Lespedeza bicolor Turcz. Honey Prevents Inflammation Response and Inhibits Ferroptosis by Nrf2/HO-1 Pathway in DSS-Induced Human Caco-2 Cells

Lespedeza bicolor Turcz. (L. bicolor) honey, a monofloral honey, has garnered increased attention due to its origin in the L. bicolor plant. A previous study has shown that L. bicolor honey can ameliorate inflammation. In this study, we aimed to investigate the effects of L. bicolor honey extract and its biomarker (Trifolin) on DSS-induced ulcerative colitis (UC). Our results demonstrated that L. bicolor honey extract and Trifolin significantly increased the expression levels of the tight junction cytokines Claudin-1 and ZO-1. Additionally, they decreased the pro-inflammatory factors TNF-α and IL-6 and enhanced the antioxidant factors NQO1 and GSTA1. Based on metabolomic analyses, L. bicolor honey extract and Trifolin regulated the progression of UC by inhibiting ferroptosis. Mechanistically, they improved the levels of SOD and iron load, increased the GSH/GSSG ratio, reduced MDA content and ROS release, and upregulated the Nrf2/HO-1 pathway, thereby inhibiting DSS-induced UC. Moreover, the expression levels of ferroptosis-related genes indicated that they decreased FTL, ACSL4, and PTGS2 while increasing SLC7A11 expression to resist ferroptosis. In conclusion, our study found that L. bicolor honey improves DSS-induced UC by inhibiting ferroptosis by activating the Nrf2/HO-1 pathway. These findings further elucidate the understanding of anti-inflammatory and antioxidant activities of L. bicolor honey.


Introduction
Honey is a sweet substance collected by bees from plant nectar, secretions, or insect secretions, which they store and fully brew into the hive [1].It contains various nutrients, including different types of sugars, proteins, organic acids, and phenolic compounds [2].Consequently, many types of honey have been used to treat diseases such as IBD, atherosclerosis, stomatitis, and sinusitis [3][4][5][6].
In recent years, monofloral honey has gained significant attention from consumers due to its unique nutritional properties.Lespedeza bicolor Turcz.(L.bicolor) honey, derived from the L. bicolor plant, is considered a typical medicinal honey.This plant is known for its functions in clearing heat, detoxifying, promoting blood circulation, removing blood stasis, moisturizing the lungs, and relieving cough [7].According to our previous study, L. bicolor honey exhibited rich in phenolic active ingredients (such as chlorogenic acid, ferulic acid, vitexin, rutin, gallic acid, myricitrin, morin, trifolin, glycitein, wogonin, butin and liquiritigenin) for playing anti-inflammatory activity by regulating sphingolipid metabolism and necroptosis pathway in an LPS-induced mouse macrophage RAW 264.7 cell model [8].Additionally, several studies have found that adding wildflower honey to oral rehydration solutions can effectively improve the symptoms of gastroenteritis in children under five years old [9].Through in vitro intestinal simulations using human fecal microbiota, wildflower honey has been shown to regulate intestinal inflammation by increasing the abundance of beneficial Lactobacillus, reducing the abundance of harmful Gram-negative bacteria, decreasing the production of short-chain fatty acids, and inhibiting the expression of pro-inflammatory factors [10].These studies indicate that specific honey is effective in preventing and treating human inflammatory diseases.
Ulcerative colitis (UC) is a chronic inflammatory disease of the gastrointestinal tract that primarily affects the colon [11].The exact cause of UC remains unclear, but it often presents with symptoms such as abdominal pain and diarrhea [11].UC is challenging to cure and carries the risk of progressing to cancer [12].At present, Caco-2 cells induced by DSS are widely used as a cell model for exploring UC.It has been reported that DSStreated Caco-2 cells disrupt cell homeostasis, accompanied by membrane and protein trafficking impairment, which leads to membrane integrity, cell polarity alteration, and barrier dysfunction [13].Several studies have reported that honey can improve DSSinduced UC by significantly reducing the levels of the pro-inflammatory factors IL-6, TNF-α, and TGF-β1 while upregulating the expression level of IkB-α [14].Additionally, research has shown that honey can alleviate UC by enhancing the antioxidant capacity of cells, including increasing the levels of SOD and GSH-Px [15].This improvement helps resist intestinal oxidative stress and removes reactive oxygen species (ROS) that accumulate due to disruption of the intestinal epithelial barrier [15].
Ferroptosis is a unique form of cell death linked to oxidative stress and dysregulated iron metabolism [16].The primary mechanism of ferroptosis involves an imbalance between the generation and degradation of lipid ROS in cells [17].In this process, the inhibition of glutathione peroxidase (GPX4) reduces the antioxidant capacity of cells, leading to ROS accumulation and oxidative cell death [18].During inflammation, cells secrete inflammatory mediators, such as TNF-α, which contribute to ROS formation, phospholipid peroxidation, and the continuous downregulation of GPX4, thereby accelerating ferroptosis [18].Recent studies have shown that ferroptosis can alleviate DSS-induced colitis by interfering with the Nrf2/HO-1 signaling pathway [15].Additionally, it can inhibit endoplasmic reticulum stress through the NF-κB pathway, significantly reducing ferroptosis and alleviating colitis [19].These findings suggest that blocking the pathogenic pathway of ferroptosis can lead to UC remission [19].Honey contains functional active substances, such as flavonoids, polyphenols, and amino acids, which exhibit strong antioxidant and anti-inflammatory effects [2].Studies have found that kaempferol can inhibit ferroptosis by activating the Nrf2/SLC7A11/GPX4 signaling pathway, while galanin can do so by activating the SLC7A11/GPX4 axis [20,21].Baicalin and safflomin A have been shown to inhibit ferroptosis by upregulating GPX4, inhibiting lipid peroxidation, and reducing iron accumulation [22].Given the abundance of these functional substances in honey, which can significantly inhibit ferroptosis, it is essential to further explore honey as a natural product with potential therapeutic value for combating ferroptosis.
In this study, we used a DSS-induced Caco-2 cell model to investigate the protective effects of L. bicolor honey against UC.Specifically, we focused on how L. bicolor honey activates the Nrf2/HO-1 signaling pathway to inhibit ferroptosis.Our evaluation confirmed the anti-inflammatory properties of L. bicolor honey in the context of UC.This research provides foundational scientific data on the medicinal value of L. bicolor honey and offers a new perspective for UC treatment.

Honey Sample Preparation
Lespedeza bicolor Turcz.honey samples were collected from the northern margin and southeastern hilly area of the Great Khingan Mountains (Heilongjiang, China) from 1 August to 31 August 2020.Based on our previous research, we have confirmed L. bicolor honey samples as monofloral honey [23].The extraction procedure for L. bicolor honey was modified based on a previously published method [23].We started by fully diluting a 5 g L. bicolor honey sample with 10 mL of ultrapure water and vortexed for 10 min.Subsequently, the sample underwent centrifugation at 8000× g for 5 min to collect the supernatant.Polyphenol compounds in the honey were then collected using SPE-C 18 cartridges, followed by elution using MeOH.The eluted solution was dried using nitrogen gas.Finally, the dried extracts were dissolved in ultrapure water to obtain a stock solution (at 200 mg/mL) for further study.

Cell Culture and Cell Viability Assay
Caco-2 cells were obtained from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China).The cells were incubated in DMEM supplemented with 10% FBS (v/v), 100 U/mL penicillin, and 100 µg/mL streptomycin and maintained in a 5% CO 2 incubator at 37 • C. Cell viability was assessed using the cell counting kit-8 (CCK-8) from Dojindo, Inc. (Kumamoto, Japan), following the manufacturer's instructions.

Total RNA Isolation and Quantitation
Caco-2 cells were seeded in 6-well plates at a density of 1 × 10 5 cells/mL.When cell confluence reached ≥70%, the cells were pretreated with Fer-1 (10 µM), Trifolin (10 µM), and various concentrations of L. bicolor honey extract (50, 100, and 200 µg/kg).After 2 h, the cells were stimulated with 2.5% DSS (wt%) for 24 h.The cell medium was discarded, and the cells were washed twice with PBS.Total RNA was extracted from the cells using an RNA extraction system with TRIzol reagent.RNA concentration was measured using a NanoDrop 2000 ultra-micro spectrophotometer from Thermo Fisher Scientific (Pittsburgh, PA, USA).cDNA was synthesized from 1000 ng of total RNA in a 10 µL reaction volume using the PrimeScript RT Reagent Kit (Dalian, China).Quantitative real-time PCR was performed using a 7500c Real-Time PCR Detection System (Hangzhou, China).Gene primers used to amplify selected cytokine transcripts are listed in Table S1.β-actin served as the housekeeping gene for normalization of expression levels.The reaction efficiency of RT-qPCR was shown in Figure S1.The 2 −∆∆Ct method was employed to calculate the relative expression levels of the target genes.

Collection of Metabolites from Cells
Caco-2 cells were seeded and pretreated as described in Section 2.3.2.The cell medium was discarded, and cells were washed twice with PBS.Subsequently, 1 mL of a mixed solution of methanol, acetonitrile, and ultrapure water (2:2:1, v/v/v) was added to the cells, vortexed for 1 min, and centrifuged at 10,000× g for 5 min at 4 • C. The supernatant was transferred to a new tube and dried using nitrogen gas.Finally, the cellular extracts were re-dissolved in 200 µL of a mixed solution of acetonitrile and ultrapure water (1:1, v/v) for UHPLC/Q-TOF-MS analysis.
For mass spectrometry, the parameters in the negative ionization mode were set as follows: gas temperature, 325 • C; gas flow, 10 L/min; nebulizer pressure, 35 psi; sheath gas temperature, 370 • C; sheath gas flow, 12 L/min; VCap, 3500 V; fragmentor voltage, 135 V.The acquisition range was m/z 100-1700.The reference ions were set at 112.985587 and 1033.988109 in the negative mode.

Protein Extraction and Analysis of Oxidative Stress Indexes
Caco-2 cells were seeded and pretreated as described in Section 2.3.2.Cells were washed twice with PBS.Caco-2 cells were lysed using 80 µL of NP40 Buffer with a phosphatase inhibitor cocktail.The protein concentration was determined using the BCA Protein Assay Kit from Beyotime Biotechnology Co., Ltd.(Shanghai, China), according to the manufacturer's instructions.
Oxidative stress markers included superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, total iron content, and glutathione (GSH) content.Kits used for analysis included the Total Superoxide Dismutase Assay Kit with WST-8, Lipid Peroxidation Assay Kit, and Cellular Glutathione Peroxidase Assay Kit with DTNB, all from Beyotime Biotechnology Co., Ltd.(Shanghai, China).The total iron content in the cells was determined using an iron assay kit from Yuanye Biological Technology Co., Ltd.(Shanghai, China).These kits were used to measure SOD activity, MDA content, iron ion content, and GSH content in Caco-2 cells, according to the manufacturer's instructions.

Detection of Cellular ROS Generation
Caco-2 cells were seeded and pretreated as described in Section 2.3.2.The cell slides were washed with PBS and then incubated with 10 µM DCFH-DA for 30 min at 37 • C. Subsequently, the cells were washed twice with PBS and examined using a confocal laser scanning microscope from Leica Camera AG (Hessian, Germany) with excitation/emission at 480/520 nm.

Statistical Analysis
Variance testing was performed using IBM SPSS version 26.0 software (New York, NY, USA).Data were presented using GraphPad Prism 8.0 (San Diego, CA, USA).Agilent MassHunter Profinder B.10.0 software from Agilent Technologies Co., Ltd.(Santa Clara, CA, USA) was used to convert the data acquisition.dfiles into .ceffiles.Data analysis was conducted using Agilent Mass Profiler Professional 15.0 software (MPP) from Agilent Technologies Co., Ltd.(Santa Clara, CA, USA), with the parameters set according to previous research [23].

Effect of Different Treatments on Caco-2 Viability
To ensure that all treatments did not cause obvious toxic effects on Caco-2 cell growth and metabolism, we used the CCK-8 assay to assess the impact of different concentrations of L. bicolor honey extract (50, 100, 200, and  Technologies Co., Ltd.(Santa Clara, CA, USA), with the parameters set according to previous research [23].

Effect of Different Treatments on Caco-2 Viability
To ensure that all treatments did not cause obvious toxic effects on Caco-2 cell growth and metabolism, we used the CCK-8 assay to assess the impact of different concentrations of L. bicolor honey extract (50, 100, 200, and

Effect of Different Treatments on Expression of Relative Tight Junction Cytokines
DSS-induced UC stimulates the colonic mucosa and increases intestinal barrier permeability, resulting in downregulation of tight junction proteins that act as intestinal barriers, such as ZO-1, ZO-2, Occludins, and Claudins [24].We established a model of DSS-induced intestinal barrier dysfunction in Caco-2 cells.L. bicolor honey extract, which contains Trifolin as its primary biomarker [23], was used to pretreat Caco-2 cells to assess its effect.
The mRNA transcription levels of tight junction cytokines ZO-1 and Claudin-1 were measured by qPCR to investigate the protective effect of L. bicolor honey on intestinal barrier integrity.The results are shown in Figure 1B.Initially, cells were pretreated with 2.5% DSS, which disrupted the integrity of the monolayer and significantly downregulated ZO-1 and Claudin-1 expression (p < 0.001).In the Blank group, their expression levels were significantly higher than those in the DSS-treated group (p < 0.001).Subsequently, Caco-2 cells were incubated with different concentrations of L. bicolor honey extract (50, 100, and 200 µg/kg) and 10 µM Trifolin.Compared to the DSS group, the expression of the tight junction cytokine was significantly upregulated in the treatment groups (p < 0.001), with a concentration-dependent effect observed for L. bicolor honey.A decrease in the expression levels of Claudin-1 and ZO-1 can lead to the impairment of tight junction function, improve the permeability of intestinal tissue, and eventually lead to the occurrence of various systemic infectious diseases and even tumors.The increase in the expression levels of Claudin-1 and ZO-1 means a significant inhibitory effect on the development of inflammatory diseases.Additionally, the effect of 10 µM Trifolin was not significantly different from that of 100 µg/kg L. bicolor honey extract, suggesting similar efficacy between the two treatments.In conclusion, L. bicolor honey extract and Trifolin have a significant protective effect on the tight junctions of Caco-2 cells.

Effect of Different Treatments on Inflammation Cytokines Expression
UC pathogenesis stimulates an inflammatory response, causing damage to the intestinal mucosa, compromising the intestinal mucosal barrier, and facilitating the invasion of external toxins and microorganisms, resulting in the release of endotoxins.This process stimulates the expression of inflammatory factors, such as TNF and interleukin (IL); compared with the Blank group, its expression was significantly increased (p < 0.001), thereby exacerbating intestinal damage [25].TNF-α and IL-6 are critical transcription factors that regulate these inflammatory responses [14].TNF-α, in particular, is the main pro-inflammatory factor contributing to intestinal barrier dysfunction, while IL-6 is synthesized under inflammatory conditions and exacerbates intestinal inflammation [14].In the 2.5% DSS-induced Caco-2 cell monolayer inflammation model, Caco-2 cells were pretreated with different concentrations of L. bicolor honey extract (50, 100, and 200 µg/kg) and 10 µM Trifolin.The mRNA transcription levels of the inflammatory mediators IL-6 and TNF-α were assessed using qPCR technology to investigate the inhibitory effect of L. bicolor honey on the intestinal inflammatory response.The results are presented in Figure 1C.Initially, 2.5% DSS was used to induce an inflammatory response in the Caco-2 cells.
Compared to the Blank group, the inflammatory mediators IL-6 and TNF-α were significantly upregulated (p < 0.001).Subsequently, Caco-2 cells were treated with different concentrations of L. bicolor honey extract (50, 100, and 200 µg/kg) and 10 µM Trifolin.The expression of both inflammatory mediators was significantly downregulated compared to that in the DSS group (p < 0.001), with the effect showing a concentration-dependent response to L. bicolor honey.Additionally, the effect of 10 µM Trifolin and 100 µg/kg L. bicolor honey extract on IL-6 showed no significant difference, and similarly, the effects of 10 µM Trifolin and 50 µg/kg L. bicolor honey extract on TNF-α were not significantly different.These results indicate that Trifolin and L. bicolor honey extract have similar effects in reducing inflammatory factors.TNF-α and IL-6 are important cytokines involved in the maintenance and dynamic balance of the immune system, and they can regulate inflammation and host defense.The expression of TNF-α and IL-6 was disordered under the stimulation of DSS but tended to be normal after treatment with L. bicolor honey and Trifolin.In conclusion, L. bicolor honey extract and Trifolin significantly downregulate the expression levels of the inflammatory mediators IL-6 and TNF-α during the inflammatory response, thereby regulating the inflammatory process.

Effect of Different Treatments on Antioxidant Factors Expression
The pathogenesis of UC is closely linked to the oxidative stress response [26].Upon activation by oxidative stress, cells produce numerous reactive oxygen species and electrophiles, disrupting the original redox balance [27].Quinone oxidoreductase 1 (NQO1) plays a crucial role in reducing oxidative stress by regulating reduced coenzyme Q and vitamin E [28].GSTA1, a member of the glutathione transferase (GST) family, is one of the most abundant detoxification enzymes.Its expression enhances cellular fusion, influencing the cell's response to oxidative stress [29].
To establish a monolayer oxidative stress model, Caco-2 cells were induced with 2.5% DSS.Different concentrations of L. bicolor honey extract (50, 100, and 200 µg/kg) and 10 µM Trifolin were used for pretreatment.The mRNA expression levels of the antioxidant factors NQO1 and GSTA1 were assessed using qPCR technology.The results are shown in Figure 1C.The expression levels of NQO1 and GSTA1 in Caco-2 cells induced by 2.5% DSS were significantly upregulated compared to those in the Blank group (p < 0.001).Furthermore, compared to the DSS group, the expression of NQO1 and GSTA1 was significantly upregulated in Caco-2 cells treated with different concentrations of L. bicolor honey extract and Trifolin (p < 0.01), demonstrating a concentration-dependent effect of L. bicolor honey.Cells under the effect of DSS stimulate the oxidative stress reaction.GSTA1 can convert and metabolize reactive oxygen species (ROS) to control cell damage by free radicals and peroxides in the oxidative stress response.GSTA1 and NQO1 are antioxidant factors that maintain redox homeostasis.The antioxidant effect of 10 µM Trifolin and 100 µg/kg L. bicolor honey extract on NQO1 and GSTA1 showed no significant difference, indicating a similar antioxidant effect between the two treatments.In conclusion, L. bicolor honey extract and Trifolin play a regulatory role in the antioxidant response to DSS-induced oxidative stress and maintain cellular redox homeostasis.

Effect of Different Treatments on Cell Metabolism
The PLS-DA model in Figure 2A demonstrated significant changes in Caco-2 cells after 2.5% DSS induction compared to the Blank group (p < 0.001).Compared to the DSS group, cell metabolites were significantly altered following treatment with 100 µg/kg L. bicolor honey extract (p < 0.001) and 10 µM Trifolin (p < 0.001).This indicates that L. bicolor honey extract and Trifolin can mitigate the effects of UC induced by DSS.Subsequently, we utilized a volcano plot to compare differential metabolites between L. bicolor honey extract and Trifolin-treated groups with the DSS group, as well as between the Blank group and DSS group, as shown in Figure 2B.Differential metabolites were filtered based on FoldChange ≥ 2 or FoldChange ≤ 0.5 and differential metabolites with VIP ≥ 1, as determined by the PLS-DA model [30].Additionally, differential metabolites were further filtered based on p < 0.05 (ANOVA) to identify compounds, as shown in Table 1.The distribution of content in each group of these differential metabolites is depicted in Figure 2C.
The differential metabolites were mapped to the KEGG Pathway database to elucidate their involvement in cellular pathways.The top 20 enriched pathways are depicted in Figure 2D.The significance of these enriched pathways was determined by calculating their p value and RichFactor using the KEGG Pathway database.A smaller p value and a higher RichFactor indicate a greater degree of influence of the pathway [31].Additionally, the numerical value assigned to each pathway indicates the number of compounds affected, with a higher number reflecting a more substantial impact on the pathway [31].Through this analysis, pathways highly influential and associated with intestinal inflammation were scrutinized, ultimately identifying ferroptosis as a significant pathway [15].
Table 1 presents the differential metabolites observed in Caco-2 cells induced by DSS, in which pretreatment with 100 µg/kg L. bicolor honey extract and 10 µM Trifolin primarily influenced the levels of glutamate, cystine, GSH, and GSSG.Compared to the Blank group, the expression of cystine was downregulated, while the expression of other substances was upregulated in the DSS group.Following pretreatment with L. bicolor honey extract and Trifolin, the expression of these substances was downregulated compared to that in the DSS group.These findings suggest that both L. bicolor honey extract and Trifolin attenuate the ferroptosis process in DSS-induced colitis.The differential metabolites were mapped to the KEGG Pathway database to elucidate their involvement in cellular pathways.The top 20 enriched pathways are depicted in Figure 2D.The significance of these enriched pathways was determined by calculating their p value and RichFactor using the KEGG Pathway database.A smaller p value and a higher RichFactor indicate a greater degree of influence of the pathway [31].Additionally, the numerical value assigned to each pathway indicates the number of compounds affected, with a higher number reflecting a more substantial impact on the pathway [31].Through this analysis, pathways highly influential and associated with intestinal inflammation were scrutinized, ultimately identifying ferroptosis as a significant pathway [15].
Table 1 presents the differential metabolites observed in Caco-2 cells induced by DSS, in which pretreatment with 100 µg/kg L. bicolor honey extract and 10 µM Trifolin primarily influenced the levels of glutamate, cystine, GSH, and GSSG.Compared to the Blank group, the expression of cystine was downregulated, while the expression of other substances was upregulated in the DSS group.Following pretreatment with L. bicolor honey extract and Trifolin, the expression of these substances was downregulated compared to that in the DSS group.These findings suggest that both L. bicolor honey extract and Trifolin attenuate the ferroptosis process in DSS-induced colitis.Amino acid metabolism plays a pivotal role in the intricate pathways of ferroptosis [32].Oxidative stress induced by excess glutamate stands as a key initiator in the activation of ferroptosis pathways [33].Fueled by heightened concentrations of glutamate within cells, System Xc, situated on the cell membrane, orchestrates the transport of extracellular cystine and intracellular glutamate [34].When cells encounter oxidative stress, the extracellular glutamate levels surge, thereby hindering the transport functionality of System Xc [34].As shown in Table 1 and Figure 2C, following DSS induction in Caco-2 cells, there was a downregulation in cystine expression and an upregulation in glutamate expression.These findings underscore that escalated extracellular glutamate under oxidative stress curtails the transport capabilities of System Xc, consequently diminishing cystine uptake rates and impeding glutamate exportation from cells, leading to intracellular accumulation.
Table 1 exhibits the downregulation of intracellular and extracellular glutamate expression subsequent to treatment with L. bicolor honey extract and Trifolin.This observation suggests mitigation of the oxidative stress response in Caco-2 cells, resulting in reduced extracellular glutamate levels and restoration of System Xc transport functionality, thereby decreasing intracellular glutamate levels.However, compared to the DSS group, the downregulation of intracellular cystine expression suggests that L. bicolor honey extract and Trifolin may not fully restore Caco-2 cells to their normal state.

Effect of Different Treatments on Oxidative Stress
Previous studies have demonstrated that MDA, GSH, and iron ion loading serve as three markers of ferroptosis [35].A key characteristic of ferroptosis is lipid peroxidation, resulting in the cytotoxic byproduct MDA [35].As illustrated in Figure 3B, compared to the Blank group, the MDA content in Caco-2 cells significantly increased following induction with 2.5% DSS (p < 0.001).Pretreatment of Caco-2 cells with 100 µg/kg L. bicolor honey extract and 10 µM Trifolin markedly decreased MDA content compared to the DSS group (p < 0.001).Moreover, there was no significant difference in MDA inhibition between the L. bicolor honey extract and Fer-1 groups.These findings suggest that L. bicolor honey extract and Trifolin can alleviate DSS-induced colitis by reducing MDA content and inhibiting lipid peroxidation induced by inflammation.
Antioxidants 2024, 13, x FOR PEER REVIEW 11 of 17 Caco-2 cells was significantly higher compared to that in the Blank group (p < 0.001).However, pretreatment with 100 µg/kg L. bicolor honey extract and 10 µM Trifolin significantly decreased the iron ion content compared to the DSS group (p < 0.001).Furthermore, there was no significant difference between the L. bicolor honey extract treatment group and the Fer-1 treatment group.These findings suggested that L. bicolor honey extract and Trifolin protect against DSS-induced colitis by reducing intracellular iron ion levels and inhibiting ferroptosis.SOD is a crucial antioxidant metalloenzyme in the body, playing a pivotal role in maintaining the balance between oxidation and antioxidation [40].Previous studies have demonstrated its significant role in colitis by scavenging superoxide free radicals, thereby inhibiting inflammation and apoptosis and ameliorating colitis [41].A colitis model was established in Caco-2 cells by stimulation with 2.5% DSS.Compared to the Blank group, the SOD content in the DSS group was significantly decreased (p < 0.001).Pretreatment of Caco-2 cells with 100 µg/kg L. bicolor honey extract and 10 µM Trifolin resulted in a substantial increase in SOD content compared to the Blank group.Moreover, L. bicolor honey extract significantly increased the SOD content compared to that in the Fer-1 group (p < 0.05).These findings indicate that L. bicolor honey extract can modulate SOD content, Excessive accumulation of peroxide in cells is directly associated with ferroptosis [36].As shown in Table 1, the expression of GSH and GSSG was upregulated following DSS induction, which is attributable to the oxidative stress response that generates peroxide in cells [37].Consequently, GSH levels were elevated, providing hydrogen electrons and participating in redox reactions, thereby leading to increased GSSG production.Pretreatment of Caco-2 cells with L. bicolor honey extract inhibited the inflammatory response, resulting in the downregulation of GSH and GSSG expression.These findings indicate that L.
bicolor honey possesses antioxidant capacity, promoting peroxidase removal from cells and adjusting GSH biosynthesis and metabolic disorders to resist DSS-induced oxidative stress.
Additionally, we evaluated the GSH/GSSG ratio, as shown in Figure 3E.After DSSinduced Caco-2 cells, compared to the Blank group, higher levels of oxidative stress were observed, leading to a decreased GSH/GSSG ratio (p < 0.001).Pretreatment with L. bicolor honey extract and Trifolin significantly increased the GSH/GSSG ratio compared to the Blank group (p < 0.001).These results further validate the findings of Section 3.5, demonstrating that L. bicolor honey extract and Trifolin have a detoxifying effect on DSS-induced Caco-2 cells and enhance cellular redox levels.It has been shown that L. bicolor honey extract and Trifolin protect GSH against oxidative and free radical-mediated cell damage.
Iron ions play a critical role in ferroptosis and are essential components of lipid peroxidases, catalyzing redox reactions in cells [38].Fe 2+ , due to its instability and high reactivity, can generate hydroxyl radicals via the Fenton reaction and directly react with polyunsaturated fatty acids in membranes, thereby producing a large number of ROS that lead to ferroptosis [38,39].Following induction with 2.5% DSS, the iron ion content in Caco-2 cells was significantly higher compared to that in the Blank group (p < 0.001).However, pretreatment with 100 µg/kg L. bicolor honey extract and 10 µM Trifolin significantly decreased the iron ion content compared to the DSS group (p < 0.001).Furthermore, there was no significant difference between the L. bicolor honey extract treatment group and the Fer-1 treatment group.These findings suggested that L. bicolor honey extract and Trifolin protect against DSS-induced colitis by reducing intracellular iron ion levels and inhibiting ferroptosis.
SOD is a crucial antioxidant metalloenzyme in the body, playing a pivotal role in maintaining the balance between oxidation and antioxidation [40].Previous studies have demonstrated its significant role in colitis by scavenging superoxide free radicals, thereby inhibiting inflammation and apoptosis and ameliorating colitis [41].A colitis model was established in Caco-2 cells by stimulation with 2.5% DSS.Compared to the Blank group, the SOD content in the DSS group was significantly decreased (p < 0.001).Pretreatment of Caco-2 cells with 100 µg/kg L. bicolor honey extract and 10 µM Trifolin resulted in a substantial increase in SOD content compared to the Blank group.Moreover, L. bicolor honey extract significantly increased the SOD content compared to that in the Fer-1 group (p < 0.05).These findings indicate that L. bicolor honey extract can modulate SOD content, restore cellular redox balance, and mitigate the ferroptosis process during DSS-induced cellular inflammation.
One characteristic of ferroptosis is the imbalance between the generation and degradation of lipid ROS in cells [38].This imbalance is primarily caused by the excessive accumulation of intracellular iron-dependent ROS and the reduced scavenging effect of GPX4 [42].When the antioxidant capacity of cells decreases and becomes insufficient to remove the excessive accumulation of lipid ROS, ferroptosis occurs [38].To evaluate this, we measured intracellular ROS levels.As depicted in Figure 4, fluorescence intensity was significantly higher in the DSS group compared to the Blank group, indicating activation of ferroptosis in Caco-2 cells under DSS induction.However, fluorescence intensity markedly decreased following pretreatment with 10 µM Fer-1, 100 µg/kg L. bicolor honey extract, and 10 µM Trifolin.This suggests that these treatments can inhibit lipid oxidation and reduce ROS levels, thereby regulating ferroptosis and mitigating the inflammatory response induced by DSS.

Effect of Different Treatments on Ferroptosis through Nrf2/HO-1 Pathway
The Nrf2/HO-1 pathway is a critical signaling pathway involved in the antioxidative stress response, and its upregulation can protect intestinal cells [43].When DSS-induced oxidative stress occurs in monolayer enterocytes, Nrf2 dissociates from KEAP1 and translocates to the nucleus, where it becomes activated [44].Once in the nucleus, Nrf2 binds to ARE and promotes the expression of downstream genes and antioxidant enzymes, such as NQO1 and HO-1, thereby regulating cellular homeostasis [45].measured intracellular ROS levels.As depicted in Figure 4, fluorescence intensity was significantly higher in the DSS group compared to the Blank group, indicating activation of ferroptosis in Caco-2 cells under DSS induction.However, fluorescence intensity markedly decreased following pretreatment with 10 µM Fer-1, 100 µg/kg L. bicolor honey extract, and 10 µM Trifolin.This suggests that these treatments can inhibit lipid oxidation and reduce ROS levels, thereby regulating ferroptosis and mitigating the inflammatory response induced by DSS.

Effect of Different Treatments on Ferroptosis through Nrf2/HO-1 Pathway
The Nrf2/HO-1 pathway is a critical signaling pathway involved in the antioxidative stress response, and its upregulation can protect intestinal cells [43].When DSS-induced oxidative stress occurs in monolayer enterocytes, Nrf2 dissociates from KEAP1 and translocates to the nucleus, where it becomes activated [44].Once in the nucleus, Nrf2 binds to ARE and promotes the expression of downstream genes and antioxidant enzymes, such as NQO1 and HO-1, thereby regulating cellular homeostasis [45].
As depicted in Figure 5, the induction of Caco-2 cells with 2.5% DSS significantly increased the expression of Nrf2 compared to the Blank group (p < 0.001).Treatment with 10 µM Fer-1, 100 µg/kg L. bicolor honey extract, and 10 µM Trifolin further increased Nrf2 expression compared to that in the DSS group.Moreover, there was no significant As depicted in Figure 5, the induction of Caco-2 cells with 2.5% DSS significantly increased the expression of Nrf2 compared to the Blank group (p < 0.001).Treatment with 10 µM Fer-1, 100 µg/kg L. bicolor honey extract, and 10 µM Trifolin further increased Nrf2 expression compared to that in the DSS group.Moreover, there was no significant difference in Nrf2 expression between the groups treated with 10 µM Fer-1 and 100 µg/kg L. bicolor honey extract.HO-1 is an important antioxidant enzyme that degrades heme, plays an antioxidant role, and breaks down biliverdin and its reduced products, such as bilirubin and iron ions [46].
HO-1 can remove ROS and participate in the process of ferroptosis through its antioxidant action [47].As illustrated in Figure 5, following stimulation with 2.5% DSS, the expression of HO-1 was significantly increased compared to that in the Blank group (p < 0.001).Subsequent pretreatment of cells with 10 µM Fer-1, 100 µg/kg L. bicolor honey extract, and 10 µM Trifolin further significantly increased HO-1 expression compared to the DSS group (p < 0.01).Moreover, there was no significant difference in HO-1 expression between the groups treated with 10 µM Fer-1 and 100 µg/kg L. bicolor honey extract.The expression level of Nrf2 can affect the expression of HO-1.In addition, HO-1, the product of the degradation of bilirubin nitric oxide and other Nrf2 regulation of genes for UC, has a regulatory role.These findings suggested that L. bicolor honey extract and Trifolin could regulate the ferroptosis process and protect against DSS-induced colitis by upregulating the Nrf2/HO-1 pathway to exert antioxidant effects.
difference in Nrf2 expression between the groups treated with 10 µM Fer-1 and 100 µg/kg L. bicolor honey extract.HO-1 is an important antioxidant enzyme that degrades heme, plays an antioxidant role, and breaks down biliverdin and its reduced products, such as bilirubin and iron ions [46].HO-1 can remove ROS and participate in the process of ferroptosis through its antioxidant action [47].As illustrated in Figure 5, following stimulation with 2.5% DSS, the expression of HO-1 was significantly increased compared to that in the Blank group (p < 0.001).Subsequent pretreatment of cells with 10 µM Fer-1, 100 µg/kg L. bicolor honey extract, and 10 µM Trifolin further significantly increased HO-1 expression compared to the DSS group (p < 0.01).Moreover, there was no significant difference in HO-1 expression between the groups treated with 10 µM Fer-1 and 100 µg/kg L. bicolor honey extract.The expression level of Nrf2 can affect the expression of HO-1.In addition, HO-1, the product of the degradation of bilirubin nitric oxide and other Nrf2 regulation of genes for UC, has a regulatory role.These findings suggested that L. bicolor honey extract and Trifolin could regulate the ferroptosis process and protect against DSS-induced colitis by upregulating the Nrf2/HO-1 pathway to exert antioxidant effects.

Effect of Different Treatments on Intestinal Epithelial Cell Ferroptosis
Furthermore, we detected the expression levels of several key factors (FTL, ACSL4, SLC7A11, and PTGS2) in ferroptosis by qPCR technology to validate the above results and evaluate the regulatory effects of L. bicolor honey extract and Trifolin on the ferroptosis process.
For FTL, as one of the subunits of ferritin, excess intracellular iron ions can be stored in ferritin, isolating them and preventing them from participating in ROS generation reactions [48].When disruption of ferritin leads to elevated ROS and activation of ferroptosis, FTL bound to iron ions dissociates and its content increases [48].As shown in Figure 5, following induction with 2.5% DSS, the expression level of FTL in Caco-2 cells significantly increased

Effect of Different Treatments on Intestinal Epithelial Cell Ferroptosis
Furthermore, we detected the expression levels of several key factors (FTL, ACSL4, SLC7A11, and PTGS2) in ferroptosis by qPCR technology to validate the above results and evaluate the regulatory effects of L. bicolor honey extract and Trifolin on the ferroptosis process.
For FTL, as one of the subunits of ferritin, excess intracellular iron ions can be stored in ferritin, isolating them and preventing them from participating in ROS generation reactions [48].When disruption of ferritin leads to elevated ROS and activation of ferroptosis, FTL bound to iron ions dissociates and its content increases [48].As shown in Figure 5, following induction with 2.5% DSS, the expression level of FTL in Caco-2 cells significantly increased compared to that in the Blank group (p < 0.001).However, its expression level decreased in the treatment group compared to that in the DSS group (p < 0.01).
For ACSL4: Its mechanism involves promoting the esterification of polyunsaturated fatty acids (PUFA) to acyl-coenzyme A (acyl-CoA), thereby promoting ferroptosis [49].As depicted in Figure 5, following induction with 2.5% DSS, the expression of ACSL4 in Caco-2 cells significantly increased compared to that in the Blank group (p < 0.001), indicating induction of ferroptosis.Compared to the DSS group, the expression level of ACSL4 was decreased in the treatment group (p < 0.01).Therefore, as an important target for treating ferroptosis-related diseases, the expression level of ACSL4 can be reduced by the intervention of L. bicolor honey and Trifolin to slow down the progression of ferroptosisrelated diseases.
For SLC7A11, as the transport functional unit of the Xc − system, it plays a crucial role in the reverse transport of cystine and glutamate across the cell membrane, facilitating the uptake of cystine and its conversion into cysteine inside the cell, a key step in GSH synthesis [50].GSH, in turn, participates in reducing lipid peroxides to mitigate ROS production, thereby preventing ferroptosis [37].
As depicted in Figure 5, following stimulation of Caco-2 cells with 2.5% DSS, the expression of SLC7A11 was significantly decreased (p < 0.001).This result indicated that DSS induction led to reduced SLC7A11 expression, impaired Xc − system function, and disruption of lipid redox balance, ultimately leading to ferroptosis [50].Following intervention with different treatment groups, the expression levels of SLC7A11 were significantly higher compared to those in the DSS group (p < 0.001).
These results indicated that the Xc − system restored its function, intracellular lipid redox returned to normal, and ferroptosis was inhibited.PTGS2 is considered one of the markers of ferroptosis and is involved in the inflammatory response, enhancing the release of TNF-α, IL-1β, IL-6, and other inflammatory factors and promoting ferroptosis [51].As shown in Figure 5, Caco-2 cells stimulated with 2.5% DSS showed significantly decreased expression (p < 0.001).After intervention with different treatment groups, its expression levels were significantly lower than those in the DSS group (p < 0.001).These results, consistent with Figure 1C, indicated that L. bicolor honey extract and Trifolin inhibited ferroptosis.Additionally, after intervention with different treatment groups, the expression of the inflammatory factors TNF-α and IL-6 in Caco-2 cells significantly decreased (p < 0.01), as shown in Figure 1C.Therefore, L. bicolor honey extract and Trifolin could alleviate DSSinduced colitis by inhibiting the release of inflammatory factors and thereby inhibiting ferroptosis.

Conclusions
This study further explores the anti-inflammatory effects and regulatory mechanisms of L. bicolor honey and its biomarker, Trifolin.Using a DSS-induced Caco-2 model, we found that pretreatment with L. bicolor honey extract and Trifolin increased the tight junction integrity of damaged cells and maintained the integrity of the monolayer.Additionally, L. bicolor honey extract and Trifolin demonstrated anti-inflammatory effects by reducing the expression of inflammatory factors TNF-α and IL-6 and antioxidant effects by increasing the expression of antioxidant factors NQO1 and GSTA1.Metabolomic analysis revealed that L. bicolor honey extract and Trifolin could regulate ferroptosis by reducing SOD, MDA, iron ion load, and ROS release and by modulating the gene expression levels of ferroptosis-related factors FTL, ACSL4, SLC7A11, and PTGS2 to alleviate the DSS-induced ferroptosis-related inflammatory response.Furthermore, our results suggest that L. bicolor honey extract and Trifolin inhibit ferroptosis by activating the Nrf2/HO-1 pathway.These findings provide foundational scientific data supporting the potential application of L. bicolor honey as a natural anti-inflammatory agent for human health.

Figure 2 .
Figure 2. Effects of different treatments on cellular metabolites in DSS-induced Caco-2 cells.(A) Partial least squares discrimination analysis (PLS-DA) based on the metabolites of Caco-2 cells among Blank, DSS, 100 µg/kg Honey + DSS, and 10 µM Trifolin + DSS groups (n = 4 for each group).(B) Volcano plot based on the differential metabolites of Caco-2 cells among DSS vs. Blank, 100 µg/kg Honey + DSS vs. DSS, and 10 µM Trifolin + DSS vs. DSS.(C) Log2 value of differential metabolites based on p value < 0.05, VIP ≥ 1 from PLS-DA, and FoldChange ≥ 2 or ≤0.5 from the volcano plot among each group.(D) Top-20 metabolic pathway mappings with KEGG databases based on FoldChange ≥ 2 or ≤0.5 for the difference in metabolites among each group.

Figure 2 .
Figure 2. Effects of different treatments on cellular metabolites in DSS-induced Caco-2 cells.(A) Partial least squares discrimination analysis (PLS-DA) based on the metabolites of Caco-2 cells among Blank, DSS, 100 µg/kg Honey + DSS, and 10 µM Trifolin + DSS groups (n = 4 for each group).(B) Volcano plot based on the differential metabolites of Caco-2 cells among DSS vs. Blank, 100 µg/kg Honey + DSS vs. DSS, and 10 µM Trifolin + DSS vs. DSS.(C) Log 2 value of differential metabolites based on p value < 0.05, VIP ≥ 1 from PLS-DA, and FoldChange ≥ 2 or ≤0.5 from the volcano plot among each group.(D) Top-20 metabolic pathway mappings with KEGG databases based on FoldChange ≥ 2 or ≤0.5 for the difference in metabolites among each group.

Figure 3 .
Figure 3. (A) Schematic diagram of the mechanism of ferroptosis in DSS-induced human Caco-2 cells through the Nrf2/HO-1 pathway.Effects of different treatments on oxidative stress in DSS-induced Caco-2 cells.Content of (B) SOD, (C) MDA, (D) free iron, and (E) GSH/GSSG ratio in DSSinduced Caco-2 cells.a, b, c, and d indicate significant differences (p < 0.05).The groups containing differently labeled letters indicate significant differences (p < 0.05).The groups with the same letter labeled exhibit no significant differences.

Figure 3 .
Figure 3. (A) Schematic diagram of the mechanism of ferroptosis in DSS-induced human Caco-2 cells through the Nrf2/HO-1 pathway.Effects of different treatments on oxidative stress in DSS-induced Caco-2 cells.Content of (B) SOD, (C) MDA, (D) free iron, and (E) GSH/GSSG ratio in DSS-induced Caco-2 cells.a, b, c, and d indicate significant differences (p < 0.05).The groups containing differently labeled letters indicate significant differences (p < 0.05).The groups with the same letter labeled exhibit no significant differences.

Figure 4 .
Figure 4. Effects of different treatments on the ROS expression level in DSS-induced Caco-2 cells.'DCFH-DA' indicated that ROS was captured via a confocal laser scanning microscope at a 488 nm excitation wavelength and a 525 nm emission wavelength.The scale bar is 5 μm and the magnification is 200×.'Brightfield' indicated that images were captured by a normal light source.'Merge' indicated the superposition of two images, 'DCFH-DS' and 'Brightfield'.

Figure 4 .
Figure 4. Effects of different treatments on the ROS expression level in DSS-induced Caco-2 cells.'DCFH-DA' indicated that ROS was captured via a confocal laser scanning microscope at a 488 nm excitation wavelength and a 525 nm emission wavelength.The scale bar is 5 µm and the magnification is 200×.'Brightfield' indicated that images were captured by a normal light source.'Merge' indicated the superposition of two images, 'DCFH-DS' and 'Brightfield'.

Table 1 .
Comparison of the variations in cellular metabolites across Caco-2 cells for each of the three designated groups.