Atractylodin inhibits ferroptosis in sepsis‑induced acute gastrointestinal injury via SIRT3/PRDX3

Introduction

Sepsis is characterized by life-threatening organ dysfunction resulting from an aberrant host response to infection (1). Gastrointestinal injury (GI) is prevalent among individuals with sepsis and has been linked to increased mortality rates (2). In 2017 alone, an estimated global sepsis incidence was 48.9 million cases (3). GI complications can stem from either underlying causes of sepsis, such as peritonitis originating from the abdominal cavity, or systemic pro-inflammatory responses seen in cases of sepsis and septic shock (4). A potential explanation for GI disease in sepsis could be attributed to disruptions in bowel peristalsis due to extensive use of sedatives and prolonged mechanical ventilation. The pathology of sepsis involves a complex interplay between different biological systems that results in severe dysregulation of the inflammatory network (5). Despite advances in understanding sepsis, the mechanisms underlying sepsis-induced GI remain complex and poorly understood, highlighting the need for further research (6).

Mitochondria are critical organelles for energy production via oxidative phosphorylation, a reaction conjugated with producing reactive oxygen species (ROS). Tadokoro et al (7) showed that doxorubicin inhibited mitochondrial phospholipid hydroperoxide glutathione peroxidase (GPX4) expression, resulting in mitochondrial-dependent ferroptosis. Ferroptosis is an iron-dependent cell death that differs from apoptosis and necrosis due to excessive accumulation of peroxidized polyunsaturated fatty acids, which are principally oxidized polyunsaturated fatty acids by ROS (8). Ferroptosis occurs in various cells and is vital in sepsis-induced multiple organ injury (9,10). It is particularly crucial to inhibit ferroptosis caused by oxidative damage.

In the repair process of acute GI, diverse molecular signals are involved in regulating mitochondrial malfunction. Sirtuin 3 (SIRT3), a mitochondrial deacetylase, mitigates mitochondrial oxidative damage and apoptosis by peroxiredoxin-3 (PRDX3). SIRT3 is known to regulate the acetylation status of several mitochondrial proteins, thereby influencing their activity and stability (11). PRDX3 functions as an efficient scavenger of hydrogen peroxide (H2O2) to safeguard cells against oxidative damage, especially in intestinal ischemia/reperfusion (I/R) injury (11). PRDX3 functions by oxidizing and forming its inactive dimer form to clear H2O2 and previous studies have demonstrated its effective inhibition of oxidative stress, apoptosis, and mitigation of cellular damage (11,12). Transgenic mice overexpressing PRDX3 exhibit reduced mitochondrial H2O2 production and oxidative damage compared with control mice (13).

Atractylodin is a bioactive compound derived from Atractylodes lancea (Thunb.) DC., which has been widely used to treat various gastrointestinal diseases, including dyspepsia, flatulence, nausea and diarrhea (14). Atractylodin has been reported to exert anti-inflammatory effects in various inflammatory diseases. Lipopolysaccharide-induced acute lung injury was ameliorated by atractylodin inhibiting the nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome and Toll-like receptor 4 pathways (15). Lipopolysaccharide- and D-galactosamine-induced acute liver failure was also attenuated by atractylodin via suppressing inflammation and oxidative stress (13). While investigating the effects of atractylodin on the gastrointestinal tract, a study demonstrated the ameliorative effects of atractylodin on intestinal dysmotility, constipation, and diarrhea in an experimental rat model (15). Studies have highlighted its potential therapeutic effects, particularly in the context of immune modulation and anti-inflammatory activities (16,17). Despite the obvious anti-inflammatory effects of atractylodin, few studies have been conducted on its molecular targets.

Based on the aforementioned backgrounds, it was hypothesized that atractylodin might attenuate mitochondrial dysfunction in sepsis-induced GI by activating SIRT3 to deacetylate PRDX3. Thus, the present study aimed to perform molecular biological experiments to verify the potential of atractylodin in GI. It is hoped that the present study will provide a new strategy for alleviating GI.

Materials and methods

Antibodies and reagents

Isoflurane was obtained from RWD Life Science. Anhydrous ethanol analytical reagent (AR), xylene (AR), paraffin (56–58°C), water-soluble eosin Y staining solution, Tween 20, sodium chloride (AR), trichloromethane (AR) and 3% H2O2 were purchased from Sinopharm Chemical Reagent Co., Ltd. Hematoxylin was obtained from MilliporeSigma. Dimethyl sulfoxide (DMSO), 10 mM of phosphate-buffered saline buffer (PBS), loading buffer, 4′,6-diamidino-2-phenylindole (DAPI) solution, anti-fluorescence attenuation sealant, IL-4, IL-6, IL-10, IL-1β and IL-1α, enzyme-linked immunosorbent assay (ELISA) kits and H2O2, malondialdehyde, Fe2+ and mitochondrial respiratory chain complex activity assay kits were purchased from Beijing Solarbio Science & Technology Co., Ltd. GSH/GSSG detection kit was from Nanjing Jiancheng Bioengineering Institute. Protease phosphatase inhibitor mixture, RIPA lysate (strong), BCA protein assay kit, JC-1 mitochondrial membrane potential assay kit and dihydroethidium (DHE) assay kit were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. The details of the antibodies used are listed in Table SI.

Animal treatment

Male C57 BL/6 mice aged 6–8 weeks, weighing 22–25 g, free from specific pathogens (n=90), were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. The mice were housed at 23°C and 50% relative humidity on a 12/12-h light/dark cycle. Basic feed was processed by Beijing Huanyu Zhongke Biotechnology according to the national standard (GB 14924.3-2010). The model of sepsis was induced by performing cecal ligation and perforation. Briefly, the mice were anesthetized using 4% isoflurane, followed by a sterile midline laparotomy of ~2 cm to expose the cecum. Half of the distal end of the cecum was ligated at its center, and then a 21-gauge needle was inserted between the ligature site and the end of the cecum to extrude a small amount of cecal contents. The cecum was gently reduced and the laparotomy site was sutured. In the Sham group, animals underwent laparotomy and intestinal manipulation to expose the cecum without ligation or puncture. Monitoring was performed twice a day (once in the morning and once in the evening) and recorded every 4 h for 48 h after surgery. All mice received resuscitation through subcutaneous injection of normal saline at a 24 ml/kg body weight dose. Subsequently, all mice were anesthetized with pentobarbital sodium (40 mg/kg) and sacrificed for further analysis, including collection of serum as well as stomach and colon tissue. The animal experiments in the present study was conducted in the Liyang Hospital of Chinese Medicine and approved by the Experimental Animal Ethics Committee of Liyang Hospital of Chinese Medicine (Jiangsu, China; approval no. 2024LY-02-02-03).

The solvent DMSO was employed to dissolve atractylodin and prepare a storage solution. Prior to administration, the solution was diluted to the desired concentration using saline, ensuring that the final concentration of DMSO did not exceed 0.1%. The mice were randomly allocated into five groups: Sham operation group (Sham group), the cecal ligation perforation sepsis group (Model group), and the atractylodin treatment group (low, medium, and high doses), with 12 mice in each group.

One hour before surgery, the atractylodin treatment group received intraperitoneal injections of atractylodin at a concentration of 10 mg/kg/d for the low-dose group, a concentration of 20 mg/kg/d for medium-dose group, and a high-dose concentration of 40 mg/kg/d for high dose group. Post-surgery, mice received once-daily intraperitoneal injection doses of the designated atractylodin dese for 7 days. The dosage of the drug was obtained from the previous studies (18,19).

Clinical evaluation was performed with no pulsation on palpation of the carotid artery, detection of absence of corneal reflex, and secondary confirmation. In addition, the animals that died naturally were subjected to pathological analysis to exclude experimental interference factors. The present study strictly adhered to the following criteria to determine the timing of sacrifice through daily weight monitoring, a behavioral scoring system, and veterinary assessment, ensuring that animals did not suffer avoidable pain. Sacrifice were performed when one of the following situations occurred: i) Weight loss: A rapid weight loss of 15–20%; ii) weakness and loss of mobility: Unable to stand for more than 24 h, loss of appetite, dehydration; iii) infection and wound problems: A board-like abdomen upon abdominal palpation (indicating diffuse peritonitis); bloody discharge around the anus (indicating intestinal ischemic necrosis); iv) abnormal body temperature: A deviation of 4°C from the normal body temperature for more than 24 h; v) pain and behavioral abnormalities: Obvious signs of pain (such as aggression, aimless running), neurological symptoms (convulsions, paralysis).

A total of 90 C57BL/6 mice were used in the present study, of which six died naturally (autopsy showed sepsis), and the remaining mice were sacrificed at the end of the experiment. Isolfurane 4% (oxygen flow 2 l/min) was used for induction, and the oxygen flow rate was adjusted to 1.8% during the maintenance phase. The depth of anesthesia was verified by blood gas analysis after operation. Animals were sacrificed with an overdose of sodium pentobarbital (150 mg/kilogram) by intraperitoneal injection. Clinical evaluation was performed with no pulsation on palpation of the carotid artery, detection of absence of corneal reflex, and secondary confirmation. In addition, the animals that died naturally were subjected to pathological analysis to exclude experimental interference factors.

Histological examination

The stomach and colon tissue of the mice was fixed for 12–24 h at room temperature in 4% paraformaldehydeTissue embedded in paraffin was cut into 3 µm thick sections. The tissue sections were deparaffinized with xylene (twice, each for 5 min), followed by hydration with decreasing concentrations of ethanol. The sections were stained with hematoxylin for 15 min, then rinsed with tap water. The sections were then incubated in acid-alcohol for 30 sec, immersed in tap water for 15 min, and stained with eosin for 5 min. The sections were dehydrated with a gradient of ethanol (95, 95, 100, 100%, each for 2 sec) and cleared with xylene twice (each for 1 min). Finally, the sections were cleared with xylene, mounted with neutral resin, and observed under an inverted microscope.

Western blotting

After extracting total protein from the tissue using RIPA lysis buffer (Beyotime Institute of Biotechnology; cat. no. P0013), the BCA method is used to detect protein concentration. The protein extraction of stomach and colon tissues was boiled with gel-loading buffer for 10 min at 100°C, 50 µg protein was loaded per lane, and resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. The membrane was blocked with 5% BSA in 1X PBST buffer (0.01% Tween-20) for 90 min at room temperature and was probed with GAPDH) antibody (1:5,000; Proteintech Group, Inc., 10494-1-AP) or GPX4 antibody (1:1,000; Abcam, ab125066), Mitochondrial import receptor subunit TOM20 homolog (1:1,000; Abcam, ab186735), transferrin receptor protein 1 (TFR1) antibody (1:1,000; Abcam, ab214039), PRDX3 antibody (1:1,000; Abcam, ab73349), SIRT3 antibody (1:1,000; Proteintech Group, Inc., 10099-1-AP), SLC7A11 antibody (1:1,000; Proteintech Group, Inc., 32384-1-AP), occludin antibody (1:1,000; Abcam, ab216327), and zona occludens protein 1 (ZO-1) antibody (1:1,000; Proteintech Group, Inc.; cat. no. 21773-1-AP), for 2 h at room temperature. The membrane was washed three times (10 min each in 1X PBST) and incubated with secondary anti-rabbit IgG (H+L) antibody, (horseradish peroxidase conjugate) (1:5,000; Cell Signaling Technology, Inc., #7074), for 90 min at room temperature. After being washed three times (15 min each in 1X PBST), the membrane was incubated in a 2.0 mM DAB solution prepared in PBS for 5 min. The image of the immunoblot was digitalized with the GelDoc XR+ system and saved in TIF format. The protein levels were quantified by ImageJ software (National Institutes of Health, version 1.8.0) and normalized to GAPDH as an internal control.

Co-immunoprecipitation (Co-IP)

For immunoprecipitation assays, Co-Immunoprecipitation kit (Beyotime Institute of Biotechnology; cat. no. P2175) was used following manufacturer instructions. Briefly, the tissue was collected and rinsed with PBS. Subsequently, tissue was lysed in the EBC buffer supplemented with protease inhibitors [50 mM tris (pH 7.5), 120 mM NaCl, and 0.5% NP-40]. Following ultrasonic lysis (power: 60%, ultrasonic intermittent time: 1 min, ultrasonic frequency: three times, ultrasonic exposure 10 s/time, 4°C), Protein A beads conjugated with anti-PRDX3 antibody (1:500, Abcam, cat. no. ab222807) were added to the lysate at a ratio of 20 µl bead suspension per 500 µl protein sample, followed by overnight immunoprecipitation at 4°C. The next day, the immunoprecipitate was pelleted by centrifugation at 500 × g for 3 min at 4°C. Then, the resulting product was washed in the lysis buffer. The immunoprecipitate was denatured at 100°C for 15 min with 50 µl 2X SDS protein loading buffer. The immunoprecipitate, input samples, and other lysates (10 µl) were separated by 10% SDS-PAGE and transferred to a PVDF) membrane for subsequent Western blotting. To detect acetylation of PRDX3, anti-Peroxiredoxin 3/PRDX3 antibody (Abcam; cat. no. ab222807) to precipitate PRDX3 from the samples. Subsequently, the Acetylated-lysine Antibody (Cell Signaling Technology, Inc.; cat. no. 9441) was used to assess the acetylated levels of PRDX3 through immunoimprinting.

Measurement of ROS levels

DHE, a ROS-level indicative fluorescence probe (λex=535 nm, λem=610 nm), was used to detect intracellular superoxide anions. Fresh tissues are frozen (−20°C) and then cut into 10 µm thick sections using a DAKEWE 6250 cryostat microtome. The probe of DHE was incubated with tissue slices and the intensity of red fluorescence could reflect the level of ROS under a fluorescence microscope.

TdT-mediated dUTP Nick-End Labeling (TUNEL) staining

One Step TUNEL Apoptosis Assay Kit (Beyotime Institute of Biotechnology; cat. no. C1090) was used to detect apoptotic cells. After incubating with the TUNEL regent in the dark for 1 h at 37°C, cells were stained with DAPI (10 min at room temperature). Apoptotic cells showed red fluorescence. Cells were counted in three randomly selected fields of view using an inverted fluorescence microscope (20X magnification, Olympus IX83, Olympus Corporation).

Determination of H2O2 content

H2O2 content was determined by H2O2 Content Detection Kit (Beijing Solarbio Science & Technology Co., Ltd.). The H2O2 detection reagent was melted in an ice bath. Next, the sample extraction solution or standard was added to the detection well, followed by the H2O2 detection reagent. This was gently mixed and allowed to rest at room temperature (15–30°C) for 5 min. A volume of 200 µl was aliquoted into a 96-well plate to measure the absorbance at 415 nm. Subsequently, the concentration of H2O2 in the sample was determined using a standard curve. Absorbance values were measured at 415 nm using a 96-well plate according to the manufacturer's instructions.

Detection of markers of oxidative stress

To clarify the effect of atractylodin on sepsis-induced mitochondrial oxidative damage, we detected the activity of catalase (CAT) in tissues using the Catalase (CAT) Activity Assay Kit (Solarbio, BC0205), determined the activity of superoxide dismutase 2 (SOD2) in tissues with the Superoxide Dismutase (SOD) Isoenzyme Activity Assay Kit (Solarbio, BC5255), and measured the content of glutathione/oxidized glutathione (GSH/GSSG) in tissues by means of the Total Glutathione (T-GSH)/Oxidized Glutathione (GSSG) Assay Kit (Nanjing Jiancheng Bioengineering Institute, A061-1-2), all in accordance with the instructions provided by the kit manufacturers. In addition, the contents of hydrogen peroxide (H2O2) and malondialdehyde (MDA) in tissues were detected using the Hydrogen Peroxide (H2O2) Content Assay Kit (Solarbio, BC3595) and the Malondialdehyde (MDA) Content Assay Kit (Solarbio, BC025), respectively.

Measurement of mitochondrial membrane potential

The mitochondrial membrane potential was assessed utilizing an advanced mitochondrial membrane potential assay kit incorporating JC-1 (cat. no. C2003S; Beyotime Institute of Biotechnology) according to the manufacturer's instructions. In summary, mitochondria were isolated using a tissue mitochondria isolation kit in accordance with the manufacturer's protocol. The isolated mitochondria were subsequently incubated with 0.5 ml of JC-1 fluorescent dye for 30 min at 4°C in the absence of light, followed by analysis via flow cytometry (BECKMANCOULTER, CytoFLEX) using FlowJo software (v10.6.2, FlowJo, FlowJo Enterprise).

Measurement of mitochondrial complexes activity

The activity of complex I–IV in mitochondrial was determined with the micro mitochondrial respiratory chain complex I, II, III or IV activity assay kit (Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. Briefly, stomach and colon tissues isolated from two respective mice in the same group were pooled and suspended in the mitochondrial complex extraction buffer, followed by gentle homogenization. The homogenate was centrifuged at 600 × g for 10 min at 4°C to remove cell debris and nuclei, and the supernatant was centrifuged again at 11,000 × g for 15 min at 4°C. The resultant pellet was resuspended in the extraction buffer and crushed by ultrasonication (power: 60%; intermittent time: 1 min; frequency: three times; 10 s/time, 4°C). The complex activity of mitochondrial homogenates in the respective reaction buffer was then measured spectrophotometrically at 340 nm (complex I), 605 nm (complex II), and 550 nm (complex III and IV), respectively.

Immunofluorescence

Immunofluorescence staining was performed on paraffin-embedded tissue sections. Following dewaxing and hydration, antigen retrieval was performed using citrate buffer (pH 6.0), microwave treatment for 3 min. The sections are blocked with 1% goat serum (Beijing Solarbio Science & Technology Co., Ltd.; cat. no. SL038) in PBS at room temperature for 1 h and incubated with the primary antibody overnight at 4°C (Table SI. Subsequently, the sections are washed with PBST, incubated with goat anti-rabbit Alexa Fluor 647 secondary antibody (Thermo Fisher Scientific, Inc.) at a 1:400 dilution at room temperature for 1 h, followed by DAPI (10 µg/ml) staining for 10 min at room temperature, and mounting. Finally, the sections are observed under a fluorescence microscope (20X, Olympus, Tokyo, Japan) and quantified using. ImageJ software (National Institutes of Health, version 1.8.0).

Transmission electron Microscopy (TEM)

TEM analyses were accomplished using HT7700 Hitachi Transmission Electron Microscope (Hitachi High-Technologies Corporation). TEM was used to observe the mitochondrial state of tissues. Freshly stomach and colon tissues were quickly cut into 1 mm cubes fixed overnight in 2.5% glutaraldehyde (4°C) and then post-fixed with 1% osmium tetroxide (4°C, 2 h), dehydrated through a graded ethanol series. Embedding was performed in a 1:1 resin (EMBed-812) and propylene oxide (Electron Microscopy Services) mix for 1 h at room temperature, followed by a 2:1 resin:propylene oxide mixture overnight at room temperature. Tissue were then placed in 100% resin for 3 h at room temperature. Ultrathin sections (70 nm) were collected and double-stained with uranyl acetate and lead citrate (room temperature for 15 min). Finally, the sections were visualized with a JEM1200ex Electron Microscope (JEOL).

Adeno-associated virus (AAV) vector experimental protocols

AAV serotype 9 (AAV9)-short hairpin (sh)SIRT3 and AAV9-shNC were synthesized by Jikai Gene Chemical Technology Co., Ltd. Male WT C57Bl/6J mice (8-week-old) were injected in the tail vein with 2.5×1011 viral genome particles of AAV 9. AAV9-mediated gene transfer and expression were allowed for 2 weeks before subsequent experiment.

Statistical analysis

Data are expressed as mean ± standard deviation. Comparisons among groups were tested with a one-way analysis of variance followed by the Tukey test. An unpaired t-test is used between the two groups (GraphPad Prism version 5; Dotmatics). P<0.05 was considered to indicate a statistically significant difference.

Results

In vivo safety

The present study initiated dose safety studies in mice to determine safety of atractylodin. C57BL/6 mice were treated with atractylodin (10, 20, 40 mg/kg/day) for 2 weeks to simulate long-term administration. Following common discovery-stage practices, multiple indices of liver function and renal function were examined. As shown in Fig. S1 atractylodin treatment did not markedly impair liver or renal function. The results showed that there was no significant difference in body weight, which proved the safety of the three groups of doses.

Atractylodin mitigates sepsis-induced GI

To evaluate the effects of atractylodin on acute GI, the present study created a model for sepsis-induced GI. The model of sepsis was induced by performing cecal ligation and perforation. Then, the corresponding treatments were performed in each group. The stomach and colon tissue morphology of each group were observed by H&E staining. The Sham group exhibited no evidence of intestinal mucosal injury in the pathological examination results. However, in the model group, there was an increase in epithelial space, a decrease in the number of villous epithelial cells and crypt cells, a disordered arrangement of villous epithelium, and some overflow of villous tips. Atractylodin treatment markedly attenuated sepsis-induced acute GI (Fig. 1A).

Figure 1. Atractylodin mitigates sepsis-induced GI. (A) H&E staining of mousee stomach (scale bar, 200 µm) and colon tissue. (B) The immunohistochemical detection of tissue damage in the stomach; scale bar, 100 µm. (C) Relative expression of Occludin in the stomach. (D) Relative expression of ZO-1 in the stomach. (E) The immunohistochemical detection of tissue damage in the colon, scale bar 100 µm. (F) Relative expression of occludin in the colon. (G) Relative expression of ZO-1 in the colon. The (H) stomach tissue and (J) colon tissue cell apoptosis were analyzed via TUNEL assay. Blue is nuclei stained with DAPI, while red is TUNEL-positive cells stained with TUNEL; scale bar 50 µm. The percentage of TUNEL-positive cells in (I) stomach tissues and (K) colon tissues. TUNEL-positive cells (%) were calculated by the number of positive cells divided by the total number of cells. Data are combined from n=3 biological repeats. *P<0.05, **P<0.01 vs. the model group, ##P<0.01 vs. the Sham group. GI, gastrointestinal injury; H&E, hematoxylin-eosin; TUNEL, terminal dUTP nick end labeling; ZO-1, Zona occludens protein 1.

ZO-1 and Occludin are essential intestinal epithelial tight junction proteins. The present study investigated occludin and ZO-1 expression in the stomach and colon tissue with immunohistochemistry (Fig. 1B and E). The groups treated with atractylodin in low, medium and high doses appeared to relieve the pathological injuries to different degrees compared with the model group. The results demonstrated that the levels of ZO-1 and occludin in the model group were markedly lower than those in the Sham group, indicating impaired colon barrier function. Compared with the model group, the expression levels of ZO-1 and Occludin were markedly higher in all atractylodin treated groups (Fig. 1C, D, F and G).

The present study then assessed the role of atractylodin in modulating apoptosis of stomach and colon tissues in the mice using TUNEL staining. The number of TUNEL-positive cells was increased in mice's stomach and colon tissues in the model group, whereas atractylodin blocked this elevation (P<0.01; Fig. 1H-K). these results demonstrated that atractylodin represses the apoptosis of stomach and colon tissues in mice.

To further evaluate the effects of atractylodin on sepsis-induced GI mice, the level of pro-inflammatory cytokines were determined in the serum and tissues. The cytokines of IL-1β, IL-6, TNF-α, IL-4, and IL-10 were evaluated in the blood, stomach tissue, and colon tissue using ELISA (Fig. S2). Atractylodin effectively prevented the increase in pro-inflammatory cytokines, while increasing levels of anti-inflammatory cytokines IL-4, and IL-10.

Atractylodin improves mitochondrial dysfunction

Fig. 2A and B illustrates changes in antioxidant defense system indicators such as SOD2 and CAT levels in experimental mice. In the treatment group, there was a significant increase in CAT, SOD 2, and GSH/GSSG levels when compared with the model group in stomach and colon tissues. As shown in Fig. 2A-B, atractylodin treatment markedly decreased levels of H2O2 and MDA in a dose-dependent manner. In addition, mitochondrial morphology changes and function were evaluated by TEM. TEM revealed more disorganized, swollen, and damaged mitochondria in the model group. This mitochondrial damage was mitigated in the atractylodin-treated group in stomach and colon tissues (Fig. 2C). Semiquantitative western blotting analysis showed a trend to increase in TOM20/GAPDH ratio, which suggests an increase in mitochondrial number or mitochondrial dimensions in atractylodin-treated group compared with the model group (Fig. 2D-G).

Figure 2. Effect of atractylodin on regulating mitochondrial dysfunction. (A) Regulation of mitochondrial oxidative damage markers: CAT, SOD 2, GSH/GSSG, H2O2, MDA in the stomach, and (B) colon tissue. (C) TEM images of mitochondrial morphology of each group. Western blot analysis of mitochondrial membrane TOM20 markers in (D) stomach and (E) colon tissue. Scale bar 500 nm. Relative quantitation of the intensity of TOM20 in the (F) stomach and (G) colon tissue. Values are means ± SD of n=6 (A, B, F and G) or n=3 (D and E) experiments. *P<0.05, **P<0.01 vs. the model group, ##P<0.01 vs. the Sham group. CAT, catalase; SOD 2, superoxide dismutase; GSH/GSSG, glutathione/glutathione (oxidized); MDA, malondialdehyde TEM, transmission electron microscopy; TOM20, mitochondrial import receptor subunit TOM20 homolog; MDA, malondialdehyde.

The mitochondrial function was further examined. To assess the transmembrane potential, a flow cytometric-based assay was performed to measure transmembrane potential in each group in the stomach and colon tissue. Cells of the gastric and colon tissue of mice in each group were isolated and the mitochondrial membrane potential was detected using a mitochondrial membrane potential detection kit. We found that the potential for the dissipation of mitochondrial transmembrane could be attenuated by atractylodin in each group of stomach and colon tissue (Fig. 3A-D). To examine the ROS levels, stomach and colon tissue were stained with DHE, a superoxide indicator. The intensity of DHE fluorescence was markedly decreased in all atractylodin concentration groups compared with the model group (Fig. 3E-H). Mitochondrial complexes I, II, III, IV, and V activities were also markedly enhanced in each atractylodin concentration group compared with those in the model group (Fig. 3I and J).

Figure 3. Results of stomach and colon tissue mitochondrial membrane potential and ROS. Flow cytometric diagram of cell mitochondrial membrane potential in each group in (A) stomach and (C) colon tissue. Green fluorescence represents the monomeric form of JC-1 (JC-1 monomers), indicating mitochondrial transmembrane potential dissipation. Analysis of cell mitochondrial membrane potential in each group in (B) stomach and (D) colon tissue. DHE fluorescence (red) images in the (E) stomach and (G) colon tissue; scale bar 50 µm. Statistical analysis results of mitochondrial ROS in (F) stomach and (H) colon tissue. The activity of mitochondrial respiratory chain complex I, II, III, IV, V in (I) stomach and (J) colon tissue. Values are means ± SD of n=3 (A-H) or n=6 (I and J) experiments. *P<0.05, **P<0.01 vs. the model group, ##P<0.01 vs. the Sham group. ROS, reactive oxygen species; DHE, dihydroethidium.

Atractylodin inhibits ferroptosis in mice with sepsis-induced GI

To address the role of atractylodin in ferroptosis of sepsis-induced GI, the Fe2+ levels in stomach and colon tissues were determined. The results showed that the level of Fe2+ in stomach and colon tissues of mice was markedly decreased in the atractylodin-treated group (Fig. 4A and F). Based on the aforementioned results, the essential proteins related to ferroptosis were detected in each group. Western blotting showed that the levels of GPX4 and Solute carrier family 7 member 11 (SLC7A11) in the atractylodin-treated group were markedly higher compared with those of the model group. At the same time, TFR1 was decreased in both stomach tissues (Fig. 5B-E) and colon tissues (Fig. 5G-J). these results showed that atractylodin inhibited ferroptosis in mice with sepsis-induced GI.

Figure 4. Atractylodin treatment improves the prognosis of sepsis-induced ferroptosis in the stomach and colon tissues. (A) Determination of the expression levels of Fe2+ in stomach tissues. (B) Protein expression levels of GPX4, SLC7A11, TFR1 and GAPDH in stomach tissue in each group. Quantification of relative protein expression of (C) GPX4, (D) SLC7A11 and (E) TFR1in the stomach tissue. (F) Determination of the expression levels of Fe2+ in colon tissues. (G) Protein expression levels of GPX4, SLC7A11, TFR1 and GAPDH in colon tissue in each group. (H-I) Quantifying relative protein expression of (H) GPX4, (I) SLC7A11 and (J) TFR1 in the colon tissue. Values are means ± SD of n=6 (A and F) or n=3 (B-E and G-J) experiments. *P<0.05, **P<0.01 vs. the model group, ##P<0.01 vs. the Sham group. GPX4, Phospholipid hydroperoxide glutathione peroxidase; SLC7A11, Solute carrier family 7 member 11; TFR1, transferrin receptor protein 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 5. Atractylodin suppresses the expression of Ac-PRDX3 while inducing the expression of SIRT3 in stomach and colon tissues. (A) The western blot assay of Ac-PRDX3 and PRDX3 in stomach tissue and (E) colon tissue of each group. Quantifying relative protein expression of Ac-PRDX3/PRDX3 in the (B) stomach and (F) colon tissue. The western blot assay of SIRT3 in (C) stomach and (G) colon tissue of each group. Quantification of relative protein expression of SIRT3/GAPDH in the (D) stomach and (H) colon tissue. Confocal immunofluorescence of the mice cochlear tissues in different groups. Confocal immunofluorescence of SIRT3 (green) in (I) stomach tissue and (K) colon tissue; scale bar, 50 µm. the cell nuclear parts were labeled with blue. Quantification of relative expression of SIRT3 in the (J) stomach and (L) colon tissue. Co-IP of PRDX3 with SIRT3 in (M) stomach and (N) colon tissue. (O-R) Relative quantitative evaluation of the western-blot analysis for SIRT3/PRDX3, PRDX3/GAPDH expression was obtained using ImageJ software. Values are means ± SD of n=3 (A-R) experiments. *P<0.05, **P<0.01 vs. the model group, ##P<0.01 vs. the Sham group. PRDX3, peroxiredoxin-3; SIRT3, NAD-dependent protein deacetylase sirtuin-3, mitochondrial; Co-IP, coimmunoprecipitation.

Atractylodin upregulate SIRT3 while suppress acetylation of PRDX3

It has been suggested that SIRT3-mediated deacetylation of PRDX3 could alleviate mitochondrial oxidative injury (11). The present study examined the expression of Ac-PRDX3 and SIRT3 in the stomach and colon tissues of mice. The expression of Ac-PRDX3/PRDX3 in the stomach tissues of mice in the atractylodin-treated group was lower than that in the model group. The high-dose atractylodin-treated group showed the lowest Ac-PRDX3/PRDX3 levels in stomach tissues (Fig. 5A and B). As shown in Fig. 5C and D, the SIRT3 expression markedly increased in the high-medium- and low-dose groups compared with the model group. The same trend was observed in the colon tissues (Fig. 5E-H). The expression of SIRT3 increased in the stomach and colon tissues of mice in the atractylodin-treated group compared with the model group (Fig. 5C, D, G and H). To explore how atractylodin regulates SIRT3 expression in cells, SIRT3 was colocalized with the nuclei marker DAPI using immunofluorescence in the stomach and colon tissues. As shown in Fig 5I and K, The increased SIRT3 expression with the atractylodin treatment groups was shown by immunofluorescence. The expression of SIRT3 increased with an increased dose of atractylodin in the stomach and colon tissues of mice in atractylodin treated group compared with the model group (Fig. 5J and L).

To assess whether atractylodin promotes the binding of SIRT3 to PRDX3, a co-immunoprecipitation assay was performed. The results showed that the binding of SIRT3 with PRDX3 was increased in all atractylodin treatment groups (Fig. 5M and N). The high doses of the atractylodin group showed increased SIRT3/PRDX3 and PRDX3/GAPDH in the stomach and colon tissues (Fig. 5N and O).

Atractylodin abolishes sepsis-induced mitochondrial dysfunction in GI through mediation of SIRT3/PRDX3 signaling

The downstream regulatory mechanism of the SIRT3 and PRDX3 was explored in sepsis-induced GI. To confirm the involvement of SIRT3 in the reparative effect on sepsis-induced GI, shSIRT3 was intravenously injected into the mice through the tail vein to knock down SIRT3. Fig. 6A and B showed that the protein levels of SIRT3 were markedly downregulated by 68% following injection with shSIRT3, as compared with the mice injected with shNC. Mice were divided into five groups: sham group, model group, model + atractylodin group, AAV-shSIRT3 group, and AAV-shSIRT3 + atractylodin group. The expression of GPX4 in the stomach and colon tissue was measured by eastern blotting. The results demonstrated that in the stomach and colon tissue, the GPX4 expression in the model group was markedly reduced compared with the sham group (Fig. 6C and E). However, it was obviously upregulated in the model mice treated with high-atractylodin. Compared with the model combined with atractylodin group, the GPX4 level of both the AAV-shSIRT3 and the AAV-shSIRT3 + atractylodin group was markedly decreased (Fig. 6D and F). These results suggested that SIRT3 plays a key role in ferroptosis-mediated GI. Western blotting also demonstrated that the protein expression of TOM20, ZO-1 and occludin was markedly decreased in the AAV-shSIRT3 + atractylodin treated group compared with the model combined with atractylodin group in the stomach (Fig. 6G-J) and colon tissues (Fig. 6K-N). These results showed that atractylodin inhibited mitochondrial oxidative stress by increasing SIRT3 expression. Moreover, mitochondrial complexes I, II/III, IV and V activities were also markedly attenuated in AAV-shSIRT3+ atractylodin-treated mice, as compared with model combined with atractylodin mice in the stomach (Fig. 6O-S) and colon tissues (Fig. 6T-X).

Figure 6. Regulatory mechanism of SIRT3 and PRDX3 in sepsis-induced ferroptosis-mediated mitochondrial oxidative damage. (A) Western blot analysis of SIRT3 protein level in the stomach and colon tissue. Mice were injected with sh-SIRT3 or control shRNA (shRNA-NC). (B) Western blot analysis of GPX4 protein level in stomach and (C) Western blot analysis of GPX4 protein level in colon tissue. (D) Relative SIRT3/GAPDH expression obtained using ImageJ software. Relative quantitative evaluation of the GPX4/GAPDH expression in (E) stomach and (F) colon tissue of each group. (G) Western blot analysis of TOM20, occludin, and ZO-1 protein level in stomach tissue of each group. Relative quantitative evaluation of (H) TOM20, (I) occludin and (J) ZO-1 protein expression level in stomach tissue in each group. (K) Western blot analysis of TOM20, occludin, and ZO-1 protein level in stomach and colon tissue of each group. Relative quantitative evaluation of (L) TOM20, (M) occludin and (N) ZO-1 protein expression level in colon tissue. Activity of mitochondrial respiratory chain complex (O) I, mitochondrial respiratory chain complex II (P), mitochondrial respiratory chain complex III (Q), mitochondrial respiratory chain complex IV (R), mitochondrial respiratory chain complex V (S) in the stomach tissue. (T-X) The activity of mitochondrial respiratory chain complex I (T), mitochondrial respiratory chain complex II (U), mitochondrial respiratory chain complex III (V), mitochondrial respiratory chain complex IV (W), mitochondrial respiratory chain complex V (X) in the colon tissue. Values are means ± SD of n=3 (A-N) or n=6 (O-X) experiments. **P<0.01. SIRT3, NAD-dependent protein deacetylase sirtuin-3, mitochondrial; PRDX3, peroxiredoxin-3; sh, short hairpin; GPX4, Phospholipid hydroperoxide glutathione peroxidase; TOM20, mitochondrial import receptor subunit TOM20 homolog; ZO-1, zona occludens protein 1.

Discussion

Sepsis is a clinical syndrome characterized by an aberrant inflammatory response to infection, leading to organ dysfunction. Target organ dysfunction caused by sepsis and multiple organ dysfunction syndrome are the primary causes of patient mortality, with acute GI being particularly prevalent (3). The gastrointestinal tract is the target for inflammatory mediators in sepsis and is a significant source of these mediators (20). Studies have reported varying degrees of GI in patients with sepsis (21), making it the most common complication associated with this condition. Impaired gastrointestinal function results in bacterial translocation and toxin transfer into the bloodstream, exacerbating inflammation and impairing multiple organ function. Consequently, sepsis and gastrointestinal dysfunction mutually reinforce each other, creating a vicious cycle. Furthermore, since the gastrointestinal tract is often the initial site affected by multiple organ dysfunction syndrome resulting from sepsis, actively improving gastrointestinal function of patients is significant in enhancing the success rate of sepsis treatment.

Atractylodin is classified as an acetylene compound, and modern pharmacological studies have demonstrated its anti-inflammatory and antioxidant properties (22). The present study constructed a cecum ligation perforation model to investigate how atractylodin alleviates sepsis-associated gastrointestinal damage. ZO-1 and occludin are crucial components of tight junctions, and their downregulated expression or reduced activity can impair the formation of tight junctions between cells, compromising the vital defense barrier function of the intestinal mucosa and increasing the risk of enteroborne infections caused by harmful bacteria and toxins penetrating the bloodstream (23). The present study indicated that the administration of atractylodin upregulated the expression of ZO-1 and occludin in gastrointestinal tissue, suggesting its potential to enhance gastrointestinal tissue barrier function and reduce inflammation occurrence. Histological examination using H&E and immunohistochemical staining also revealed improved intestinal damage repair and enhanced barrier function in both stomach and colon tissues among mice in the all-administration group.

The concept of ferroptosis was initially proposed as a form of iron-dependent programmed cell death, distinct from apoptosis, cell necrosis, and autophagy (24). Various cellular metabolic events including redox homeostasis, iron load, mitochondrial function and lipid metabolism regulate ferroptosis. Mitochondria play crucial regulatory roles in the process of iron death and are essential for cellular resistance against it (25). Maintaining mitochondrial integrity is an effective strategy for preventing iron death across different cell types. The present study used electron microscopy to demonstrate structural disorder, swelling and damage in the model group mice; however, treatment with atractylodin reduced mitochondrial damage while increasing the number and size of mitochondria in the administration group. Upregulation of CAT, SOD2 and GSH/GSSG proteins indicated that atractylodin could mitigate cell damage; meanwhile, downregulation of H2O2 and MDA proteins suggested an improvement in mitochondrial oxidative stress.

Mitochondria are the primary sites for ATP generation in animal and plant cells. During respiratory oxidation, asymmetric protons and other ions are distributed on both sides of the inner membrane of mitochondria, resulting in mitochondrial membrane potential (MMP) (26). Maintaining a normal MMP is essential for sustaining mitochondrial oxidative phosphorylation and ATP production, which contributes to maintaining cellular physiological functions (27). The present study employed flow cytometry and mitochondrial membrane potential detection kits to investigate the effects of atractylodin on mitochondrial transmembrane energy consumption and membrane potential in stomach and colon tissues. The results revealed that atractylodin can decrease mitochondrial transmembrane energy consumption while enhancing the membrane potential.

The mitochondrial respiratory chain is a key component of cellular energy metabolism, functioning as a continuous reaction system that consists of a series of hydrogen transfer reactions and electron transfer reactions in a specific sequence, commonly referred to as the electron transport chain (28). This respiratory chain reaction efficiently synthesizes abundant ATP molecules while simultaneously removing hydrogen atoms from metabolites to generate water (29). Assessing the activity of mitochondrial complexes can provide insights into the effectiveness of electron transport in redox processes involved in oxidative phosphorylation and cell death. The present study demonstrated significant enhancements in mitochondrial complex I, II, III, IV and V activities among mice exposed to different concentrations of atractylodin. Furthermore, elevated levels of mitochondrial ROS induce ferritin autophagy and increase intracellular iron content, ultimately leading to ferroptosis (30). Gastric and colon tissues were stained with DHE, a superoxide indicator. Compared with the model group, each concentration group treated with atractylodin exhibited markedly reduced fluorescence intensity of DHE, indicating that atractylodin effectively mitigates mitochondrial ROS levels.

The accumulation of large quantities of free Fe2+ can also induce ferroptosis (31) due to its role as a cofactor for various metabolic enzymes, such as lipid oxygenase, thereby enhancing their activity and promoting the production of lipid peroxides. Fe2+ level catalyzed by the Fenton reaction leads to the generation of peroxy and hydroxyl radicals, which further react with lipid peroxides, resulting in the substantial production of lipid ROS. Ultimately, these processes culminate in the induction of ferroptosis (31). The present study assessed the Fe2+ level and observed a significant reduction in the atractylodin-administration group. The SLC7A11-GPX4 axis is the pivotal system involved in resistance against ferroptosis (31). GPX4 is a glutathione peroxidase that utilizes glutathione to detoxify lipid peroxides and inhibit ferroptosis. TFR1 is a membrane protein widely expressed across various cell and tissue types within the human body. Western blotting analysis revealed markedly higher levels of GPX4 and SLC7A11 in the administration group treated with atractylodin compared with those in the model group. It also downregulated TFR1 expression.

PRDX is a potent thiol peroxidase family that comprises at least six subtypes in mammalian cells (32). Isoforms PRDX1, PRDX2 and PRDX6 are localized in the cytoplasm, while PRDX4 is found in the endoplasmic reticulum. PRDX5 is located in both the peroxisome and mitochondria, whereas PRDX3 is the dominant species within the mitochondria (33). As PRDX3 is the most abundant and effective enzyme for H2O2 elimination in mitochondria, it serves as an important mitochondrial antioxidant protein and acts as a target for nearly 90% of HO produced in the matrix (34). The clearance of HO by PRDX3 occurs through its oxidation to an inactive dimer form (35), with previous studies reporting its ability to effectively inhibit oxidative stress, and apoptosis (11–13,36), and decrease cell damage. Transgenic mice overexpressing PRDX3 have shown reduced production of H2O2 and decreased oxidative damage within their mitochondria compared with control mice (37). SIRT3, a highly conserved nicotinamide adenine dinucleotide (NAD)-dependent deacetylase primarily expressed in mitochondria, regulates several mitochondrial proteins involved in fatty acid oxidation, oxidative phosphorylation, and antioxidant reaction systems (38). The immediate clearance of ROS by SIRT3 is not feasible, while PRDX3 undergoes reversible acetylation (39). Therefore, it was hypothesized that SIRT3 plays a role in sepsis combined with GI through deacetylation of PRDX3. The present study revealed the downregulation of PRDX3 expression along with upregulation of SIRT3 expression in gastric tissues treated with atractylodin. To investigate how atractylodin regulates SIRT3 expression within cells, immunofluorescence was employed to co-localize SIRT3 with nuclear marker DAPI within gastric and colon tissues. Immunofluorescence analysis demonstrated increased SIRT3 expression upon treatment with atractylodin. Co-IP revealed an enhanced interaction between SIRT3 and PRDX3 in the group treated with atractylodin. Moreover, when SIRT3 was downregulated using AAV-shSIRT3, the regulatory effect of atractylodin on mitochondrial and cellular ferroptosis was attenuated. These findings suggested that SITR3 mediated the inhibitory effects of atractylodin on cell ferroptosis in sepsis-induced GI.

Research indicates that SIRT3 within mitochondria undergoes SUMOylation under physiological conditions, markedly inhibiting its deacetylase activity (40). Under conditions of metabolic stress, the desumoylation enzyme SENP1 translocates to mitochondria, restoring the deacetylation activity of SIRT3 by removing its SUMOylation modification (41). This process regulates the acetylation levels of mitochondrial proteins, thereby influencing metabolic functions (41). Notably, resveratrol, a natural activator of SIRT3, has been shown to upregulate the expression of SOD2 and catalase via the SIRT3/FoxO3a signaling axis, ROS and lipid peroxide levels, and inhibit ferroptosis by enhancing the GSH/GPX4 pathway activity. This activity effectively mitigates intestinal I/R injury (42). These findings demonstrate the regulatory mechanisms of the dynamic post-translational modifications of SIRT3 and provide a theoretical foundation for the cytoprotective effects of monomeric components of traditional Chinese medicine by targeting the downstream effector molecules of SIRT3. Based on these findings, it was hypothesized that atractylotin may enhance the deacetylation of PRDX3 by SIRT3 through the desumoylation of SIRT3, which ultimately exerts its organ-protective effect by restoring mitochondrial redox homeostasis and inhibiting ferroptosis during sepsis-induced GI. However, the precise molecular regulation mechanism remains to be elucidated by future studies.

The present study acknowledges several limitations. First, the investigation did not incorporate an analysis of the effect of interval dosing on efficacy, as comparisons between short and long treatment courses were not conducted. As a mechanistic study exploring the role of atractylodin in sepsis treatment, the primary objective was to verify its fundamental efficacy and dose-response relationship. To optimize dosing frequency and duration, it is essential to integrate pharmacokinetic data. Secondly, further research is required to elucidate the precise molecular regulatory mechanisms involved in downstream processes.

The present basic science study offers essential preclinical evidence supporting the potential therapeutic efficacy of atractylodin in the treatment of sepsis and establishes a mechanistic basis that underscores the need for further translational research. Prior to initiating clinical evaluation, it is imperative to conduct comprehensive preclinical safety and toxicology studies in relevant animal models to evaluate potential systemic toxicity, organ-specific adverse effects and establish safe dosage ranges for initial human trials. Subsequent rigorous investigations into pharmacokinetics and pharmacodynamics (PK/PD) are necessary to fully elucidate the absorption, distribution, metabolism and excretion profile of atractylodin in relevant preclinical species and to understand how drug exposure levels correlate with the observed therapeutic outcomes and potential toxicities. These PK/PD data are crucial for informing initial dose selection for human studies. Following successful preclinical validation, the potential of atractylodin would be evaluated in a structured clinical trial program. This typically begins with Phase 1 trials in a small group of healthy volunteers or patients with stable conditions to assess safety, tolerability and basic human PK/PD (43). If deemed safe, Phase 2 trials would then enroll a larger cohort of sepsis patients to investigate preliminary efficacy, explore dose-response relationships, and further evaluate safety in the target population. Efficacy endpoints in Phase 2 sepsis trials commonly include changes in organ dysfunction scores, inflammatory markers, or surrogate outcomes (44). Based on promising Phase 2 results, Phase 3 trials would be necessary to confirm efficacy on clinically relevant endpoints such as 28-day mortality and time to organ failure resolution, in large, multi-center, randomized controlled studies comparing atractylodin to current standard of care (45). Successfully navigating these rigorous clinical trial phases is required to ultimately assess the safety, efficacy, and optimal dosing of atractylodin for potential clinical use in human patients with sepsis.

In summary, atractylodin can ameliorate mitochondrial dysfunction in the acute gastrointestinal tract of sepsis and mitigate ferroptosis. The underlying mechanism involves the SIRT3/PRDX3 signaling pathway. Atractylodin holds promise as a potential therapeutic agent for the treatment and prevention of gastrointestinal disorders.

GI is a critical illness associated with high morbidity and mortality. Mitochondrial oxidative stress and ferroptosis are the key pathogenic events resulting from GI. The present study first reported the following observations: i) Atractylodin mitigated sepsis-induced GI. ii) PRDX3 protected against sepsis-induced acute GI, mitochondrial oxidative damage and ferroptosis. iii) SIRT3 deacetylates PRDX3 and can therefore alleviate sepsis-induced acute GI mitochondrial oxidative damage and ferroptosis.

The present study revealed that atractylodin can alleviate mitochondrial dysfunction and decrease ferroptosis in sepsis-induced acute GI. In addition, it was found that the mechanisms of atractylodin are associated with SIRT3/PRDX3 signaling. Together, the findings of the present study demonstrated that atractylodin may serve as a promising therapeutic agent for treating and preventing GI.

Supplementary Material

Supporting Data

Supporting Data

Acknowledgements

Not applicable.

Funding

The present study was supported by the National Natural Science Foundation of China (grant no. 81804035), the Project of Jiangsu Provincial Administration of Chinese Medicine (grant no. MS2023092), the Changzhou Sci and Tech Program (grant no. CJ20239006) and the Youth Talent Technology Project of Changzhou Health Commission (grant no. QN202137).

Availability of data and materials

The data generated in the present study are included in the figures and/or tables of this article.

Authors' contributions

MQC was responsible for investigation and resources. JLW, HDZ and TTL were responsible for investigation. TW was responsible for resources. YXH was responsible for validation, investigation and writing the original draft. YB was responsible for writing, reviewing and editing, resources, methodology, investigation, funding acquisition and conceptualization. HG was responsible for writing, reviewing and editing, validation, resources and investigation. All authors read and approved the final manuscript. YXH and MQI confirm the authenticity of all the raw data.

Ethics approval and consent to participate

The animal experiments in the present study was conducted in the Liyang Hospital of Chinese Medicine and approved by the Experimental Animal Ethics Committee of Liyang Hospital of Chinese Medicine (Jiangsu, China; approval no. 2024LY-02-02-03).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References