Sepsis is a systemic inflammatory response syndrome (SIRS) caused by infection with a complex pathogenesis, and it is one of the main causes of death in critically ill patients (Meyer and Prescott, 2024; Klingensmith and Coopersmith, 2023). According to the international consensus (Sepsis-3 definition), sepsis is defined as a dysregulated host response to infection, accompanied by a Sequential Organ Failure Assessment (SOFA) score ≥2 (Seymour et al., 2016). The core pathological mechanism of sepsis is that after the invasion of pathogens, the body excessively releases inflammatory mediators and cytokines, triggering an imbalance between systemic inflammatory and anti-inflammatory responses, causing vascular endothelial damage, microcirculation disorders, and abnormal cell metabolism, ultimately leading to multiple organ failure (Dobson et al., 2024; Hotchkiss and Karl, 2003; Hunt, 2019; Shankar-Hari et al., 2016). In addition, patients with sepsis often have reduced intestinal microbiota diversity, an increased proportion of pathogenic bacteria (such as Escherichia coli and Fusobacterium nucleatum), and a decrease in beneficial bacteria (such as Bifidobacterium and Lactobacillus) (Haak and Wiersinga, 2017). Many studies have reported that the intestinal immunity plays an important role in the development of many diseases like systemic inflammatory response syndrome, and sepsis, and (multiple organ dysfunction) MODS (Brandt et al., 2022; Oami et al., 2024; Ge et al., 2020). Moreover, it was reported that Claudin-2 upregulation enhanced the intestinal permeability, immune activation, dysbiosis, and mortality in sepsis (Oami et al., 2024). Therefore, it is increasingly urgent to study the effects of sepsis on the intestinal immunity.

Intestinal microbiota is an important factor affecting intestinal immunity, imbalance of microbiota causes pathogen-associated molecular patterns (such as LPS) to enter the blood circulation through the damaged intestinal barrier, activate the Toll-like receptor (TLR4/2) signaling pathway, trigger excessive inflammatory response (such as TNF-α, IL - 6 release), and then cause systemic inflammatory response syndrome (SIRS) and MODS (Adelman et al., 2020; Kullberg et al., 2021). In addition, intestinal flora imbalance can lead to reduced expression of tight junction proteins and increased intestinal permeability, allowing bacteria and endotoxins to translocate to extraintestinal organs (Gai et al., 2021). For example, LPS released by Gram-negative bacteria enters the liver through the portal vein, activates Kupffer cells to release inflammatory factors, and aggravates sepsis-related liver damage and systemic inflammatory responses (Liu et al., 2023; Luan et al., 2024). Short-chain fatty acids (SCFAs, such as butyrate) produced by intestinal metabolism have anti-inflammatory effects, inhibit the NF-κB pathway and promote the differentiation of regulatory T cells. The level of SCFAs in the intestine of patients with sepsis is reduced, leading to imbalanced immune regulation and uncontrolled inflammatory response (Lou et al., 2023). And it was reported that the composition of the intestinal microbiome is affected by sepsis, and might contribute to the development of organ failure (Haak and Wiersinga, 2017). Therefore, restoring the balance of the microbiota is a key target for the treatment of sepsis.

Shenfu injection (SFI) is a traditional Chinese medicine containing extracts of red ginseng (Panax), aconite root (Radix aconiti lateralis preparata) and black monkshood (Aconitum) (Yang et al., 2014; Xu et al., 2024). It has been reported that SFI has a variety of anti-inflammatory, anti-apoptosis, anti-oxidation and regulation of innate immunity (Hong et al., 2015; Zhao et al., 2022). When administered clinically, it has been reported to inhibit excessive inflammatory responses (such as reducing TNF-α and IL - 6 levels) and alleviate the “inflammatory storm” in sepsis (Yang et al., 2014; Luo et al., 2021; Li et al., 2022; Xu et al., 2022). Traditionally, SFI is used to enhance myocardial contractility, dilate peripheral blood vessels, and increase blood pressure, and has the effects of improving circulation and resisting shock (Xu et al., 2024). It is reported that the mechanism of action of SFI includes restoring hemodynamic stability, increasing tissue oxygen partial pressure and oxygen content, and improving microcirculation and tissue metabolism. Therefore, SFI can promote shock resuscitation (Liu et al., 2015; Hua et al., 2024). SFI has also been shown to improve tissue function and hemodynamic status in heart failure and exert potent anti-endotoxin, anti-inflammatory effects and act as a potent oxygen free radical scavenger (Li et al., 2014; Zhao et al., 2025). In addition, studies have shown that SFI can alleviate the “inflammatory storm” in sepsis by inhibiting excessive inflammatory responses (such as reducing TNF-α and IL - 6 levels) (Jin et al., 2018; Wu et al., 2015; Xing et al., 2015; Yang et al., 2025). However, the effects of SFI on the gut microbiota remain largely unknown.

In this study, SFI was administered to septic mice to investigate its effects on gut microbiota alterations. A severe sepsis model was established via cecal ligation and puncture (CLP), followed by intravenous SFI administration. We systematically assessed intestinal epithelial integrity, inflammatory cytokine levels (e.g., IL - 1β, IL - 6) to evaluate SFI’s potential intestinal protective effects. High-throughput sequencing was employed to characterize gut microbiome dynamics, with particular attention to changes in dominant taxa. Our multimodal approach combining histopathological analysis, cytokine profiling, and 16S rDNA sequencing collectively contributes to a deeper understanding of SFI’s therapeutic mechanisms in sepsis management, particularly its role in microbiota-host crosstalk. This study provides valuable insights for developing microbiota-targeted adjuvant therapies for septic patients.

In this study, a model of severe sepsis was established in mice using CLP (Rittirsch et al., 2009), and the effects of drug administration on intestinal epithelial cells and inflammation to assess whether SFI can restore the balance of the microbiota. Cecal ligation and puncture resulted in elevated heart rate, body temperature, and white blood cell, indicating systemic inflammation, which was not detected in the sham surgery. It also significantly increased serum levels of IL - 1β and IL - 6, liver and kidney function markers BUN and SCr levels. This model meets the criteria of the Diagnosis and Treatment Guidelines for Sepsis: 2012 (Dellinger et al., 2013), indicating that a severe sepsis mice model was successfully established.

Severe trauma or sepsis can induce accelerated apoptosis of intestinal mucosal epithelial cells, lamina propria lymphocytes, and eosinophils, thereby destroying the integrity of the intestinal barrier, leading to abnormally increased mucosal permeability and translocation of intestinal microbiota (Hotchkiss et al., 2016; Tao et al., 2024; Wang et al., 2024a; Yan et al., 2025). In the severe sepsis model that we established, the intestinal morphology changed significantly 16 hours after CLP surgery, with a large amount of necrotic cell fragments, necrotic and detached epithelial cells, and a small amount of capillary congestion observed in the intestinal cavity. These observations may be attributed to sepsis-induced systemic blood flow redistribution, which can lead to intestinal microcirculatory hypoperfusion, followed by hypoxic injury of intestinal mucosal epithelial cells (Duess et al., 2023). This pathological process further leads to increased mucosal endothelial permeability, enhanced leukocyte-endothelial cell adhesion, and degradation of tight junction proteins, ultimately resulting in dual dysfunction of the intestinal mechanical barrier and immune barrier (King et al., 2014).

In addition, in the early stage of sepsis, the NF-κB signaling cascade, triggers the positive feedback release of pro-inflammatory factors such as IL - 1β, IL - 6, and PAF, and upregulates the secondary infiltration of neutrophils mediated by IL - 8, forming an “inflammation-coagulation vicious cycle”, which ultimately leads to intestinal barrier collapse and multiple organ dysfunction (Kurt et al., 2007; Sun et al., 2021; Wang et al., 2021). As observed in our model, injury and bleeding may accelerate the inflammatory response of intestinal epithelial cells. At 8 h after CLP surgery, the levels of IL - 1β and IL - 6 in intestinal epithelial cells in the CLP group were significantly higher than those in the control group. SFI has previously been reported to alleviate inflammatory responses by inhibiting the NF-κB pathway (Liu et al., 2019; Wang et al., 2024b). In our model, SFI improved CLP-induced inflammatory responses by reducing IL - 1β and IL - 6 levels.

In patients with sepsis, serum creatinine (SCr) and blood urea nitrogen (BUN) are important indicators of renal function damage and are closely related to the severity and prognosis of the disease. Studies have shown that the BUN level of patients with sepsis is significantly higher than that of patients without sepsis (Gao et al., 2012; Min et al., 2022). Although there is no statistical difference in SCr between the two groups, it is positively correlated with oxidative stress factors such as malondialdehyde (MDA) and nitric oxide (NO), indicating that oxidative stress may aggravate renal function damage. The systemic inflammatory response and microcirculatory disorders caused by sepsis can lead to acute kidney injury (AKI), which in turn increases SCr and BUN, reflecting decreased glomerular filtration rate and azotemia (Costa et al., 2016; Qiu et al., 2019). In addition, the increase in BUN may also be related to a high metabolic state, increased protein breakdown, and insufficient renal perfusion. In our model, SCr and BUN levels in the blood were significantly increased by 5.5-fold and 4.4-fold at 16 h after CLP surgery. SFI significantly reduced SCr and BUN levels. These results suggest that SFI can prevent renal injury by regulating SCr and BUN levels, an observation previously reported in a rat model of intestinal ischemia-reperfusion (Hua et al., 2024) and heart failure (Zhao et al., 2020) after SFI into intestinal and cardiac tissues.

The intestine has long been considered a key factor in multiple organ dysfunction syndrome (MODS) (Klingensmith and Coopersmith, 2016; Mittal and Coopersmith, 2014; Van Coillie et al., 2022). Maintaining intestinal and systemic immune homeostasis depends on a balanced microbiota, and an imbalance in the intestinal microbiota may increase an individual’s risk of sepsis (Haak and Wiersinga, 2017). Regulating the intestinal flora may become a new direction for the treatment of sepsis (Haak et al., 2018). In critically ill patients, dysbiosis is common, manifested by a decrease in the number of “beneficial” commensal bacteria (such as Firmicutes or Bacteroidetes) and an enrichment of potentially pathogenic intestinal bacteria (such as Proteobacteria) (Sun et al., 2022; Wozniak et al., 2022). The abundance of Proteobacteria may become a potential indicator for disease diagnosis (Rizzatti et al., 2017; Shin et al., 2015). In addition, the intestinal microbiota is able to prevent foreign microorganisms from colonizing the gastrointestinal tract, a phenomenon known as “colonization resistance” (Kim et al., 2017). Studies have shown that Escherichia coli, Proteobacterium, and Enterobacter may cause bacteremia in frail patients because these intestinal bacteria are more prone to translocation, especially obligate anaerobes (Girard and Ely, 2007; Hyernard et al., 2019). Our study showed that there were significant differences in the intestinal microbial composition between the sham and CLP groups. As expected, Shenfu could regulate the abundance of Muribaculaceae, Lachnospirales, Escherichia-Shigella, and other bacteria, restoring them to levels similar to those in healthy mice. Escherichia-Shigella may cause sepsis-associated inflammation due to its ability to invade and damage the human colonic epithelium (Azimirad et al., 2020). Lachnospirales are butyrate-producing bacteria, anaerobes with probiotic properties, and play specific roles in metabolic diseases, inflammatory environments, and biotransformations (Xia et al., 2023; Yang et al., 2024). Muribaculaceae was significantly reduced in abundance in colitis mice and has an important role in microbiota homeostasis (Niu et al., 2022; Zhu et al., 2024). In addition, Interleukins can also interact with the microbiota in certain circumstances. For example, gut microbiota can influence the concentration of bile acid and the level of interleukin-22 to orchestrate polycystic ovary syndrome (Qi et al., 2019), Akkermansia muciniphila can improves cognitive function in aged mice by reducing the proinflammatory cytokine IL - 6 (Zhu et al., 2023). In this study, Enterococcus were significantly positive correlated with IL - 1β, IL - 6, SCR. A previous study has reported that antimicrobial overproduction sustains intestinal inflammation by inhibiting Enterococcus colonization (Jang et al., 2023), which demonstrate the relationship between Enterococcus and inflammation. In addition, the relative abundance changes of Staphvlococcus, Ruminococcus, Odoribacten were also significantly correlated with the expression of IL - 6, indicating that IL6 might be able to regulate the diversity changes of the bacterial community. As mentioned above, Shenfu can regulate the intestinal microbiota of septic mice by increasing beneficial bacteria and reducing pathogenic bacteria. However, although this article has to some extent expounded on the role of SFI in regulating the intestinal flora, the 16-hour observation window can only capture the acute-phase response and cannot reflect the long-term results. Therefore, in subsequent research and applications, if SFI is to be used clinically, further in-depth studies on its mechanism of action are still necessary.

In conclusion, this study established a cecal ligation and puncture (CLP) model to evaluate the therapeutic effects of Shenfu Injection (SFI) in sepsis management. The experimental results demonstrated that SFI administration significantly attenuated intestinal inflammation and reduced serum levels of pro-inflammatory mediators, including IL - 1β, IL - 6, SCr, and BUN. Furthermore, microbial analysis revealed that SFI treatment effectively modulated gut microbiota composition by decreasing the relative abundance of pathogenic bacteria (particularly Escherichia-Shigella) while enhancing probiotic populations, notably Akkermansia and Lactobacillus species. These findings collectively indicate the therapeutic potential of SFI in sepsis treatment through dual mechanisms of inflammatory response mitigation and gut microbiota regulation. Notably, this investigation provides a valuable foundation for future mechanistic studies exploring SFI-mediated sepsis management via intestinal microbiome modulation, potentially informing the development of novel therapeutic strategies for critical care medicine.