction has been revealed by previous studies, the mechanism by which gut microbiota−associated bile acid imbalance disrupts vascular endothelial structure in distant organs through interactions between nuclear receptors and pattern recognition receptors remains incompletely untangled. Moreover, there is a lack of systematic experimental evidence on how multi−component traditional Chinese medicine formulas can simultaneously modulate the microbiome and bile acid metabolism to alleviate SI−ALI.

Furthermore, in the context of ALI and sepsis triggered by bacterial infection and LPS stimulation, TLR4 recognizes LPS and activates downstream pro-inflammatory cascades via the adaptor MYD88, leading to IκB degradation and NF-κB translocation. This process upregulates inflammatory cytokines such as IL-1β, IL-6, and TNF-α, resulting in alveolar epithelial and vascular endothelial injury, increased permeability, and inflammatory exudation (Zhang et al., 2021; Guo et al., 2024). Concurrently, TLR4 activation also induces phosphorylation of JNK, a member of the MAPK family, which plays a critical role in LPS-induced inflammatory responses and neutrophil function. The TLR4-JNK axis has been demonstrated to determine the occurrence and extent of neutrophil NETosis upon LPS stimulation (Khan et al., 2017). The reviews and experimental evidence have extended and confirmed this mechanism. LPS activates TLR4, which subsequently triggers NADPH oxidase 2 (NOX2)-dependent oxidative stress via c-Jun N-terminal kinase (JNK) signaling(Wang H, et al., 2024; Espiritu and O’Sullivan, 2025), thereby promoting the release of neutrophil extracellular traps (NETs). In sepsis models, blocking TLR4 or inhibiting JNK significantly reduces NET formation and tissue injury (Xia et al., 2024).Studies have shown that MYD88 drives detrimental intravascular NET formation in sepsis and exacerbates disease progression; MYD88 deficiency significantly reduces lethal NET formation and improves outcomes, indicating that MYD88 is a key upstream regulator of excessive NET activation (Englert et al., 2025). Therefore, in the pathogenesis of SI-ALI, TLR4/MYD88-mediated NF-κB and JNK signaling not only amplify the pro-inflammatory cytokine storm but also promote NET generation and oxidative stress, collectively contributing to endothelial dysfunction and lung injury. Blocking this pathway effectively alleviates inflammation and lung injury, underscoring its significance as a therapeutic target.

The reviews and methodological evaluations have indicated that although existing clinical studies on TCM for sepsis have limitations in randomization and sample size, a growing body of animal model research and increasingly standardized clinical trials are demonstrating the potential effects of TCM compound formulations in improving sepsis−related organ function, alleviating inflammation, and restoring the intestinal barrier. This suggests that this field is entering a phase of more rigorous clinical validation (Fu et al., 2024; Ji et al., 2025).Preclinical studies suggest that the therapeutic effect of MDD on sepsis may be achieved through synergistic regulation of pulmonary and gastrointestinal functions (Chen et al., 2016, 2018). This finding provides modern clinical evidence for the classical theory of TCM that “the lung and intestine are interiorly-exteriorly related.” It indicates that this formula can exert favorable comprehensive therapeutic effects via the approach of “simultaneous treatment of the lung and intestine,” as demonstrated in years of clinical practice at Guangdong Provincial Hospital of Chinese Medicine and Guizhou Provincial Hospital of Traditional Chinese Medicine, with its protective effect on gastrointestinal function being particularly notable (Chen et al., 2016, 2018). The composition of MDD includes Rheum palmatum L (Dahuang), Na₂SO₄·10H₂O (Mangxiao), Magnolia officinalis var. biloba (Houpo), Forsythia suspensa (Lianqiao), Scutellaria baicalensis Georgi (Huangqin), Prunus armeniaca var. armeniaca (Xingren), Bletilla striata (Baiji), and Panax notoginseng (Sanqi). The plant names have been checked with http://www.worldfloraonline.org (04/09/2025).

The major constituents of MDD have been reported to be involved in sepsis, acute lung injury, and the regulation of intestinal microbiota. Further microbiome and metabolome studies have demonstrated that baicalin alleviates intestinal inflammation by modulating the composition of the gut microbiota and enhancing intestinal barrier function, which complements its effects on inhibiting inflammatory pathways and promoting the polarization of macrophages toward an anti-inflammatory phenotype (Wan et al., 2024; Zhang et al., 2025). Emodin has been reported to alleviate LPS-induced lung injury by inhibiting the JNK/Nur77/c-Jun pathway and to ameliorate sepsis-associated lung injury both in vitro and in vivo through modulation of SIRT1 (Liu et al., 2022; Xie et al., 2022). Furthermore, honokiol, a major active component of magnolia officinalis, significantly alleviates pulmonary edema and histopathological damage, and improves survival rates in LPS- and CLP-induced acute lung injury or sepsis models by inhibiting inflammation and oxidative stress, protecting the pulmonary microvascular endothelial barrier, and suppressing NLRP3-mediated pyroptosis (Weng et al., 2011; Liu et al., 2021, 2023).Ginsenoside-related metabolites are associated with the regulation of immune homeostasis (Zhuang et al., 2021).It is interesting that Rheum palmatum L(Dahuang), Na₂SO₄·10H₂O(Mangxiao), Magnolia officinalis var. biloba(Houpo) improve ALI by regulating the expression of HIF-1α, downregulating glycolysis, and reducing inflammation (Shang et al., 2024). Besides, multiple major components in MDD have been reported to modulate the gut microbiota. Baicalin can alleviate intestinal inflammation and improve intestinal barrier function by inhibiting inflammatory pathways and remodeling the gut microbiota composition (Zhang S, et al., 2024).Emodin significantly alters the structure of the gut microbial community, reduces the Firmicutes/Bacteroidetes ratio, and promotes the production of beneficial metabolites, thereby ameliorating inflammation and intestinal permeability (Mabwi et al., 2023).Ginsenosides simultaneously increased short-chain fatty acids (SCFAs) and significantly modulated bile acids in the blood and tissues, with changes in the microbiota being correlated with alterations in bile acids (Li et al., 2025). The study demonstrated that ginsenoside Rk3 enriched butyrate-producing bacteria and elevated butyrate levels. As a key microbial metabolite, butyrate mediated the anti-inflammatory and neuroprotective effects (Zhou S, et al., 2025).Honokiol has been shown to ameliorate non-alcoholic fatty liver disease in mice, accompanied by remodeling of the ileal gut microbiota and alterations in the serum bile acid profile; notably, significant correlations between microbial taxa and bile acid levels suggest that honokiol exerts its protective effects through coordinated regulation of gut microbiota and bile acid metabolism (Zhai et al., 2023). Therefore, the multi-component combination of MDD possesses the potential to simultaneously target the gut microbiota ecosystem, bile acid metabolism, and pulmonary cell signaling pathways, which constitutes the theoretical basis for selecting MDD as an interventional agent.

This study systematically analyzed the gut microbiota characteristics and associated bile acid profiles in the control group, model group, and MDD group, aiming to investigate how gut microbiota and its mediated bile acid metabolic disorders influence systemic inflammation and SI-ALI. The results are intended to elucidate the microbiological and metabolomic basis of the TCM principle of “simultaneous treatment of lung and gut” in SI-ALI. We hypothesized that MDD alleviates SI-ALI by modulating the gut microbiota–bile acid metabolism, activating the FXR, thereby inhibiting the TLR4/MYD88 pathway, reducing NETs formation, and protecting the pulmonary vascular endothelium. These findings provide a theoretical foundation for developing novel therapeutic strategies targeting the microbiota-bile acid axis.

In this study, we employed a comprehensive approach combining 16S rRNA sequencing and targeted bile acid metabolomics to investigate the gut-lung axis correlation and the therapeutic role of anti-SI-ALI. Our mechanistic findings demonstrated that MDD exerts protective effects against LPS-induced SI-ALI through multiple pathways. Specifically, MDD downregulates NETs activation primarily via modulation of the FXR/TLR4/MYD88 signaling pathway (Figures 10, 11). Furthermore, our results show that MDD significantly decreases fecal levels of primary and secondary bile acids and modulates gut microbiota composition in SI-ALI mice, particularly by reducing the abundance of Parabacteroides and Bacteroides genus. These combined effects contribute to SI-ALI alleviation through two primary mechanisms: mitigation of intrapulmonary NETs formation and reduction of pulmonary endothelial dysfunction. Importantly, our findings establish that the therapeutic efficacy of MDD in SI-ALI is significantly associated with its dual regulatory effects on gut microbiota composition and bile acid metabolism.

In this study, MDD significantly improved the 72-hour survival rate, indicating its comprehensive protective effect against acute inflammatory outburst and its downstream complications. The increase in survival was consistent with reductions in inflammatory factors, decreased NETs, and recovery of pulmonary function, suggesting that MDD alleviates SI-ALI not through a single pathway but via multi-target synergistic mechanisms. Improvements in respiratory parameters such as tidal volume and peak inspiratory flow reflected the restoration of gas exchange and lung compliance, which likely resulted from reduced pulmonary interstitial edema and inflammatory cell infiltration, thereby lowering alveolar–capillary barrier permeability. Decreases in the W/D weight ratio and BALF protein further demonstrated the alleviation of microvascular permeability and pulmonary edema, supporting its protective effect on the microvascular barrier. Meanwhile, downregulation of TNF-α and IL-6 indicated suppression of the early inflammatory amplification loop. This anti-inflammatory effect corresponded with improvements in endothelial markers—MDD reversed the LPS-induced upregulation of VCAM-1 and downregulation of VE-cadherin, demonstrating that it not only reduces pro-inflammatory stimuli but also preserves cell–cell adhesion and endothelial integrity. To explore the upstream factors underlying these outcomes, the following section will focus on alterations in gut microbiota and bile acid metabolism, as well as evidence supporting their roles as potential mediating mechanisms. A recent study has identified that phenylpyruvate, secreted by intestinal Candida albicans, may target SIRT2 in the context of sepsis (Gu et al., 2023). Moreover, the production of hyodeoxycholic acid (HDCA) functions as an endogenous inhibitor of TLR4, potentially mitigating systemic inflammation associated with sepsis (Li J. et al., 2023). A recent study revealed that rhamnose, a bacterial carbohydrate metabolite, could enhance the phagocytic capacity of macrophages by directly binding to and activating SLC12A4 within these cells, which protect the host against sepsis (Li D. et al., 2024). The above findings overlap in their assertion of the significant role of gut microbiota and their associated metabolites in the pathogenesis of sepsis. Nonetheless, the application of fecal microbiota transplantation(FMT) in patients presents considerable challenges (Biemond et al., 2023), emphasizing the need to investigate straightforward and practical approaches for modulating gut microbiota within the framework of sepsis treatment. To address this challenge, the present study employed a TCM formulation to evaluate its effects on gut microbiota, aiming to untangle the mechanism underlying lung-gut co-therapy and develop an alternative rapid strategy for FMT.

Previous studies have demonstrated that MDD exhibits promising efficacy in clinical disease treatment (Chen et al., 2016, 2018). However, its specific clinical mechanisms remain unclear, and direct evidence for the role of traditional Chinese medicine in alleviating pulmonary and intestinal complications associated with sepsis is relatively limited. In this study, we found that MDD could improve Parabacteroides and Bacteroides on genus level. Studies have revealed that Parabacteroides exhibits a dual role. Lower abundance is showed in patients with obesity, inflammatory bowel disease (IBD), and metabolic syndrome, whereas it is increased in individuals with psoriasis, neonatal cholestasis, alopecia areata, hypertension, and polycystic ovary syndrome (PCOS) (Ezeji et al., 2021). This suggests that Parabacteroides may display both probiotic and pathogenic properties depending on virous host. Notably, it promotes the synthesis of secondary bile acids (Wang et al., 2019), which aligns with the findings of this study.

Similarly, Bacteroides, a prominent genus within the gut microbiota, functions as an opportunistic pathogen like Parabacteroides (Zafar and Saier, 2021). It is now understood that Bacteroides play a crucial role in maintaining gut health and facilitating digestion in healthy individuals. However, under compromised immune function, broad-spectrum antibiotic employment, or other adverse conditions, Bacteroides may overgrow, increasing the risk of sepsis (van der Poll et al., 2021; Mu et al., 2022). Such over-proliferation can lead to bacterial translocation and toxin release, triggering a systemic inflammatory response that may ultimately result in multi-organ dysfunction, posing a serious threat to patient survival. In this respect, a high abundance of Bacteroides has been associated with increased likelihood of achieving a Sequential Organ Failure Assessment (SOFA) score of 10 or more during hospitalization, a higher proportion of patients with ICU stays exceeding 30 days, and elevated mortality rates (Sun et al., 2023). Given that our results indicated that MDD could reduce the abundance of Bacteroides in mice, we hypothesize that the alleviation of SI-ALI by MDD may be associated with Parabacteroides and Bacteroides.

The role of bile acids in lung injury exhibits a double-edged effect. On one hand, certain bile acids demonstrate significant therapeutic potential. For instance, GCDCA significantly suppress NLRP3 inflammasome activation by activating the TGR5 receptor pathway (Zhang Z. Y. et al., 2024). On the other hand, bile acids can induce lung injury through various mechanisms, including interaction with the secretory phospholipase A2 (sPLA2) pathway, disruption of alveolar surfactant function, modulation of inflammatory responses and local immunity, as well as direct cytotoxic effects (De Luca et al., 2022).A high concentrations-bile acids exhibit cytotoxicity, Deoxycholic acid (DCA) and CDCA were found to induce robust secretion of pro-inflammatory cytokines IL-1α and IL-1β from bone marrow-derived dendritic cells (Oleszycka et al., 2023). Our findings demonstrate that MDD reduces the concentrations of both primary and secondary bile acids, particularly the pathogenic bile acid CDCA. This reduction in concentration markedly attenuates the cytotoxic effects of bile acids on cells.

Significant alterations in gut microbiota composition were observed in the SI-ALI, characterized by a marked increase in the abundance of opportunistic pathogens such as Parabacteroides and Bacteroides. These microbiota harbor abundant BSH and 7α-dehydroxylase, which accelerate the conversion of primary bile acids to secondary bile acids. Notably, at physiological concentrations, bile acids exert anti-inflammatory and metabolic regulatory roles by activating receptors such as FXR and TGR5. However, elevated bile acid levels can trigger the explosive release of pro-inflammatory cytokines IL-1β and TNF-α, directly damaging tissue barriers. This study demonstrated that increased secondary bile acids in the intestinal tract of the LPS group were positively correlated with pulmonary inflammatory infiltration and endothelial injury, suggesting that microbiota-mediated excessive bile acid accumulation may be a key driver of SI-ALI.

Notably, it was observed that the protective effect of MDD against lung injury was not limited to intestinal microenvironment regulation. Further investigations revealed that MDD significantly suppressed the formation of NETs in the lungs-a key effector mediating endothelial injury and pulmonary microcirculatory dysfunction. The NETs markers CitH3 and MPO-DNA complexes were markedly elevated in the LPS group but were substantially inhibited following MDD intervention. This change was probably associated with MDD-mediated reduction in Parabacteroides, activation of FXR expression in both lung and intestinal tissues, and suppression of the TLR4/MYD88 signaling pathway. Previous studies have demonstrated that FXR, functioning as a bile acid nuclear receptor, can attenuate excessive inflammatory responses by antagonizing the TLR4 pathway (Xiao et al., 2025). Importantly, TLR4/MYD88 activation has been identified as the core driver of NETs formation (Suprewicz et al., 2025).

NETs, released by neutrophils, are web-like structures primarily composed of DNA, histones, and granular proteins. They play a pivotal role in defending against infections by physically entrapping and chemically killing pathogens (Brinkmann et al., 2004). However, excessive formation or clearance impairment of NETs can lead to tissue damage and inflammatory responses, contributing to pathological processes such as autoimmune diseases, thrombosis, and cancer progression (Papayannopoulos, 2018). Research indicated that NETs play a vital part in developing sepsis and the associated endothelial dysfunction (Zhang et al., 2023). Although a moderate level of NETs is conducive to immune function, excessive and prolonged NETs release can amplify inflammatory processes (Doring et al., 2020; Yang et al., 2020). The primary components of NETs, including extracellular DNA, histones, neutrophil elastase (NE), and MPO, can directly damage pulmonary microvascular endothelium, increase vascular permeability, and induce cell death (Zhou et al., 2024). Concurrently, they upregulate adhesion molecules such as VCAM-1 (Folco et al., 2018; Zhang et al., 2023), recruit inflammatory cells, and provide a scaffold for platelets and coagulation factors, thereby promoting immunothrombosis (Middleton et al., 2020).This cascade leads to impaired microcirculatory perfusion and tissue hypoxia. Clinical and experimental data indicate that NETs levels are positively correlated with disease severity and prognosis in severe lung injuries such as ARDS and COVID-19 (Middleton et al., 2020; Cesta et al., 2023).Inhibition of NETs formation or acceleration of their clearance has been shown to mitigate endothelial injury and improve lung function. NETs contribute to endothelial dysfunction, further worsening sepsis-induced lung injury.

FXR, a nuclear receptor belonging to the nuclear receptor superfamily, is primarily expressed in the liver, intestine, and kidney, although studies have confirmed its expression in lung tissue as well (Li Z. et al., 2023). This process can be inhibited by the highly abundant Parabacteroides (Zhao et al., 2023), which may represent the core mechanism through which MDD modulates FXR signaling. As a critical regulatory factor in bile acid metabolism, FXR plays a significant role in inflammation and metabolic regulation. Research indicates that mice overexpressing FXR demonstrate significant resistance to endotoxemia (Hao et al., 2017), suggesting a potential role of FXR in inhibiting systemic inflammatory responses. TLR4, a pattern recognition receptor, primarily recognizes LPS, a component of the outer membrane of Gram-negative bacteria. TLR4 binds to the adaptor protein MYD88 through its Toll/IL-1 Receptor homologous domain, initiating downstream signaling pathways and inducing the expression of proinflammatory cytokines such as TNF-α and IL-6 (Kawai and Akira, 2011). Overactivation of TLR4 is closely associated with a high incidence of sepsis (Kondo et al., 2012). Studies have shown that FXR can effectively inhibit inflammatory responses by suppressing the TLR4/MYD88 signaling pathway (Xiao et al., 2025). Furthermore, previous research has confirmed that the TLR4-dependent pathway can significantly enhance neutrophil migration and promote the formation of NETs (Suprewicz et al., 2025).This suggests that the FXR/TLR4/MYD88 pathway serves as a critical axis through which MDD modulates NETs and their downstream responses.

Based on the above findings, this study proposes that MDD alleviates SI-ALI through a dual synergistic mechanism: first, by directly reshaping the gut microbiota to reduce the accumulation of pro-inflammatory and cytotoxic bile acids, thereby lowering the systemic inflammatory burden at its source; second, by indirectly restoring FXR activity, which suppresses the TLR4/MYD88 signaling pathway, reduces PAD4-mediated NETs release, and consequently protects pulmonary microvascular endothelium, decreases permeability, and improves lung function. The composition of MDD is diverse. We detected the Anthraquinones, anthrones, Flavones and fatty acids, which are closely related to sepsis and ALI. Studies have shown that the delivery of baicalin in a sepsis mouse model via nanomaterials could significantly promote the polarization of macrophages from the proinflammatory M1 phenotype to the anti-inflammatory M2 phenotype, thereby effectively inhibiting inflammatory responses and exerting a therapeutic effect (Zhou M. et al., 2025). Emodin was also found to significantly alleviate LPS-induced ALI and exert a protective effect by regulating the JNK/Nur77/c-Jun signaling pathway (Xie et al., 2022). Besides, it was reported that emodin regulate the level of severe acute pancreatitis-related exosomes and their proinflammatory components, inhibit the proinflammatory polarization of alveolar macrophages, and thereby alleviate ALI (Hu et al., 2022).MDD may exhibit its unique therapeutic advantages based on multi-component synergistic interactions by regulating the gut-lung axis function through multiple targets and intervening in the pathological process of SI-ALI.

However, this study has certain limitations that should be acknowledged. Firstly, MDD, being a multi-component traditional Chinese medicine, comprises a complex mixture of bioactive compounds. Although some components have been identified through UPLC-MS analysis, the interactions among these components and their specific contributions to the overall therapeutic effect remain incompletely understood, requiring further investigation. Secondly, this study represents a preliminary exploration primarily aimed at untangling the potential mechanisms through which MDD alleviates SI-ALI by modulating gut microbiota and bile acid metabolism. Therefore, the findings of this study need to be further validated through larger-scale clinical studies and more in-depth experimental verification to confirm its efficacy and mechanisms. It should be noted that 16S rRNA sequencing is inherently a relative quantification method, which may introduce limitations in interpreting changes in community abundance. Subsequent studies could further employ qPCR to perform targeted quantification of key genera and species to strengthen the conclusions. Besides, the MDD-M group exhibited the optimal therapeutic efficacy, while the high- and low-dose groups showed relatively weaker effects. This observation suggests that the action of MDD may not follow a strictly dose-dependent pattern. Similar phenomena are not uncommon in multi-component TCM formulations (Lin et al., 2025; Liu et al., 2025), which may be attributed to their multi-target, multi-pathway regulatory characteristics and saturation effects in vivo metabolism. On the one hand, certain components at higher doses may lead to competition among pharmacological pathways or increased metabolic burden, thereby attenuating the overall synergistic effect. On the other hand, insufficient exposure to active constituents at lower doses may also limit efficacy. From the perspective of TCM theory, a high dose of MDD may exert overly strong purgative effects, potentially impairing the body’s healthy qi (zhengqi), whereas a low dose may provide insufficient purgative action to effectively eliminate the pathogenic factors. In summary, while this study primarily focused on the therapeutic effects of MDD, it did not systematically evaluate the safety and potential side effects of high doses, which represents an important direction for future research. More detailed histopathological examinations and monitoring of biochemical indicators such as liver function in the high-dose group will help clarify its safety window.

The study demonstrates that MDD can significantly alleviate SI-ALI, as evidenced by improved histopathology, reduced inflammatory cytokines, and attenuated pulmonary edema. The remodeling of the intestinal microbiota and alterations in targeted bile acids, combined with molecular biology validation, suggest that MDD may modulate the gut microbiota–bile acid axis, activate FXR, and inhibit the TLR4/MYD88 inflammatory pathway, thereby reducing PAD4-mediated NETs formation and ultimately ameliorating endothelial dysfunction.

However, this study has several limitations. First, the interactions among the components of MDD and their respective contributions to therapeutic efficacy remain unclear, necessitating further research on the interactions between individual compounds and the complete formula. Second, more rigorous mechanistic validation is required regarding dose-response relationships, safety assessments, and the translation of findings from animal models to clinical applications. Third, the limitations of the relative quantification method using 16S rRNA sequencing suggest that key bacterial genera or species should be further validated using targeted approaches such as quantitative polymerase chain reaction (qPCR) or metagenomics.