Patients diagnosed with sepsis in the Department of Critical Care Medicine in Shanghai Ruijin Hospital from October 1, to 31 May, 2024, were prospectively enrolled. This study protocol conformed to the ethics guidelines of the Declaration of Helsinki and was approved by the ethics committee of Ruijin Hospital (No. 20210101). Informed consent was obtained from each participant.

The enrollment criteria were as follows: (1) patients aged 18–90 years, (2) meeting sepsis 3.0 definition, and (3) hospital stay >24 h. Exclusion criteria were: (1) discharge or death within 24 h after admission; (2) participation in other clinical research; (3) emergency surgery after admission; (4) malignant tumor; (5) pregnant or lactating patients; (6) lack of necessary clinical data; and (7) allergy to SFI or the ingredients of SFI. The SFI was administered to the patients according to the decision of the attending physician. Finally, 41 patients with sepsis were enrolled, including 20 patients who used SFI (50 mL continuous intravenous infusion every 6 hours for 5 days) while 21 patients who did not.

Peripheral blood was withdrawn from patients with or without SFI treatment on the fifth day of diagnosis. Plasma samples were obtained after centrifugation at 2,000 rpm for 10 min and were stored at −80°C for further analysis (Supplementary Table S1). D-lactate plasma concentration was measured by detecting absorbance at 450 nm following the manufacturer’s introductions (D-Lactate Assay Kit, Abcam, ab83429).

Shenfu injection (SFI, batch number: 221101AK01) was obtained from Huarun Sanjiu Pharmaceutical Co., Ltd. (Ya’an, Sichuan, P.R. China). Its quality was analyzed using fingerprint technology during production and was controlled according to the standards of the China Food and Drug Administration (WS3-B-3427-98-2013 and P. ZL. 205-001) (Table 1). The voucher specimens were stored at the herbarium center of Huarun Sanjiu Pharmaceutical Co., Ltd. (Panax ginseng C.A. Mey: Voucher No. A17201208001, Ratio 47%; Aconitum carmichaelii Debeaux: Voucher No. A17220921003, Ratio 17.1%).

Male C57BL/6J mice (8–10 weeks, 20–25 g) were obtained from Charles River (Beijing, China) and randomly divided into experimental groups (n = 6 per group). Before induce sepsis, mice were intraperitoneally injected with pentobarbital (New Asia Pharma, Shanghai, China) for anesthesia (50 mg/kg). Sepsis was induced by surgically performing cecal ligation and puncture (CLP). Briefly, after anesthesia, the abdominal wall was incised for 2–3 cm. The cecum was carefully exposed and ligated with a 5–0 silk thread 15 mm from the apex and passed through the distal part of the cecum using a 20-gauge needle. After removing a small amount of faecal content, the cecum was reset into the abdominal cavity and the exposed abdominal wall was closed. Mice in the sham group underwent the same procedure but without cecum ligation and puncture. All mice were rehydrated with 1 mL 0.9% saline subcutaneous injection at the end of surgery and were free to move around to obtain water and food. In the intervention groups, mice were intraperitoneally injected with a low dose of SFI (LSF) (10 mL/kg) and HSF (20 mL/kg) 1 h after CLP surgery. 48 h after CLP challenge, all mice were anesthetized with the animal anesthetic pentobarbital via intraperitoneal injection at a dose of 125 mg/kg (Pang and Laferriere, 2020). After the depth of anesthesia is ensured the mice were rapidly euthanized by spinal dislocation method to collect tissue. Fecal samples close to the anus were collected for microbiome analysis. Blood samples were collected from retro-orbital sinus for further analysis. For proliferation and migration analysis, 5-Ethynyl-2′- deoxyuridine (EdU) was i.p. injected before CLP challenge or 90 min before the mice were sacrificed. The intestines were collected, cleaned, and fixed in 4% formalin for further examination.

After deparaffinization and dehydration, intestinal sections were incubated in Ethylenediaminetetraacetic acid (EDTA) (PH 9.0) for 1 min 30 s for antigen retrieval, and then blocked with 10% normal goat serum for 1 h at room temperature. Sections were incubated with antibodies against EpCAM (ab221552, Abcam, Cambridge, United Kingdom) at 4°C overnight. Then sections were incubated with horseradish peroxidase (HRP)-conjugated Goat Anti-rabbit secondary antibody for 1 h at 37°C. After washing, sections were incubated with Fluorescein isothiocyanate (FITC) solution for 10 min at room temperature. After washing, the sections were incubated in citric acid (PH 6.0) for 5 min. After blocking with 10% normal goat serum for 20 min at room temperature, sections were incubated with OLFM4 (39141, CST, Danvers, MA, United States) at 4°C overnight. Then sections were incubated with HRP-conjugated Goat Anti-rabbit secondary antibody for 1 h at 37°C. After washing, sections were incubated with Cyanine3 (Cy3) for 10 min at room temperature. After washing, sections were stained with EdU in Click-iT EdU Reaction Buffer (Thermo Fisher Scientific). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) for 5 min in the dark at room temperature, and then stained with diaminobenzidine for microscopic examination (DS-U3; Nikon, Tokyo, Japan). The slides were mounted with mounting media for further scanning (Sigma, Missouri, United States).

After deparaffinization and dehydration, intestinal sections were incubated in EDTA (PH 9.0) for 1 min 30 s for antigen retrieval and then blocked with 10% normal goat serum for 1 h at room temperature. Sections were incubated with antibodies against ATF4 (11815, CST) and OLFM4 at 4°C overnight. The sections were then incubated with HRP-conjugated Goat Anti-rabbit secondary antibody for 1 h at 37°C. After washing, sections were incubated with Cy3 for 10 min at room temperature. Nuclei were stained with DAPI for 5 min in the dark at room temperature, and then stained with diaminobenzidine for microscopic examination (DS-U3; Nikon, Tokyo, Japan). The slides were mounted with mounting media for further scanning (Sigma, Missouri, United States).

Mouse intestinal tissues were lysed using RIPA buffer (#89901, Thermo fisher Scientific) supplemented with PhosSTOP and complete protease inhibitors (Merck, Beijing, China). Protein samples were subjected to 12.5% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (162-0177, BioRad, Shanghai, China). Blots were probed with appropriate primary antibodies against ATF4 (11815, CST), Sox9 (3579, CST), OLFM4 (39141, CST), and phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2) (9101, CST) at 4°C overnight. The blots were then incubated with the appropriate secondary antibodies (1:5000, Proteintech) and detected using HRP substrate (WBLUF0500, Millipore, United States). For internal controls of equal loading, the blots were stripped with stripping buffer (100 mmol/L 2-mercaptoethanol, 2% SDS, 62.5 mmol/L, Tris pH 6.8) and re-probed with ERK1/2 or Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) antibodies.

Paraffin blocks of intestinal tissues were cut into sections with a thickness of 5 µm. After deparaffinization and hydration, the sections were stained with hematoxylin and eosin solution. H&E images were obtained randomly from the stained sections. Pathological scores were analyzed based on histopathological criteria for intestinal damage (Chen et al., 2021).

Mouse intestinal tissue samples were fixed in 4% formalin and embedded in Paraffin. After deparaffinization and dehydration, sections were antigen retrieved and incubated with ZO-1 (ab221547, Abcam), Occludin antibodies (ab216327, Abcam), and cleaved-caspase 3 (9664, Cell Signaling Technology) at 4°C overnight, and then incubated with biotinylated secondary antibody at 37°C for 30 min. Finally, the sections were visualized using 3,3′-diaminobenzidine (DAB) solution and counterstained with hematoxylin. Images were captured using a light microscope (BX50, Olympus). IHC staining was analyzed and quantified by pathologists.

Mouse intestinal tissue samples were fixed in 4% formalin and embedded in Paraffin. After deparaffinization and dehydration, the sections were incubated with Proteinase K (20 μg/mL, A113-03, Vazyme, Jiangsu, China) for 10 min at room temperature. After washing, sections were incubated with 1xEquilibration buffer for 30 min at room temperature. Discard 1xEquilibration buffer on the sections and incubate sections with recombinant TdT enzyme (Vazyme) for 60 min at 37°C in dark. After washing, the sections were stained with DAPI solution (1:500) for 5 min at room temperature in the dark. Finally, the sections were mounted and scanned.

The plasma levels of intestinal-type fatty acid-binding protein (I-FABP) in septic patients were measured using an ELISA kit (CSB-E08024h, CUSABIO Technology, Wuhan, China). Lactate levels in mouse plasma were detected using a lactate assay kit according to the manufacturer’s instructions (L-Lactate Assay Kit, 1200011002, Eton Bioscience). The absorbance was measured at 490 nm. The levels of diamine oxidase (DAO) in mouse plasma were measured using a DAO assay kit (E-EL-M0412c, Elabscience, Shanghai, China) according to the manufacturer’s instructions.

Total RNA was extracted from intestinal tissue lysates using the TRIzol reagent (R401, Vazyme, Jiangsu, China) following the manufacturer’s protocol. The RNA concentration and purity were measured using a Gen5 Microplate Spectrophotometer (BioTek, VT, United States). Reverse transcription (RT) of RNA was performed using the HiScript III RT SuperMix (R323, Vazyme). ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme) was used for quantitative polymerase chain reaction to analyze the mRNA levels.

Sequencing of the cecal microbiota was performed on an Illumina NovaSeq6000 (Illumina Inc., San Diego, CA, United States) and 250 bp paired-end reads were generated. Raw sequencing data were provided in the FASTQ format. Cutadapt software was used to remove sequences without primers and cut off the adapter. Uparse v7.0.1001 was used to obtain operational taxonomic units (OTUs) for species annotation and microbial diversity analysis. Alpha diversity was analyzed using the Chao1, Shannon, and Simpson indices. Beta diversity was displayed using unweighted UniFrac Non-metric Multidimensional Scaling (NMDS) analysis. The 16S rRNA gene sequencing and relevant data analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China).

All data are expressed as mean ± standard deviations (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparisons test. Figures were prepared using GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA, United States).

The clinical data showed that the plasma levels of D-lactate and I-FABP in septic patients with intestinal dysfunction were significantly reduced upon SFI administration (Figures 1A,B). No SFI-related adverse events had been reported in our study. To evaluate the effects of SFI on intestinal injury during sepsis, mice were challenged with CLP and treated with different doses of SFI (10 mL/kg and 20 mL/kg). The plasma levels of DAO were significantly higher in the septic group than in the control group. SFI treatment significantly decreased the levels of DAO (Figure 1C). The H&E staining showed that the intestinal mucosal structures were obviously damaged in the septic mice (Figures 1D,E). The morphology of the ileal mucosal villi and the intestinal glands was severely disintegrated. In addition, the length of the villi in the intestinal tissues was significantly reduced in septic mice (Figure 1F). Both LSF and HSF effectively improved the morphologies of the intestine according to the Chiu’s pathological scores (Figures 1D,E). The length of the villi was also significantly increased by both LSF and HSF (Figure 1F). Additionally, Upon SFI administration, CLP-induced increased levels of lactate were significantly reduced (Figure 1G). These data indicated that both LSF and HSF have beneficial effects on the intestinal injury associated with sepsis.

To investigate the effect of SFI on crypt proliferation, mice were i.p. injected with EdU before sacrifice. The results showed that the proliferation of the crypts was significantly decreased in septic mice, which was dramatically increased by both LSF and HSF (Figures 2A,B). Furthermore, to test the effect of SFI on the migration of enterocytes in the intestine, mice were i.p. injected with EdU before CLP challenge to allow EdU + cells to migrate for identical length of time along the villus. The results showed that sepsis-related decreases in the migration rate and distance of enterocytes were significantly increased by SFI (Figures 2C,D). Additionally, the number of the crypts in the intestine was significantly decreased in the septic mice and was restored by SFI administration (Figure 2E). These data indicated that SFI can promote crypt proliferation and enterocyte migration for the repair of villus damage induced by sepsis.

ATF4 has been reported to positively modulate self-renewal maintenance of intestinal stem cells (Guo et al., 2023). It has been reported that ATF4 is associated with cell survival and proliferation (Williams et al., 2020). Therefore, we analyzed the expression of ATF4 and stemness-related markers, OLFM4 and Sox9, in the intestinal tissues of septic mice. The results showed that SFI significantly upregulated the expression of ATF4 both in OLFM4-stained crypt stem cells and intestinal tissues (Figures 3A,B). Additionally, the protein levels of ATF4, OLFM4, and Sox9 in the intestinal tissues of septic mice were significantly upregulated after HSF treatment (Figures 3C,D). These data imply that SFI-induced self-renewal of crypt stem cells might be mediated by ATF4-related activation of stemness, thereby alleviating sepsis-induced intestinal injury.

The barrier function of the intestine is maintained by the expression of tight junction proteins (Marincola Smith et al., 2021; Wang J. et al., 2019). Therefore, we analyzed the effect of SFI on the expressions of tight junction proteins in the intestinal tissues of septic mice. The results showed that the expressions of ZO-1 and Occludin were significantly decreased in the intestinal tissues of septic mice, while LSF and HSF administration effectively upregulated ZO-1 and Occludin protein expressions in the mucosal villi (Figures 4A–D). Additionally, TUNEL staining showed that the apoptosis of the epithelial cells in the intestinal tissues of septic mice was significantly inhibited by SFI administration (Figures 4E,F). These data reveal the protective effects of SFI on the intestinal barrier function in sepsis.

We further investigated the effects of SFI on systemic and local pro-inflammatory responses of septic mice. The plasma levels of Interleukin 1β (IL-1β), Interleukin 6 (IL-6), and tumor necrosis factor-⍺ (TNF-⍺) were significantly reduced by both LSF and HSF (Figures 5A–C). Additionally, the expression levels of pro-inflammatory cytokines in the intestinal tissues were significantly reduced by both LSF and HSF administration (Figures 5D–F). We then analyzed the effects of SFI on classical pro-inflammatory signaling pathways including mitogen-activated protein kinase (MAPK) signaling and nuclear factor kappa B (NF-κB) signaling. The results showed that the levels of phosphorylated ERK1/2 were significantly increased in the intestinal tissues of septic mice and were greatly inhibited by SFI treatment (Figures 5G,H). These data indicated that the activation of ERK1/2 signaling plays an important role in the anti-inflammatory effect of SFI on intestinal tissues during sepsis.

The gut microbiota is an important modulator of the host response to sepsis (Haak and Wiersinga, 2017). Therefore, we speculated that SFI alleviates intestinal injury by modulating the gut microbiota. 16S rRNA sequencing analysis was performed on cecum fecal samples to assess the composition of gut microbiota and the impact of SFI on the gut microbiota of septic mice. The results showed that the alpha diversity indices (Chao1, Shannon, and Simpson) of gut microbiota were reduced in septic mice compared to the control mice (Figures 6A–C). HSF restored the slight reduction in alpha diversity induced by sepsis (Figures 6A–C), suggesting that the alpha diversity of gut microbiota can be reversed by HSF treatment. We then examined the beta diversity of the gut microbiota to determine the variation in gut microbial composition among the groups. The gut microbial composition of the septic group was slightly different from that of the control group, whereas the gut microbial composition of the HSF group was more similar to that of the control group than that of the septic group (Figure 6D).

Moreover, the gut microbiota exhibited differences between the control, septic, and SFI-treated groups at the phylum, family, and genus levels (Figures 7A–C). In particular, the relative abundance of Escherichia-Shigella and Proteobacteria which was related to lipopolysaccharide (LPS) production significantly increased in the septic group compared to the control group, which was slightly reduced by HSF administration (Figures 7D,E). In addition, the relative abundance of Alloprevotella and Butyricimonas, which are associated with the production of short chain fatty acids (SCFA) and the anti-inflammatory function, was slightly decreased in the septic mice and significantly increased in the high-dose SFI-treated group (Figures 7F,G). Linear discriminant analysis Effect Size (LEfSe) (LDA score (log10) > 4) was performed to identify the critical biomarkers of gut microbiota among various groups. In the septic group, Proteobacteria, Enterobacterales, Enterobacteriaceae, Escherichia Shigella, and Gammaproteobacteria was more abundant than the others. Additionally, in the high-dose SFI-treated group, Butyricimonas and marinifilaceae were more abundant compared to the others (Figures 7H,I). Collectively, these data revealed the regulatory importance of SFI in the gut microbiota during sepsis.