Introduction

Sepsis as a life-threatening organ dysfunction is caused mainly by a dysregulated host response to infection, with a mortality rate of 17–30% and 30.9–87.9% in developing and industrialised countries, respectively1,2 .The further development of sepsis leads to multiple organ dysfunction syndrome (MODS), whereby the intestine is considered the “engine” of septic MODS and the barrier function of the intestine plays a key role in this process3.

The intestinal barrier, acting as a crucial interface between the human body and external environment, must strike a delicate balance between permeability for nutrient absorption and tightness to prevent harmful microorganisms from breaching its defenses. While serving as the body’s first line of defense, the intestine is also particularly vulnerable during sepsis4.

During sepsis and septic shock, decreased blood flow, ischemia-reperfusion injury, oxidative stress, and altered energy metabolism can compromise the intestinal barrier function, leading to the translocation of gut bacteria and subsequent systemic inflammation5. Preserving the integrity of the intestinal barrier is essential in sepsis management to avert the onset and progression of MODS and decrease patient mortality.

RSV is a polyphenolic compound naturally found in seeds or plants like Veratrum, Polygonum cuspidatum and grapes. It exhibits various biological activities, including antioxidant and anti-inflammatory properties6. Animal studies have indicated that RSV can protect against sepsis by reducing structural damage and dysfunction in vital organs such as the lungs, kidneys, and liver7,8,9.Toll-like receptor 4 (TLR4) acts as the transmembrane signal transduction receptor for LPS. When LPS binds to LPS binding protein (LBP) in serum, it forms a complex that interacts with TLR4 on the surface of intestinal epithelial cells, leading to the upregulation of downstream molecules like myeloid differentiation factor 88 (MyD88) and nuclear transcription factor κB (NF-κB). This activation results in an increase in proinflammatory cytokines such as IL-6, IL-1β, and TNF-α, which exacerbate the inflammatory response, induce cell apoptosis, and contribute to organ dysfunction10,11,12.

Studies have shown that RSV can have a positive impact on LPS-induced sepsis by influencing the TLR4/NF-κB/TNF-α signaling pathway13. We previous research has indicated that inhibiting TLR4 can reduce inflammation, apoptosis, and improve LPS-induced intestinal barrier dysfunction by regulating intestinal flora14. In order to further understand the beneficial effects and underlying mechanisms of RSV on intestinal barrier function in septic rats, a septic rat model was established using LPS. Cytokine levels, markers of intestinal injury, changes in the ultrastructure of the intestinal mucosa, and expression levels of tight junction proteins (ZO-1, Claudin-2, and Occludin) were measured. By focusing on the LPS/TLR4/MyD88/NF-κB signaling pathways, this study aims to offer new insights into the treatment of sepsis-related intestinal barrier dysfunction.

Results

RSV reduced inflammatory response and intestinal injury markers.

Related inflammatory factors expression in the intestine were assessed via ELISA to investigate the impact of intravenous RSV administration on LPS-induced inflammatory responses. The levels of IL-1β, IL-6, TNF-α and IL-10 were significantly different among the three groups by one-way ANOVA (F = 367.085, 354.999, 387.691, 99.880, respectively. All P < 0.01, η2 = 0.984, 0.983, 0.984, 0.943, respectively). Rats injected with LPS exhibited elevated proinflammatory cytokines and reduced anti-inflammatory factors compared to control group rats (P < 0.01). Conversely, rats treated with RSV showed decreased IL-1β, IL-6, and TNF-α levels and increased IL-10 levels compared to model group rats (P < 0.01), indicating the effective attenuation of pro-inflammatory cytokine production by RSV (Fig. 1A-D). The levels of I-FABP and D-LA as markers of intestinal tissue injury were significantly different among the three groups (F = 103.040, 56.891, respectively. All P < 0.01, η2 = 0.945, 0.905, respectively).With levels in the model group significantly higher than those in the control group (P < 0.01), while in the RSV intervention group were lower than those in the model group (Fig. 1E, F, P < 0.01).

Fig. 1
Fig. 1
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The effects of RSV on inflammatory responses and intestinal injury markers. (A) IL-1β, (B) IL-6, (C) TNF-α, (D) IL-10, (E) I-FABP, (F) D-LA levels in rats intestinal tissue were detected by enzyme linked immunosorbent assay. We used Gel-Pro analysis software for quantification. Data were presented as mean ± SD, n = 5 rats per group. Compared with the control group, *P < 0.01, compared with the model group, **P < 0.01.

RSV attenuated LPS-induced intestinal injury

The impact of RSV intervention was assessed by examining the pathological changes in intestinal tissue using HE staining and glycogen PAS staining(Fig. 2). In the control group, the intestinal tissue exhibited intact morphology with a rich number of intestinal glands that were regularly arranged, and no noticeable pathological structural changes or inflammatory cell infiltration were detected. Conversely, in the model group, there was focal loss of mucosal epithelial cells replaced by proliferative connective tissue (black arrow), sparse and disordered distribution of intestinal glands (blue arrow), and significant inflammatory cell infiltration in the interstitium (yellow arrow). Following RSV intervention, the intestinal tissue maintained its integrity with abundant and regularly arranged intestinal glands, and there was a reduction in inflammatory cell infiltration in the interstitium (yellow arrow) (Fig. 2A). The histological score of the intestinal tissue showed a significant different among the three groups (F = 28.5, P < 0.01, η2 = 0.826). In which the histological score showed a significant decrease with RSV treatment compared to the model group (Fig. 2B, P < 0.01). Glycogen staining PAS was utilized to examine the glycocalyx, a vital component of the ileum mucus layer, which was noticeably disrupted in the model group. Conversely, in the RSV group, the mucus was evenly distributed throughout the colon, preserving the integrity of the intestinal mucosa (Fig. 2C). Quantitative analysis revealed a significant different among the three groups (F = 8.792, P < 0.01, η2 = 0.594). In which, compared with the model group, the number of goblet cells in the RSV group increased (Fig. 2D, P < 0.01). Similar results were observed in TEM analysis, showing that microvilli maintained a normal architecture and tight junctions in the control group. Conversely, the intestinal microvilli structure in the model group showed clear abnormalities. Following treatment with RSV, the intestinal villi appeared relatively complete, well-organized, and densely arranged, with a tightly connected structure (Fig. 2E). These findings suggest that RSV can mitigate sepsis-induced intestinal injury and provide certain level of intestinal protection.

Fig. 2
Fig. 2
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The effects of RSV on pathological changes in ileum tissue. (A) Ileum tissue from different groups were stained using HE (200×). (B) Chiu’s scores. (C) PAS staining (200×). (D) Quantitative analysis of PAS staining. (E) Transmission electron microscopy images. Scale bar = 1 μm. Data were presented as mean ± SD, n = 5 rats per group. Compared with the control group, *P < 0.01, compared with the model group, **P < 0.01.

RSV increased the expression of tight junction proteins

TJ proteins play a crucial role in connecting epithelial cells to form the intestinal barrier defense, which is essential for maintaining intestinal barrier function. Using immunofluorescence techniques, the expression of key tight junction proteins was observed. The results showed that the model group had lower red-blue fluorescent intensity from Claudin-2, Occludin, and ZO-1 compared to the control group. As anticipated, RSV enhanced the red-blue fluorescence (Fig. 3A-C). Further analysis of immunofluorescence images in Claudin-2, Occludin, and ZO-1 were significantly different among the three groups (F = 211.874, 743.991, 201.279, respectively. All P < 0.01, η2 = 0.972, 0.992, 0.971, respectively). Compared with the control group, the optical density was significantly decreased in the model group, while the RSV group showed an increase compared with the model group(Fig. 3D-F, P < 0.01). These findings suggest that RSV has a protective effect in a sepsis-induced intestinal injury model by upregulating the expression of TJ proteins.

Fig. 3
Fig. 3
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The effects of RSV on intestinal tight junction proteins expression. (A) Claudin-2, (B) Occludin, (C) ZO-1 was measured by immunofluorescence (400×). (D-F) IPP6.0 software was used to analyze the optical density of immunofluorescence photos. We used Gel-Pro analyzer software to quantify. Data were presented as mean ± SD, n = 5 rats per group. Compared with the control group, *P < 0.01, compared with the model group, **P < 0.01.

RSV prevented the apoptosis of intestinal mucosal cells

The expression of apoptosis-associated proteins in the ileum tissue of the different groups was analyzed using WB and IHC. The expression of Caspase-3 and Bax in intestinal tissue were significantly different among the three groups (F = 217.990, 281.324, respectively. All P < 0.01, η2 = 0.973, 0.979, respectively). Compared with the control group, the expression of Caspase-3 and Bax in the model group was significantly increased, and in the RSV intervention group was significantly lower than that in the model group(Fig. 4A-C, P < 0.01). Consistent results were obtained from qRT-PCR data (Fig. 4D, E, F = 36.47, 52.792, respectively. All P < 0.01, η2 = 0.859, 0.898, respectively). IHC findings indicated noticeable apoptosis of intestinal mucosal cells in the model group, with partial alleviation observed in the RSV group (Fig. 4F-I, F = 16.328, 3.697, respectively. All P < 0.01, η2 = 0.731, 0.381, respectively). Hence, RSV demonstrates a specific anti-apoptotic effect primarily through the inhibition of relevant apoptotic proteins.

Fig. 4
Fig. 4
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The effect of RSV on the expression of apoptotic proteins in the ileum. (A) Western blotting was used to detect Caspase-3 and Bax expression levels. (B, C) Quantitative analysis of the protein expression levels of Caspase-3 and Bax. (D, E) RT-PCR was used to dectect Caspase-3 and Bax mRNA level. Gel-Pro analyzer software was used to quantify these levels. (F, G) IHC was used to measure apoptosis-related proteins Bax and Caspase-3 in ileum tissue (400×). (H, I) IPP6.0 software was used to analyze the relative density of Bax and Caspase-3.We used Gel-Pro analyzer software to quantify. Data were presented as mean ± SD, n = 5 rats per group. Compared with the control group,*P < 0.01, compared with model group,**P < 0.01.

RSV can inhibit the inflammatory response mediated by TLR4/MyD88/NFκB p65 protein pathways

WB and IHC were employed to assess the expression levels of TLR4, MyD88, and NF-κB p65 in the intestinal tissue of rats, aiming to further elucidate the mechanism of RSV in sepsis. The results indicated significant differences in the expression of TLR4, MyD88, and NF-κB p65 among the three groups(F = 113.323, 86.811, 16.463, respectively. All P < 0.01, η2 = 0.950, 0.935, 0.733, respectively). Among them, the expression of histone pathways was significantly increased in the model compared with the control group. Conversely, the RSV group exhibited a decrease in the expression of these proteins (Fig. 5A-D, P < 0.01). These findings were further supported by qRT-PCR data (Fig. 5E-G, F = 49.391, 20.144, 48.063, respectively. All P < 0.01, η2 = 0.892, 0.771, 0.889, respectively). IHC results showed high levels of TLR4, MyD88, and NF-κB p65 proteins in the ileum tissue of the model group, while their expression was significantly reduced in the RSV group (Fig. 5H-M, F = 24.038, 15.866, 11.642, respectively. All P < 0.01, η2 = 0.800, 0.726, 0.660, respectively). Collectively, these results suggest that RSV may protect the intestinal barrier function in septic rats by inhibiting the inflammatory response mediated by TLR4/MyD88/NF-κB signaling pathway.

Fig. 5
Fig. 5
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The effects of RSV on the expression of TLR4/MyD88/NF-κB p65 signaling pathway. (A) The protein levels of TLR4, MyD88, and NF-κB p65 in ileum tissue were evaluated through WB analysis. (B-D) Quantitative analysis of the protein expression levels of TLR4, MyD88, and NF-κB p65. (E-G) The mRNA levels of TLR4, MyD88, and NF-κB p65 were detected using RT-PCR. (H) TLR4, (I) MyD88, (J) NF-κB p65 proteins in the ileum tissue was measured by IHC (400×). (K-M) IPP6.0 software was used to analyze the relative density of TLR4, MyD88, and NF-κB p65.We used Gel-Pro analyzer software to quantify. Data were presented as mean ± SD, n = 5 rats per group. Compared with the control group,*P < 0.01, compared with model group,**P < 0.01.

Discussion

Sepsis, a common disease in the intensive care unit, is characterized by shock, a systemic inflammatory response, and MODS15. The intestinal tract is frequently impacted in critical illnesses like sepsis, often leading to multiple organ failure. Early stages of sepsis can result in damage, necrosis, and degeneration of the gastrointestinal mucosa and epithelial cells, possibly due to the vulnerability of the intestinal blood supply. Moreover, a large number of bacteria accumulate in the intestinal cavity, which form a unique balance under normal physiological conditions16,17.However, the intestinal environment is disturbed during sepsis and the balance between the bacteria is broken, and the imbalance of bacteria can aggravate the development of the disease18,19. Currently, there is no effective treatment for septic intestinal dysfunction.

The TLRs pathway plays an important role in the process of mediated inflammation. Inhibition of TLR4 signaling pathway can significantly reduce the expression of various inflammatory factors in sepsis20,21,22. Our previous research has indicated that TLR4 deficiency can reduce inflammatory responses, decrease intestinal apoptosis, enhance the expression of TJ proteins to preserve intestinal mucosal integrity, and alleviate sepsis14.Therefore, TLR4 may represent a promising therapeutic target for sepsis.

RSV demonstrates a range of protective mechanisms against sepsis, including anti-inflammatory, antiviral, antibacterial, and antifungal capabilities23,24,25. Currently, it is believed that its primary role is to reduce inflammation, enhance energy metabolism, and decrease oxidative stress, indicating promising clinical applications in the treatment of sepsis26.For instance, it has been shown to protect mice from acute lung injury induced by cecal ligation and puncture (CLP) by inhibiting the expression of the NF-κB and MAPK pathways27. Additionally, it improves sepsis-induced acute kidney injury by inhibiting the activation of the endoplasmic reticulum stress-activated inositol-requiring enzyme 1 (IRE1)/NF-κB pathway28.Through its antioxidant, anti-inflammatory, non-adrenergic, and non-cholinergic mechanisms, it enhances ileal smooth muscle reactivity in septic rats29.Furthermore, RSV can protect septic mice from multimicrobial septic shock caused by CLP by inhibiting the TLR4-mediated inflammatory response. It also safeguards the myocardial cells of septic rats by activating the PI3K/AKT/mTOR signaling pathway and inhibiting the TLR4-mediated pro-inflammatory response30,31. Therefore, the therapeutic potential of RSV in sepsis has attracted the attention of researchers. This study aimed to investigate RSV’s efficacy in sepsis treatment and its underlying mechanism. Our findings suggest that RSV can mitigate systemic inflammatory responses and enhance the integrity of the intestinal mucosal barrier by suppressing the TLR4/MyD88/NF-κB signaling pathway in the intestine, providing protective effects in septic rats. These results are consistent with the previous study conducted by Song et al.32. In their research, they found that RSV can enhance the intestinal immune barrier function in mice by increasing the mRNA and protein expression of tight junction proteins involved in intestinal barrier integrity. Additionally, RSV was shown to inhibit the TLR4/NF-ĸB signaling pathway, thereby reducing the expression of associated inflammatory factors.

The excessive inflammatory reaction and cytokine storm during sepsis is widely recognized as primary contributors to high mortality rates33. When the body is infected by pathogenic microorganisms, immune cells and epithelial/lymphatic endothelial cells at the site of infection produce a variety of cytokines. These cytokines can be categorized into pro-inflammatory factors (such as IL-1β, IL-6, and TNF-α) and anti-inflammatory factors (such as IL-10). These inflammatory mediators play a crucial role in the inflammatory response of sepsis. Prolonged inflammatory response can disrupt the body’s immune balance, ultimately leading to multiple organ failure34,35.

In the study, LPS was found to increase IL-1β, IL-6, and TNF-α levels while decreasing IL-10 in the model group, however, these expressions were reduced in the RSV rats. I-FABP is closely linked to intestinal inflammatory diseases, tissue ischemia, and injury, serving as a marker for assessing intestinal tissue damage36.The presence of blood D-LA, a bacterial metabolite from the intestine, indicates compromised intestinal barrier function and increased permeability, making it an indicator of changes in mucosal permeability37.The levels of I-FABP and D-LA were significantly elevated in the model group, confirming LPS-induced intestinal injury. Conversely, RSV intervention notably decreased I-FABP and D-LA levels. These findings suggest that RSV can mitigate the LPS-induced inflammatory response associated with sepsis and alleviate sepsis-induced intestinal injury.Similarly, loading resveratrol into silk fibroin nanoparticles showed that, through both in vivo and in vitro experiments, RSV could alleviate intestinal injury and enhance the expression of markers associated with intestinal epithelial barrier function in colitis rats, yielding results comparable to those of glucocorticoids38. Histological studies using H&E, PAS, and TEM further confirmed varying degrees of intestinal tissue damage in septic rats, including inflammatory cell infiltration, sparse distribution of intestinal glands, and disordered intestinal microvilli structure. However, RSV intervention appeared to alleviate these effects, indicating a protective role against sepsis-induced intestinal injury.

Apoptosis involves the activation, expression, and regulation of a series of genes that are essential for the renewal of intestinal mucosal cells. Excessive apoptosis can further damage the intestinal epithelial barrier function, leading to the invasion of endotoxin and bacteria, which in turn aggravates the inflammatory response39.Caspase-3 is a crucial executor in the apoptosis process, with its activation considered a biomarker of apoptosis40.The Bcl-2 protein family, consisting of over 20 proteins, can be categorized into those that resist apoptosis and those that promote cell apoptosis, with Bax being one of the proteins that promote apoptosis41.Our findings demonstrate that RSV attenuates LPS-induced apoptosis of intestinal mucosal cells by reducing the levels of apoptosis-associated proteins Bax and Caspase-3. Furthermore, qRT-PCR and IHC were utilized to evaluate apoptosis in ileum tissue, yielding consistent results.Similar studies42,43 have confirmed that RSV, as a sirtuin 1 (SIRT1) agonist, not only promotes autophagy but also protects against intestinal damage by inhibiting the expression of pro-apoptotic proteins, such as Bax and Caspase-3.

The TJ proteins, including ZO-1, Claudin-2, and Occludin, form the fundamental structure of the mechanical barrier that regulates paracellular permeability and prevents the entry of macromolecular toxins into the body44.When pathogenic microorganisms invade, damage to these proteins leads to heightened permeability of the intestinal mucosa, facilitating the passage of endotoxins and bacteria into the bloodstream, ultimately promoting sepsis45.

Our previous study demonstrated that the upregulation of tight junction proteins effectively mitigated LPS-induced intestinal injury in sepsis46.In the current study, fluorescence microscopy images revealed alterations in the TJ of rats in the model group, which were subsequently reversed by RSV treatment. We noted a reduction in ZO-1, Claudin-2, and Occludin levels in septic rats, while the opposite trend was observed in RSV-treated rats. Optical density analysis further supported these observations. Collectively, our findings suggest a beneficial impact of RSV in preserving the integrity of the intestinal mucosal barrier through the enhancement of TJ protein expression.Consistent with the findings of Chen et al.47, RSV appears to preserve the integrity of intestinal barrier function by enhancing the mRNA expression levels of ZO-1, Claudin-2, and Occludin.

LPS serves as a critical mediator in sepsis and is recognized by TLR4. Upon activation, TLR4 recruits downstream MyD88 to target the NF-κB signaling cascade, thereby mediating various inflammatory processes11.Previous studies have shown that inhibiting the TLR4/MyD88/NF-κB pathway can effectively reduce the expression of inflammatory factors in the intestinal tissues of septic rats48,49.To investigate the impact of RSV on intestinal barrier function in septic rats, we examined the expression of TLR4, MyD88, and NF-κB in the ileum tissue through WB, qRT-PCR, and IHC. Our results revealed that LPS intervention triggered the activation of the TLR4/MyD88/NF-κB pathway, leading to increased protein expression levels associated with this pathway. Conversely, treatment with RSV resulted in the downregulation of TLR4 expression, inhibition of MyD88, and NF-κB p65 activation. The consistency of these findings was confirmed through qRT-PCR and IHC analysis of protein expression levels in ileum tissues. RSV, as reported by Guan et al., has the potential to modulate the TLR4/MyD88/NF-κB signaling pathway, which is crucial for maintaining intestinal barrier integrity50. The protective effects of natural compounds such as RSV against inflammatory challenges underscore the importance of investigating their mechanisms in relation to intestinal health.

Limitations of the study

One limitation of this study is the small sample size, which may prevent the detection of certain differences. Additionally, while RSV shows potential as a treatment for intestinal damage and dysfunction of the intestinal mucosal barrier in sepsis, research on its effects in this context remains in the early stages. Currently, only in vivo experiments have been conducted, with a lack of accompanying in vitro studies. Therefore, further in vitro experiments are necessary for a more comprehensive understanding. Moreover, we selected a single dose of RSV based on previous studies, without evaluating the effects of varying doses, which could influence the results. In future research, we aim to elucidate the therapeutic effects of RSV on sepsis through the establishment of a dose-response curve.

Conclusion

TLR4-mediated inflammation is a key factor in the development of sepsis. Our research has confirmed TLR4 activation in a rat model of sepsis induced by LPS, which worsens the inflammatory response. RSV shows promise in reducing this response by blocking the activation of the TLR4/MyD88/NF-κB signaling pathway triggered by LPS. Additionally, it protects against damage to the intestinal mucosal barrier in septic rats by increasing the levels of intestinal tight junction proteins and reducing intestinal cell apoptosis.

Materials and methods

Animals

Twenty-one in all Sprague Dawley rats weighing 180–220 g (No.422023100009987) were purchased from Hubei Enbet Biotechnology Co. LTD with license number SCXK (E) 2021-0027. Rats were housed in the Laboratory Animal Center of Zhejiang Academy of Traditional Chinese Medicine at a temperature of 22℃ with a 12-hour light-dark cycle. They were provided with ad libitum access to food and water and were acclimated for one week under standard conditions before the experiment. After 7 days of feeding, the rats were injected intraperitoneally with 10 mg/kg of LPS to establish the model. RSV was administered intravenously at a dose of 8 mg/kg, 10 min prior to modeling, and then euthanized rats were sampled 24 h later.Power analyses, along with G Power software, were utilized to determine the appropriate sample size. Subsequently, the animals were randomly assigned to three groups, each consisting of five rats, with two additional rats retained from each group as spares.

Ethics declaration

All animal care and experimental procedures followed the guidelines set by the Chinese Pharmacological Society and the ARRIVE guidelines. The study was approved by the Ethics Committee of the Zhejiang Academy of Traditional Chinese Medicine, (Hangzhou, Zhejiang, China) (Approval number:2024-035).

Experiment grouping and sepsis model building

Fifteen male Sprague Dawley rats of specified pathogen-free status were randomly assigned to three equal groups (n = 5). Group 1, the Control group, received an intraperitoneal injection of 0.9% NaCl saline solution, equivalent in volume to the experimental drug. Group 2, the LPS group, was administered a single dose of 10 mg/kg LPS intraperitoneally. Group 3, the RSV + LPS group, received a tail vein injection of 8 mg/kg RSV (MedChemExpress, USA) 10 min prior to LPS injection. The LPS used was sourced from E. coli extract (Sigma Aldrich Shanghai Trading Co., Ltd.) to induce sepsis in the rat model.

Due to the very low oral bioavailability of RSV and its poor solubility in water, we utilized an organic solvent, dimethyl sulfoxide (DMSO), for its dissolution. Following the modeling process, the observed behaviors in the rats—such as mental lethargy, agitation, increased breathing and heart rate, and piloerection—indicated a successful modeling outcome.

Euthanasia and sampling

In our study, we conducted in vivo experiments while adhering to the relevant ethical standards (American Veterinary Medical Association Guidelines) for euthanasia procedures involving rats. At the conclusion of the trial period, the animals were anesthetized. Initially, we administered isoflurane at a concentration of 2.5% to the rats to mitigate their stress response during treatment. Following anesthesia, the rats were placed in a quiet and comfortable environment to further alleviate restlessness and anxiety. Subsequently, the rats were anesthetized with phenobarbital at a dose of 45 mg/kg body weight via intraperitoneal injection. After the injection, we monitored physiological indicators, including heart rate and respiration. Euthanasia was confirmed when there was a cessation of heartbeat and breathing.Finally, the ileum tissue was collected and stored at -80℃ for further experiments.

ELISA assay

The tissue was washed with pre-cooled PBS (0.01 M, pH = 7.4) to remove residual blood. After weighing, the tissue was cut into pieces and placed in a tissue grinder along with the corresponding volume of PBS (usually in a ratio of 1:9, i.e. 1 g tissue sample to 9 mL PBS), and ground in an ice bath. Because the homogenate of ileal tissue may not be as easy to separate as serum, the homogenate was centrifuged at 5000 rpm for 15 min and the supernatant collected for assay. The concentrations of IL-1β (MM-00477R2), IL-6 (MM-0190R2), IL-10 (MM-0195R2), TNF-α (MM-0180R2), D-LA (E-BC-K002-M) and I-FABP (MM-0051R2) in the supernatant were detected by ELISA. The procedure was performed in strict accordance with the corresponding instructions of the kit (Shanghai Enzyme-Linked Immunobiology Co., Ltd. Shanghai, China).

Histological analysis

The ileum was harvested 4 cm from the ileocecal junction and the tissue was fixed, dehydrated, and embedded for sectioning. HE staining (H9627, Sigma) was used to observe morphological and pathological changes in intestinal tissues under a microscope (Fi3, Nikon, Japan, 400×).The intestinal epithelial damage was scored according to the degree of intestinal damage51.Additionally, glycogen staining with Periodic Acid-Schiff (PAS) was used to examine pathological changes in ileum tissue. The ileal tissues were dehydrated using graded alcohol solutions and subsequently cleared with the transparent agent xylene. Following this, the cleared tissues were immersed in wax for embedding. The wax-embedded blocks were then sectioned using a pathological microtome. The sections were stained first with a periodate acid staining solution for 15 to 20 min, followed by Schiff staining solution (BA4114B) for 20 to 30 min, and finally with Mayer’s hematoxylin staining solution for 2 min.

Transmission electron microscope

The fresh ileum tissue was fixed in 2.5% glutaraldehyde at room temperature for 24 h. This was followed by post-fixation, dehydration, infiltration, embedding, and sectioning procedures. Subsequently, the tissue sections were double stained with uranyl acetate and lead citrate before being examined under a transmission electron microscope (HT7800, Hitachi, Japan).

Western blot

The ileum tissue blocks were placed in 2 ml EP tubes and 200 µl of lysate was added for lysis, homogenization, and centrifugation to extract proteins. Subsequently, the bicinchoninic acid (BCA) assay was utilized for quantitative protein determination. Following denaturation through boiling for 10 min, 40 µg of proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes. These membranes were then blocked with 5% skim milk for 2 h at room temperature and labeled with TLR4 (1:1000,Bs-20594R, Bioss, Beijing, China), MYD88 (1:1000, Bs-1047R, Bioss, Beijing, China), NF-кB (1:1000, Bs-20159R, Bioss, Beijing, China), Caspas-3 (1:3000, 22915-1-AP, Proteintech, Wuhan, China), Bax (1:1000, Bs-0623R, Bioss, Beijing, China), β-actin (1:5000) overnight at 4 ℃. The next day, after washing with TBST 5 times, the membrane was incubated with HRP-labeled goat anti-rabbit secondary antibody (1:10000, BA1054, Boster, Wuhan, China) or HRP-labeled goat anti-mouse secondary antibody (1:10000, BA1051, Boster, Wuhan, China) for two hours at room temperature. The ECL chemiluminescence kit (KF8003, Affinity, Changzhou, China) was used to detect the protein bands. The intensity of the bands was determined using the Gel-pro analyzer software (version: 4.0.0.4).

qRT-PCR

The ileum tissue (100 mg) was placed in 1.5 mL EP tubes and 1 mL Trizol (15596-026) was added for RNA extraction. RNA concentration was measured using the ratio of the OD260 to OD280. Subsequently, the RNA was reverse transcribed into cDNA with the Reverse Transcription Kit (R233-01, VAZYME, Nanjing, China), followed by RT-qPCR amplification using SYBR Green Master Mix (Q111-02, VAZYME, Nanjing, China) under specific conditions: Pre-degeneration at 95 ℃ for 10 min, degeneration at 95 ℃ for 15 s, annealing and extension at 60 ℃ for 60 s. The primer sequences can be found in Table 1. The fold change was calculated using the 2−ΔΔCt method. The endogenous reference gene for TLR4, Bax, Caspase-3, MyD88, and NF-кB was β-actin.

Table 1 Primer sequence.
Full size table

Immunohistochemistry analysis

The ileal tissue was initially dehydrated using a gradient of alcohol. Following this process, the tissue blocks should become transparent after alcohol dehydration. The clearing agent, xylene, can be simultaneously mixed with the dehydrating agent and paraffin. Once the xylene replaces the dehydrating agent, paraffin can effectively penetrate the tissue. The transparent tissue block is then immersed in wax and embedded in succession. It is essential that the temperature of the embedded wax is slightly higher than that of the immersed wax to ensure complete integration of the tissue block with the embedded paraffin. The embedded wax blocks were sectioned using a Leica pathology microtome. Finally, the paraffin sections were sequentially subjected to xylene, absolute ethanol, and alcohol for deparaffinization.We utilized an electric pottery oven for antigen repair, with a repair duration of 15 min. During this process, the repair solution (0.01 M EDTA buffer, pH 9.0) was sufficient to prevent the tissue from drying. Subsequently, a 3% hydrogen peroxide solution was applied to the sectioned tissue to inhibit endogenous peroxidase activity. This was followed by incubation at room temperature for 15 min, after which the tissue was washed three times with PBS for 3 min each. Following blocking, the primary antibody was applied and left to incubate overnight (15 h) at 4℃ in a humidified chamber. Subsequently, the HRP-labeled iVision™ poly-HRP goat anti-rabbit/mouse secondary antibody (DD13, Talent Biomedical, Xiamen, China) was added drop by drop and incubated at 37 °C for 30 min. Finally, a fresh DAB (G1212-200-t, Servicebio, Wuhan, China) color solution was added, followed by staining, dehydration, film sealing, imaging, and analysis.

Immunofluorescence staining

Following deparaffinization, hydration, and antigen repair, tissue sections were treated with diluted goat serum (AR1009, Boster, Wuhan, China) and blocked for 30 min at room temperature. Subsequently, the sections were incubated with primary antibodies against ZO-1 (1:100, PB9234, Boster, Wuhan, China), Claudin-2 (1:100, GTX54535, Genetex, USA), and Occludin (1:100, bs-10011R, Bioss, Beijing, China) overnight at 4 °C. The next day, the primary antibodies were removed and the sections were incubated with a fluorescently labeled secondary antibody (CY3) sheep anti-rabbit IgG (1:100, BA1032 Boster, Wuhan, China) for 1 h, followed by staining with DAPI for 5 min to visualize the nuclei. Images were then captured using a fluorescence microscope (Fi3, Nikon, Japan, 400×) for observation.

Statistical analysis

Statistical analysis was conducted using SPSS 25.0 software (IBM Company, USA). Data that followed a normal distribution are expressed as “mean ± standard deviation” (\( {\bar{\text{X}}} \)± s). An F-test was employed, and one-way analysis of variance was utilized for comparisons among groups and the least significant difference (LSD)-t test was used for further pairwise comparisons. A p-value of less than 0.05 was considered statistically significant.