Loganin alleviates oxidative stress-induced apoptosis by modulating mitochondrial function and STAT3 signaling in DSS-induced colitis and H₂O₂-injured Caco-2 cells

Abstract

Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by recurrent inflammation and mucosal damage. Loganin, a natural iridoid glycoside with anti-inflammatory and antioxidant properties, has shown protective effects in experimental colitis. However, its role in regulating mitochondrial dysfunction, oxidative stress-induced epithelial cell apoptosis, and barrier injury in UC remains incompletely understood. A dextran sulfate sodium (DSS)-induced colitis model was established in mice, and an H₂O₂-induced epithelial injury model was established in Caco-2 cells to evaluate the protective effects of loganin in vivo and in vitro. Colonic injury, oxidative stress, apoptosis, and barrier integrity were assessed by histological analysis, biochemical assays, immunofluorescence, Western blotting, flow cytometry, TEER measurement, and FITC-dextran permeability analysis. Mitochondrial dysfunction was evaluated by measuring mitochondrial membrane potential, mtROS accumulation, cytochrome c redistribution, and mitochondrial ultrastructure. In addition, network pharmacology, molecular docking, and IL-6 supplementation experiments were performed to explore the involvement of IL-6/STAT3/Bcl-2-related signaling in the anti-apoptotic effects of loganin. Loganin significantly alleviated histological damage and preserved colonic architecture in DSS-treated mice. It reduced oxidative stress, as evidenced by lower MDA levels, enhanced antioxidant enzyme activity, and improved intestinal barrier function. Loganin treatment also suppressed epithelial cell apoptosis, as shown by decreased BAX and cleaved caspase-3 levels and increased Bcl-2 expression. In H₂O₂-injured Caco-2 cells, loganin attenuated mitochondrial dysfunction, preserved mitochondrial membrane potential, and reduced mtROS intensity from 16.54% to 10.11%. Loganin also reduced the apoptosis rate from 29.05% to 12.68% and improved epithelial integrity. These findings support the involvement of IL-6/STAT3/Bcl-2-related signaling in the protective effects of loganin. Our findings suggest that loganin alleviates oxidative stress-induced epithelial cell apoptosis and mitochondrial dysfunction in experimental colitis models. These protective effects are associated with modulation of IL-6/STAT3/Bcl-2-related signaling. Collectively, this study extends previous evidence on the protective effects of loganin in colitis and provides additional mechanistic insight into its role in epithelial homeostasis and barrier integrity.

Introduction

Ulcerative colitis (UC) is a chronic and relapsing inflammatory disease of the colon characterized by diffuse mucosal inflammation and epithelial barrier disruption¹. The incidence and prevalence of UC have been steadily increasing worldwide, posing a substantial healthcare burden and affecting patients’ quality of life². Persistent inflammation leads to epithelial cell apoptosis, mucosal ulceration, and compromised intestinal integrity, which further aggravate the inflammatory microenvironment and perpetuate a vicious cycle of tissue injury³. Accumulating evidence highlights that epithelial homeostasis and barrier restoration are essential for the remission and long-term management of UC⁴.

The intestinal epithelium serves as the first line of defense against luminal pathogens and metabolites. Disruption of epithelial integrity is a hallmark of UC pathogenesis and has been closely linked to oxidative stress and mitochondrial dysfunction⁵. Excessive production of reactive oxygen species (ROS) under inflammatory conditions leads to mitochondrial depolarization, impaired ATP generation, and activation of apoptotic signaling cascades⁶. Mitochondrial outer membrane permeabilization facilitates cytochrome c release and downstream activation of caspases, thereby exacerbating epithelial cell loss and barrier failure⁷. Recent studies have emphasized that restoring mitochondrial homeostasis not only mitigates oxidative damage but also preserves tight-junction assembly and epithelial regeneration, highlighting mitochondria as a promising therapeutic target in UC⁸.

Loganin, an iridoid glycoside widely present in Cornus officinalis and other medicinal plants, has attracted increasing attention because of its anti-inflammatory, antioxidant, and anti-apoptotic properties ⁹. Previous studies have demonstrated that loganin alleviates tissue injury in models of neuroinflammation, hepatic fibrosis, and diabetic nephropathy by modulating oxidative stress and cell survival pathways^10–12. In the context of UC, existing studies have also shown that loganin exerts protective effects in experimental colitis, mainly through anti-inflammatory mechanisms, including regulation of macrophage polarization and sirtuin 1 (Sirt1)/nuclear factor kappa B (NF-κB), signal transducer and activator of transcription 3(STAT3)/NF-κB, and Toll-like receptor 4 (TLR4)/NF-κB/JAK-STAT3-related signaling pathways^13–15. In addition, emerging evidence indicates that loganin can influence mitochondrial function, including suppression of NLRP3 inflammasome activation and promotion of mitophagy, suggesting a broader role in the control of oxidative stress and mitochondrial damage^16,17. However, these previous studies have focused primarily on inflammatory regulation, and whether loganin protects intestinal epithelial homeostasis by modulating mitochondrial dysfunction, oxidative stress-associated epithelial cell apoptosis, and barrier disruption in UC remains insufficiently characterized.

Given the central role of oxidative stress and mitochondrial dysfunction in the pathogenesis of UC, therapeutic strategies aimed at preserving mitochondrial homeostasis may be beneficial for maintaining epithelial integrity and barrier function. Thus, although previous studies have supported the anti-inflammatory effects of loganin in colitis, its influence on mitochondrial injury-associated epithelial cell apoptosis and barrier dysfunction remains to be further defined. Therefore, this study was designed to investigate whether loganin alleviates experimental colitis by modulating mitochondrial function, oxidative stress-induced epithelial cell apoptosis, and STAT3-related signaling in vivo and in vitro.

Methods

In vivo experimental procedures

Forty male C57BL/6 mice (8 weeks old, 20–25 g) were housed under specific pathogen-free (SPF) conditions with ad libitum access to food and water, maintained on a 12-hour light/dark cycle. Loganin was purchased from MedChemExpress (MCE, Monmouth Junction, NJ, USA; catalog no. HY-N0512) with a reported purity of 99.85%. For in vivo administration, loganin was first dissolved in dimethyl sulfoxide (DMSO) and then diluted with normal saline to the required concentration for oral gavage, with a final DMSO concentration of 2% (v/v). Fresh working solutions were prepared daily before use.

After a 5-day acclimatization period, the mice were randomly assigned to five groups: Control, dextran sulfate sodium (DSS), DSS + low-dose loganin (DSS + LL), DSS + high-dose loganin (DSS + HL), and loganin (n = 8 per group). Group size was determined with reference to previous studies employing similar DSS-induced colitis models¹⁴, together with ethical considerations to minimize animal use while maintaining sufficient biological replicates for statistical analysis. Based on established acute DSS-induced colitis protocols, mice in the DSS, DSS + LL, and DSS + HL groups received 2.5% DSS in drinking water for 7 consecutive days to induce colitis¹⁸. For loganin intervention, 20 and 60 mg/kg/day were selected as low and high doses for exploratory in vivo evaluation, with reference to previous studies of loganin in ulcerative colitis models¹³. The DSS solution was freshly prepared and replaced daily throughout the 7-day administration period. Loganin was administered by oral gavage at 20 mg/kg/day or 60 mg/kg/day in the DSS + LL and DSS + HL groups, respectively. To ensure drug exposure at the onset of DSS challenge and to evaluate the potential protective effect of loganin during the early phase of colitis induction, loganin treatment was initiated 1 day before DSS exposure and continued throughout the 7-day DSS administration period. Mice in the loganin-only group received 60 mg/kg/day loganin by oral gavage for the same total duration of 8 days, but without DSS administration, whereas the Control group received normal drinking water.

Body weight and disease activity index (DAI) scores were recorded daily. The DAI was calculated as a composite score based on body weight loss, stool consistency, and fecal bleeding according to established DSS colitis scoring criteria¹⁸. Each parameter was scored on a 0–4 scale, and the final DAI score was calculated as the sum of the three subscores. At the end of the 8-day experimental period, the animals were euthanized via CO₂ inhalation, and colon tissues were collected for further analysis. All animal procedures were performed in accordance with the ARRIVE guidelines and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Xinjiang Medical University (Approval No. K202307-21).

Cell culture and treatment

Caco-2 cells were cultured in alpha minimum essential medium (α-MEM) supplemented with 20% fetal bovine serum (FBS), 1% non-essential amino acids, and 1% penicillin–streptomycin at 37 °C in a humidified atmosphere containing 5% CO₂. The working concentration of loganin was determined based on preliminary cell viability evaluation using the Cell Counting Kit-8 (CCK-8) assay. For loganin intervention, cells were pretreated with loganin (20 µM) for 1 h before hydrogen peroxide (H₂O₂) exposure under the corresponding experimental conditions. To establish the oxidative stress model, Caco-2 cells were exposed to H₂O₂ (100 µM) for 12 h. For interleukin-6 (IL-6) supplementation experiments, recombinant human IL-6 (MedChemExpress, HY-P7044) was added at 10 ng/mL for 24 h in the H₂O₂ + loganin group before subsequent analyses, with reference to previous studies using IL-6 stimulation in Caco-2 cells¹⁹.

Histological analysis

Colon tissues were harvested from mice at the end of the experiment and fixed in 4% paraformaldehyde for 24 h at 4 °C. After fixation, tissues were embedded in paraffin and sectioned into 4 μm thick slices. The sections were deparaffinized in xylene, rehydrated through graded ethanol solutions, and then stained with hematoxylin and eosin (HE) to evaluate histopathological changes. Histological damage was assessed in a blinded manner by two independent investigators based on epithelial cell loss, crypt loss, inflammatory cell infiltration, and the extent of ulceration, with minor modifications from established DSS colitis histopathological scoring criteria²⁰. The mean score was used for subsequent analysis.

Enzyme-linked immunosorbent assay (ELISA)

ELISA was used to measure the levels of malondialdehyde (MDA), heme oxygenase-1 (HO-1), superoxide dismutase (SOD), and glutathione (GSH) in colonic tissues. After euthanizing the mice, colon tissues were collected, homogenized in cold phosphate-buffered saline (PBS), and centrifuged to collect the supernatants. The levels of MDA, HO-1, SOD, and GSH were quantified using Elabscience ELISA kits according to the manufacturer’s protocol (Catalog Nos. E-EL-0060, E-EL-H2172, E-EL-H6188, and E-EL-0026 for MDA, HO-1, SOD, and GSH, respectively). Absorbance was measured at the appropriate wavelengths, and concentrations were calculated using standard curves.

Network pharmacology and molecular docking analysis

Potential targets of loganin were collected from TCMSP, SwissTargetPrediction, and STITCH. UC-related genes were retrieved from GeneCards and DisGeNET using the keyword “ulcerative colitis”. After duplicate removal, all targets were standardized to official gene symbols (Homo sapiens), and the overlapping targets were identified for subsequent analysis. The common targets were imported into STRING (Homo sapiens, confidence score > 0.7) to construct a protein–protein interaction (PPI) network, which was visualized and analyzed using Cytoscape (version 3.10.4). For molecular docking, the three-dimensional structure of loganin and the protein structures of selected targets, including IL-6 and STAT3, were obtained from public databases. Docking was performed using AutoDock Vina (version 1.2.7), and the binding conformations were visualized using PyMOL (version 3.2.0a0).

TUNEL staining

TUNEL staining was performed to assess epithelial cell apoptosis in colon tissues. After paraffin embedding and sectioning, 4 μm thick tissue sections were deparaffinized, rehydrated, and treated with proteinase K solution. The sections were then incubated with the TUNEL Detection Kit (Beyotime, China, catalog no. C1086) according to the manufacturer’s instructions. After the reaction, the sections were counterstained with DAPI and observed under a fluorescence microscope. The number of TUNEL-positive cells was quantified to evaluate apoptosis in each group.

Western blot

Proteins from colon tissues and cultured cells were extracted using RIPA buffer containing protease and phosphatase inhibitors. Equal amounts of protein (30 µg) were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% BSA in TBST and incubated overnight at 4 °C with primary antibodies against Occludin (Santa Cruz, sc-133256, 1:1000), zonula occludens-1 (ZO-1) (Affinity, AF5145, 1:1000), Bcl-2 (Abmart, T40056S, 1:1000), BAX (Abmart, T40051S, 1:1000), pro-caspase-3 and cleaved caspase-3 (Huabio, ST1608-64, 1:1000), p-STAT3 (CST,9145,1:1000), STAT3 (CST,9139,1:1000), and GAPDH (Proteintech, 66009-1-AP, 1:5000) as a loading control. After washing, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were detected using the MCE ECL detection system and band intensities were quantified using ImageJ software.

JC-1 analysis

Mitochondrial membrane potential (MMP) was determined using the JC-1 assay kit (Beyotime, China, catalog no. C2003S). After treatments, cells were incubated with JC-1 dye at 37 °C for 30 min according to the manufacturer’s instructions, followed by washing with PBS. Fluorescence was observed under a fluorescence microscope: red fluorescence (JC-1 aggregates) indicates high MMP, and green fluorescence (JC-1 monomers) indicates low MMP. The ratio of red-to-green fluorescence was quantified using ImageJ software to assess mitochondrial membrane potential.

TEER measurement, FITC-dextran permeability assay, and immunofluorescence staining of ZO-1 and Occludin

To evaluate epithelial barrier function, Caco-2 cell monolayers were established under the indicated treatment conditions. Transepithelial electrical resistance (TEER) was measured at the end of treatment using an epithelial voltohmmeter according to the manufacturer’s instructions, and the values were used to assess changes in barrier integrity. For the FITC-dextran permeability assay, Caco-2 monolayers were incubated with FITC-dextran (4000 Da, 40 mg/mL) for 4 h at 37 °C. After incubation, the cells were washed with PBS and fixed with 4% paraformaldehyde. Representative fluorescence images were captured using a fluorescence microscope, and FITC-dextran fluorescence intensity was quantified using ImageJ software as an indicator of epithelial permeability. For immunofluorescence staining, cells were permeabilized and incubated overnight at 4 °C with primary antibodies against Occludin (1:200) and ZO-1 (1:200). After washing, cells were incubated with fluorescent secondary antibodies (1:500) for 1 h at room temperature, and nuclei were counterstained with DAPI (1:1000). Fluorescence images were acquired using a fluorescence microscope, and the fluorescence intensities of Occludin and ZO-1 were quantified using ImageJ software.

Flow cytometry

Apoptosis was analyzed by flow cytometry using the Apoptosis Detection Kit (Vazyme, China, catalog no. A211). After treatment, cells were harvested, washed with PBS, and resuspended in binding buffer. Cells were incubated with FITC-labeled Annexin-V and PI at room temperature for 15 min in the dark. After staining, apoptotic cells were analyzed using a BD FACSCanto II flow cytometer (BD Biosciences). Early apoptotic cells were identified as Annexin V-positive/PI-negative, while late apoptotic and necrotic cells were identified as Annexin V-positive/PI-positive. Data were processed using FlowJo software to determine the percentage of apoptotic cells in each group.

Data analysis

All data were expressed as the mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 10 software. Body weight across time was analyzed using two-way repeated-measures ANOVA followed by Tukey’s multiple comparisons test. DAI scores at individual time points and histological injury scores, as score-based variables, were analyzed using the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Comparisons among multiple groups for end-point continuous variables were performed using one-way ANOVA followed by Tukey’s post hoc test. A p-value < 0.05 was considered statistically significant.

Results

Loganin attenuates DSS-induced colonic injury in mice

A DSS-induced colitis mouse model was established to evaluate the protective effects of loganin. As shown in Fig. 1A–B, mice exposed to DSS exhibited progressive body-weight loss starting from day 3, accompanied by a significant increase in DAI from day 5. Loganin treatment, particularly at the high dose, significantly attenuated DSS-induced body-weight loss and reduced DAI scores throughout the modeling period, with a more pronounced effect observed in the high-dose group.

Fig. 1.

Loganin-mediated protection against DSS-induced colonic injury in mice. (A) Body weight of mice during the experimental period. (B) Disease activity index (DAI) scores of mice during the experimental period. (C) Representative macroscopic images of the colon. (D) Colon length measurement. (E) Representative hematoxylin and eosin (HE)-stained colonic sections, shown at 5× (scale bar = 200 μm) and 20× (scale bar = 50 μm) magnification. (F) Histological injury scores based on HE staining. (G) Immunofluorescence staining for Occludin (red) and DAPI (blue) in colonic tissue. (H) Quantification of Occludin fluorescence intensity. (I) Immunofluorescence staining for ZO-1 (red) and DAPI (blue) in colonic tissue. (J) Quantification of ZO-1 fluorescence intensity. Data are presented as mean ± SD (n = 8 per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant. DSS was compared with Ctrl, treatment groups were compared with DSS, and the loganin group was compared with Ctrl.

DSS administration also caused marked colon shortening, as shown in Fig. 1C–D. Histopathological examination further revealed epithelial erosion, crypt destruction, and inflammatory cell infiltration in the DSS group (Fig. 1E–F). These pathological features were alleviated in the loganin-treated groups, as indicated by reduced histological injury scores (Fig. 1E–F).

To further assess epithelial barrier integrity, the expression of tight-junction proteins was examined by immunofluorescence. DSS exposure led to decreased expression and disrupted distribution of Occludin and ZO-1 along the epithelial surface, suggesting impaired barrier integrity. Loganin treatment partially restored the continuity and fluorescence intensity of Occludin and ZO-1 (Fig. 1G–J). Together, these findings indicate that loganin attenuates DSS-induced colonic injury, accompanied by improvements in clinical parameters, histological features, and epithelial barrier-associated proteins.

Loganin reduces oxidative stress and apoptosis in DSS-induced colitis

UC progression is closely associated with excessive oxidative stress and apoptosis, both of which contribute to colonic epithelial damage. To evaluate whether loganin could alleviate these pathological changes, we assessed key oxidative stress markers and apoptosis-related parameters in DSS-induced colitis mice. As shown in Fig. 2A, DSS treatment markedly increased MDA levels, indicating enhanced lipid peroxidation and oxidative damage. In contrast, loganin treatment reduced MDA levels in a dose-dependent manner. In addition, loganin significantly increased the levels of the antioxidant markers SOD, HO-1, and GSH compared with the DSS group (Fig. 2B–D), supporting a protective effect of loganin against oxidative stress.

Fig. 2.

Loganin reduces oxidative stress and apoptosis in DSS-induced colitis. (A–D) Levels of oxidative stress markers in colonic tissue: MDA, SOD, HO-1, and GSH were measured by ELISA. (E) Representative TUNEL staining of colonic tissue sections. (F) Quantification of TUNEL-positive cells as a percentage of total cells. (G) Western blot analysis of apoptosis-related proteins (Bcl-2, BAX, pro-caspase-3, cleaved caspase-3) in colonic tissue. (H–J) Quantification of BAX, Bcl-2, and cleaved caspase-3 protein levels by Western blot. Data are presented as mean ± SD (n = 8 per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant. DSS was compared with Ctrl, treatment groups were compared with DSS, and the loganin group was compared with Ctrl.

Epithelial cell apoptosis was assessed by TUNEL staining. DSS treatment markedly increased the number of TUNEL-positive cells, whereas loganin treatment significantly reduced apoptotic cell numbers, indicating an anti-apoptotic effect in vivo (Fig. 2E–F).

To further validate these findings at the protein level, we examined the expression of apoptosis-related markers by western blot. DSS treatment increased the expression of the pro-apoptotic proteins BAX and cleaved caspase-3, while reducing the expression of the anti-apoptotic protein Bcl-2. Loganin treatment reversed these changes, as evidenced by restored Bcl-2 expression and reduced BAX and cleaved caspase-3 levels (Fig. 2G–J). Together, these findings suggest that loganin mitigates DSS-induced colonic injury by attenuating oxidative stress and apoptosis.

Network pharmacology and molecular docking insights

In our in vivo experiments, we observed that loganin effectively alleviated oxidative stress and apoptosis, as well as preserved intestinal barrier integrity in DSS-induced colitis mice. To explore the underlying molecular mechanisms responsible for these protective effects, network pharmacology and molecular docking analyses were performed. This approach aimed to identify potential targets and key signaling pathways through which loganin exerts its therapeutic action.

First, a Venn diagram (Fig. 3A) was generated to identify common targets between loganin and UC. This analysis revealed 72 overlapping targets, suggesting that loganin might regulate multiple pathways associated with UC pathogenesis. These potential targets were further analyzed using a protein-protein interaction (PPI) network (Fig. 3B), highlighting several critical hub genes, such as TNF, IL6, STAT3, EGFR, and Bcl-2, which are involved in inflammation, apoptosis, and epithelial barrier dysfunction.

Fig. 3.

Network pharmacology and molecular docking analysis of loganin-associated targets in UC. (A) Venn diagram showing the overlap between loganin-related targets and UC-associated genes. (B) Protein-protein interaction (PPI) network of the overlapping targets, highlighting key hub genes involved in loganin’s potential mechanisms, including TNF, EGFR, IL6, STAT3, and BCL2. (C) Molecular docking of loganin with IL-6. (D) Molecular docking of loganin with STAT3.

Next, molecular docking simulations were conducted to assess the binding affinities of loganin with key targets identified through the network analysis. The docking results revealed that loganin exhibited strong binding with IL-6 (Fig. 3C), with a binding energy of -7.98 kcal/mol, indicating a stable interaction. Similarly, loganin showed favorable binding with STAT3 (Fig. 3D), further supporting the hypothesis that loganin may modulate the IL-6/STAT3/BCL2 signaling pathway, which plays a pivotal role in inflammatory response and apoptosis.

Loganin reduces mitochondrial damage in H₂O₂-induced epithelial cell injury

To evaluate the effects of loganin on mitochondrial injury under oxidative stress, mitochondrial membrane potential (MMP), mitochondrial ROS production, cytochrome c redistribution, and mitochondrial ultrastructure were examined in H₂O₂-injured Caco-2 cells. JC-1 staining showed that H₂O₂ exposure markedly reduced the red-to-green fluorescence ratio, indicating loss of MMP, whereas loganin treatment partially restored this ratio (Fig. 4A and B).

Fig. 4.

Loganin protects against mitochondrial dysfunction in H₂O₂-induced epithelial cell injury. (A) Representative JC-1 staining images showing mitochondrial membrane potential (MMP) in Ctrl, H₂O₂ -treated, loganin-treated, and H₂O₂ + loganin-treated Caco-2 cells. (B) Quantification of the JC-1 red/green fluorescence ratio. (C) Representative MitoSOX staining images showing mitochondrial reactive oxygen species (mtROS) levels. (D) Quantification of mtROS fluorescence intensity. (E) Immunofluorescence staining of TOM20 (red), cytochrome c (Cyt c, green), and DAPI (blue) in Caco-2 cells. (F) Representative fluorescence intensity profile of TOM20 and Cyt c signals. (G) Representative transmission electron microscopy (TEM) images of mitochondrial ultrastructure. Data are presented as mean ± SD (n = 4 per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant. H₂O₂ was compared with Ctrl, H₂O₂ + loganin was compared with H₂O₂, and the loganin group was compared with Ctrl.

MitoSOX staining further showed that mtROS levels were substantially elevated after H₂O₂ exposure but were attenuated by loganin treatment (Fig. 4C and D). To assess mitochondrial membrane permeabilization and cytochrome c redistribution, TOM20/cytochrome c immunofluorescence staining was performed (Fig. 4E). In control cells, cytochrome c largely overlapped with TOM20, whereas H₂O₂ exposure reduced this overlap, consistent with cytochrome c release from mitochondria into the cytosol. In the presence of loganin, the overlap between TOM20 and cytochrome c appeared to be partially preserved. This pattern was further illustrated by the fluorescence intensity profile in Fig. 4F, which showed a closer correspondence between TOM20 and cytochrome c signals in the control and loganin-treated groups than in the H₂O₂ group.

Mitochondrial ultrastructure was further examined by TEM (Fig. 4G). Compared with the control group, H₂O₂-treated cells displayed mitochondrial swelling, disrupted cristae, and loss of membrane integrity, whereas these structural abnormalities were alleviated in the loganin-treated group. Taken together, these findings indicate that loganin alleviates H₂O₂-induced mitochondrial injury by preserving MMP, reducing mtROS accumulation, limiting cytochrome c redistribution, and maintaining mitochondrial ultrastructure.

Loganin promotes intestinal barrier integrity in H₂O₂-induced epithelial cell injury

To investigate the protective effects of loganin on epithelial barrier function at the cellular level, we examined the tight junction proteins ZO-1 and Occludin in H₂O₂-injured Caco-2 cells. As shown in Fig. 5A and B, H₂O₂ exposure weakened the immunofluorescence signals of both ZO-1 and Occludin and disrupted their distribution along the cell borders. In contrast, loganin treatment preserved the staining pattern of these proteins and partially restored their expression. Quantitative analysis was consistent with these observations, showing higher fluorescence intensities of ZO-1 and Occludin in the loganin-treated group than in the H₂O₂ group (Fig. 5E and F).

Fig. 5.

Loganin promotes intestinal barrier integrity in H₂O₂-induced epithelial injury. (A) Representative immunofluorescence images of ZO-1 (green) and DAPI (blue) in Caco-2 cells under different treatment conditions. (B) Representative immunofluorescence images of Occludin (green) and DAPI (blue) in Caco-2 cells under different treatment conditions. (C) Representative images of the FITC-dextran permeability assay in Caco-2 cells. (D) TEER measurement used to assess epithelial barrier integrity. (E–G) Quantitative analysis of ZO-1 fluorescence intensity, Occludin fluorescence intensity, and FITC-dextran fluorescence intensity, respectively. (H) Western blot analysis of ZO-1, Occludin, and GAPDH protein expression in Caco-2 cells. (I, J) Densitometric quantification of ZO-1 and Occludin protein expression. Data are presented as mean ± SD (n = 4 per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant. H₂O₂ was compared with Ctrl, H₂O₂ + loganin was compared with H₂O₂, and the loganin group was compared with Ctrl.

Barrier function was further evaluated by TEER and FITC-dextran permeability assays. H₂O₂ treatment reduced TEER, indicating impaired epithelial barrier function, whereas loganin treatment partially restored TEER (Fig. 5D). A similar trend was observed in the permeability assay: FITC-dextran fluorescence increased after H₂O₂ exposure but decreased following loganin treatment, suggesting attenuation of barrier dysfunction (Fig. 5C and G).

These findings were further supported by Western blot analysis (Fig. 5H). H₂O₂ treatment lowered the protein levels of ZO-1 and Occludin, while loganin treatment reversed these changes. Densitometric quantification (Fig. 5I and J) confirmed that both tight junction proteins were expressed at higher levels in the loganin-treated group than in the H₂O₂ group. Taken together, these results indicate that loganin preserves epithelial barrier integrity under oxidative stress conditions by maintaining tight junction protein expression and reducing epithelial permeability.

Loganin inhibits apoptosis through the IL-6/STAT3/BCL2 signaling pathway in H₂O₂-induced epithelial cell injury

To investigate the mechanism by which loganin inhibits apoptosis in H₂O₂-induced epithelial cell injury, network pharmacology analysis identified the IL-6/STAT3/BCL2 signaling pathway as a potential regulator of apoptosis and cell survival. Western blot analysis showed that H₂O₂ treatment increased the levels of pro-apoptotic proteins BAX and cleaved caspase-3, and decreased the anti-apoptotic protein Bcl-2 (Fig. 6A–D). p-STAT3 levels also increased, indicating activation of the STAT3 pathway. In contrast, loganin treatment reduced BAX and cleaved caspase-3 levels, and restored Bcl-2, demonstrating its anti-apoptotic effect. Additionally, loganin decreased p-STAT3 levels, suggesting that loganin inhibits the STAT3 pathway, contributing to its anti-apoptotic action in H₂O₂-induced epithelial cell injury (Fig. 6E–F).

Fig. 6.

Loganin inhibits apoptosis through the IL-6/STAT3/BCL2 signaling pathway in H₂O₂-induced epithelial cell injury. (A) Western blot analysis of apoptosis-related proteins Bcl-2, BAX, pro-caspase-3, and cleaved caspase-3. (B–D) Quantification of BAX, Bcl-2, and cleaved caspase-3 protein levels. (E) Western blot analysis of STAT3 and p-STAT3 protein levels. (F) Quantification of p-STAT3 protein levels. (G) Flow cytometry analysis of apoptosis in Ctrl, H₂O₂, H₂O₂ + loganin, and H₂O₂ + loganin + IL-6 groups. (H) Quantification of apoptosis rate. (I) Western blot analysis of apoptosis-related proteins Bcl-2, BAX, pro-caspase-3, and cleaved caspase-3 in Ctrl, H₂O₂, H₂O₂ + loganin, and H₂O₂ + loganin + IL-6 groups. (J–L) Quantification of BAX, Bcl-2, and cleaved caspase-3 protein levels, respectively. (M) Western blot analysis of STAT3 and p-STAT3 protein levels. (N) Quantification of p-STAT3 protein levels. Data are presented as mean ± SD (n = 4 per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant. For panels A–F, H₂O₂ was compared with Ctrl, H₂O₂ + loganin was compared with H₂O₂, and the loganin group was compared with Ctrl. For panels G–N, H₂O₂ was compared with Ctrl, H₂O₂ + loganin was compared with H₂O₂, and H₂O₂ + loganin + IL-6 was compared with H₂O₂ + loganin.

To further investigate the role of the IL-6/STAT3/BCL2 signaling pathway, IL-6 was added to the loganin treatment to assess its impact on apoptosis. Flow cytometric analysis showed that H₂O₂ treatment significantly increased apoptosis, as indicated by an increased proportion of Annexin V-positive cells (Fig. 6G–H).Treatment with loganin significantly reduced apoptosis compared to H₂O₂ treatment alone. However, when IL-6 was added to the H₂O₂ + loganin group, the apoptosis rate significantly increased, indicating that IL-6 partially counteracted the anti-apoptotic effects of loganin. Western blot analysis (Fig. 6I–L) was used to examine the expression of apoptosis-related proteins. In the H₂O₂ + loganin group, BAX and cleaved caspase-3 levels were significantly reduced, while Bcl-2 expression was restored, confirming the anti-apoptotic effects of loganin. In contrast, in the H₂O₂ + loganin + IL-6 group, BAX and cleaved caspase-3 levels were significantly elevated, and Bcl-2 expression was decreased compared to the H₂O₂ + loganin group, suggesting that IL-6 reduced the protective effects of loganin. Consistently, p-STAT3 levels were significantly lower in the H₂O₂ + loganin group than in the H₂O₂ group, whereas IL-6 supplementation increased p-STAT3 levels in the H₂O₂ + loganin + IL-6 group (Fig. 6M–N). These findings demonstrate that loganin inhibits apoptosis in H₂O₂-induced epithelial cell injury by suppressing p-STAT3 activation. The addition of IL-6 reverses part of loganin’s protective effects by enhancing STAT3 activation, leading to an increase in apoptosis.

Discussion

UC is a chronic inflammatory bowel disease that causes recurrent inflammation, leading to complications such as mucosal injury, increased risk of colorectal cancer, and a reduction in quality of life²¹. While current treatments, including corticosteroids, immunosuppressants, and 5-ASA, provide symptom relief, they often fail to address the underlying causes of UC and are associated with long-term side effects²². Therefore, the development of alternative therapies is essential. Loganin, a naturally occurring compound with known anti-inflammatory and antioxidant properties, has gained attention as a potential therapeutic agent²³. Our study suggests that loganin effectively mitigates DSS-induced colonic injury in mice by reducing oxidative stress, inhibiting epithelial cell apoptosis, and improving intestinal barrier integrity, highlighting its potential as a therapeutic candidate for UC (Fig. 7).

Fig. 7.

Mechanistic overview of loganin’s protective effects in oxidative stress-induced apoptosis and mitochondrial dysfunction in UC. Oxidative stress induced by DSS or H₂O₂ promotes ROS/mtROS accumulation, mitochondrial dysfunction, apoptosis-related protein imbalance, epithelial apoptosis, and barrier disruption. In contrast, loganin attenuates oxidative stress-associated injury, accompanied by reduced p-STAT3 levels, modulation of apoptosis-related proteins, decreased epithelial apoptosis, and preserved barrier integrity. The figure summarizes the working model proposed on the basis of the present in vivo and in vitro findings.

Oxidative stress plays a central role in the progression of UC by damaging cellular components, exacerbating inflammation, and hindering mucosal repair, as prolonged redox imbalance promotes chronic mucosal injury and barrier dysfunction in colitis models and patients²⁴. Apoptosis of epithelial cells further compromises the integrity of the intestinal barrier, contributing to a vicious cycle of injury and inflammation by disrupting tight junctions and facilitating immune cell infiltration²⁵. Previous studies have shown that excessive oxidative stress and apoptosis are key drivers of epithelial cell damage in UC, making them potential therapeutic targets²⁶. Inhibition of apoptosis has been shown to improve intestinal integrity and reduce inflammation in UC models, suggesting that modulating these processes may offer therapeutic benefits^27,28. In this context, loganin emerges as a promising candidate. Previous studies have shown its protective effects in various models, including its ability to reduce testicular damage and apoptosis induced by advanced glycation end products (AGEs) in a diabetic model²⁹. Loganin also inhibits hydrogen peroxide-induced apoptosis in SH-SY5Y cells, highlighting its broad potential in modulating apoptotic signaling pathways and protecting against cellular damage³⁰. These findings align with our results, where loganin significantly reduced oxidative stress markers, including MDA, and enhanced antioxidant enzyme activity in DSS-induced colitis models. Moreover, loganin suppressed apoptosis by regulating key proteins such as Bcl-2, BAX, and cleaved caspase-3, suggesting that its protective effects in UC are mediated through the modulation of oxidative stress and apoptotic pathways. Together, these results highlight loganin’s potential as a therapeutic agent for UC and other diseases characterized by oxidative damage and excessive apoptosis.

Mitochondrial dysfunction is a key pathological feature of UC, contributing to epithelial damage, apoptosis, and disruption of the intestinal barrier³¹. In our study, we observed that loganin protects against mitochondrial damage induced by oxidative stress, as evidenced by reduced mtROS and preserved MMP in H₂O₂-induced epithelial cell injury. These results are consistent with previous studies showing that loganin can regulate mitochondrial function across different disease models. For example, in a paclitaxel-induced skeletal muscle toxicity model, loganin alleviated mitochondrial dysfunction by enhancing mitochondrial function and boosting antioxidant defenses³². Similarly, loganin has been shown to protect against mitochondrial damage in other models by modulating key mitochondrial pathways³³. These findings suggest that loganin may modulate mitochondrial function, reduce oxidative damage, and help preserve mitochondrial homeostasis. In the context of UC, our study demonstrates that loganin exerts protective effects through similar mechanisms, helping to restore mitochondrial function and preserve the integrity of the intestinal epithelial barrier. These results further underscore the potential of loganin as a therapeutic agent for UC, particularly by targeting mitochondrial dysfunction and oxidative stress.

The involvement of IL-6/STAT3/BCL2-related signaling in the anti-apoptotic effects of loganin is an important finding of the present study. H₂O₂ treatment increased p-STAT3 levels and was accompanied by enhanced apoptosis, whereas loganin treatment reduced p-STAT3 activation. These results suggest that suppression of STAT3 signaling may contribute to the protective effect of loganin under oxidative stress conditions. This interpretation is consistent with previous studies showing that STAT3 plays an important role in inflammation and apoptosis in UC. For example, Grivennikov et al. reported that STAT3 is involved in both inflammatory and apoptotic responses in intestinal epithelial cells and contributes to disease progression³⁴. In our rescue experiments, addition of IL-6 to the H₂O₂ + loganin group partially reversed the anti-apoptotic effect of loganin, as reflected by increased p-STAT3 levels and enhanced apoptosis. This finding supports the idea that activation of IL-6/STAT3-related signaling can counteract the protective effect of loganin. It is also in line with previous reports showing that IL-6-induced STAT3 activation contributes to epithelial injury and inflammatory responses in UC^35,36. Taken together, these findings suggest that modulation of IL-6/STAT3/BCL2-related signaling is associated with the anti-apoptotic effect of loganin. However, IL-6 levels themselves were not directly quantified in the present study, and therefore the current data do not directly demonstrate that loganin downregulates IL-6 expression.

Our study has several limitations that should be acknowledged. First, although the DSS-induced colitis model is widely used to study experimental colitis, it may not fully recapitulate the complexity of human UC. Second, only colonic tissue samples were analyzed in vivo, whereas circulating markers in serum or plasma were not assessed, which limited the evaluation of systemic changes associated with loganin treatment. Third, although our data support the involvement of IL-6/STAT3/Bcl-2-related signaling in the protective effects of loganin, IL-6 levels were not directly quantified, and upstream regulators of STAT3 activation, including gp130 receptor activation and JAK phosphorylation, were not directly assessed. In addition, the upstream regulation of STAT3, its potential crosstalk with other inflammatory pathways, particularly NF-κB signaling, and additional downstream targets were not systematically investigated in the present study. Moreover, the H2O2-induced epithelial injury model represents a simplified in vitro oxidative stress system and does not fully recapitulate the complex inflammatory and immune microenvironment of UC in vivo. The temporal relationship between oxidative stress reduction and STAT3 inhibition, as well as whether the anti-apoptotic effect of loganin is secondary to its antioxidant activity or directly mediated through signaling modulation, remains to be clarified. Therefore, the mechanistic interpretation should be considered with appropriate caution. Finally, although loganin was administered in a low-concentration DMSO-containing vehicle, a separate vehicle control was not included for the DSS group; therefore, a possible confounding effect of DMSO on colonic injury and related in vivo readouts cannot be completely excluded.

Conclusion

In conclusion, our findings suggest that loganin attenuates oxidative stress-associated epithelial cell apoptosis while preserving intestinal barrier integrity in experimental colitis models. These protective effects are associated with improved mitochondrial function and modulation of IL-6/STAT3/Bcl-2-related signaling. Collectively, our study extends previous evidence on the protective effects of loganin in colitis and provides additional mechanistic insight into its actions on epithelial homeostasis and barrier integrity.

Abbreviations

BAX

Bcl-2-associated X protein

Bcl-2

B-cell lymphoma 2

Cyt c

Cytochrome c

DAI

Disease activity index

DSS

Dextran sulfate sodium

GSH

Glutathione

HE

Hematoxylin and eosin

HO-1

Heme oxygenase-1

H₂O₂

Hydrogen peroxide

IL-6

Interleukin-6

MDA

Malondialdehyde

MMP

Mitochondrial membrane potential

mtROS

Mitochondrial reactive oxygen species

PPI

Protein–protein interaction

ROS

Reactive oxygen species

SOD

Superoxide dismutase

STAT3

Signal transducer and activator of transcription 3

TEER

Transepithelial electrical resistance

TEM

Transmission electron microscopy

TOM20

Translocase of outer mitochondrial membrane 20

TUNEL

Terminal deoxynucleotidyl transferase dUTP nick end labeling

UC

Ulcerative colitis

ZO-1

Zonula occludens-1

Author contributions

Guofeng Li was responsible for the conceptualization, methodology, data curation, formal analysis, validation, visualization, and the writing of the original draft. Shuai Zhou was responsible for investigation and data analysis.Xing Zhao contributed to the methodology. Yongfeng Cheng participated in the review and editing of the manuscript. Zhihua Ma supervised the project, provided resources, managed the administration, acquired funding, and contributed to the review and editing of the manuscript. All authors have read and approved the final manuscript.

Funding

We are grateful for the fund support of the Xinjiang Uygur Autonomous Region Natural Science Foundation (Grant No. 2022D01C217).

Data availability

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

The study was approved by the Institutional Animal Care and Use Committee (IACUC) of Xinjiang Medical University (Approval No. K202307-21) and conducted in accordance with the ethical standards outlined in the 8th edition of the Guide for the Care and Use of Laboratory Animals (National Research Council, USA, 2011). Additionally, we confirm that the study is reported in compliance with the ARRIVE guidelines.