Lycopene Attenuates T2 Mycotoxin-Induced Hepatotoxicity and Dysbiosis by Activating PPAR Signaling

TL;DR

Lycopene helps protect the liver and gut from T-2 toxin damage by reducing inflammation and restoring healthy bacteria.

Contribution

Lycopene's protective effects against T-2 toxin are linked to PPAR signaling and dose-dependent efficacy.

Findings

Lycopene reduces oxidative stress and inflammation caused by T-2 toxin in mice.

Low-dose lycopene is more effective than high-dose in restoring gut microbiota balance.

Lycopene activates PPAR signaling and improves liver detoxification pathways.

Abstract

T-2 toxin, a widespread mycotoxin, induces hepatotoxicity and gut dysbiosis, with limited mitigation strategies. This study demonstrates that lycopene protects against T-2 mycotoxicosis in mice by integrating microbiota restoration with hepatic metabolic reprogramming. Lycopene suppressed pro-inflammatory cytokines, alleviated oxidative stress, and enriched beneficial Muribaculum while suppressing pathogenic Escherichia. Mechanistically, lycopene de-repressed Peroxisome proliferator-activated receptor (PPAR) signaling and restored fatty acid oxidation and phase I detoxification gene expression. A hormetic dose–response was evident: low-dose lycopene outperformed high-dose lycopene, underscoring non-linear efficacy. These findings position lycopene as a candidate feed additive targeting the gut–liver–PPAR axis, though dose optimization remains critical for translation.

Exposure to T2 toxin is known to induce hepatotoxicity and gut dysbiosis, yet effective dietary interventions remain underexplored. This study investigates the hepatoprotective and microbiota-modulating effects of lycopene against T2 toxin-induced toxicity in mice. Mice were exposed to T2 toxin with or without lycopene supplementation at low and high doses. The hepatic function, oxidative stress markers, inflammatory gene expression, detoxification pathway activity, and gut microbiota composition were assessed using histological, biochemical, and molecular analyses. T2 toxin exposure resulted in significant weight loss, oxidative liver damage, and gut dysbiosis—marked by a decline in beneficial phyla and an increase in pathogenic bacteria. Hepatic injury was accompanied by upregulated pro-inflammatory genes and downregulated PPAR pathway genes, leading to impaired lipid metabolism and disrupted liver histology. Lycopene supplementation effectively attenuated these effects: it reduced oxidative stress, enhanced antioxidant defense, lowered inflammatory markers, and restored gut microbial balance. Furthermore, lycopene upregulated PPAR pathway and phase I detoxification genes. Notably, the low-dose lycopene regimen demonstrated superior efficacy compared to the high-dose regimen. In conclusion, lycopene, particularly at a low dose, confers significant protection against T2 toxin-induced hepatotoxicity and gut dysbiosis, highlighting its potential as a dietary strategy for mitigating mycotoxin-induced health risks.

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1. Introduction

The Fusarium genus produces T2 toxin as a secondary metabolite [1], and it is commonly present in cereals in various nations [2]. Human and animal tissues and organs will be harmed if food or feed tainted with T2 toxin is consumed [3]. International guidelines state that a person’s daily acceptable intake of T2 toxin should not exceed 100 ng/kg body weight (BW) [4]. According to the 2018 Biomin global study, the average detection quantity of T2 toxin was 25 mg/kg, with a detection rate of up to 23% among 8721 types of agricultural products from 75 countries [5]. As a risk factor for deadly alimentary toxic aleukia and Kashin–Beck disease in humans, T2 toxin can persist in animal tissues, meat, eggs, and milk, endangering human health through the food chain [6]. T2 toxin is harmful to the kidney, according to earlier research [7], although the precise mechanism is yet unknown.

One of the mycotoxin’s harmful mechanisms is oxidative stress. Studies have revealed that T2 toxin induces renal oxidative stress in common carp [8], chicken [9], rabbit [7], and rats [7]. It has been established that oxidative stress is an upstream activator of the apoptosis signal cascade, and that excessive reactive oxygen species (ROS) can cause apoptosis [10]. Through oxidative stress, T2 toxin triggered mitochondrial apoptosis, which subsequently caused harm to mouse embryonic stem cells [11]. Human renal tubular epithelial cells experienced oxidative stress and death as a result of T2 toxin [12]. It is unclear, however, whether oxidative stress and apoptosis are connected in the renal damage caused by T2 toxin.

Through changes in the composition of the gut bacteria and interactions between gut microorganisms and the host, mycotoxins impair the growth performance of animals [13]. The gut microbiota is affected by prolonged subclinical doses of T2 toxin [14]. Moreover, this interaction has a significant impact on the integrity of the intestinal barriers and the composition of the microbiota. Mycotoxins have been shown to increase intestinal permeability (‘leaky gut’) and disrupt intestinal barrier function [15], which is associated with a range of health problems, such as inflammatory bowel disease (IBD), obesity, and metabolic disorders [16,17]. This disruption can lead to the translocation of toxins and harmful bacteria from the gut into the bloodstream, which can cause inflammatory responses and systemic health problems [18,19]. In addition, mycotoxins can alter the diversity of the gut microbiota, from phylum to species, disrupting the balance between beneficial and pathogenic microorganisms. These changes in the gut microbiome can have far-reaching consequences for overall health, as the microbiota plays a crucial role in various physiological processes, including digestion, the immune system, and metabolism [20]. Previous research has shown that a total contamination of 12 mg/kg T2 toxin altered the composition of the bacterial community and reduced the microbial diversity index of pigs [21].

The liver is an extremely important organ in the human and animal body, coordinating a large number of essential functions that are fundamental to maintaining overall health. One of the liver’s many tasks is detoxification, a crucial process that protects the body from the harmful effects of xenobiotics, environmental toxins, and metabolic processes [22]. In addition to its direct role in neutralizing exogenous toxins and as a central hub in the body’s metabolic network, the liver influences energy metabolism, nutrient utilization, and blood regulation. Furthermore, it is intrinsically linked to the metabolism of endogenous substances [23,24]. Disruptions in these detoxification processes can therefore lead to the accumulation of harmful intermediates, which can contribute to a range of health disorders, including liver disease, hormonal imbalances, and increased susceptibility to chronic diseases [25].

The liver receives about 70% of its blood supply from the portal vein, which drains the GI tract, pancreas, and spleen [26]. This direct blood flow means that substances absorbed from the gut, including nutrients, toxins, and microbial products, are transported to the liver for processing. The gut microbiome plays a pivotal role in influencing liver function, highlighting the intricate interplay between the two organs through the gut–liver axis [27]. This interplay begins with intestinal dysbiosis, which increases intestinal permeability by disrupting the intestinal barriers, and then the toxic substances reach the liver via the bloodstream. This bidirectional communication system involves an array of molecular signals, such as inflammatory cytokines [28,29]. Microbial metabolites such as short-chain fatty acids (SCFAs) and bile acids can modulate the expression and activity of hepatic detoxification enzymes, thereby impacting the efficiency of toxin processing [30]. Moreover, the immune modulators Interleukin-6 (IL-10), Toll-like receptors (TLRs), and Macrophages play a pivotal role in this complex interplay [31]. The gut microbiome residing in the gastrointestinal tract exerts significant effects on liver physiology and function, with implications for various health conditions, like non-alcoholic fatty liver disease (NAFLD), liver cirrhosis, and metabolic syndrome [32,33,34].

The carotenoid family member lycopene is mostly found in tomatoes and guavas. It possesses several biological properties, including potent antioxidant, anti-inflammatory, anti-cancer, and anti-apoptotic effects [35]. Reports indicate that lycopene enhanced the activity of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in L02 cells while reducing the production of reactive oxygen species (ROS) [36,37]. Additionally, lycopene lowers malondialdehyde (MDA) levels in the liver and plasma [38]. However, it is believed that lycopene’s antioxidative activity [39], which has been demonstrated to be a strong defense against oxidative damage to DNA, proteins, and lipids, is mostly to blame. There may be further roles for lycopene in regulating cell–cell communication [40], suppressing cell division [41], and defending against bacterial infections [40]. According to Lin et al. [42], lycopene effectively activates AMP-activated protein kinase (AMPK) signaling in mice.

While the hepatotoxic and microbiota-disruptive effects of T2 toxin are established, no study has examined whether lycopene can confer protection along the entire gut–liver axis. Understanding this intricate connection is crucial for assessing the impact of environmental exposures on health, as the gut and liver work together to metabolize and detoxify various substances, including dietary components, drugs, and environmental toxins. This study aimed to provide the first integrated assessment of low and high doses of lycopene supplementation to concurrently alleviate hepatic oxidative damage, restore detoxification and lipid metabolism pathways, and reverse T2 toxin-induced gut dysbiosis. By targeting the bidirectional gut–liver interface rather than the liver in isolation, this work introduces a novel systems-level approach to mycotoxin mitigation to increase our understanding of the influence of exposure to environmental factors on health.

2. Materials and Methods

2.1. Animals and Treatments

Thirty-six male BALB/c mice were purchased from Yangzhou University’s Model Animal Center in Yangzhou, China, aged 4 weeks (24.7 ± 0.32 g), were included in this study and underwent 7 days of adaptation before they were randomly divided into 6 groups (6 mice per group, 2 mice per cage): the control group (C) fed with a (basal diet); the T2 group fed with a basal diet with 1.6 mg/kg (T2); the low-lycopene-dose (Ly L) group fed with a (basal diet with lycopene at 100 mg/kg per day); the high-lycopene-dose (Ly H) group fed with a (basal diet with lycopene at 400 mg/kg per day); the low-lycopene-dose-with-T2 (Ly L + T2) group fed with a (basal diet with T2 at 1.6 mg/kg and 100 mg/kg lycopene per day); the high-lycopene-dose-with-T2 (Ly H + T2) group fed with a (basal diet with T2 at 1.6 mg/kg and 400 mg/kg lycopene per day), and corn oil was used as a vehicle in all experimental groups, including the control group [43,44]. The detailed concentrations of the basal diet are shown in Table 1. The current experimental design is an acute-exposure model. The total experimental period lasted 5 weeks following the 7-day adaptation phase. During this time, all treatments (T2 toxin and/or lycopene) were administered daily via oral gavage. Briefly, T2 toxin and lycopene were administered at the same time each day (09:00–10:00 AM) to maintain consistency and minimize circadian variation, while the combination groups (Ly L + T2 and Ly H + T2) were administered the lycopene in the morning and the T2 dose in the afternoon (04:00–05:00 PM), as shown in (Figure 1). The lycopene powder (10% purity) was purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). Trichothecene (T2) was obtained from Qingdao Pribolab Biotech Co., Ltd. (Qingdao, China), and the corn oil was obtained from the market (Yangzhou, China). Mice cages were equipped with a one-sided self-feeder and a nipple water-feeder for ad libitum access to feed and water, and mice were raised in a room at constant temperature (25 ± 2 °C) and humidity (50% ± 10%) with a 12 h light–dark cycle throughout the experiment. These procedures were conducted under the guidelines for the ethical treatment of animals and with the approval of the Institutional Animal Care and Utilization Committee (IACUC) of the Animal Experimental Ethics Committee of Yangzhou University (Permit Number: (SYXK [Su] 2023-0089).

2.2. Sampling Procedure

The body weight and feed and water consumptions were measured daily for 5 weeks until the experiment finished. The data related to the severity degree of the clinical appearance and activity/behavior were monitored and recorded daily. Briefly, both scores progress from score 0 to score 4, where 0 indicates normal and 4 indicates severe, according to previous methods [45,46]. At the end of the experiment, blood samples were collected from the orbital venous plexus and centrifuged for 15 min at 3000 g at 4 °C to obtain serum samples, and then the samples were stored at −20 °C for further serum biochemical studies. The jejunum and liver samples were flushed with PBS, and then the segments were cut off and fixed in 4% paraformaldehyde solution for histomorphology analysis. Another set of liver samples were quickly frozen in liquid nitrogen and stored at −80 °C for RNA and protein extraction or oxidative status. The jejunum content was collected and stored at −80 °C for further analysis of the microbiota and metabolites. These procedures were conducted under the guidelines for the ethical treatment of animals and with the approval of the Animal Care and Use Committee of the institution.

2.3. Measurement of Liver Function Biomarkers in Serum

After we cryopreserved the serum samples at −20 °C, we centrifuged them at 4 °C. We measured the levels of total protein, Aspartate aminotransferase (AST), Alanine transaminase (ALT), low-density lipoprotein (LDL), lactate dehydrogenase (LDH), and high-density lipoprotein (HDL) in serum by using the corresponding commercial reagent kits as follows: (The Total protein assay kit, coded (A045-4-2)), (Aspartate aminotransferase test kit, coded (C010-2-1)), (Alanine aminotransferase test kit, coded (C009-2-1)), (Low-density lipoprotein cholesterol assay kit coded (A113-1-1), (Lactate dehydrogenase assay kit, coded (A020-2-2)), and (High-density lipoprotein cholesterol assay kit, coded (A112-1-1)) (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and we used an automatic clinical biochemistry analyzer (Shanghai Kehua Bio-engineering Co., Ltd. (KHB) Polaris C2000, Shanghai, China). The whole experimental procedure was strictly performed according to the manufacturer’s instructions. The measurement of the lycopene concentrations was performed according to our previous study [47].

2.4. Histological Analysis

For the H&E staining, the jejunum and liver tissues were fixed in 4% paraformaldehyde solution for 48 h, and then the sections were dehydrated in a graded series of ethanol and xylene and embedded in paraffin. The jejunum and liver sections were then sectioned at 5 μm using a Lecia RM2235 microtome (Leica Biosystems Inc., Buffalo Grove, IL, USA). The sections were deparaffinized with xylene and rehydrated through a graded dilution of ethanol for H&E staining. The images of the jejunal sections were obtained using an Olympus Simon-01 microscope (Olympus Optical Co., Ltd., Beijing, China). The villus height and crypt depth values were measured 6 times from different villi and crypts per section from each tissue using ImageJ software (Fiji) version 2.17.0 [47].

2.5. Determination of Liver Oxidative Status

An amount of 0.2 g frozen jejunal mucosae was weighed and homogenized in 2 mL of ice-cold saline. After being centrifuged at 12,000 g for 10 min at 4 °C, the supernatants were separated to measure the oxidative status in the jejunal mucosae. Commercial reagent kits (S0101, S0056, and S0131, Beyotime Biotechnology Co., Ltd, Shanghai, China) were assessed for superoxide dismutase activity, glutathione peroxidase activity, and malondialdehyde (MDA). All experimental procedures were performed according to the manufacturer’s instructions. The final results were normalized to the protein concentration in each sample.

2.6. Isolation of Microbial Genomic

Thirty jejunum content samples (five per experimental group) were collected for 16S rRNA gene sequencing. Total genomic DNA was extracted using a QIAamp DNA Stool Mini Kit (Qiagen, CA, USA), according to the manufacturer’s instructions. The DNA concentration and purity were assessed using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and all DNA samples were stored at −80 °C until further processing. The V3–V4 hypervariable region of the bacterial 16S rRNA gene was amplified using universal primers (341F: ACTCCTACGGGAGGCAGCA; 805R: GGACTACHVGGGTWTCTAAT) coupled with unique barcodes. PCR products were purified and quantified using the QuantiFluor-ST fluorometric system (Promega, Durham, NC, USA). Sequencing libraries were constructed with the Illumina TruSeq DNA sample preparation kit (Illumina, San Diego, CA, USA), followed by cluster generation and paired-end sequencing (2 × 300 bp) on the Illumina MiSeq platform. Raw sequencing data were processed using the DADA2 pipeline, which included primer removal, quality filtering, denoising, read merging, and chimera removal. This approach resolves amplicon sequence variants (ASVs) at single-nucleotide resolution, eliminating the need for traditional similarity-based clustering. To ensure robustness, an alternative workflow based on operational taxonomic unit (OTU) clustering at 97% similarity was also implemented using Vsearch. Both methods were employed to accommodate potential methodological limitations and to allow for comparison with earlier studies.

2.7. Quantitative Rt PCR

Total RNA was extracted from the liver using TRIzol Reagent (Invitrogen, 15596026, Waltham, MA, USA), and a NanoDrop 1000 spectrophotometer was used for quantification (F-3100, Suizhen, Hangzhou, China). According to the manufacturer’s protocol, 1 μg of RNA was reverse-transcribed into cDNA using HiScript II Q RT SuperMix (Vazyme biotech, R222-01, Nanjing, China). Quantitative qRT-PCR analysis was performed using AceQ qPCR SYBR Green Master Mix (Vazyme Biotech, Q111-02, Nanjing, China) on an ABI QuantStudio 3 Real-Time PCR Instrument. Gene expression levels (fold changes) were normalized to the internal reference Gapdh and calculated using the 2^−ΔΔCT^ method. Primer sequences were as presented in Table 2.

2.8. RNA Extraction and Transcriptomic Analysis

The total RNA was extracted from liver tissues. After RNA extraction, the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA) was used for RNA quality evaluation. Thereafter, an Illumina TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, USA) was used to construct the RNA-seq libraries. Then, the libraries were constructed using an Illumina HiSeq 2000 sequencer (BGI Tech, Wuhan, China) for the high-throughput sequencing. The sequencing data were filtered with SOAPnuke (v1.5.6) to obtain clean reads, and Bowtie2 (v2.5.0) was used to align high-quality clean reads to GRCm39. For subsequent data processing, cufflinks software was used to obtain quantitative fragments per kilobase of exon model per million mapped fragments (FPKM) values. Differential expression analysis was performed using DESeq2, with the threshold differentially expressed for genes set at log2(fold change) > 1 and adjusted p < 0.05. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment studies and Gene Ontology (GO) annotation were performed to gain a deeper understanding of the functions of DEGs induced by different treatments.

2.9. Statistical Analysis

Statistical calculations were carried out by conducting tests using the SPSS software package (SPSS version 23, SPSS, Inc., Chicago, IL, USA). Then, one- or two-way ANOVA was conducted using Tukey’s post hoc tests. GraphPad Prism 7 software was used to analyze the data with multiple comparisons among groups. Data were presented as the mean and standard error of the mean. Differences were considered to be statistically significant at (p < 0.05).

3. Results

3.1. Phenotypic Changes, Water and Feed Consumption, and Body Weight Changes

At the end of the experiment, phenotypic changes were observed in the experimental groups. Groups C, Ly L, and Ly H exhibited normal behavior and normal clinical appearance, while group T2 exhibited abnormal behavior with high-scoring clinical appearance, including skin inflammation on the face and around the mouth, compared to the control group. Interestingly, group Ly L + T2 was normal and healthy, indicating an effective protective effect of lycopene at a low dose. However, the Ly H + T2 group showed a less protective effect of lycopene at a high dose (Figure 2A–C).

Feed and water consumption were decreased in the T2 group compared to the control group, while in the experimental groups treated with lycopene, whether at a high or low dose, this decrease was alleviated; however, group Ly L + T2 showed more normal effects compared to group Ly H + T2 (Figure 2D,E). The healthy mice in group C showed normal body weight; moreover, groups Ly L, Ly H, and Ly L + T2 showed no significant change and a normal increase in body weight, nearly close to that of the control, compared to the C group. In contrast, the group that received T2 showed a significant decrease in final weight compared to the control group and other experimental groups. Mice that received T2 and were treated with lycopene (groups Ly L + T2 and Ly H + T2) showed a significant increase in their final weight compared to the T2 group and were able to successfully reduce the weight loss resulting from exposure to T2 (p < 0.05) (Figure 2F).

3.2. Blood Biochemistry

The T2 group showed a significant decrease in its total protein, HDL, and LDL levels compared to the control group and other experimental groups, while its AST, ALT, and LDH levels were significantly increased, indicating the harmful effects of T2 on the liver function. In contrast, the lycopene treatment group (Ly L + T2) showed protective roles and alleviated all the negative changes significantly more than the Ly H + T2 group compared to the T2 group (p < 0.05) (Figure 3). The levels of lycopene show that it appeared in the groups treated with lycopene. Moreover, the Ly L + T2 group had higher levels of lycopene compared to the Ly H + T2 group, indicating that the low dose has a higher absorption efficiency. In contrast, the lycopene levels were decreased significantly in the groups treated with lycopene and T2 (Ly L + T2 and Ly H + T2) compared to the groups treated with lycopene only (Ly L and Ly H); however, the Ly L + T2 group had significant levels of lycopene compared to the Ly H + T2 group (p < 0.05) (Figure 3G).

3.3. Jejunum Morphology and Characteristics

Sections from the jejunum segments showed short villi and some denuded villus parts in the lumen, besides marked adhesions of the villi in the T2 group compared to the C, Ly L, and Ly H groups. A healthier villus with a gradual increase was observed in the tall villi, which may indicate an increase in absorptive surfaces, which were represented by brad tips in the group treated with lycopene (Ly L + T2) compared to the T2 group, while group Ly H + T2 failed to show positive results compared to the T2 group (Figure 4A). The differences in the villus heights are shown in (Figure 4B), with significant differences between the C group and T2 group. However, the Ly L and Ly L + T2 groups showed significant differences compared to the T2 and Ly H + T2 groups, while the crypt depth was not significant among the experimental groups (Figure 4C). Furthermore, the V/C ratio showed significant differences in the control and Ly L + T2 groups compared to the T2 and Ly H + T2 groups, indicating that the absorption process might be disrupted due to T2 exposure and that the lycopene low dose had significantly alleviated this change back to normal compared to the control group (Figure 4D) (p < 0.05).

3.4. Lycopene Protects Against Intestinal Microbiota Dysbiosis Induced by T2

T2 exposure disrupted the structure of the gut microbiota, as evidenced by the altered clustering of bacterial communities in the PCA plots (weighted UniFrac distance) (Figure 5A). The alpha diversity analysis Chao describes the microbial consortium in a medium (Figure 5B, p < 0.05). The notable increase in the Chao in groups C, Ly L, and Ly L + T2 indicated the efficacy of lycopene in promoting the microbial population, confirming the claim that dietary carotenoids contribute to the promotion of beneficial gut microbiota, among other direct health benefits. However, there was no significant difference in the alpha diversity (Chao) between the experimental groups T2 and Ly H + T2. Further taxonomic analysis showed that Bacillota decreased and Pseudomonadota increased in group T2 at the phylum level, while Bacillota increased and Pseudomonadota decreased in groups Ly L and Ly L + T2 (Figure 5C, p < 0.05). At the genus level, Escherichia was significantly enriched in the T2 group compared to the other experimental groups, while Muribaculum was significantly decreased in the T2 group compared to the C, Ly L, and Ly L + T2 groups (Figure 5D, p < 0.05). Furthermore, Lactobacillus_jonsonii was significantly enriched in the Ly L + T2 group compared to the other groups. Interestingly, Desulfovibrio_porci and Streptococcus_danieliae were significantly increased in the T2 group compared to the other groups (Figure 5E p < 0.05).

3.5. Lycopene Protects Against T2 Exposure to Liver Transcriptome Alterations on KEGG Pathway

Since the liver receives blood directly from the intestine via the portal vein, hepatotoxic molecules resulting from intestinal toxin exposure are readily transported to the liver. Our results indicate that the T2 group exhibited intestinal inflammation, dysbiosis, and barrier dysfunction, all of which collectively increased the risk of secondary liver impairment. To delineate the hepatoprotective effects of lycopene against T2-induced intestinal damage, we conducted RNA-Seq analysis on liver tissues across the experimental groups. Volcano plot analysis revealed substantial transcriptomic remodeling: 293 upregulated and 264 downregulated genes in the T2 vs. control comparison; 152 upregulated and 166 downregulated genes in the Ly L + T2 vs. T2 comparison; and 148 upregulated and 564 downregulated genes in the Ly H + T2 vs. T2 comparison (Figure 6A). Rather than enumerating individual differentially expressed genes in isolation, we grouped them into functionally relevant categories. Notably, Gene Set Enrichment Analysis (GSEA) demonstrated that the most enriched pathway in Ly L + T2-treated livers was the PPAR signaling pathway, which was conversely dysregulated in the T2 group relative to controls (Figure 6B; FDR q-value < 0.01). Given the centrality of PPAR signaling in hepatic lipid metabolism, fatty acid transport, and anti-inflammatory responses, we next examined the expression patterns of individual PPAR pathway components. Hierarchical clustering of all PPAR-associated genes across the experimental groups confirmed the coordinated transcriptional regulation of this pathway (Figure 6C,D). Specifically, T2 exposure significantly downregulated a core set of PPAR target genes involved in fatty acid uptake and oxidation, including Fabp1, Fads2, Fabp2, Cyp4a12b, Fabp4, Acsl1, Ehhadh, Plin2, Angptl4, Acsl3, Pck1, and Cpt1b (Figure 6E). Conversely, lycopene supplementation (Ly L + T2) partially reversed this suppression, upregulating Fabp1, Fads2, Fabp2, Cyp4a12b, Fabp5, Cyp4a31, and Sorbs1 compared to the T2 group (Figure 6F). Importantly, three of these genes, Fabp5, Cyp4a31, and Sorbs1, were exclusively upregulated in the Ly L + T2 group and not detected in either the control or T2 groups, suggesting that lycopene may recruit additional PPAR-related effectors to restore metabolic homeostasis. These findings demonstrate that lycopene mitigates T2-induced hepatic transcriptomic alterations by restoring and selectively enhancing the PPAR signaling pathway, thereby counteracting the metabolic dysregulation triggered by gut-derived toxins.

3.6. Lycopene Protects Against T2 Exposure to Liver Transcriptome Alterations in Biological Processes

In the above results, the T2 group showed that the exposure of T2 altered the PPAR pathway, which is linked to the liver’s biological processes. To determine the protective roles of lycopene treatment against T2 exposure, we further analyzed RNA-Seq data. In (Figure 7A), the GSEA of biological processes shows that the Ly L + T2 group was significantly enriched in lipid metabolism compared to the T2 group, while it was dysregulated significantly in the T2 group compared to the control group (FDR q-value < 0.01). The heatmap analysis further determined all the genes associated with lipid metabolism in all the groups (Figure 7B) or among the C, Ly L + T2, and T2 groups (Figure 7C). The genes IDI1, ACSM2, DGKA, INSIG1, PIK3C2G, MSMO1, CROT, HSD17B7, PIK3C2A, AGPAT4, FADS2, ARV1, LRAT, IP6K2, B3GALNT1, ELOVL5, ACSL1, ELOVL2, ACSL3, CYP7B1, CPT1B, CYP51, SQLE, PIKFYVE, CYP2U1, EHHADH, ACOT1, ANGPTL4, ST6GALNAC3, and PLPP1 were the downregulated genes associated with lipid metabolism in T2 versus the control (Figure 7D), while the upregulated genes in the Ly L + T2 versus T2 group were IDI1, ABHD2, INSIG1, PCSK9, MSMO1, HSD17B6, SGMS2, PIK3C2A, AACS, FADS3, FADS2, LIPG, ENPP2, LDLR, B3GALNT1, ELOVL5, CYP4A31, MID1IP1, ELOVL2, ERLIN1, CYP7B1, CYP51, SQLE, PIKFYVE, CYP2U1, DGKQ, and B4GALT5 (Figure 7E). Interestingly, the ERLIN1, HSD17B6, MID1IP1, AACS, CYP4A31, and FADS3 genes are also associated with lipid metabolism, and they were upregulated in the Ly L + T2 group but not in the control, Ly H + T2, and T2 groups. The results of the KEGG pathway analysis showed clearly that the PPAR pathway and its associated genes were altered by the exposure to T2; moreover, as is evidenced, the PPAR pathway is linked directly to lipid metabolism and can cause chronic liver disorders and dysfunction.

3.7. Liver Morphological Changes and Antioxidant Enzyme Activities

The histopathological analysis of liver tissues showed normal morphology in the C, Ly L, and Ly H groups. The T2 group revealed mononuclear cell infiltration, vascular occlusions, and disorganized hepatic cords compared to the C, Ly L, and Ly H groups. In contrast, in the mice treated with lycopene (Ly L + T2 group), all the morphological changes due to T2 exposure were mitigated; however, group Ly H + T2 still showed abnormalities, such as vascular occlusions and disorganized hepatic cords, compared to the C, Ly L, and Ly H groups (Figure 8A). Moreover, the antioxidant enzyme activity was also affected by T2 exposure (Figure 8B). The enzymes SOD and GSH-Px were decreased significantly in the T2 groups compared to the other experimental groups. Moreover, the MDA levels were increased significantly compared to those in the C and Ly L groups, indicating that the liver had acute oxidative stress due to T2 exposure. Treatment group Ly L + T2 had significantly increased levels of SOD and GSH-Px and decreased MDA levels compared to the T2 group, indicating the protective roles of lycopene supplementation against oxidative stress induced by T2 exposure (p < 0.05).

3.8. RT-qPCR Analysis of Pro-Inflammatory Gene Expression in Liver

Based on previous results, we selected groups C, Ly L, T2, and Ly L + T2 for the current analysis. We selected overlapping genes in enrichment pathways for up- and downregulated DEGs for further qRT-PCR measurements. In response to inflammation, the results showed that mice exposed to T2 exhibited significantly increased expressions of Cxcl10, Cxcr3, ICAM1, Il-β1, Il-6, Ptger1, TLR2, and NFkbil1 compared to the experimental groups C, Ly L, and Ly L + T2 (Figure 9) (p < 0.05). The low-dose lycopene group showed no changes and was normal compared to the control group. Furthermore, the experimental group treated with lycopene and T2 (Ly L + T2) showed a significant decrease in the expressions of the Cxcl10, Cxcr3, Il-β1, Il-6, and NFkbil1 genes compared to the T2 group (p < 0.05). However, the Ly L + T2 group also showed a decrease in the expressions of the ICAM1 and Ptger1 genes, and the p value was equal to 0.05, while the TLR2 gene p value was equal to 0.06. Therefore, exposure to T2 stimulated pro-inflammatory-related genes in response to oxidative stress, while lycopene treatment reduced the overexpression of pro-inflammatory genes, indicating its protective role against oxidative stress caused by exposure to T2.

4. Discussion

T2 toxin is a type A trichothecene mycotoxin that has strong immunosuppressive and cytotoxic effects on animals. The dosage and technique for animal exposure determine the earliest symptoms and signs of T2 toxin exposure [49]. The T-2 toxin can act as a hazardous agent, as it can be absorbed through intact skin or contaminated food/feed, causing blistering, irritation, systemic toxicity, and toxic Epidermal Necrolysis (TEN) on the skin [50]. The T2 mycotoxin also causes physical weakness, especially in cases of severe poisoning or chronic exposure, which can manifest itself as exhaustion, general weakness, and lethargy. Weakness is a common systemic symptom, alongside other symptoms such as nausea, gastrointestinal complaints, and neurological disorders [49], which agrees with our findings. In addition, the present study showed that T2 toxin exposures in mice resulted in a significant decrease in their body weight, which was consistent with previous reports [51]. Moreover, it is reported in several studies that lycopene could not always increase the body weight, since it is not a growth factor promoter, which agrees with our results [52,53]. However, lycopene treatment significantly reduced T2 toxin-induced weight loss, weakness, and phenotypic changes, showing the significant protective effect of lycopene in reducing T2 effects.

There is evidence that disrupted intestinal barriers, inflammatory responses, and gut microbiota dysbiosis have a strong relationship [54]. The diversity of the gut microbiota plays a crucial role in maintaining systemic health. A higher species diversity is believed to indicate greater stability within the microbiota, help prevent pathogen colonization and boost immunity [55]. In the current study, T2 toxin exposure significantly lowered the alpha diversity in the mouse gut microbiota, based on the Chao indices in fecal rDNA analyses. These findings are consistent with the reports of T2 toxin induced in mice [56]. Furthermore, the Chao indices in the mouse gut microbiota decreased after dietary T2 toxin induction [8]. Current evidence indicates that T2 toxin alters the structure of the gut microbiota, and these changes are closely associated with disease risk [57]. The gut microbiota serves as the primary barrier for the host, protecting against harmful metabolites found in feed [58]. The gut microbes of the host possess the capability to detoxify or modify mycotoxins through the secretion of enzymes into the gastrointestinal tract. The majority of the transformation consists of the hydrolysis of mycotoxins, leading to the creation of metabolites that exhibit lower toxicity compared to the parent compound [59]. In animals, the gut microbiota can de-epoxidize T2 toxin [60]; however, animals have a less effective system [61]. Moreover, the recovery rate of elevated levels of T2 toxin from various sections of the intestinal tract and the feces of animals consuming a diet contaminated with T2 toxin suggests that the gut microbiota of animals demonstrates reduced efficacy in hydrolyzing T2 toxin [62]. Various bacterial species within the phylum Baciltota have been identified for their capacity to detoxify mycotoxins [63]. In the current study, exposure to T2 toxin significantly lowered the relative abundance of Baciltota and significantly increased the relative abundance of Pseudomonadota. The elevated presence of Baciltota is regarded as crucial for health preservation and overall balance, as well as in inflammatory bowel disease and colon cancer [64]. The T2 toxin group had a lower abundance of Baciltota compared with the other groups; lycopene supplementation reversed this phenomenon. The present study showed that lycopene inclusion can improve the relative abundance of Baciltota in T2 toxin-induced mice. At the genus level, the T2 toxin induced an increase in the relative abundance of Escherichia, and it decreased the relative abundance of Muribaculum. Lycopene supplementation reversed the T2 toxin-induced decreases in the relative abundances of Muribaculum, as well as the increased abundance of Escherichia. Muribaculum is considered a beneficial bacterium known for its gut-protective effects; its increased abundance indicates that it plays an active role in maintaining the gut microbiota in T2 toxin-induced mice. Escherichia coli is a common pathogen in the intestines of people who have diarrhea. It disrupts the function of the gut epithelial barrier by drastically increasing inflammation and decreasing the expression of tight junction proteins in intestinal epithelial cells [65]. Our findings collectively demonstrate that lycopene supplementation notably enhanced the populations of beneficial bacteria, contributing to the repair of the intestinal barrier and offering protection against T2 toxin-induced intestinal inflammation.

Given the compromised intestinal barrier and gut microbiota dysbiosis induced by T2 toxin, the liver, as a central organ in detoxification, becomes the secondary target of both the mycotoxin and pro-inflammatory microbial metabolites translocated from the gut. According to previous studies, T2 toxin is the toxin that animals absorb most rapidly and easily. It also spreads rapidly throughout the body’s organs, accumulating little, or not accumulating at all, in any one of them. After absorption in rodent species, the highest concentration of T2 toxin in plasma may be detected twenty to thirty minutes later [21]. Furthermore, a previous study shows that T2 toxicity, which increases ROS production, disrupts cell membranes, generates MDA, and releases LDH while simultaneously reducing intracellular antioxidant defenses, including GSH and antioxidant enzymes (SOD and CAT), leading to oxidative damage [8]. The present study found that the mice treated with T2 toxin showed significantly increased AST, ALT, LDH, and MDA, along with significantly decreased HDL, LDL, SOD, and GSH-Px, consistent with the previous report [66]. In contrast, lycopene treatment significantly lowered the levels of AST, ALT, LDH, and MDA and improved the antioxidant capacity and enzyme activities, indicating that lycopene enhanced antioxidant defenses and reduced oxidative liver damage caused by T2 toxin.

Moreover, the toxic substances produced by the gut microbiota due to dysbiosis resulted in increased intestinal permeability, which assists these substances in reaching the liver through the vein and can significantly impair liver detoxification mechanisms [67]. A study showed that intestinal immune dysregulation driven by dysbiosis exhibited barrier disruption and bacterial translocation in rats with cirrhosis disease [68]. In the liver, toxins bind to Toll-like receptor 2 (TLR2) on hepatocytes and Kupffer cells [69]. This binding triggers an inflammatory response, activating nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs), leading to the production of pro-inflammatory cytokines such as IL-1β and IL-6 [70]. Another study analyzed the inflammations associated with gut microbiota dysbiosis with non-alcoholic fatty liver disease and found that the gut-derived toxins led to chronic inflammation, which elevated the levels of cytokines such as ICAM1, IL-1β, and IL-6 and the levels of chemokines such as Cxcl10, which activated CXC receptor 3 (Cxcr3), as well as NFbil1, which regulate the NF-κB signaling pathway [71]. This agrees with our study results, where the gene expressions of Cxcl10, Cxcr3, ICAM1, Il-β1, Il-6, Ptger1, TLR2, and NFkbil1 were highly expressed in the T2 group, while the supplementation of lycopene inhibited this upregulation to the normal levels compared to the control group. These cytokines interfere with the expressions of detoxification enzymes and transporters, reducing the liver’s detoxification capacity and promoting fibrosis and cirrhosis [72,73,74].

Chronic inflammation hampers the liver’s detoxification processes by causing hepatocellular injury and dysfunction [75]. ROS accumulation leads to oxidative stress, causing damage to cellular components such as lipids, proteins, and DNA, contributing to tissue injury and dysfunction [76]. ROS can also damage liver cells and mitochondria, impairing the cellular machinery responsible for detoxification, including enzymes involved in phase I and phase II detoxification pathways [77]. The liver’s phase I detoxification involves CYP450 enzymes that oxidize toxins, making them more water-soluble. Inflammation and oxidative stress can downregulate the expression and activity of CYP450 enzymes, reducing the liver’s ability to metabolize and clear toxins [78], which agrees with our results obtained by downregulating the CYP4A12B gene expression in the T2 group, indicating that the disruption of phase I detoxification involves CYP450, while it was upregulated in the lycopene-treated group Ly L + T2, in addition to the CYP4A31 gene.

The present study allows us to propose a more integrated mechanistic axis linking the observed gut microbiota modulation to hepatic PPAR activation. Specifically, T2 toxin-induced dysbiosis, characterized by a significant reduction in beneficial Baciltota and Muribaculum and an expansion of pathogenic Escherichia, likely compromises the intestinal barrier integrity. This disruption facilitates the translocation of bacterial products and toxins to the liver via the portal vein. In the liver, these ligands activate Toll-like receptors (e.g., TLR2), triggering the upregulation of pro-inflammatory mediators (IL-1β, IL-6, ICAM1, Cxcl10) observed in our transcriptomic data. This hepatic inflammatory milieu is known to directly suppress PPAR signaling. Therefore, we propose that lycopene exerts its hepatoprotective effects not solely via direct antioxidant activity but indirectly through a two-step mechanism: first, by restoring gut eubiosis (increasing Baciltota, Muribaculum, and decreasing Escherichia), thereby reducing the portal endotoxin load, and second, by relieving the inflammation-mediated suppression of PPARα/γ, thereby restoring fatty acid oxidation (CYP4A31) and lipid metabolism gene expression (FABP5).

The suppression of the PPAR pathway observed in the T2 toxin group can be mechanistically linked to the upstream events of gut dysbiosis and endotoxin translocation. Hepatic inflammation driven by gut-derived LPS and bacterial ligands activates NF-κB signaling [79]. The pro-inflammatory cytokines secreted downstream of this signaling (IL-1β, IL-6) interfere with the transcriptional activity of PPARα, a nuclear receptor crucial for fatty acid oxidation [80]. This aligns with our observed downregulation of PPAR target genes such as CYP4A31 and FABP5. Crucially, there is evidence that lycopene, independent of PPAR, may alter ATP synthesis, raise the synthesis of reactive oxygen species (ROS), or induce mitochondrial uncoupling [81]. In specific cell types or under certain conditions, such effects could hold therapeutic potential; however, they might also lead to cytotoxicity. Recent research indicates that lycopene could impact mitochondrial activity in addition to having the significant function of initiating the PPAR pathway [82]. Lycopene treatment appears to disrupt this pathological cascade at its origin. By reversing T2-induced dysbiosis and reducing the Escherichia abundance and increasing Muribaculum, lycopene lowers the inflammatory stimulus reaching the liver. This attenuation of hepatic inflammation subsequently ‘de-represses’ PPAR signaling, allowing for the recovery of lipid metabolism gene expression. This interpretation positions the gut microbiota as the primary effector and the PPAR pathway as a secondary, but critical, downstream executor of lycopene’s protective function. Therefore, our findings support the existence of a ‘gut–immune–liver’ axis in T2 mycotoxicosis, wherein lycopene disrupts the pathological flow of toxicity by first stabilizing the intestinal ecosystem, thereby alleviating hepatic inflammatory stress and restoring PPAR-mediated metabolic homeostasis.

In contrast, the high lycopene dose showed very limited protective roles during the experiment and no roles in some parts compared to the lycopene low dose against T2 exposure. In alignment with the present study, it has been demonstrated that high lycopene levels alter bioenergetics, oxidative phosphorylation, and mitochondrial dynamics, sometimes leading to mitochondrial stress [37]. Our previous study showed that lycopene has demonstrated an impact on the composition of the gut microbiota, promoting the growth of Lactobacillus and other beneficial bacteria while potentially inhibiting pathogenic microorganisms [47]. According to our present results, the high dose of lycopene has the potential to disrupt the balance of microorganisms and cause unexpected effects, such as dysbiosis or modifications in the production of short-chain fatty acids.

Last but not least, there are very limited studies that have been conducted on the potential of natural products such as lycopene in the treatment of diseases caused by mycotoxins, particularly T2 mycotoxins, and this needs to be investigated further [83]. Lycopene, like many other carotenoids and phytochemicals, is known to exhibit hormetic dose–response characteristics, where low doses induce beneficial adaptive responses, while high doses may trigger cellular stress, pro-oxidant effects, or the feedback inhibition of detoxification and antioxidant pathways. In our study, although high-dose lycopene still conferred some protective effects, its diminished efficacy relative to the low dose may reflect such hormetic modulation. The current dataset does not allow us to definitively establish these mechanisms; therefore, future investigation is needed with an emphasis on their application in food, animal feed, and supplements. Effective collaboration between researchers and the food/feed industry is crucial for the successful implementation of these solutions. By addressing the current gaps in the research and adopting a multidisciplinary approach, our knowledge of how natural products can support gut–liver axis health amid mycotoxin challenges can be significantly improved.

5. Conclusions

This study systematically investigated the significant hepatoprotective and microbiota-modulating effects against T2 toxin-induced toxicity in mice, demonstrating that lycopene protects against T2 mycotoxicosis through a coordinated gut–liver axis mechanism, wherein restoration of intestinal eubiosis, particularly enrichment of Muribaculum and suppression of Escherichia, alleviates hepatic inflammatory stress and de-represses PPAR signaling, thereby restoring fatty acid oxidation and phase I detoxification gene expression. The striking dose–response relationship observed, wherein low-dose lycopene conferred superior protection relative to high-dose lycopene, carries important translational implications. This non-linear efficacy aligns with the hormetic properties documented for many dietary phytochemicals and suggests that empirical dose selection, rather than maximally tolerated dosing, is critical for therapeutic development. These findings position the gut–liver–PPAR axis as a targetable pathway for mycotoxin mitigation and establish lycopene as a promising feed additive candidate. However, the diminished efficacy at higher doses underscores the necessity for rigorous dose optimization studies before translational application in animal or human nutrition.