Monotropein (CAS# [5945-50-6], HPLC 98%) was purchased from Baoji Herbest Bio-Tech Co., Ltd. (China). Molecular formula: C₁₆H₂₂O₁₁. The structure of the compound is shown in Figure 1A.

Male Kunming (KM) mice were obtained from the Laboratory Animal Center of Guangzhou University of Traditional Chinese Medicine (License No. SCXK 2013-0002). All experimental mice were bred in specific pathogen-free conditions of 20 ∼ 25°C temperature, 55% ± 10% humidity, and 12-h/12-h light/dark cycle with 24-h free access to standard feed and sterilized water. The experimental scheme was permitted by the Animal Ethics Committee of Guangzhou University of Chinese Medicine (License No. 20210824012) and was conducted in accordance with the US National Institutes of Health guidelines for humane animal use (NIH Publications, No. 8023, revised 1978).

Dextran sodium sulfate (DSS, MW 36,000–50,000) was purchased from HuicH (China). After 1 week of animal adaptation feeding, the weighted random sampling method was used for grouping. Specifically, six containers were prepared, each containing an equal number of balls numbered from 1 to the total number of animals. The animals are weighed in turn, and a corresponding number of pellets are drawn from the container according to their weight. The extracted numbers were used to assign the animals to six groups: one normal group and five model groups (Gellerstedt, 2002). One group was randomly selected as the normal group, and the remaining five groups of animals were remixed to establish a chronic colitis model.

The model building was divided into three cycles. In the first cycle, all animals in the modeling group were given 2% DSS for 10 days, followed by sterilized water for 10 days. In the second cycle, animals in the DSS group were given 2%DSS for 5 days, followed by sterilized water for 5 days. In the third cycle, animals in the DSS group were given 2%DSS for 4 days, followed by sterilized water for 4 days. In all cycles, normal group animals were given sterilized water every day. The dosage of monotropein was referred to in a previous report (Chen et al., 2020) and was combined with the effective dose of the pre-experiment. Monotropein was dissolved in sterilized water. Administration began with the second cycle. After the first cycle of modeling, the modeling group was randomly divided into a DSS group, a 5-ASA (80 mg/kg) group, a monotropein low-dose (0.125 mg/kg) group, a monotropein medium-dose (0.5 mg/kg) group, and a monotropein high-dose (2 mg/kg) group according to the weight random principle (n = 11). The 5-ASA and monotropein treatments were administered daily throughout the second and third cycles (18 days). The DSS group received DSS according to the schedule mentioned above. From the second cycle to the end of the third cycle, 0.1 mL/10 g of the drug was given daily by intragastric administration (Figure 1B). The animals’ weights were recorded daily. At the end of modeling and administration, the animals were anesthetized with pentobarbital sodium (40 mg/kg), blood was collected, and the colon tissues were separated.

The disease activity index (DAI) was determined by referring to the method of Tohru (Funakoshi et al., 2012) and our previously described method (Zhang et al., 2022).

Colon tissues were made into tissue slices (4 μm) and stained using an H&E kit (BKMAM, China). The extent of colon tissue injury was observed under a microscope (BX53, Japan). The scoring for colon injury was performed according to the previous studies (Zhang et al., 2022; Zhou et al., 2019).

At the end of the third model-building cycle, animal serum was collected for the detection of cytokines. Animal serum was separated at 4°C and centrifuged at 3500 r/min for 15 min. The levels of various cytokines (TNF-a, IL-6, and IL-10, Meimian Biotechnology, China) were detected in the serum according to the ELISA kit instructions.

The mice were fasted for 12 h and given FITC-dextran (50 mg/kg) (MedChemExpress, United States). Four hours later, peripheral blood was collected and left in the dark for 1 h. Animal serum was separated at 4°C and centrifuged at 3,500 r/min for 15 min. Serum fluorescence intensity was measured, and the concentration of FITC-dextran in serum was calculated by a standard curve.

Slices were dewaxed with xylene and hydrated in different concentrations of alcohol for 5 min. The slices were used to repair antigens in citric acid repair solution and blocked with BSA (0.1%) for 30 min. The tissues were incubated with a suitable concentration of primary antibodies (Table 1) at 4°C overnight and then incubated with secondary antibody for 2 h in the dark. Finally, DAPI was added to the tissue and incubated for 5 min against light, and the slices were observed under a confocal Zeiss microscope (Carl Zeiss, Germany).

IEC-6 cells were treated with relevant reagents. Then, the corresponding primary antibody (E-cadherin, Beclin1) and fluorescently coupled secondary antibody were incubated for immunofluorescence staining. Nuclei were stained with DAPI. A confocal Zeiss microscope (Carl Zeiss, Germany) was used to examine and photograph cells in a random field of view.

The experiment was carried out following our previously described method (Xu et al., 2024; Chen Y.-E. et al., 2021).

The slices were dewaxed to water and were used to repair antigens in a citric acid repair solution. The tissues were washed two times, 3 min each time with PBS, and then were incubated with hydrogen peroxide block for 15 min. The tissues were washed with PBS two times, 5 min each time, and blocked with BSA (3%) for 15 min. The tissues were incubated with a-SMA antibody at 37°C for 2 h. The tissues were washed with PBS three times, 3 min each time, and incubated with HRP polymer at room temperature for 20 min. The tissues were incubated with streptomyces vitellin working solution labeled with horseradase at room temperature for 20 min. The tissues were washed with PBS three times, 3 min each time, and DAB was used to develop color for 30 s. The tissues were rinsed with running water, dehydrated, sealed with neutral gum, and finally observed under a microscope (BX53, Japan).

Rat small intestine crypt epithelial cells (IEC-6) were cultured in DMEM (Gibco, United States) containing 10% fetal bovine serum (Gibco, United States) and a 1% mixture of 100 U/mL streptomycin and penicillin (Gibco, United States) in a 5% CO₂ incubator at 37°C.

IEC-6 cells were implanted into six-well plates at a density of 3 × 10⁵/well and divided into five groups (n = 3): control group, TGF-β1-treated group (TGF-β1), and three monotropein-treated TGF-β1 groups (MON + TGF-β1 (5 µM, 10 µM, and 20 µM)). We used TGF-β1 (PeproTech, China) to induce EMT in IEC-6. The dosage of monotropein referred to the result of the cell viability test experiment and was combined with the effective dose of the pre-experiment. When the cells grew to 30%, they were cultured with TGF-β1 (10 ng/mL) to induce EMT for 72 h. During the establishment of the EMT model, cells were cultured with DMEM with 1% fetal bovine serum and a 1% mixture of 100 U/mL streptomycin and penicillin, along with gradient concentrations of monotropein, for 72 h.

A cell counting kit-8 (CCK-8; GLPBIO, United States) assay was used to measure cell viability. The experiment followed our previously described method (Lu et al., 2022).

In order to detect the expression levels of proteins related to EMT and the mTOR/P70S6K pathway, the colonic tissue and IEC-6 cells were processed by the general Western blot steps. The equivalent proteins were separated with a 10% SDS-polyacrylamide gel and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were cut horizontally at the appropriate location according to the molecular weight of the target proteins. Next, the membranes were blocked for 2 h and incubated with a suitable concentration of primary and secondary antibodies (Table 1). Finally, bands were washed with TBST and examined by ECL (Millipore, United States). The intensities of bands were calculated by ImageJ.

The total RNA of cells was extracted with the RNAex Pro reagent (Accurate Biotechnology, China). Next, an Evo M-MLV RT Kit with gDNA Clean for qPCR II (Accurate Biotechnology, China) was used to reverse-transcribe RNA samples to cDNA. The cDNA was amplified with the SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology, China) and target gene primers (Table 2) in a detection system (CFX96 TouchTM, Bio-Rad, United States). The reaction condition was as follows: 95°C for 30 s, 40 cycles of 95°C for 5 s, and 60°C for 30 s. Quantification was conducted using the 2^−△△Ct method. All target genes were normalized with GAPDH (Sangon Biotech, China).

The CETSA experiment followed our previously described method (Chen et al., 2024; Liu et al., 2024).

The DARTS assay followed our previously described method (Chen et al., 2024).

Molecular docking was performed with AutoDock Vina 1.0.2. Molecular docking and kinetic simulations were performed as described in previous articles (Chen et al., 2024; Chen et al., 2022).

All data were expressed as mean ± standard deviation (SD). The differences within the group comparison were analyzed by a one-way ANOVA test followed by Tukey’s test. P < 0.05 was considered to indicate significant differences.

To investigate the impact of monotropein on chronic colitis, a mouse model of the disease was established using 2% DSS, with 5-ASA or monotropein administered during the second and third modeling cycles (Figure 1B). During the second cycle of modeling, the DSS-treated group exhibited significant weight loss compared with the control group (Figure 1C). However, both 5-ASA and various doses of monotropein significantly mitigated this weight loss when compared to the model group (Figure 1C). In both the second and third cycles, the DSS group experienced severely loose and bloody stools, with a marked increase in DAI scores compared to the control group (Figure 1D). Conversely, the mice treated with 5-ASA or monotropein dramatically reduced hemorrhage and exhibited decreased loose, bloody, and DAI scores (Figure 1D). In the long-term intake of DSS, the DSS-treated group showed severe colon atrophy, congestion, and edema, in which the length of the colon was significantly shorter, and the thickness of the colon significantly increased compared to that of the NC group (Figures 1E–G). However, 5-ASA or monotropein dramatically alleviated colon shortening and thickening. In chronic colitis mice, the arrangement of colon epithelial cells was disordered, and their morphology was changed. The recess structure was destroyed, resulting in the loss of colon structure. These changes led to an increased histopathological score (Figure 1H). Treatment with 5-ASA or monotropein markedly improved colonic injury, as evidenced by an increased number of normal epithelial cells and intact crypts (Figure 1H). These results suggest that monotropein can potentially alleviate colonic injury in mice with chronic colitis.

To investigate the impact of monotropein on modulating chronic colitis in mice, we assayed the serum levels of inflammatory factors in each group. Results indicated that the concentrations of TNF-a and IL-6 in the DSS-treated group significantly increased, and the concentration of IL-10 significantly decreased compared to those in the control group (Figures 2A–C). Conversely, both 5-ASA and monotropein could substantially decrease the expression of TNF-α and IL-6 and increase the expression of IL-10 in mice with chronic colitis (Figures 2A–C). Furthermore, we assessed the effect of monotropein on intestinal permeability in mice with chronic colitis. As illustrated in Figure 2D, compared to the control group, the levels of serum FITC-dextran were significantly increased in DSS-induced chronic colitis mice. Immunofluorescence results showed that the expressions of occludin and ZO-1 in the DSS-treated group were markedly reduced compared to that in the control group (Figure 2E). Monotropein treatment in chronic colitis mice could increase the expression of occludin and ZO-1 in a dose-dependent manner (Figure 2E). These findings suggested that monotropein could ameliorate inflammation and decrease intestinal permeability in mice with chronic colitis.

Chronic inflammation of the intestine is characterized by repetitive damage and subsequent repair of intestinal tissue, ultimately resulting in the initiation and progression of intestinal fibrosis. Sirius red staining and Masson staining were performed on colon tissue to study the degree of intestinal fibrosis in each group of mice. The findings revealed that the colon tissue of mice exposed to DSS for an extended period exhibited a notable increase in fiber deposition. Among all groups, the DSS-treated group displayed the most severe symptoms (Figures 3A–C). Compared to the DSS-treated group, the fiber deposition of colon tissue in the 5-ASA or monotropein treatment groups improved to different degrees (Figures 3A–C). Immunohistochemical methods were used to analyze the signature proteins of the epithelial–mesenchymal transition. The results indicated that, compared to the control group, the DSS group exhibited a significant increase in the positive expression of α-smooth muscle actin (α-SMA) protein (Figures 3A, D). Compared to the DSS group, the positive expression of a-SMA protein in the monotropein treatment groups decreased to different degrees (Figures 3A, D). These findings imply that monotropein mitigates intestinal fibrosis and epithelial–mesenchymal transition in mice with chronic colitis.

To elucidate the mechanism through which monotropein inhibits epithelial–mesenchymal transition, we assessed the expression of proteins within the mTOR/P70S6K pathway, as well as the autophagy-associated protein Beclin1. Results showed that the phosphorylation of mTOR and its downstream proteins P70S6K and 4EBP1 were markedly increased in the DSS-treated group (Figures 4A–D). After monotropein treatment, the ratios of p-mTOR/mTOR, p-P70S6K/P70S6K, and p-4EBP1/4EBP1 significantly decreased (Figures 6A–D). Immunofluorescence results showed that the expression of autophagy-associated protein Beclin1 significantly decreased in the DSS-treated group, while monotropein treatment led to an increase in Beclin1 expression (Figure 4E). It should be noted that 5-ASA did not significantly regulate the expression of mTOR/P70S6K pathway proteins and Beclin1 (Figures 4A–E). These findings suggest that monotropein may inhibit epithelial–mesenchymal transition in chronic colitis by modulating autophagy through the mTOR/P70S6K pathway.

We investigated the inhibitory effect of monotropein on epithelial–mesenchymal transition (EMT) in vitro, as well as the cytotoxicity of the drug on IEC-6 cells. Throughout the modeling process, the growth rate of IEC-6 cells accelerated, and they progressively adopted a fibroblast-like morphology (Figure 5A). The results demonstrated that TGF-β1-induced IEC-6 cells lost their epithelial phenotype and expressed a mesenchymal cell phenotype. Monotropein mitigated the loss of the epithelial phenotype in TGF-β1-induced IEC-6 cells (Figure 5A). The results indicated that TGF-β1-induced IEC-6 cells lost the epithelial phenotype and expressed a mesenchymal cell phenotype. Monotropein improved the loss of the epithelial phenotype in TGF-β1-induced IEC-6 cells (Figure 5A).

The cell viability test results showed that there was no cytotoxicity for monotropein to IEC-6 cells at any concentration up to 200 µmol/L (Figure 5B). Accordingly, the relative protein levels of E-cadherin markedly decreased, and the relative protein levels of vimentin and a-SMA increased (Figures 5C–F). The relative mRNA levels of E-cadherin significantly reduced, and the relative mRNA levels of Vimentin and a-SMA remarkably increased in the TGF-β1 group (Figures 5G–I). Monotropein could significantly upregulate the levels of E-cadherin mRNA and downregulate the levels of vimentin and a-SMA mRNA (Figures 5G–I). Consequently, it markedly increased the expression in the α-SMA protein (Figures 5C–F). Corresponding to the effects observed in vivo, these results revealed that monotropein could also effectively inhibit EMT in vitro.

Molecular docking was conducted using AutoDock to explore the interaction between MON and mTOR. The results revealed that the mTOR-MON complex exhibited a robust binding affinity, with an average binding energy of −7.24 ± 0.12 kcal/mol (Figures 6A, B). To evaluate the binding stability of mTOR and MON, 100-ns MD simulations were executed on the docking-optimized mTOR-MON complex. The radius of gyration (Rg) affirmed the tightness of the complex, remaining relatively constant throughout the 100-ns simulations, with minor fluctuations observed between 50 and 80 ns (Figure 6C).

The overall conformational change was quantified by the root mean square deviation (RMSD). Lower RMSD values suggest a more stable protein–ligand complex. Figures 6D, E show that the RMSD of AS ranged between 1.50 Å and 2.00 Å throughout the simulation, while the RMSD of the protein varied between 2.50 Å and 8.00 Å. The relatively minor RMSD fluctuations during the MD simulations indicated the reliability of the docking poses (Figures 6D, E). Root mean square fluctuation (RMSF) analysis demonstrated that most protein residues remained stable (Figure 6F).

Solvent-accessible surface area (SASA) characterized the accessible surface area of the protein solvent that could be directly contacted over time (Figure 6G). Hydrogen bonding also plays a pivotal role in stabilizing protein–ligand interactions. During MD simulations, MON and mTOR formed stable hydrogen bonds, with the number of hydrogen bonds fluctuating between two and five, averaging four within 100 ns (Figure 6H), further validating the stability of the mTOR-MON complex. To pinpoint the key amino acid residues involved in mTOR protein binding, energetic decomposition of amino acid residues was performed. Figure 6I displayed the amino acid residues with energy contribution values below −1 kcal/mol, highlighting PHE316 as the primary contributor to binding free energy (Figure 6I).

The binding of drugs to target proteins induces conformational changes that alter the proteolytic and thermal stability of the target proteins. To further confirm the interaction potential between MON and mTOR, we used the DARTS and CETSA assays. The DARTS assay showed that mTOR undergoes approximately 70% proteolytic hydrolysis when MON is exposed to a pronase-to-total-protein ratio of 1:100. MON inhibited the proteolytic breakdown of mTOR in a dose-dependent manner until MON reached 20 μM (Figure 6J). CETSA results showed that MON significantly enhanced the thermal stability of mTOR under different temperature changes in a dose-dependent manner compared with the control. MON incubation also resulted in a significant increase in the thermal stability of mTOR compared with the control (Figure 6K).

We evaluated the expression of the mTOR/P70S6K pathway and autophagy-associated proteins and genes, and the relationship between inhibition of EMT and regulation of autophagy was detected by immunofluorescence. Our findings indicated that TGF-β1 significantly induced the phosphorylation of mTOR and upregulated the ratios of p-p70S6K/p70S6K and p-4EBP1/4EBP1, effects that were reversed by monotropein (Figures 7A–D). Accordingly, the protein expression of autophagy marker genes BECN1 and LC3 remarkably decreased (Figures 7E–G). Conversely, monotropein significantly increased their protein expression compared to the TGF-β1 group (Figures 7E–G). Immunofluorescence analysis revealed that in the TGF-β1 group, the expression of E-cadherin protein was diminished, and Beclin1 protein expression was also reduced (Figure 7H). After monotropein administration, the expressions of E-cadherin and Beclin1 protein increased in a dose-dependent manner (Figure 7H). Collectively, these results imply that TGF-β1 exposure suppressed autophagy in IEC-6 cells and that monotropein can counteract this suppression to inhibit EMT.