Healthy Specific Pathogen-Free (SPF)-grade male C57BL/6 J mice (aged = 6–8 weeks; average body weight = 20 ± 2 g) were sourced from the Experimental Animal Center of Guangxi Medical University (License number: SCXK Gui 2020-0003). All animal experimental procedures were approved by the Ethics Committee of Guangxi Medical University. One week before the experiment, the animals were acclimatized and housed in a barrier-level animal room with controlled temperature (22–25°C) and relative humidity (50–70%) and a 12 h/12 h light/dark cycle. During the experiment, the mice were fed an SPF-grade growth and reproduction diet and had ad libitum access to food and water.

Zinc gluconate (analytical grade, purity = 98%) was acquired from Macklin (Shanghai, China). Ampicillin (A9518-25G-9), neomycin sulfate (N6386-5G), metronidazole (M1547-25G), gentamicin sulfate (E003632-1 g), vancomycin (1404-93-9) and LPS (O55:B5, L2880-25MG) were all purchased from Sigma-Aldrich (United States, purity > 95%). The electron microscope fixative solution (G1102), Bovine Serum Albumin (BSA, GC305010), and the 4% paraformaldehyde fixative solution were purchased from Servicebio (Wuhan, China).

The main experimental instruments included a high-speed low-temperature tissue grinder (Beijing, China), an ultra-trace Ultraviolet (UV) spectrophotometer (ThermoFisher Scientific, United States), an enzyme marker (Elx-808, Bio-Tek, United States), an electrophoresis and electrostatic transducer (Bio-Rad, USA), a Transmission Electron Microscope (TEM; HT7800, Hitachi), a dual-color infrared imaging system (ODYSSEY, Licor), an optical microscope (BX53F + DP73, OLYMPUS, Japan), and a 7,500 Real-Time Fluorescence Polymerase Chain Reaction (RT-PCR) Instrument (Applied Biosystems, United States).

In our previous study, we successfully constructed an AAC mouse model via the Antibiotic cocktail (ABX) gavage approach (Yang et al., 2022). Herein, the ABX formulation scheme was as previously described (Hill et al., 2010; Liu J. et al., 2023). Specific ingredients included ampicillin (1 mg/mL), metronidazole (1 mg/mL), gentamicin (1 mg/mL), neomycin sulfate (1 mg/mL), and vancomycin hydrochloride (0.5 mg/ mL).

The LPS-induced intestinal infection was defined as in previous literature (Ding et al., 2004). The specific preparation approach was as follows. First, 25 mg LPS crystals were dissolved fully in 5 mL sterile physiological saline to obtain a stock solution with a concentration of 5 mg/mL. Subsequently, the solution was stored separately in a −20°C refrigerator in the dark. During the experiment, the appropriate amount of the stock solution was suctioned and diluted to obtain a final concentration of 0.5 mg/mL.

Herein, the ZG solution was prepared as previously described (Lai et al., 2016; Hsieh et al., 2017). Briefly, ZG was dissolved in ultrapure sterile water to obtain the Zn(C₆H₁₁0₇)₂ stock solution (with a concentration of 9.11 mg/mL). The stock solution was then sterilized using a 0.22 μm filter and stored in the dark at 4°C. To ensure mice from each dosage group received the same volume of the ZG solution, experimental concentrations were obtained with an equal-volume dilution of the stock solution. We previously determined the median lethal dose (LD₅₀) of ZG and evaluated its safety in mice (Wang et al., 2024). Herein, 0.57 mg/mL, 1.14 mg/mL, and 3.42 mg/mL were selected as the low, medium, and high dose concentrations of the ZG solution, respectively.

We sourced 30 SPF-grade C57BL/6 J male mice and then acclimatized and fed them for 1 week before the experiment. The animals were randomly divided into five groups (N = 6): Normal Control (NC), model control (NS + ABX/LPS), low-dose ZG (ZG(L) + ABX/LPS), medium-dose ZG (ZG(M) + ABX/LPS), and high-dose ZG (ZG(H) + ABX/LPS). The NC group was left untreated throughout the experiment but received a normal diet and sufficient water. The remaining four groups were first subjected to continuous ABX gavage for 7 days (twice/day) to create an AAC mouse model. On day eight, LPS (5 mg/kg) was intraperitoneally injected to further induce the infection and exacerbate intestinal mucosal injury. Following that, on days 9, 11, and 13, the NS + ABX/LPS group received a tail vein injection of 0.9% Normal Saline (NS), while the ZG(L) + ABX/LPS, ZG(M) + ABX/LPS, and ZG(H) + ABX/LPS groups received tail vein injections of low (5.13 mg/kg), medium (10.26 mg/kg), and high (30.78 mg/kg) doses of the ZG solution, respectively.

The injection doses of ZG and NS were both set at 9 mL/kg but were adjusted per body weight changes in experimental mice on each day as the study progressed. Mice in all groups received adequate food and water. The body weights of mice were determined and recorded on days 1, 4, 8, 9, 10, 12, and 14. Furthermore, diarrhea was assessed in mice at fixed time points each day after ABX gavage (days 4 and 8), after LPS administration (day 9), and after ZG treatments (days 10, 12, and 14). Figure 1A shows the study program and design.

On day 14, the animals were anesthetized via inhalation of 1.5% isoflurane. Following that, blood was collected by removing the eyeballs, separating the serum, and storing it at −80°C. Subsequently, the mice were euthanized via cervical dislocation. The intestinal segment 5 cm proximal to the ileocecal junction was dissected and cut into ~1–2 cm sample tissues. All intestinal contents were then gently washed with pre-cooled physiological saline, and a portion of the sample tissue was fixed in a 4% paraformaldehyde solution for Hematoxylin & Eosin (H&E) staining and Immunohistochemistry (IHC) analysis. Another portion was immersed in the electron microscope fixative solution for TEM observation. The remaining sample tissues were labeled and stored in portions at −80°C for Western Blotting (WB) and RT-PCR assays. Furthermore, the contents of the cecum were collected and immersed in liquid nitrogen for gut microbiological testing.

Ileum specimens were obtained, routinely dehydrated, paraffin-embedded, sliced into 5 μm sections, deparaffinized with xylene, dehydrated with gradient alcohol, and subjected to H&E staining. The morphology of the intestinal tissues was then observed and photographed using a light microscope. The height of five intact villi (villus length, V) and the depth of five crypts (crypt depth, C) in each section were measured separately, and then the data were recorded. Following that, the chorionic ratio [Villous to Capillary Ratio (VCR or V/C)] was determined.

Ileal specimens were obtained, sliced into 1 mm × 1 mm × 1 mm tissue blocks, rinsed and fixed in 0.1 M Phosphate Buffered Saline (PBS; PH7.4), dehydrated in gradient alcohol, osmotically embedded, polymerized, and ultra-thinly sliced (60–80 nm). Following that, the slices were stained with alcohol-saturated 2% uranyl acetate and 2.6% lead citrate solution and then visualized and photographed using TEM.

The paraffinized sections were recovered through heating to 98°C in 10 mM citrate buffer (PH 6. 0) for 10 min. Endogenous peroxidase was blocked with 10% (v/v) H₂O₂ for 30 min, while nonspecific antigens were blocked with serum at Room Temperature (RT) (20–25°C) for 30 min. The paraffinized sections were then incubated with rabbit anti-JAMA (1:400; GB111265-100; Servicebio, Wuhan, China) and rabbit anti-MLCK (1:400; GB113358-100; Servicebio, Wuhan, China) primary antibodies at 4°C overnight and then treated with Horseradish Peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibodies (1:200; GB23303; Servicebio, Wuhan, China). Photographic images were captured and analyzed using an OLYMPUS microscope and Image-Pro Plus 6.0 software, respectively.

Total RNA was extracted from ~30 mg of colon tissue using NucleoZol reagent (740404.200, Gene Co. Ltd). Subsequently, RNA was quantified using a spectrophotometer at a wavelength of 260 nm. Complementary DNA (cDNA) was obtained via reverse transcription using the HiScript III RT SuperMix for qPCR (+gDNA wiper) Kit (R323-01, Vazyme, Nanjing, China). The ChamQ Universal SYBR qPCR Master Mix Kit (Q711-03, Vazyme, Nanjing, China) was used to perform qPCR on the ABI 7500 system (Applied Biosystems, CA, United States). Sangon Biotech Co. Ltd. (Shanghai) synthesized the primers used herein (Supplementary Table S1). The RT-QPCR data were analyzed using the 2^−ΔΔCT method, with the GAPDH gene expression as the endogenous control.

Total protein was isolated from the ileum and then quantified using the BCA Protein Assay kit. Protein (20 μg) from different samples was separated using 8 ∼ 12% Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) (Supplementary Table S2) and then transferred to Polyvinylidene Difluoride (PVDF) membranes. Supplementary Table S3 details the WB-related gene transfer conditions. The membranes were blocked with 5% skimmed milk for 4 h at RT and then probed overnight at 4°C with primary antibodies, including rabbit anti-ZO-1 (1:800, ab96587, Abcam), rabbit anti-Occludin [1:1,000, Cat #91131, Cell Signaling Technology (CST)], rabbit anti-Claudin-1 (1:1,500, AF0127, Affinity), rabbit anti-TLR4 (1:1,500, AF7017, Affinity), rabbit anti-NF-κB/p65 (1:2,000, Cat #8242, CST), and rabbit anti- β-actin (1:2,500, Cat #4970, CST), which was used as the loading control. Subsequently, the membranes were incubated with a goat anti-rabbit HRP-linked secondary antibody (1:10,000, Cat #bs-0295G-HRP, Bioss antibodies) for 1 h. The membranes were visualized using the Western BrightTM ECL kit (Advansta, CA, United States).

The concentrations of DAO, D-LA, and ET in serum were determined using ELISA kits (FANKEW, Shanghai, China; and Shanghai Kexing Trading Co., Ltd., China) per the manufacturers’ instructions.

Genomic DNA was extracted from the collected samples using the CTAB approach (Yu C. et al., 2023). A Nanodrop 2000 UV–Vis spectrophotometer (Thermofisher Scientific, Wilmington, MA, United States) and 1% agarose gel electrophoresis were used to examine the concentrations and quality of the DNA samples. The forward primer 341F (5′-CCTAYGGGRBGCASCAG-3′) and reverse primer 806R (5′-GGACTACNNGGGTATCTAAT-3′) were designed to amplify the V3 + V4 hypervariable regions of the 16S rDNA gene on a thermocycler PCR system. The PCR products were detected using 2% agarose gel electrophoresis and were gelled and recovered using an AxyPrep DNA gel recovery kit (Axygen Biosciences, Union City, CA, United States). Following quantification and homogenization, the DNA products underwent paired-end sequencing on an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, United States). Statistical analyses were performed using R (Version 2.15.3). Mice ileum contents were obtained via 16S rRNA high-throughput sequencing, commissioned by Wekemo Tech Group Co., Ltd. (Shenzhen, China).

Data were presented as Mean ± Standard Error of the Mean (mean ± SEM). The data were first collated using Excel 2019. One-way ANOVA was performed using SPSS27.0 software, and the Tukey method (for normally distributed data with equal variances) and the Kruskal-Wallis test (Dunn’s method; for non-normally distributed data) were used for multiple pairwise comparisons. The test value was set to p = 0.05, and results or differences with p < 0.05 were considered statistically significant. Drawings were generated using GraphPad Prism 9.0 software.

Supplementary Table S4 and Figure 1C show the changes in body weight in mice during the experiment. No statistically significant difference (F = 0.29, p = 0.884) was found in baseline body weight (day 0) of mice in each group, indicating that they were comparable across all five groups. As the experiment progressed, the weight of mice in the NC group exhibited a continuous growth trend. In contrast, the overall trend of weight changes in the other four groups remained basically the same, although a decrease was noted on day 4, which later gradually increased to the baseline level by day 8. Furthermore, a linear decline was found on day 9, while a gradual increase was noted from day 10 onwards. Notably, the NC group exhibited a higher final body weight (day 14) compared with the other four groups.

On day 4, there was no significant change in body weight in the NS + ABX/LPS group compared with the NC group (p > 0.05). However, on day 8, the NS + ABX/LPS group exhibited a significant decrease in body weight compared with the NC group (p < 0.05), with a more significant reduction found on Day 9 (p < 0.0001). On day 10, no significant increase in the body weight of mice was noted in all the ZG treatment groups compared with the NS + ABX/LPS group (p > 0.05). Furthermore, on days 12 and 14, the ZG(H) + ABX/LPS group exhibited a significant increase in body weight compared with the NS + ABX/LPS group (p < 0.01 or p < 0.001). These results suggested that the concurrent induction of antibiotics and LPS-induced intestinal barrier damage had a substantial negative impact on the body weight of mice, representing an effect that was effectively reversed by ZG treatment.

Figure 1B and Table 1 illustrate the changes in mice feces and diarrhea scores during the experiment, respectively. The NC group exhibited normal feces throughout the experiment. On the other hand, the NS + ABX/LPS group developed diarrhea from day 4 onwards, which manifested as a significant increase in diarrhea scores as the study progressed (p < 0.05). On the other hand, the ZG treatment group exhibited a significant reduction in diarrhea scores as the study progressed (p < 0.05).

On day 10, no significant difference in diarrhea scores was identified between the ZG treatment group and the NS + ABX/LPS group. On day 12, diarrhea scores in the ZG(M) + ABX/LPS and ZG(H) + ABX/LPS groups significantly decreased (p < 0.05). On day 14, ZG treatment group exhibited significantly lower diarrhea scores (p < 0.05), with some demonstrating even greater reductions (p < 0.01). Compared with the NC group, diarrhea scores were significantly higher in the other four groups on day 10 (p < 0.05). On day 12, diarrhea scores were significantly elevated in the NS + ABX/LPS and ZG(L) + ABX/LPS groups (p < 0.05), and they were also significantly escalated in the NS + ABX/LPS group on day 14 (p < 0.05). Notably, the diarrhea scores in the ZG treatment groups were consistent with those in the NC group on day 14. These findings suggested that the dual induction of antibiotics and LPS-induced intestinal dysfunction caused diarrhea in mice, in which ZG treatment significantly ameliorated. However, the amelioration of diarrheal symptoms exhibited variability across different doses of ZG.

Ultrastructural changes in the mouse ileum tissue were observed under a TEM (Figure 3). Ileal IECs of NC mice were columnar, with a long, tightly arranged and neatly distributed microvilli morphology. Other notable features encompassed precisely delineated TJ and intermediate junction structures, as well as the presence of bridging granule formations. Furthermore, cellular gaps were tightly packed, with clearly defined epithelial cell membranes and nuclear membranes. The cell nuclei were regular, with homogeneous cytoplasmic distribution and scattered organelles. Additionally, mitochondria, rough endoplasmic reticulum (RER), and lysosomes were visible, with intact membrane structures. Conversely, the IECs of NS + ABX/LPS mice exhibited cone-shaped morphology, with numerous microvilli shedding and disappearing. Additionally, TJs, intermediate junctions, and desmosome structures were absent, accompanied by widened intercellular spaces, partial damage and dissolution of epithelial cell membranes, and decreased cytoplasmic density. Furthermore, the nucleus was sunken (with an abnormal shape), the mitochondria were swollen, the membrane structure was blurred, the matrix was partially dissolved and cavitated, and the cristae were broken and invisible. Additionally, the RER expanded, with ribosomes sparsely distributed on the endoplasmic reticulum (ER), and lysosomes were visible. Compared with the NS + ABX/LPS group, with the increase of the ZG treatment dose, the morphology of IECs was gradually improved, the microvilli were arranged neatly, the ileum structure was gradually restored, and the epithelial cell membrane gradually became intact. Furthermore, organelle structures, such as the nucleus, mitochondria, and RER were gradually improved. These findings demonstrated that ZG exerted a therapeutic effect on the dual induction of antibiotics and LPS-induced intestinal mucosal damage.

The TLR4/NF-κB signaling pathway is an essential immune response regulatory system that is involved primarily in the perception and response to external pathogens (e.g., bacterial LPS, viral RNA, etc.) in vivo. In the intestines, this signaling pathway plays a critical role in maintaining intestinal barrier integrity and immune homeostasis (Samuelson et al., 2022).

According to TLR4 WB results (Figures 9A,B), TLR4 protein expression level was significantly higher in the NS + ABX/LPS group than that in the NC group (p < 0.05). Furthermore, compared with the NS + ABX/LPS group, TLR4 protein expression level was significantly lower (p < 0.05) in the ZG treatment groups (except ZG(L) + ABX/LPS), with one group demonstrating even a lower expression level (p < 0.0001). Notably, ZG(H) + ABX/LPS exerted the greatest inhibitory effect on TLR4 protein expression level. According to NF-κB/p65 WB results (Figures 9C,D), NF-κB/p65 protein expression level was significantly higher in the NS + ABX/LPS group than that in the NC group (p < 0.0001). Furthermore, compared with the NS + ABX/LPS group, the ZG treatment groups exhibited a significantly lower NF-κB/p65 protein expression level (p < 0.05), with some demonstrating even a lower expression level (p < 0.0001). Notably, ZG(H) + ABX/LPS exerted the greatest inhibitory effect on NF-κB/p65 protein expression level. These findings suggest that the dual induction of antibiotics and LPS-induced ileal mucosal injury in mice could be associated with the activation of TLR4 and NF-κB/p65 protein levels, and ZG could play an anti-inflammatory and barrier-protective role against antibiotics and LPS-induced intestinal mucosal injury.

Measurement of intestinal mucosal permeability can reveal the integrity and maturity of the gut. Increased intestinal mucosal barrier permeability may reflect disrupted barrier function. To assess this impairment, the levels of DAO, D-LA, and ET in mouse serum were quantified by ELISA.

Figure 10A indicates that compared with NC mice, the DAO content in the serum of NS + ABX/LPS mice was extremely significantly elevated (p < 0.0001). Compared with NS + ABX/LPS group, the DAO content in the serum of mice in each ZG treatment group was significantly or extremely significantly lower (p < 0.01 or p < 0.0001), reaching the lowest in the ZG(H) + ABX/LPS group. As illustrated in Figure 10B D-LA content in the serum of NS + ABX/LPS mice was extremely significantly higher than that in NC mice (p < 0.0001). Moreover, D-LA content in the serum of mice in each ZG treatment group was significantly or extremely significantly lower compared with the NS + ABX/LPS group (p < 0.01 or p < 0.0001), reaching the lowest in the ZG(H) + ABX/LPS group. Figure 10C showed that compared with NC, the ET content in the serum of NS + ABX/LPS mice was extremely significantly higher (p < 0.0001). The ET content in the serum of mice in each ZG treatment group was extremely significantly lower compared with the NS + ABX/LPS (p < 0.001 or p < 0.0001), being lowest in the ZG(H) + ABX/LPS. These findings indicate that the combined induction of antibiotics and LPS disrupted the intestinal barrier, resulting in heightened permeability of the intestinal mucosal barrier and inflammation. Treatment with ZG may alleviate the rise in intestinal mucosal barrier permeability and alleviate intestinal inflammation.

The weight of mice in the normal control (NC) group exhibited a consistent upward trend, consistent with typical physiological patterns. However, mice in the NS + ABX/LPS group experienced weight loss as the duration of antibiotic (ABX) usage extended, suggesting that prolonged administration of broad-spectrum antibiotics could result in weight reduction in mice. Exogenous intraperitoneal injection of LPS linearly decreased the weight of the mice, indicating that acute inflammation and intestinal barrier damage induced by LPS adversely affected the overall health of mice. Studies have explored the relationship between inflammation and weight regulation in animal models and human studies. Research by Patterson et al. showed that LPS could destroy the intestinal barrier, causing intestinal microecological components, endotoxins, and other substances to enter the bloodstream, triggering a systemic inflammatory response. In such cases, the mice might have experienced loss of appetite and inadequate energy intake, resulting in weight loss (Patterson et al., 2022). A study by Medeiros et al. showed that zinc supplementation reduced the fecal shedding of Shigella flexneri in zinc-deficient mice, improved body weight, and reduced intestinal inflammation (Medeiros et al., 2019). These findings are consistent with those obtained in this study, suggesting that ZG treatment can promote weight gain in mice to a certain extent, especially at high doses. Our results demonstrate the potential of ZG to confer intestinal protection and repair.

It has been shown that zinc has several intestinal health benefits. Zinc is not only a cofactor of various enzymes and regulates cell metabolism, but also maintains and repairs the intestinal barrier (Ohashi and Fukada, 2019; Sarkar et al., 2019). Thus, ZG can potentially alleviate diarrhea symptoms by enhancing the repair of IECs and attenuating the intestinal damage caused by the dual induction of antibiotics and LPS. Our results indicated that high-dose ZG was the most effective in reducing diarrhea scores on the 12th and 14th days of the experiment. These findings are consistent with several studies indicating that higher doses of zinc may improve intestinal mucosal barrier and facilitate the restoration of IECs (Su et al., 2023). Existing literature has highlighted the anti-inflammatory, antioxidant, and epithelial cell repair properties of ZG (DiSilvestro et al., 2015; Bücker et al., 2020). These benefits might be attributed to mechanisms such as the regulation of cell adhesion, stimulation of cell proliferation, and mitigation of inflammatory reactions. Such actions of ZG could contribute to preserving intestinal barrier integrity and impeding the advancement of intestinal diarrhea.

The morphological structure of the small intestinal mucosa, intestinal injury score, and histopathological grading are often considered important evaluation indicators of the digestion and absorption, intestinal health and functional recovery processes. Among them, the height of small intestinal villi and the depth of crypts are regarded as important indicators of the digestion and absorption function in the small intestine (Ding et al., 2004; Wu et al., 2019). Shortened villus height implies lower surface area for nutrient absorption, while deeper crypt depth indicates faster turnover of new villus cells. In addition, VCR can be a robust marker for assessing intestinal function and health status (Rubin and Levin, 2016). Under light microscopy, analysis of ileal tissues revealed severe damage to the intestinal mucosa in mice subjected to dual antibiotics and LPS induction. The mucosa displayed significant shedding of epithelial cells from the intestinal villi, along with widened intercellular spaces, granulocyte infiltration, and disrupted crypt structures. Moreover, the reductions in villus height, crypt depth, and the VCR were observed, indicating impaired intestinal barrier function in mice. In this study, we found that treatment with different doses of ZG, ZG(L) + ABX/LPS slightly improved the pathological damage of the ileum tissue in mice, but did not completely restore the normal structure. In comparison, ZG(M) + ABX/LPS and ZG(H) + ABX/LPS treatment significantly improved the pathological damage of ileum tissue, as evidence by the reduced mucosal damage, increased intestinal villus height and crypt depth, and increased goblet cells. ZG is known to possess anti-inflammatory and antioxidative properties, which could contribute to its protective effects on the intestinal mucosa. Specifically, ZG has been shown to inhibit the production of pro-inflammatory cytokines and reduce oxidative stress, both of which are implicated in the pathogenesis of intestinal barrier dysfunction and mucosal damage. Additionally, ZG has been reported to modulate immune responses and promote mucosal healing, further supporting its potential in ameliorating intestinal injury. It is important to emphasize how the observed improvements in ileal morphology correlate with the known pharmacological properties of ZG. The observed protective effects of ZG on ileal morphology could be attributed to its multifaceted mechanisms of action. Firstly, ZG possesses anti-inflammatory properties, which may play a pivotal role in mitigating mucosal damage. By inhibiting the production of pro-inflammatory cytokines, such as IL-6 and tumor necrosis factor-alpha (TNF-α), ZG can attenuate the inflammatory response triggered by dual induction of antibiotics and LPS. This anti-inflammatory effect helps to reduce tissue inflammation and prevent further damage to the intestinal mucosa. Secondly, ZG exhibits antioxidative properties, which are crucial for protecting against oxidative stress-induced mucosal injury. Oxidative stress, characterized by an imbalance between ROS production and antioxidant defense mechanisms, contributes to intestinal barrier dysfunction and mucosal damage. ZG acts as a scavenger of ROS and enhances the activity of endogenous antioxidant enzymes, thereby reducing oxidative damage to the intestinal epithelium and promoting mucosal integrity. Furthermore, ZG has been reported to modulate immune responses, including the regulation of immune cell infiltration and cytokine production. By modulating the immune microenvironment in the intestine, ZG can attenuate immune-mediated damage to the mucosa and promote tissue repair and regeneration. Collectively, these results imply that ZG can protect against the damage of intestinal mucosal morphology and structure caused by dual induction of antibiotics and LPS, especially the ZG(H) + ABX/LPS formulation.

Intestinal microvilli and TJ are key structures of IECs which maintain the stability of the intracellular environment, regulating epithelial permeability, and maintaining cell polarity. IECs increase the intestinal surface area by forming finger-like protrusions, known as intestinal microvilli, which promotes the absorption of nutrients and invasion defense against antigens (Jauregi-Miguel, 2021). Under an electron microscope, normal and healthy intestinal microvilli exhibit a columnar, orderly, tightly packed, and evenly arranged appearance. This organized arrangement and consistent density are vital for the proper functioning of IECs and maintaining the stability of the intestinal environment. Therefore, both intestinal microvilli and TJs modulate the IEC function and intestinal health. TEM analysis of the ultrastructure of IECs at the microscopic level has demonstrated that IECs from NC mice exhibit normal morphology. Microvilli are neatly arranged with a tight structure, while TJs, intermediate junctions, and desmosome structures are clearly visible. Intercellular spaces are compact, and the distribution of the nucleus and cytoplasm is even. Moreover, the structures of mitochondria, rough endoplasmic reticulum, and lysosomes appear intact. The IECs of NS + ABX/LPS mice exhibited abnormal morphology, significant shedding of microvilli, structural damage to the TJ, disappearance of the intermediate junction, and desmosomal structures, widening of the intercellular space, dissolution of the epithelial cell membrane, damaged and sunken nucleus, swollen mitochondria, among other alterations. Following ZG treatment, the morphology of IECs in ZG(L) + ABX/LPS and ZG(M) + ABX/LPS mice was partially improved, but some pathological features such as partial loss of microvilli, sparse and disordered arrangement, blurred TJ structure and mitochondrial swelling could still be observed. However, the microvilli of IECs in ZG(H) + ABX/LPS mice were satisfactorily repaired, and the TJ, intermediate junction and desmosome structures could be visualized. This study demonstrates that dual induction with antibiotics and LPS can destroy the morphology and TJ structure of mouse intestinal epithelial microvilli, as well as the mechanical barrier of the mouse intestinal mucosa, thereby increasing intestinal permeability. ZG confers protection against the detrimental effect of dual antibiotics and LPS induction on IECs microvilli and TJ, thereby reducing intestinal mucosal permeability and facilitating the restoration of the compromised intestinal mucosal barrier. Zinc, as a critical micronutrient, modulates cell signal recognition and enhances intestinal barrier function. Zhou et al. demonstrated that exogenous Zn-Asp can mitigate damage induced by DON exposure through the regulation of mouse intestinal stem cell renewal and regeneration capacity, thus ameliorating intestinal morphology and barrier function (Zhou et al., 2020). Furthermore, oral administration of ZnBM has been shown to restore the integrity of LPS-damaged Caco-2 cell monolayers and significantly enhance barrier function (Liu L. et al., 2023).

IECs are the outermost cells in the intestine. They establish a tight barrier via the TJ and control the permeability of intestinal substances. These TJs contain numerous types of proteins, including structural proteins (ZOs), transmembrane proteins (Occludin and Claudins), and junction adhesion molecules (JAMAS) (Laukoetter et al., 2006; Allaire et al., 2018). The integrity of the TJ complex is crucial in preventing the infiltration of substances like bacteria, pathogens, and endotoxins into the intercellular space. Disruption of intestinal mucosal TJ proteins can elevate intestinal mucosal permeability, resulting in bacterial translocation, infection, diarrhea, and related symptoms (Kayama et al., 2020; Oda et al., 2022). Furthermore, the contractility of IECs significantly impacts intestinal mucosal permeability. Myosin and actin in the cytoskeleton modulate the contraction of IECs and are regulated by MLCK (González-Mariscal et al., 2008; He et al., 2008). It has been demonstrated that myosin function is altered by intestinal inflammation, leading to disruption of the TJ and increased intestinal permeability (Naydenov et al., 2016).

The expression level of ZO-1 influences the integrity of the mucosal barrier and thus the formation of TJs and integrity of epithelial cells. Disruption of ZO-1 may damage the mucosal barrier and increase permeability to harmful substances (Kuo et al., 2021, 2022). Evidence from prior investigations demonstrate that LPS modulates the function and structure of epithelial cells by causing excessive production of intracellular oxidative stress (ROS) and release of pro-inflammatory factors (TNF-α, IL-6), and decreasing the expression of mechanical barrier proteins (ZO-1 and Occludin) (Wu et al., 2018). The findings of our study indicated a significant reduction in ZO-1 expression in the ileal mucosa of mice subjected to both antibiotic and LPS induction. This decline in ZO-1 expression may compromise the normal function of this TJ protein within the intestinal mucosal epithelium, thereby weakening the intestinal mucosal barrier function. Previous research suggests that zinc ions may potentially enhance the integrity and functionality of the mucosal barrier. This could be attributed to zinc’s ability to modulate cell signaling pathways and exert antioxidant and anti-inflammatory effects (Ohashi and Fukada, 2019). In the Caco-2 model, Wang et al. found that zinc altered the morphology of the intestinal TJ, regulated the structure and function of the intestine, and enhanced the intestinal barrier function (Wang et al., 2013). In our study, we also found that ZG promoted the expression of ZO-1 in IECs which strengthened and repaired the intestinal barrier function.

Occludin and Claudins are TJ proteins existing between IECs and are responsible for maintaining the intestinal mucosal barrier and reducing inflammatory extravasation (Da et al., 2022). Hua et al. have showed that the down-regulation of occludin resulted in intestinal barrier impairment. The decrease in occludin expression level is driven by inflammation, antibiotic interventions, and exposure to endotoxins such as LPS, all of which disrupt the TJ among IECs (Hua et al., 2023). The present findings are consistent with this, demonstrating that the combined induction of antibiotics and LPS significantly decreased occludin gene and protein expression in the ileal tissues of mice. Furthermore, previous research suggests that zinc may positively regulate the expression of mucosal barrier proteins to produce inhibit inflammation, oxidative damage and promote epithelial cell repair (Souffriau et al., 2020; Wu et al., 2023). Yi et al. showed that zinc oxide (ZnO) increased the height of ileal villi and enhanced the expression of Occludin in the duodenum and ileum, as well as increased the mRNA expression levels of MUC-2 and ZO-1 in the jejunum and ileum. In this way, it reduced diarrhea symptoms and improved intestinal barrier function in weaned piglets (Yi et al., 2023). These findings are consistent with our results. ZG may repair and maintain the intestinal mucosal barrier by positively regulating Occludin expression. Meanwhile, the regulation of Occludin by ZG exhibits a dose-dependent pattern, with higher doses potentially being more efficacious in promoting intestinal barrier repair. Downregulation of claudin-1 expression decreases the number and complexity of TJs, thereby weakening the barrier function of the intestinal epithelium. Abnormal expression and distribution of claudin-1 results in breakage of TJ chains and formation of holes, allowing ions and macromolecular solutes to enter via the intercellular space, causing damage to the intestine and reducing the barrier function of the intestine (Schmitz et al., 1999; Fischer et al., 2014). Prior research demonstrated that Claudin-1 plays a critical role in maintaining intestinal barrier integrity by regulating paracellular permeability (Saeedi et al., 2015). Furthermore, Atsugi et al. demonstrated that Claudin-1 knockout mice exhibited severe barrier dysfunction, leading to epidermal dehydration and ultimately death, highlighting the essential role of Claudin-1 in mucosal barrier function (Atsugi et al., 2020). Claudin-1 forms the mucosal barrier, and its downregulation has been linked to intestinal inflammation and injury (Zhong et al., 2015; He et al., 2019). Our findings suggest that the combined induction of antibiotics and LPS may have disrupted Claudin-1 expression, thereby impairing the intestinal barrier. Zinc has been shown to positively regulate mucosal barrier integrity and intestinal health. It is plausible that ZG enhances tight junctions by promoting Claudin-1 expression, facilitating the repair of damaged intestinal mucosa. The differential effects of varying doses of ZG on Claudin-1 regulation may be attributed to dose-dependency.

The mRNA expression of JAMA and JAM2 is a commonly used as an indicator for assessing the intestinal barrier integrity (Wong and Kinstler, 2023). It has also been reported that JAM-A can promote the repair of mucosal damage in the JAM-A gene-deficient (JAM-A^−/−) mouse model of colitis. Overexpression of JAM-A decreases the permeability of IECs, which in turn increases the body’s immune compensation (Vetrano et al., 2008). Our findings indicate that the dual induction of antibiotic- and LPS-induced ileal mucosal damage in mice promoted a significant downregulation of JAM-A mRNA and protein expression levels, which further damaged the intestinal mucosal barrier. ZG treatment significantly increased the expression levels of JAM-A mRNA and protein, suggesting that ZG regulate the repair of intestinal mucosa barrier by regulating JAM-A.

The adhesion between IECs and the stability of the pericellular skeleton are crucial for maintaining the integrity of the intestinal barrier. MLCK is an enzyme that regulates IEC contraction and intercellular junction stability by phosphorylating myosin light chains, thereby influencing intestinal permeability. In response to inflammation or damage, the expression and activity of MLCK increase, resulting in IEC contraction and the loosening of intercellular connections. Furthermore, intestinal injury can compromise the intestinal epithelial barrier, allowing endotoxins like LPS to penetrate. LPS can activate MLCK, thereby damage TJ protein structure of IECs leading to intestinal barrier destruction and increased permeability, making it easier for bacteria, toxins, and other harmful substances to enter the intestinal wall and enter the blood circulation. This triggers inflammatory reactions and diseases (Han et al., 2017; Zhang et al., 2022). Application of the MLCK inhibitor (ML-7) enhanced LPS-induced HPAEC activity, reduced the expression of p-MLCK, and weakened the phosphorylation of myosin light chain or MLCK, thereby stabilizing vascular barrier function (Bai et al., 2009). Elsewhere, MLCK gene knock out or treating wild-type mice with MLCK inhibitors prevented the phosphorylation of MLCK in IECs, maintained TJs structure, reduced protein leakage, and alleviated diarrhea caused by T cell activation (Clayburgh et al., 2005). These findings align with our current investigation, underscoring the importance of MLCK in preserving intestinal barrier integrity and its association with diarrhea pathogenesis. Our study reveals that ZG treatment decreases MLCK expression, suggesting it as one mechanism behind its protective effects. By inhibiting MLCK, ZG may safeguard intestinal barrier function and mitigate inflammatory damage. In summary, our results suggest that ZG can restore intestinal mucosal permeability induced by the dual induction of antibiotics and LPS in mice by up-regulating expression of structural proteins, transmembrane proteins, and adhesion-linkage molecules in the TJ, as well as down-regulating the expression of myosin light-chain kinase.

Serological testing of intestinal barrier function may contribute to the diagnosis of intestinal barrier dysfunction. Studies have shown that DAO, D-LA, and ET in serum are the main biomarkers of intestinal mucosal permeability and indirectly reflect intestinal mucosal barrier damage (Yu et al., 2022).

DAO is a highly active structural enzyme expressed in IECs, and its blood levels can be an indicator of intestinal barrier integrity. DAO is usually present in blood circulation in very small amounts, and its levels are influenced by the maturity and integrity of the intestinal mucosa (Camara-Lemarroy et al., 2021). Damage to the intestinal mucosal barrier can lead to a leak of DAO into the bloodstream. This rise in serum DAO levels can be a valuable indicator of the severity of intestinal mucosal diseases (Li et al., 2010). D-LA is a metabolite of intestinal bacteria which may be produced by several types of bacteria in the intestine (such as Escherichia coli, lactobacillus, Klebsiella, etc.). Given that no other tissue can produce or metabolize D-LA, the D-LA detected in the blood -LA is only derived from the intestines (Li et al., 2016). Disruption of the intestinal barrier integrity results in an increase in intestinal permeability, allowing the passage of D-LA produced by gut bacteria through the damaged mucosa into the bloodstream. Consequently, blood D-LA level is a potential clinical indicator for assessing intestinal barrier permeability in patients and identifying the translocation of intestinal endotoxins and bacteria post-surgery. ET, a toxic molecule found in the cell walls of Gram-negative bacteria, is released upon bacterial death or rupture (Vindigni et al., 2016). ET is a component of LPS and is commonly present in the cell walls of Enterobacteriaceae. The intestine harbors large amounts of LPS, primarily secreted from intestinal bacteria. Under normal conditions, the intact intestinal barrier prevents LPS from entering the bloodstream. However, following damage of the intestinal barrier function, the permeability of the mucosal barrier is increased, allowing LPS to enter the intestinal mucosa and the bloodstream, resulting in LPSemia. Therefore, monitoring peripheral blood LPS levels serves as a vital approach for assessing bacterial translocation across the intestinal barrier (Earley et al., 2015; Li W. et al., 2023).

In this study, we comprehensively assessed the functional status of the intestinal barrier by evaluating altered intestinal mucosal permeability, the degree of damage to intestinal mucosal cells, and bacterial translocation endotoxemia using the three indices of DAO, D-LA, and ET, respectively. The results indicated that the levels of DAO, D-LA, and ET in the serum of NS + ABX/LPS mice were significantly higher compared with those of NC mice. The serum levels of DAO, D-LA, and ET in various ZG treatment groups were significantly or extremely significantly reduced. These findings indicate that the combined administration of antibiotics and LPS increased intestinal mucosal permeability in mice. However, ZG treatment significantly alleviated the increase in intestinal mucosal permeability induced by the combined use of antibiotics and LPS. Therefore, ZG may protect the intestinal tract from damage by reducing the permeability of the intestinal mucosal barrier and/or improving intestinal barrier integrity. Moreover, there is evidence that Proteobacteria can secrete endotoxins. Elsewhere, Earley et al. also reported that downregulation of S24-7 in Bacteroidetes promoted burn-induced bacterial translocation and increased intestinal permeability (Wessels et al., 2017). In this study, a correlation was observed between the rise in intestinal mucosal permeability and serum ET levels in model mice, which exhibited decreased Bacteroidetes and Firmicutes abundance and increased abundance of Proteobacteria in the microbial barrier, consistent with previous reports. This suggests that ZG may preserve intestinal barrier integrity by inhibiting dysbiosis induced by dual antibiotics and LPS, thereby reducing endotoxemia. However, further research is needed to elucidate whether ZG maintains intestinal barrier function through microbiota modulation and its underlying mechanism.

The TLR4/NF-κB signaling pathway regulates the maintenance and repair of intestinal barrier. Research indicates that activation of this pathway can improve the adhesion of IECs and decrease intestinal permeability, thereby enhancing the integrity of the intestinal barrier. However, prolonged or excessive activation of the TLR4/NF-κB signaling pathway may suppress intestinal inflammation, causing harm and intestinal barrier impairment (Samuelson et al., 2022). In previous studies, LPS increased the permeability of TJs in the gastrointestinal mucosal epithelium and damaged its integrity. This process allowed endotoxins to enter the intestine via paracellular osmosis, causing endotoxin translocation and abnormal expression of cytokines, further exacerbating intestinal inflammation and damage (Zhao et al., 2023). Moreover, our results demonstrate that dual induction by antibiotics and LPS activate the NF-κB signaling pathway via TLR4 resulting in inflammation and immune responses.

Zinc can promote the proliferation and repair of IECs and enhance the repair of intestinal damage. Indeed, zinc deficiency decreased intestinal barrier function (Haase and Rink, 2009). Immune mediators such as TLR ligands can elevate intracellular Zn²⁺ by promoting the expression of ZIP transporter proteins and down-regulating ZnT. The concentration of Zinc is a major regulator of monocytes which recognize bacterial LPS and activate TLR4. A moderate increase in free zinc levels may stimulate LPS-activated monocyte secretion of pro-inflammatory factors (Song et al., 2015; Hu et al., 2023). Song et al. reported that zinc improves the integrity of the intestinal barrier in weaned piglets via the MAPK and TGF-β signaling pathways (Lee et al., 2018). Previous research has demonstrated that inhibiting the intestinal NF-κB using zinc-based drugs can lower the levels of cytokines TNF-α and IL-6, thus protecting the intestinal mucosal barrier from damage induced by LPS in young mice (Xu et al., 2018; Xiao et al., 2019). The findings of our study also suggest that ZG may exert its anti-inflammatory properties and maintain the intestinal barrier by modulating the TLR4/NF-κB signaling pathway. However, our study did not explore the specific regulatory mechanism of ZG in the TLR4/NF-κB/p65 signaling pathway and its impact on inflammatory signaling and intestinal barrier function. Further investigations should primarily elucidate these aspects.

In this study, Illumina NovaSeq sequencing of the 16S rRNA V3 + V4 hypervariable region was employed to assess the composition and alterations of the cecal microbiota in normal mice, mice with induced intestinal mucosal barrier injury through dual antibiotic and LPS administration, and ZG-treated mice. Analysis of Shannon, Simpson, and Observed features indices indicated higher microbial diversity in normal mice compared to model mice, suggesting that the dual induction of antibiotics and LPS reduces intestinal flora diversity. However, each ZG treatment group exhibited varying degrees of increase in the Shannon index and decrease in the Simpson index. These findings demonstrate that ZG treatment contributes to the restoration of bacterial flora diversity and richness. Using the weighted UniFrac algorithm, we compared the composition of the microbiota of in mouse cecum based on the PCoA and NMDS methods. Analysis of scatter plots of PCoA and NMDS scores showed that bacterial flora separation occurred at the ASV level in the normal control group, model control group, and ZG treatment group, indicating that treatment and dosage factors can affect bacterial flora composition. There appears to be a partial overlap in bacterial communities between the NS + ABX/LPS and ZG(L) + ABX/LPS groups, although they are separate from the NC, ZG(M) + ABX/LPS, and ZG(H) + ABX/LPS groups. This suggests that low-dose ZG treatment may have limited restorative effects on the intestinal flora. In addition, there is partial overlap between the ZG(M) + ABX/LPS and ZG(H) + ABX/LPS groups, with some similarity observed with the NC group, indicating potential similarities in bacterial flora composition among the mid- and high-dose ZG treatment groups. The recovery of bacterial flora in the intestinal tract may offer some beneficial effects. Overall, we hypothesize that the therapeutic dosage of ZG may exhibit a threshold effect on modulating the composition of the intestinal flora.

Furthermore, we analyzed the distribution of intestinal microbial colony content among groups at the phylum and genus levels. In this study, Proteobacteria, Firmicutes, Bacteroidetes, Verrucomicrobia, and Actinobacteria were the dominant groups, which is consistent with the findings of previous studies (Kim et al., 2022). However, treatment of mice with a double blow of antibiotics and Escherichia coli (E. coli O55:B5) altered these dominant bacterial groups, with the relative abundance of Proteobacteria, Blautia, and Enterobacter significantly increased. The relative abundance of Firmicutes, Bacteroidetes, Verrucomicrobia, Lactobacillus, and Akkermansia was significantly reduced. These findings align with the anticipated dysbiosis of microbial communities induced by the combined use of antibiotics and LPS.

Proteobacteria are the main pathogenic bacterial phylum which contributes to symptoms such as intestinal infections, diarrhea, immune imbalance, and dysbiosis. Research shows that Proteobacteria is the main bacterial phylum in the intestine, and its high abundance may indicate a biological disorder in the microflora (Huang et al., 2019). Enterobacter is a facultative anaerobic bacterium. The excessive proliferation of Enterobacter can trigger an abnormal activation of the host’s immune system, resulting in an inflammatory response. Consequently, it is commonly viewed as a signaling indicator of pathogenic microorganisms. In this study, the relative abundance of Proteobacteria and Enterobacter in the NS + ABX/LPS group was significantly increased suggesting that dual induction of antibiotics and LPS caused imbalance in the intestinal flora of mice. However, ZG(M) + ABX/LPS and ZG(H) + ABX/LPS treatment significantly reduced the abundance of Proteobacteria and Enterobacter. These results indicate that ZG intervention may have a counter-regulatory effect on the relative abundance of Proteobacteria and Enterobacter.

Firmicutes are integral to intestinal health, contributing significantly to the digestion of complex carbohydrates by producing enzymes and generating beneficial metabolites like short-chain fatty acids (such as propionic, acetic, and butyric acids). These compounds not only supply energy to intestinal cells but also support the integrity of the intestinal mucosal barrier. Moreover, Firmicutes play a crucial role in inhibiting the proliferation and infiltration of pathogenic bacteria through various mechanisms, including the production of antimicrobial substances, regulation of intestinal pH, and reinforcement of the intestinal mucosal barrier. As a result, Firmicutes protect the intestinal tract from potential infections and maintain its overall well-being (Kapoor et al., 2023). Although glycosides cannot be digested by the body into short-chain fatty acids, Bacteroidetes can degrade them thereby providing calories and nutrients to the body. High abundance of Bacteroidetes can therefore improve the function of the intestinal mucosal barrier and enhance the innate immune response. A study by Sonnenburg J.L. showed that Bacteroidetes can inhibit bacterial growth by prompting IECs to secrete substantial mucus and binding it to peptidoglycan on the bacterial surface. Moreover, Verrucomicrobia, a group of microorganisms often found in the intestinal mucus layer, produce metabolites that stimulate IECs, exerting a regulatory influence in the intestinal environment. In addition, Verrucomicrobia can activate the immune response via activating the G protein-coupled receptors (Kim et al., 2021). Lactobacillus belongs to the group of Firmicutes and is considered a probiotic. It produces lactic acid which inhibits the colonization and growth of pathogenic bacteria in the intestinal mucosal epithelium by decreasing intestinal pH. Furthermore, lactic acid bacteria can stimulate the secretion of adhesins, prevent the apoptosis of IECs, and consequently, help uphold the stability of the intestinal mucosal barrier (Miranda et al., 2018; Choi et al., 2020). Akkermansia is a potentially beneficial gut bacterium classified as Verrucomicrobia. It can increase the thickness of the intestinal mucosa, break down the mucus on the intestinal surface, and suppress the production of inflammatory cytokines, thereby improve the intestinal barrier function and stability of the intestinal microbiota (Derrien et al., 2017; Plovier et al., 2017). In our investigation, the NS + ABX/LPS group exhibited a significant decrease in the relative abundance of Bacteroidetes, Verrucomicrobia, Firmicutes, Parabacteroides, Lactobacillus, and Akkermansia. This suggests that the combined induction of antibiotics and LPS disrupted the balance of intestinal flora in mice. Specifically, Bacteroides and Parabacteroides showed significant increases in the ZG(M) + ABX/LPS and ZG(H) + ABX/LPS groups, while Firmicutes and Lactobacillus were significantly increased in the ZG(L) + ABX/LPS and ZG(H) + ABX/LPS groups. Verrucomicrobia and Akkermansia were significantly increased in ZG(H) + ABX/LPS. Altogether, our results suggest that ZG can restore the relative abundance of the above bacteria, but the performance varied among different treatment groups, suggesting a ZG dose-dependent effect.