Evaluation of Zn-Peptides from Holothuria scabra: Gut Microbiota Profile and Cytokine Signalling Pathways in Rat Pups Born to Zinc-Deficient Parent

Evaluation of Zn-Peptides from Holothuria scabra: Gut Microbiota Profile and Cytokine Signalling Pathways in Rat Pups Born to Zinc-Deficient Parent | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of Zn-Peptides from Holothuria scabra: Gut Microbiota Profile and Cytokine Signalling Pathways in Rat Pups Born to Zinc-Deficient Parent Gita Syahputra, Fajrin Shidiq, Yustinus Maladan, Windi Susmayanti, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7478928/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Zinc is essential for skeletal development and maintaining immune homeostasis, with zinc deficiency (ZD) leading to compromised immunity and heightened infection risk. This study explores the potential of Zinc-chelating Peptides (ZCP), a novel third-generation zinc supplement sourced from Holothuria scabra , to ameliorate ZD-induced immune dysregulation and gut dysbiosis in rat offspring. Through a metagenomic approach, we demonstrate that ZD markedly increases pro-inflammatory cytokines (IL-6, IL-10, IFN-γ) and disrupts gut microbial diversity. ZCP treatment effectively modulated these cytokine profiles, with ZnSO 4 supplementation (ZDZn group) leading to the greatest restoration of microbial diversity. These results underscore the critical role of the gut microbiome in mediating zinc's immunomodulatory effects and suggest that ZCP holds promise for restoring both gut health and immune balance in ZD states, necessitating further exploration of its underlying mechanisms and long-term implications. immunity infection microbes sea cucumber supplementation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Growth retardation is a well-documented and significant consequence of zinc deficiency (ZD) in young animals [ 1 ]. This impairment in growth may arise from a combination of factors, including a reduction in the body's ability to synthesize proteins, which are the building blocks of tissues, and an increase in catabolic processes that break down existing tissues [ 2 ]. Zinc plays a crucial role in the processes of bone growth and development, influencing the proliferation and differentiation of chondrocytes, the cells responsible for cartilage formation in the growth plates [ 1 , 3 ]. Consequently, insufficient zinc can directly impede the longitudinal growth of bones, contributing to overall growth stunting. The intricate relationship between zinc and fundamental anabolic processes highlights its essential role in achieving normal growth and development in young mammals [ 1 , 2 , 4 – 6 ]. In young mammals, including rat pups which serve as a valuable model for studying early development, ZD can lead to a spectrum of serious health consequences. These include impaired growth and overall development, hindering the achievement of full physical potential[ 7 ]. Furthermore, ZD severely compromises the function of the immune system, leading to an increased susceptibility to a wide range of infections. Behavioural patterns can also be negatively affected, manifesting as impairments in activity, attention, and motor development [ 8 – 13 ]. Notably, maternal ZD during pregnancy can also have detrimental effects on offspring, influencing their growth, neurobehavioral development, and immune function [ 11 – 13 ]. The multifaceted negative impacts of ZD during this sensitive life stage underscore the critical need for effective preventative and therapeutic strategies. The immune system exhibits a particular sensitivity to fluctuations in zinc status. Zinc is vital for the proper development, differentiation, and function of a wide array of immune cells, including T cells, B cells, natural killer (NK) cells, and neutrophils.[ 14 ] Furthermore, zinc is involved in the production and regulation of cytokines, which are signaling molecules that coordinate immune responses [ 15 , 16 ]. A deficiency in zinc can disrupt the delicate balance of these immune components, leading to impaired immune function and an increased vulnerability to infections. The impact of zinc on the immune system underscores its critical role in maintaining the body's defenses against pathogens, particularly in young, developing individuals whose immune systems are still maturing [ 16 , 17 ]. Studies show that ZD in humans reduces the production of interferon-gamma (IFN-γ) and have a negative effect on cell-mediated immunity [ 18 ]. The IFN-γ increase is associated with epigenetic and redox-mediated mechanisms [ 19 ]. ZD has been demonstrated to elevate reactive oxygen species (ROS) production, which in turn leads to increased activation of p38 mitogen-activated protein kinase (MAPK). This increased activation is necessary for the synthesis of interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) [ 19 ]. Many of the adverse effects of ZD on cytokine production can be reversed through zinc supplementation. Supplementation corrects the production of both interleukin-2 (IL-2) and interferon-gamma (IFN-γ) [ 20 ]. Nonetheless, there is a paucity of research on the topic, but studies on zinc chelates have not yet been published in relation to cytokine secretion resulting from the supplementation with this Zn-peptides type. Zinc and the microbiota of the gastrointestinal tract have been demonstrated to be interrelated. Research findings indicate that zinc levels can influence the composition and function of the gastrointestinal microbiota, and vice versa. The gut microbiota, a complex ecosystem present within the gastrointestinal tract, plays a pivotal role in various physiological processes, including nutrient absorption, intestinal permeability regulation, host metabolism, and modulation of mucosal and systemic immunity [ 21 ]. Physiological and nutritional doses of zinc have been demonstrated to enhance gut wall integrity, thereby mitigating the translocation of bacteria and gut microbiome metabolites into the systemic circulation [ 22 ]. Specific bacteria caused of high zinc diets can enrich Akkermansia , Faecalibaculum , Helicobacter, Dubosiella, Caulobacter , and Bradyrhizobium . High-zinc diets are associated with a lower abundance of Sphingomonas Romboutsia, Bacteroides, Lactobacillus, and Bifidobacterium [ 23 ]. Zinc supplementation has been shown to improve the integrity of the gut wall, reduce the translocation of bacteria from the gut into the bloodstream, and influence the diversity and composition of the gut microbiota [ 24 ]. Furthermore, it can play a role in modulating the production of cytokines, often leading to a reduction in the levels of pro-inflammatory cytokines [ 25 ]. These findings indicate that addressing ZD through supplementation can have direct benefits for gut health by strengthening the intestinal barrier and promoting a more balanced inflammatory state within the gut [ 26 ]. This study employs zinc-chelating peptides (ZCPs) derived from Holothuria scabra , the characteristics of which have been established in prior investigations [ 27 ]. The evaluation of Zn-Peptides sourced from Holothuria scabra with respect to cytokine secretion and gut microbiota profiles in zinc-deficient rat offspring has not been previously documented. The present research is proposed to elucidate the comprehensive potential of third-generation zinc supplementation through a metagenomic approach and its impact on the immune system of zinc-deficient rats offspring. 2. Methods This study employed an in vivo methodology to ascertain the capacity of ZCPs to normalize Zn absorption in ZD rat offspring from the maternal uterine environment. The methodology employed to develop zinc-deficient animals during the first 1000 days of life (conversion with human age) and the composition of the zinc-deficient feed utilized to obtain the experimental animal model have been registered with Indonesian patent number P00202410265. The research flowchart is presented in Fig. 1 . 2.1 Research Ethics This study has been granted ethical approval by the Health Research Ethics Committee of the Faculty of Medicine, University of Indonesia (FKUI) - RSUPN Dr. Cipto Mangunkusumo (certificate number: Ket-309/UN2.F1/ETIK/PPM/00.02/2022; protocol number: 22-03-0342). 2.2 Animals Subject This investigation employed an adult cohort of male and female Sprague-Dawley rats, comprising a balanced population with a weight of 150–200 grams at the age of 8 weeks. The animal subjects were obtained from the Animal Research Facilities at Faculty of Medicine, University of Indonesia and maintained in a room with a constant temperature of 25–27°C and constant humidity (45–65 RH). The lighting schedule was maintained at an adequate level, with 12 hours of light and 12 hours of dark. The rats were provided with feed in the form of pellets and had access to drinking water at all times. Subsequent to this, the male and female rats were bred with previously categorized healthy rats and ZD rats, which were provided with feed supplementation. The rat pups from healthy and ZD rats were separated into six groups with males and females randomly allocated in three-week-old. The facility for animal subject experiments certified (SNI ISO/IEC 17025:2017) and accredited by the National Accreditation Committee, Republic of Indonesia No. LP-1855-IDN. 2.3 Zinc deficiency model The present study commenced with the preparation of zinc-deficient rats by feeding a zinc-deficit diet. The composition of the feed used to develop the ZD rat model was as follows (per kilo of feed): pollard 13.5%; corn 40%; soybean meal 15%; corn gluten meal 22%; tapioca starch 2%; morin mix 2.5%; vegetable oil 2%. The composition of the feed was as follows: calcium carbonate (1.3%), mineral premix (without Zn) (0.2%), and dicalcium phosphate (1.5%). The mothers were fed a zinc deficit diet during both gestation and lactation. The rat pups were weaned at 3 weeks of age and continued with zinc deficit feeding until seven weeks of age (registered Indonesian patent number P00202410265). 2.4 Zinc-chelating peptides therapy design As referenced in previous studies, therapeutic ZCPs derived from Holothuria scabra will be utilized in this investigation as a therapeutic supplement to ZD [ 27 ]. In the therapeutic intervention of ZD rat pups, ZCPs therapy are employed by dividing the variation of group doses of 4 and 40 mg/kg BW. The other group serving as the control group, which is administered with ZnSO4 at a dose of 40 mg/kg BW [ 28 – 33 ]. The study consisted of six groups: healthy rat pups (HG), healthy rat pups + ZCP 4 mg/kg BW (HG40), ZD rat pups + ZCP 4 mg/kg BW (ZD4), ZD rat pups + ZCP 40 mg/kg BW (ZD40), ZD rat pups + ZnSO 4 40 mg/kg BW (ZDZn), and ZD rat pups without any therapies (ZD). These abbreviations will be employed subsequently in the results and discussion section. ZCPs therapy were administered to 3-week-old rat pups along with a low-zinc diet for four weeks. Following the conclusion of the study, all rats were euthanized with a ketamine and xylazine ratio of 5:1 (mg/kg BW) [ 34 ]. Blood samples were obtained from the orbital plexus and from the heart, then stored in EDTA vacutainers. Samples were kept at cold temperature before being separated by 15 min centrifuge at 4000 rpm with 4 0 C to collect the serum. The serum was then stored at -80°C for the analysis of cytocine secretion. 2.5 DNA microbiota sequencing and data analysis All of groups were colected the segment of the rat pups sigmoid colon under sterile conditions and stored in temperature − 80 0 C until analysis. Total microbial DNA was extracted using the the Zymo Quick-DNA Fecal/Soil Microbe Miniprep Kit following the manufacturer’s protocol. DNA concentration and purity were assessed using a Nanodrop spectrophotometer (Thermo Fisher Scientific, USA). Microbial DNA was amplified targeting the 16S rRNA gene using universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-CGGTTACCTTGTTACGACTT-3′) [ 35 ]. PCR amplification was performed using the (16S barcoding (SQK-16SO24) Kit on a Applied Biosystems Thermal Cyclers for PCR Thermo Fisher PCR machine. Amplicon concentration was confirmed using a Qubit Fluorometer (Invitrogen, USA) Qubit 3 Fluorometer dengan KIT Qubit™ dsDNA HS Assay Kit, Invitrogen by Thermo Fisher to ensure sufficient template quantity for sequencing. Amplicon size and specificity were further verified by agarose gel electrophoresis using a 1% (w/v) agarose gel with Gel Doc™ EZ Imager. For next-generation sequencing (NGS) library preparation, the 16S amplicons were barcoded using the 16S Barcoding Kit 1–24 (SQK-16S024, Oxford Nanopore Technologies, UK) according to the manufacturer’s instructions. Libraries were pooled in equimolar concentrations and prepared for sequencing using the Ligation Sequencing Kit (SQK-LSK114, Oxford Nanopore Technologies, UK). Sequencing was conducted on a MinION Mk1C device (Oxford Nanopore Technologies, UK) equipped with a FLO-MIN106 (R9.4.1) flow cell. The sequencing run was managed using MinKNOW software (version 24.02.16) with real-time basecalling performed using Dorado (version 7.3.11), configured with the fast model optimized for 450 bp reads [ 36 ]. The sequencing workflow was carried out at the Sequencing Laboratory of the National Research and Innovation Agency (BRIN). Raw FASTQ files were processed. First. adapter sequences were trimmed using Porechop (version 0.2.4) [ 37 ] and reads were quality-filtered using NanoFilt (version 2.8.0), applying a minimum Phred quality score threshold of Q10. Reads were further filtered based on length, retaining those between 1,300 bp and 1,700 bp to match targeted amplicon of 27F and 1492R. Read quality, both pre- and post-filtering, was assessed using NanoPlot (version 1.20.0) (De Coster et al., 2018). Filtered reads were classified using Kraken2 (version 2.1.3) within the 16S pipeline (version 1.4.0) implemented in EPI2ME (Oxford Nanopore Technologies, UK) [ 38 ]. Taxonomic classification was performed against the NCBI 16S_18S reference database with default parameters, including a minimum percent identity of 95% and a minimum reference coverage of 90%. Data visualization and statistical analyses were conducted in R (version 4.3.1) 2.6 Concentration of cytokine secretion assay Cytokine secretion was quantitatively analysed in serum samples obtained from all groups of rat pups. The cytokine parameters examined included IL6, IL10, TNFα, and IFNγ levels, utilizing the ElabScience ELISA Kit for Rat. The experimental procedures were conducted in accordance with the protocols outlined in the kit's assay protocol for each parameter and previous study results [ 39 – 45 ]. In briefly, The ELISA procedure begins with preparing reagents by bringing them to room temperature and diluting the concentrated wash buffer. The standard is reconstituted, and both the biotinylated detection Ab working solution and the HRP conjugate working solution are made. Next, diluted standard, blank, and sample are added to designated wells, and the plate is incubated. Following incubation, the liquid is decanted, biotinylated detection Ab working solution is added, and the plate is incubated again. A wash step is performed, and then HRP conjugate working solution is added with another incubation. Another wash is performed before adding Substrate Reagent and incubating in the dark. The reaction is stopped with Stop Solution, and the optical density is read using a micro-plate reader. 2.7 Statistical analysis The presentation of the data was as mean ± standard deviation (SD) or median (range). The statistical analysis used a t-test and a one-way ANOVA to compare differences in treatment between groups. Tukey's multiple comparisons test was then used for post hoc analysis. A significance level of p < 0.05 was set. Correlation analysis was conducted using Pearson’s test. The analysis and calculation of the data was performed using GraphPad Prism 9.0 for Windows. 3. Results 3.1 Sequencing data and microbial diversity A total of 866,465 sequence reads were successfully assigned to taxonomic groups, spanning 667 species, 301 genera, 71 families, 33 orders, 16 classes, and 10 phyla ( Supplementary Data 1 – Abundance TSV). Table 1 Reads data before and after filtering across sample. Sample Filtering Total reads Total bases Mean read length Mean Q HG Before 199,868 310,889,655 1,555.5 11.4 After 176,462 261,778,874 1,483.5 12.2 HG40 Before 13,076 19,661,907 1,503.7 11.1 After 11,005 16,088,090 1,461.9 11.9 ZD Before 153,965 239,060,313 1,552.7 11.4 After 134,188 199,980,948 1,490.3 12.1 ZD4 Before 145,632 223,901,584 1,537.4 11.4 After 127,177 187,484,810 1,474.2 12.2 ZD40 Before 155,101 242,950,714 1,566.4 11.4 After 136,079 204,091,741 1,499.8 12.2 ZDZn Before 365,872 535,680,079 1,464.1 11.1 After 281,554 414,641,295 1,472.7 11.9 Among the samples, the HG group harbored 266 microbial species, including 24 species unique to this sample. The HG40 group contained 55 microbial species, 7 of which were exclusive to this environment. The ZD group exhibited 220 microbial species, with 16 unique species identified. In the ZD4 group, 275 microbial species were identified, including 39 species exclusive to this group. The ZD40 group demonstrated greater diversity with 346 microbial species, of which 63 were unique. Notably, the ZDZn group exhibited the highest microbial diversity, with 446 species identified, including 99 unique species absent from all other samples. Reads data for all sample summarize in Table 1 . Table 2 Sample diversity indices Sample Richness Shannon diversity index Simpson's index Inverse Simpson's index HG 266 3.6 0.94 1.06 HG40 55 2.23 0.72 1.39 ZD 220 3.22 0.92 1.08 ZD4 275 3.37 0.91 1.1 ZD40 346 3.28 0.87 1.16 ZDZn 446 4.16 0.96 1.04 The ZDZn group demonstrated the highest microbial richness, with 446 species identified, and had the greatest Shannon diversity index (4.16), indicating both a high number of species and an even distribution among them. Its Simpson's index (0.96) highlighted minimal dominance by individual species, further supported by an inverse Simpson's index of 1.04. The ZD40 group followed, with a richness of 346 species and a Shannon diversity index of 3.28, reflecting substantial diversity. Its Simpson's index (0.87) and inverse Simpson's index (1.16) indicated slightly higher species dominance compared to ZDZn (Table 2 ). The ZD4 and ZD groups displayed moderate diversity, with 275 and 220 species, respectively. The Shannon diversity indices were 3.37 for ZD4 and 3.22 for ZD, suggesting relatively even species distributions. Both groups exhibited Simpson's indices above 0.9, indicating low species dominance, with inverse Simpson's indices of 1.10 for ZD4 and 1.08 for ZD. The HG group, containing 266 species, had a Shannon diversity index of 3.6, reflecting higher species evenness compared to ZD4 and ZD. Its Simpson's index (0.94) and inverse Simpson's index (1.06) supported this observation. In contrast, the HG40 group exhibited the lowest richness (55 species) and the lowest Shannon diversity index (2.23), indicating limited diversity. Its Simpson's index (0.72) and inverse Simpson's index (1.39) suggested greater dominance by a few species compared to the other groups. The composition of microbial communities varied significantly among the samples, as visualized in a heatmap of the top 100 microbial species (Fig. 2 ). In the HG group, Enterococcus faecalis was the most abundant species (28,537 reads), followed by Ligilactobacillus murinus (10,880 reads) and Limosilactobacillus reuteri (10,784 reads). The HG40 group was dominated by Collinsella aerofaciens (5,285 reads), with lower abundances of Enterococcus faecalis (1,260 reads) and Lactobacillus hominis (483 reads). In the ZD group, Ligilactobacillus murinus (19,668 reads), Limosilactobacillus reuteri (18,582 reads), and L. hominis (15,019 reads) were the dominant taxa, collectively accounting for the majority of reads. The ZD4 group was characterized by a high abundance of Romboutsia timonensis (28,954 reads), Romboutsia ilealis (16,201 reads), and L. johnsonii (12,408 reads). The ZD40 group exhibited a significant presence of L. johnsonii (44,535 reads), Limosilactobacillus reuteri (13,716 reads), and Anaerobiospirillum succiniciproducens (8,473 reads). Finally, the ZDZn group, with the highest diversity, was enriched with Romboutsia timonensis (34,050 reads), L. hominis (23,369 reads), and R. ilealis (16,201 reads). As depicted in ZD barplot (Fig. 3 and Supplementary Data 2 - Sankey diagram) Bacillota (98%) and Pseudomonadota (2%) are the two identified bacterial phyla that make up ZD. Bacilli (64,18%) and Lactobacillales (64.24%) prevailed at the class and ordo-levels. The most prevalent family among all six families found was Lactobacillales (64.24%), which included three dominant genera from the same family: Limosilactobacillus (22.66%), Lactobacillus (21.9%), and Ligilactobacillus (21.89%). Streptococcus, Romboutsia, Clostridium , and Anaerobiospirillum are the additional genera that have been identified. Finally, at the species-level profilling showed the Limosilactobacillus reuteri (28.63%), Lactobacillus hominis (24.68%), Ligilactobacillus murinus (30.3%), Romboutsia timonensis (19.59%), and Clostridium disporicum (13.98%) dominated. Based on the diagram for ZD40 (Fig. 3 ), it shown that ZD40 was composed of three bacterial phyla, which are Bacillota, Bacteroidota, and Pseudomonadota. Bacillota (85.58%) was identified as the most abundant phylum followed by Pseudomonadota (12.67%), and Bacteroidota (1.25%). At the class level, Bacilli (69.50%) was the dominant and Lactobacillales (69.48) was the dominant for ordo level. Lactobacillaceae (65.37%) was identified to be the most dominant family among all the bacterial families ( n = 8). Genus level microbial resolution revealed the predominance of Lactobacillus (49.01%) among all identified genera ( n = 11). Finally, species-level profiling revealed the abundance of L. johnsonii (33.38%), Limosilactobacillus reuteri (10.28%), Anaerobiospirillum succiniciproducens (6.35%), and L. hominis (5.10%) among other specises (n = 14). For ZDZn (Fig. 3 ) reveals that ZDZn contains two bacterial phyla, which are Bacillota as the most abundant (93.99%) and Bacteroidota (6.01%). The dominant class of ZDZn was Clostridia (73.41%), followed by Bacilli (24.90%) and Bacteroidia (1.69%). Lachnospirales was the most abundant ordo (37.19%) while Lachnospiraceae was the most abundant family (38.47%) among all the bacterial families ( n = 8). For the genus level, Blautia was the most dominant (23.73%) followed by Romboutsia (23.33%) among all genera (n = 16). At the species level, R. timonenses revealed as the most abundant species (18.22%) followed by L. hominis (12.50%) among all identified species (n = 21). 3.2 Concentration of cytokines secretion Figure 4 showed that, Interleukin-10 (IL10) typically considered an anti-inflammatory cytokine. The lowest level is seen in the healthy control group (HG, approx. 80 pg/mL). The zinc-deficient group (ZD) shows a significantly higher level (approx. 130 pg/mL) compared to HG (p < 0.0001). All treatment groups (ZD4, ZD40, ZDZn) and the healthy treated group (HG40) also show elevated IL10 levels compared to HG (ranging approx. 110–120 pg/mL), but these levels appear slightly lower than the untreated ZD group. ZD significantly elevates IL10 levels. Treatments, including ZCP and ZnSO₄ in deficient rats and ZCP in healthy rats, also result in IL10 levels higher than healthy controls, though potentially slightly reduced compared to untreated ZD. Meanwhile, Interleukin-6 (IL6) generally considered a pro-inflammatory cytokine. The HG group has the lowest level (approx. 125 pg/mL). The ZD group shows a significantly increased IL6 level (approx. 210 pg/mL) compared to HG. The ZCP-treated ZD groups (ZD4 and ZD40) also have high IL6 levels (approx. 190–205 pg/mL). The ZnSO₄-treated group (ZDZn, approx. 155 pg/mL) and the healthy treated group (HG40, approx. 140 pg/mL) show IL6 levels lower than the ZD group but still elevated compared to the HG group. ZD significantly increases IL6 levels. ZCP treatment at both doses maintains high IL6 levels in deficient rats. ZnSO₄ treatment in deficient rats and ZCP treatment in healthy rats appear to partially moderate this increase compared to ZD, but levels remain above healthy controls. The ratio of the pro-inflammatory IL6 to the anti-inflammatory IL10. A higher ratio might suggest a more pro-inflammatory state. The ratios across all groups range from about 1.3 to 1.7. HG is around 1.5. ZD, ZD4, and ZD40 appear slightly higher (around 1.6–1.7). ZDZn and HG40 appear slightly lower (around 1.3–1.4). There are no significance markers shown on this graph. Based on the visual representation and lack of significance markers, there may not be statistically significant differences in the IL6/IL10 ratio between the groups. However, there's a slight trend towards a higher ratio in ZD, ZD4, and ZD40 groups compared to HG, ZDZn, and HG40. Result shows, Tumor Necrosis Factor-alpha (TNFα), another pro-inflammatory cytokine. All groups show similar mean levels of TNFα (around 70–75 pg/mL) and no significance markers are present. The treatments and/or ZD state did not appear to cause significant changes in TNFα levels among the different groups in this study. Interferon-gamma, a cytokine involved in immune response, often associated with Th1-type inflammation. The ZD group shows a significantly higher level (approx. 440 pg/mL) compared to the HG group (approx. 380 pg/mL) (p < 0.01). Interestingly, all treatment groups (ZD4, ZD40, ZDZn) and the healthy treated group (HG40) show markedly lower IFNγ levels (around 300–330 pg/mL) compared to both the ZD and the HG groups. ZD significantly increases IFNγ levels compared to healthy controls. All tested treatments (ZCP in deficient and healthy rats, ZnSO₄ in deficient rats) appear to reduce IFNγ levels below those seen in both untreated deficient and healthy control groups. 3.3 Correlation of cytokine release in ZD model and ZD which received the supplement model The provided heatmap illustrates (Figure 5 ) the relationships between key cytokines in zinc-deficient rat pups. Each cell in the matrix represents the Pearson correlation coefficient, indicating the strength and direction of the linear association between two cytokines. Heatmap of Figure 5 A showed, strong negative correlation is observed between IL-10 and both IL-6 (r = -0.49) and TNFα (r = -0.52), suggesting an inverse relationship where increased levels of the anti-inflammatory cytokine IL-10 are associated with decreased levels of these pro-inflammatory cytokines. IL-6 and TNFα exhibit a weak positive correlation (r = -0.12), indicating a minimal linear relationship. The correlation between IL-10 and IFNγ is weakly positive (r = -0.23). IL-6 shows a moderate negative correlation with IFNγ (r = -0.32), while TNFα and IFNγ have a weak positive correlation (r = -0.11). Overall, the data suggests a complex interplay between these cytokines in ZD, with IL-10 potentially playing a role in modulating the levels of pro-inflammatory cytokines. Meanwhile heatmap in 5B analyzing the inter-cytokine correlations reveals several relationships in ZD which received the supplement (ZD4 model). A moderate positive correlation exists between TNFα and IL6 (r = 0.40). Conversely, a notable strong negative correlation is observed between IL6 and IL10 (r = -0.63). TNFα also shows a moderate negative correlation with IL10 (r = -0.40). IFNγ exhibits weaker correlations with the other cytokines: a weak negative correlation with TNFα (r = -0.22), a weak to moderate negative correlation with IL6 (r = -0.32), and a weak positive correlation with IL10 (r = 0.23). These findings highlight potential coordination or opposition in the expression patterns of these cytokines under the conditions studied, with the IL6-IL10 relationship being the most strongly antagonistic. 4. Discussion 4.1 Zinc deficiency significantly alters the gut microbiome composition The study definitively identified microbial diversity in the intestinal organs of the colon segment through 16S rRNA sequencing in six groups of rats. HG, HG40, ZD, ZD4, ZD40, and ZDZn. The objective was straightforward: to identify the microbial diversity and associated effects in each group, with a particular focus on zinc metabolism. The comparison between the healthy (HG) and zinc-deficient (ZD) groups indicates that ZD impacts the gut microbiome primarily by causing shifts in its composition rather than drastically reducing overall diversity. The ZD group showed slightly lower species richness and Shannon diversity compared to the HG group, their Simpson's diversity index remained similar [ 46 ]. Zinc is indispensable for bacteria, acting as a cofactor for hundreds of enzymes crucial for growth and virulence. However, the host actively combats bacterial infections by limiting zinc availability at infection sites – a process called "nutritional immunity." Proteins like S100A8/A9 (calprotectin) are deployed by immune cells to sequester zinc [ 47 , 48 ]. While systemic ZD (low zinc throughout the body) isn't the same as targeted zinc withholding by the immune system, it does mean less zinc is available overall. This can directly inhibit the growth of bacteria that cannot efficiently scavenge scarce zinc. Pathogenic bacteria often possess high-affinity zinc uptake systems (like the ZnuABC transporter) specifically to overcome host zinc restriction [ 48 , 49 ]. Zinc is critical for the development and function of immune cells (T-cells, B-cells, macrophages). Deficiency impairs immune responses, leading to increased susceptibility to infections. However, it doesn't simply weaken all defences; it can dysregulate the immune system, sometimes leading to inappropriate inflammation or altered cytokine profiles, which changes the environment bacteria experience. An altered immune landscape due to ZD can shift the competitive balance between different microbial species[ 50 – 52 ]. Zinc is vital for maintaining the intestinal epithelial barrier. It supports the function of tight junctions, the structures that seal the space between gut cells. Deficiency can increase intestinal permeability ("leaky gut"). A compromised barrier allows bacterial products (like LPS) to cross into tissues, potentially triggering inflammation. It also changes the conditions within the gut lumen, affecting microbial composition (dysbiosis) [ 50 , 53 , 54 ]. Due to varying zinc requirements and acquisition capabilities (e.g., presence of high-affinity transporters), different bacterial species and strains exhibit different sensitivities to zinc limitation [ 51 , 52 , 55 , 56 ]. Therefore, ZD doesn't simply inhibit all bacteria equally. It creates a complex, altered environment where the interplay between impaired bacterial metabolism, altered host immunity, compromised barrier function, and inter-species competition for limited zinc resources can lead to significant shifts in the microbiota composition, potentially favouring certain (sometimes pathogenic) species over others. The microbial composition clearly differs substantially across the groups (HG, HG40, ZD, ZD4, ZD40, ZDZn), as expected based on distinct treatments or conditions. The stark differences between the HG (control) and ZD (zinc deficient) groups highlight the significant impact of ZD on gut microbial composition, changing the dominant species entirely. The various treatment groups (ZD4, ZD40, ZDZn) applied to the zinc-deficient state each resulted in unique microbial signatures, suggesting dose-dependent effects or different mechanisms of action if the treatments varied beyond zinc levels. Zinc is an essential micronutrient for both the host and gut bacteria. It plays critical catalytic, structural, and regulatory roles in cellular physiology [ 57 , 58 ]. The microbiota composition changes, particularly the increase in certain Lactobacillus and Romboutsia species with presumed zinc supplementation, and the differing profiles in zinc-deficient states, align with broader research indicating that zinc is a crucial modulator of gut microbiota [ 59 ]. The microbial shifts observed in this study, particularly in the zinc-related groups (ZD, ZD4, ZD40, ZDZn), can be contextualized with existing research on zinc's impact on gut microbiota. In ZD group, dominance of Ligilactobacillus murinus , Limosilactobacillus reuteri , and Lactobacillus hominis findings in zinc-deficient models, but shifts in Lactobacillus species are commonly reported. L. murinus has been shown to modulate intestinal barrier damage and gut microbiota in immunosuppressed models, often increasing short-chain fatty acid-producing bacteria [ 60 ]. L. reuteri is a well-studied probiotic with anti-inflammatory properties and the ability to modulate gut microbiota. Some studies have linked Lactobacillaceae family members abundance to metal availability, including zinc [ 61 , 62 ]. The presence of these species in the ZD group is interesting and might reflect a complex interplay where some lactobacilli might thrive or be less affected by moderate zinc scarcity compared to other bacteria. Their relative abundance increases as other zinc-dependent microbes decrease. Their persistence or increase in ZD40 (for L. reuteri ) suggests a positive response to or role during zinc therapy. In treatment groups (ZD4, ZD40, ZDZn), zinc supplementation can modulate the gut microbiota, often reversing some of the changes caused by deficiency and sometimes promoting the growth of beneficial bacteria. A systematic review highlighted that high dietary zinc could alter bacterial taxa, sometimes increasing metal resistance genes [ 63 , 64 ]. The shifts towards Romboutsia timonensis , Romboutsia ilealis , and Lactobacillus johnsonii (ZD4), increased Lactobacillus johnsonii and Limosilactobacillus reuteri (ZD40), and enrichment of Romboutsia timonensis and Lactobacillus hominis (ZDZn) in this study are specific changes that would be interesting to compare to targeted supplementation studies. Romboutsia timonensis and Romboutsia ilealis (dominant in ZD4 and ZDZn groups). Romboutsia is a genus within the Clostridiales order. While less studied than Lactobacillus or Bifidobacterium , emerging research is exploring its role. The significant increase in these species in the ZD4 and ZDZn groups is a key finding. This suggests that these Romboutsia species might be favored by or play a role in the gut environment shaped by zinc therapy. General reviews on dietary zinc mention that zinc status alters bacterial taxa, but specific effects on Romboutsia would require more targeted literature [ 65 ]. The varied profiles in ZD4, ZD40, and ZDZn groups highlight that zinc therapy can selectively promote certain bacterial taxa, such as specific Lactobacillus and Romboutsia species. The differences between these supplemented groups underscore that the dose or form of zinc may fine-tune these microbial shifts [ 66 ]. 4.2 Zinc deficiency significantly alters inflammatory cytokine profile The significant increase in the anti-inflammatory cytokine IL-10 in the zinc-deficient (ZD) group (approx. 130 pg/mL vs. 80 pg/mL in HG, p < 0.0001), and its persistence at elevated levels in all treatment groups (ZD4, ZD40, ZDZn, HG40) compared to healthy controls, is a pivotal observation. While IL-10 is a potent anti-inflammatory agent, its chronic elevation can sometimes be indicative of an immune system struggling to resolve ongoing inflammation or an attempt to counteract a strong pro-inflammatory drive [ 67 ]. In the context of ZD, which generally promotes inflammation (as seen with IL-6 and IFN-γ), this IL-10 surge could indeed be a compensatory mechanism [ 68 ]. This elevation may result from disrupted zinc homeostasis in immune cells, particularly affecting regulatory T cells (Treg), which are primary producers of IL-10. Zinc deficiency impairs Treg differentiation by downregulating Foxp3 expression, leading to compensatory IL-10 overproduction as the immune system attempts to counterbalance inflammation [ 69 ]. The fact that even zinc supplementation (both ZCP and ZnSO₄) did not normalize IL-10 levels, and ZCP in healthy rats (HG40) also increased it, suggests a more complex interaction than simple deficiency correction. It might imply that, the underlying inflammatory insult caused by the initial deficiency has long-lasting effects on immune programming. The forms of zinc used, or the act of supplementation itself, might directly stimulate IL-10 producing cells (e.g., regulatory T cells, certain macrophage subsets) [ 70 ]. This finding links to the concept of immune homeostasis and how the body attempts to restore balance. Persistent IL-10 elevation, while seemingly beneficial, warrants scrutiny as it can sometimes be associated with immune exhaustion or even contribute to pathogen persistence or tumor immune evasion in other contexts if not properly regulated [ 71 ]. Zinc deficiency robustly increased pro-inflammatory IL-6 (approx. 210 pg/mL in ZD vs. 125 pg/mL in HG). Notably, ZCP treatment in deficient rats (ZD4, ZD40) did little to reduce these high levels, whereas ZnSO₄ (ZDZn) showed a more pronounced (though incomplete) reduction. Even ZCP in healthy rats (HG40) slightly elevated IL-6. This elevation could be due to a disruption in zinc homeostasis in immune cells, particularly affecting regulatory T cells (Treg), which produce IL-10. Zinc deficiency impairs Treg differentiation by downregulating Foxp3 expression, prompting the immune system to produce more IL-10. This increase in IL-6 is driven by macrophages and intestinal epithelial cells, which are triggered by increased gut permeability (leaky gut) and NF-κB signaling. The strong IL-6 induction in ZD is well-documented and often linked to the activation of the NF-κB pathway, a critical transcription factor for many pro-inflammatory genes [ 72 ]. Zinc is known to inhibit NF-κB activation; thus, its deficiency unleashes this pathway [ 73 ]. The differential effect of ZCP versus ZnSO₄ is significant. While ZnSO₄ is a readily ionizable form of zinc, zinc-peptide chelates (like ZCP) are proposed to have different absorption pathways and bioavailability characteristics. It is suggested that ZCP may depend on peptide-mediated uptake via transporters such as ZIP4, which has a higher expression in ZD. However, this may not effectively deliver zinc to the intracellular compartments that are essential for NF-κB inhibition, constraining its anti-inflammatory effectiveness in comparison to ZnSO₄ [ 74 ]. The inability of ZCP to effectively lower IL-6 in ZD rats, possible because poorer dissociation of zinc from the peptide at relevant cellular sites, this indicates that the release of zinc from its peptides may be inefficient, potentially resulting in insufficient zinc availability for its anti-inflammatory functions within the cell. Besides that, the peptide moiety itself having some mild pro-inflammatory activity or interfering with zinc's anti-inflammatory action against IL-6 specifically. Meanwhile, ZCP need for higher or more prolonged dosing because capability to insufficient to achieve a significant decrease in IL-6 [ 75 – 77 ]. The balance between pro-inflammatory (IL-6) and anti-inflammatory (IL-10) cytokines is critical in determining the net immune status. A sustained high IL-6/IL-10 ratio is often implicated in the pathogenesis of chronic inflammatory diseases [ 78 , 79 ]. While not statistically significant in the provided data, the slight trend towards a higher IL-6/IL-10 ratio in ZD, ZD4, and ZD40 (more pro-inflammatory) versus a lower ratio in ZDZn and HG40 is suggestive. ZnSO₄ more effective at tipping the balance away from a pro-inflammatory state in deficient animals compared to ZCP. For ZCP supplementation, in healthy animals (HG40) promote a relatively more anti-inflammatory balance, primarily driven by its IL-10 elevating and IFN-γ suppressing effects, despite slightly raising IL-6. In essence, these trends suggest a differential role for ZnSO₄ and ZCP depending on the physiological state (deficient vs. healthy) of the animals. ZnSO₄ better for correcting deficiency-induced inflammation, while ZCP more subtle, immunomodulatory benefit in healthy states, potentially by promoting a regulatory (IL-10) and dampening an effector (IFN-γ) immune response. It can potentially be attributed to the slower zinc release kinetics of ZCP, which may offer preferential support to Treg function in contrast to the rapid correction of pro-inflammatory signalling. These are critical distinctions for understanding the potential therapeutic applications of each zinc form [ 75 , 77 – 79 ]. ZD is often reported to increase TNF-α (as a primary pro-inflammatory cytokine), this result is somewhat unexpected. According to previous studies, the specific model of ZD used (duration, severity) did not reach the threshold for systemic TNF-α elevation. TNF-α responses are more localized to specific tissues rather than being reflected systemically at the measured time point. Other cytokines (IL-6, IFN-γ) are the predominant drivers of inflammation in this particular context. The observed IL-10 elevation effectively dampened potential TNF-α increases [ 80 – 83 ]. Zinc deficiency significantly increased IFN-γ (approx. 440 pg/mL in ZD vs. 380 pg/mL in HG, p < 0.01). Strikingly, all treatments (ZD4, ZD40, ZDZn, HG40) not only reversed this increase but suppressed IFN-γ to levels markedly below those of healthy controls (around 300–330 pg/mL). IFN-γ is crucial for Th1-mediated immunity, vital for controlling intracellular pathogens. While its elevation in ZD suggests immune activation/dysregulation, the profound suppression by all zinc treatments below healthy baseline is a critical point [ 80 ]. The phenomenon of suppression may be attributable to zinc's modulation of T-bet and STAT1 signalling. These are pivotal for Th1 cell differentiation and IFN-γ production. Zinc overcorrection of these pathways, particularly with ZCP, may impair Th1 responses. This could be beneficial in Th1-dominant autoimmune or inflammatory conditions [ 84 ]. However, it raises concerns about potentially impairing necessary Th1 responses and increasing susceptibility to certain infections or hampering anti-tumor immunity [ 84 , 85 ]. Literature indicates zinc is essential for IFN-γ production, but excessive zinc or particular supplementation forms might lead to over-suppression. The extent of IFN-γ suppression observed here suggests a powerful modulatory effect of the administered zinc forms, perhaps impacting transcription factors like T-bet (for Th1 differentiation) or STATs involved in IFN-γ signalling [ 86 ]. The study findings collectively demonstrate that ZD induces a complex inflammatory state characterized by elevated IL-6, IFN-γ, and a possibly compensatory rise in IL-10, while TNF-α remains stable. Zinc supplementation, regardless of the form (ZCP or ZnSO₄), appears to exert potent immunomodulatory effects that go beyond simple repletion, most notably the profound suppression of IFN-γ below healthy levels. The differential impact of ZCP and ZnSO₄ on IL-6 levels further highlights that the choice of zinc supplement can lead to distinct immunological outcomes. Future studies could incorporate flow cytometry analysis to confirm changes in immune cell populations, such as Th1, Treg, and macrophages, thereby providing deeper insights into these cellular mechanisms. 4.3 Zinc deficiency leads to a dysregulated cytokine network The shifts in cytokine correlations between your zinc-deficient (ZD) rat pups and those receiving zinc supplementation (ZD4 model) provide critical insights into how zinc modulates the immune system inflammatory response. In both ZD and ZD4 groups, IL-10 (a key anti-inflammatory cytokine) showed strong to moderate negative correlations with the major pro-inflammatory cytokines IL-6 and TNF-α. IL-10 is critical for limiting host immune responses to pathogens and preventing excessive tissue damage by inhibiting the production of pro-inflammatory cytokines like TNF-α and IL-6 by macrophages and other cells [ 87 ]. IL-6 and TNF-α are pivotal pro-inflammatory cytokines often produced in concert during an inflammatory response, driven by common signalling pathways such as NF-ĸB. Their coordinated action is essential for effective pathogen clearance and initiation of adaptive immunity, but dysregulation contributes to chronic inflammatory diseases. In the ZD group, the weak negative (or essentially uncoordinated) relationship between IL-6 and TNF-α a might reflect a dysregulated and inefficient inflammatory signalling cascade characteristic of ZD. The inflammatory response may be chronic and disorganized. The shift to a positive correlation in the ZD4 group suggests that zinc supplementation helps restore a more coordinated and potentially more effective pro-inflammatory response. This could mean that when inflammation is required, these cytokines are produced in a more synchronized and controlled manner, potentially leading to more efficient resolution rather than chronic, low-grade inflammation [ 88 , 89 ]. In the ZD group, the weak negative correlation between IFN-γ and IL-10 could indicate a generally suppressed or imbalanced Th1/Treg axis. With impaired Th1 function due to zinc lack, any IFN-γ production might occur in an environment where IL-10 is also variably present, leading to a slight inverse trend as part of overall immune dysregulation. The shift to a weak positive IFN-γ /IL-10 correlation in the ZD4 group is intriguing. It doesn't suggest strong synergy but might imply a more balanced immune state where both cell-mediated immunity (potentially enhanced IFN-γ capacity due to zinc repletion) and its regulation (IL-10) can coexist and be modulated more appropriately. For instance, an appropriate immune response involves an initial IFN-γ surge followed by IL-10 to control it. This weak positive correlation might capture moments of this dynamic balance across different animals. Zinc supplementation has been shown to restore Th1/Th2 balance, which involves IFN-γ and IL-10 [ 90 , 91 ]. ZD leads to a dysregulated cytokine network. While anti-inflammatory mechanisms (IL-10) are active, their interplay with pro-inflammatory cytokines (IL-6, TNF-α) and other immune modulators (IFN-γ) appears disorganized. This aligns with human and animal studies showing ZD is associated with increased susceptibility to infections, chronic inflammation, and impaired immune cell function [ 8 , 92 , 93 ]. The lack of positive coordination between IL-6 and TNF-α in ZD rats might reflect an inefficient and prolonged inflammatory state [ 92 ]. Zinc supplementation (therapy) appears to restore a more coordinated and balanced cytokine network. This is evident in the strengthened IL-10 regulatory axis, the establishment of a positive correlation between key pro-inflammatory cytokines (suggesting more controlled responses when needed), and subtle shifts in IFN-γ interactions indicative of better immune homeostasis. Effective zinc therapy aims to normalize immune cell function, leading to appropriate cytokine production and better control over inflammatory processes [ 93 – 95 ]. 4.4 Zinc deficiency leads to a dysbiosis gut microbiome and a disorganized systemic inflammatory response The interplay between gut microbiota and host immunity is a critical determinant of health, particularly under conditions of nutritional stress such as ZD. Our findings reveal a significant modulation of both the gut microbial composition and the systemic cytokine network in a zinc-deficient rat model following zinc supplementation (ZD4 group). In the zinc-deficient (ZD) state, the gut microbiome was primarily dominated by species such as Ligilactobacillus murinus , Limosilactobacillus reuteri , and Lactobacillus hominis . This microbial profile was associated with a distinct cytokine correlation pattern characterized by strong negative correlations between the anti-inflammatory cytokine IL-10 and the pro-inflammatory cytokines IL-6 (r = -0.49) and TNF-α (r = -0.52), alongside weak, largely uncoordinated relationships between the pro-inflammatory markers IL-6 and TNF-α (r = -0.12). This suggests an immune environment under ZD where regulatory mechanisms (IL-10) are active but struggle against a backdrop of potentially disorganized inflammation. Dysbiosis increases gut permeability and allows bacterial components like lipopolysaccharides (LPS) to activate Toll-like receptor 4 (TLR4) on immune cells. This triggers the production of IL-6 and other pro-inflammatory cytokines. Upon zinc supplementation (ZD4 model), a marked shift occurred in both microbial and immune landscapes. The microbiota transitioned towards a community dominated by Romboutsia timonensis , Romboutsia ilealis , and Lactobacillus johnsonii . Concurrently, the cytokine network demonstrated evidence of enhanced coordination and regulation. Notably, the correlation between IL-6 and TNF-α shifted to moderate positive (r = 0.40), suggesting a more synchronized pro-inflammatory response. Furthermore, the negative correlation between IL-6 and IL-10 intensified (r = -0.63), indicating a more robust anti-inflammatory control loop. Additionally, the relationship between IL-10 and IFN-γ inverted from weak negative (r = -0.23) to weak positive (r = 0.23), potentially reflecting a rebalancing of Th1-type and regulatory responses. Short-chain fatty acids (SCFAs) like butyrate are produced by Lactobacillus johnsonii and Romboutsia species and may contribute to immune modulation. SCFAs bind to G protein-coupled receptor 43 (GPR43) on immune cells, promoting IL-10 production by regulatory T cells and inhibiting pro-inflammatory cytokine expression via histone deacetylase (HDAC) inhibition [ 74 ]. Zinc supplementation strengthens gut barrier integrity, reducing LPS translocation and subsequent TLR4 activation, thereby limiting systemic inflammation [ 96 ]. These parallel shifts strongly suggest a functional link between the gut microbiota and host immune regulation, mediated by zinc status. The emergence of Lactobacillus johnsonii , a species with documented immunomodulatory properties including the capacity to promote IL-10 production, likely contributes to the enhanced regulatory control observed in the ZD4 group. Similarly, the significant increase in Romboutsia species, members of the Clostridiales order, may indicate an altered production of short-chain fatty acids (SCFAs). SCFAs, particularly butyrate, are known to exert potent immunomodulatory effects, including the promotion of regulatory T cells and IL-10, which aligns with the observed cytokine changes. The restoration of gut barrier function by zinc is hypothesised to reduce dysbiosis-induced TLR signalling, thereby supporting a balanced immune response[ 74 ]. Future studies could confirm the role of microbial metabolites in modulating the cytokine network. These findings corroborate previous studies demonstrating that zinc is essential for both maintaining a healthy gut microbiome and ensuring balanced immune function. Zinc supplementation appears to not only directly support immune cell activity, improving cytokine signaling and regulation (e.g., via NF-ĸB modulation), but also indirectly reshape the gut microbial community towards a profile more conducive to immune homeostasis. Thus, the correction of ZD facilitates a transition from a dysregulated, pro-inflammatory state towards a more coordinated and effectively regulated immune environment, highlighting the therapeutic potential of zinc in modulating the gut-immune axis. 5. Conclusion The impact of zinc deficiency (ZD) in rat offspring on the gut-immune axis is significant, with the resultant consequences including gut microbiota dysbiosis and a disorganized cytokine network. The hallmark of this condition is the presence of elevated levels of pro-inflammatory cytokines, such as IL-6 and IFN-γ, accompanied by a compensatory rise in the anti-inflammatory cytokine IL-10. The correlation between pro-inflammatory markers is weak and uncoordinated in ZD rats. The supplementation of zinc, in the form of zinc sulphate (ZnSO₄) or zinc-chelating peptides (ZCP), has been demonstrated to exert an influence on both the composition of the gut microbiome and the immune response. ZnSO₄ therapy has been demonstrated to result in the most significant restoration of microbial diversity and the most effective reduction of IL-6 levels in comparison with ZCP. This finding indicates that ZnSO₄, as a readily ionizable zinc source, may be a more effective means of rapidly correcting the inflammatory state by targeting intracellular pathways such as NF-κB inhibition. Conversely, ZCP therapy, while less effective at reducing IL-6, has been shown to initiate a significant shift in the gut microbial community towards species like Romboutsia and Lactobacillus johnsonii . This microbial change has been shown to be associated with a restructuring of the cytokine network, leading to a more coordinated pro-inflammatory response and a strengthened IL-10 regulatory axis. The shift in microbial profile may contribute to immune regulation through the production of short-chain fatty acids (SCFAs). In essence, ZnSO₄ is effective in rapidly reducing inflammation in acute ZD, while ZCP more effective in the long-term modulation of the gut-immune axis and the promotion of a balanced, well-regulated immune state. The selection of supplement is contingent upon the therapeutic objectives: the expeditious management of inflammation with ZnSO₄, while the promotion of long-term immune homeostasis through gut microbiome support with ZCP. Declarations Funding: This research was supported the Vaccine and Drug Research Program of the BRIN Health Research Organization. Grant number 134/BR/VIII/2024 Author Contribution The conceptualisation of the study was a collaborative effort between G. Syahputra, F. Shidiq, and Y. Maladan. The methodology was designed by G. Syahputra, N. Gustini, Y. Hapsari and Y. Maladan, who also performed the experiments. The responsibility for the analysis and visualisation of the data was undertaken by F. Shidiq, N. Gustini, W. Susmayanti, and A. Rosyidah. The initial manuscript was drafted by G. Syahputra. A. Amanah, S. Hidayani, Y. Maladan and F. Shidqi were responsible for the review and editing of the manuscript. All authors contributed to the interpretation of results, provided critical feedback, and approved the final version of the manuscript for submission. 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Marginal Zinc Deficiency and Environmentally Relevant Concentrations of Arsenic Elicit Combined Effects on the Gut Microbiome. mSphere [Internet]. 2018 [cited 2025 Apr 20];3(6). Wessels I, Maywald M, Rink L. Zinc as a Gatekeeper of Immune Function. Nutrients 2017, Vol 9, Page 1286 [Internet]. 2017 [cited 2025 May 23];9(12):1286. Jarva H. Significance of Zinc in Innate Immune Defense Against Streptococcus pyogenes. J Infect Dis [Internet]. 2014 [cited 2025 May 23];209(10):1495–1496. Skalny A V., Aschner M, Lei XG, et al. Gut Microbiota as a Mediator of Essential and Toxic Effects of Zinc in the Intestines and Other Tissues. Int J Mol Sci [Internet]. 2021 [cited 2025 May 22];22(23):13074. Dong Y, Zhang L, Qiu D, et al. Lactobacillus murinus ZNL-13 Modulates Intestinal Barrier Damage and Gut Microbiota in Cyclophosphamide-Induced Immunosuppressed Mice. Foods [Internet]. 2025 [cited 2025 May 23];14(8):1416. Huynh U, Zastrow ML. Metallobiology of Lactobacillaceae in the gut microbiome. J Inorg Biochem [Internet]. 2022 [cited 2025 May 23];238:112023. Luo Z, Chen A, Xie A, et al. Limosilactobacillus reuteri in immunomodulation: molecular mechanisms and potential applications. Front Immunol [Internet]. 2023 [cited 2025 May 23];14:1228754. Cheng J, Kolba N, Tako E. The effect of dietary zinc and zinc physiological status on the composition of the gut microbiome in vivo. Crit Rev Food Sci Nutr [Internet]. 2024 [cited 2025 May 23];64(18):6432–6451. Wong-Chew RM, Nguyen TVH, Rogacion JM, et al. Potential Complementary Effect of Zinc and Alkalihalobacillus clausii on Gut Health and Immunity: A Narrative Review. Nutrients 2024, Vol 16, Page 887 [Internet]. 2024 [cited 2025 May 23];16(6):887. Cheng J, Kolba N, Tako E. The effect of dietary zinc and zinc physiological status on the composition of the gut microbiome in vivo. Crit Rev Food Sci Nutr [Internet]. 2024 [cited 2025 May 23];64(18):6432–6451. Skalny A V., Aschner M, Lei XG, et al. Gut Microbiota as a Mediator of Essential and Toxic Effects of Zinc in the Intestines and Other Tissues. Int J Mol Sci [Internet]. 2021 [cited 2025 May 23];22(23):13074. Iyer SS, Cheng G. Role of Interleukin 10 Transcriptional Regulation in Inflammation and Autoimmune Disease. Crit Rev Immunol [Internet]. 2012 [cited 2025 May 21];32(1):23. Coussens LM, Werb Z. Inflammation and cancer. Nature [Internet]. 2002 [cited 2025 May 21];420(6917):860. Wan Y, Zhang B. The Impact of Zinc and Zinc Homeostasis on the Intestinal Mucosal Barrier and Intestinal Diseases. Biomolecules [Internet]. 2022 [cited 2025 Aug 20];12(7):900. Hodkinson CF, Kelly M, Alexander HD, et al. Effect of zinc supplementation on the immune status of healthy older individuals aged 55-70 years: The ZENITH study. Journals of Gerontology - Series A Biological Sciences and Medical Sciences [Internet]. 2007 [cited 2025 May 21];62(6):598–608. Carlini V, Noonan DM, Abdalalem E, et al. The multifaceted nature of IL-10: regulation, role in immunological homeostasis and its relevance to cancer, COVID-19 and post-COVID conditions. Front Immunol. 2023;14:1161067. Gao H, Dai W, Zhao L, et al. The Role of Zinc and Zinc Homeostasis in Macrophage Function. J Immunol Res [Internet]. 2018 [cited 2025 Aug 20];2018:6872621. Jarosz M, Olbert M, Wyszogrodzka G, et al. Antioxidant and anti-inflammatory effects of zinc. Zinc-dependent NF-κB signaling. Inflammopharmacology [Internet]. 2017 [cited 2025 May 21];25(1):11–24. Ohashi W, Fukada T. Contribution of Zinc and Zinc Transporters in the Pathogenesis of Inflammatory Bowel Diseases. J Immunol Res [Internet]. 2019 [cited 2025 Aug 20];2019(1):8396878. Katimba HA, Wang R, Cheng C. Current findings support the potential use of bioactive peptides in enhancing zinc absorption in humans. Crit Rev Food Sci Nutr [Internet]. 2023 [cited 2025 May 21];63(19):3959–3979. Cai C, Liu Y, Xu Y, et al. Mineral-element-chelating activity of food-derived peptides: influencing factors and enhancement strategies. Crit Rev Food Sci Nutr [Internet]. 2024 [cited 2025 May 21]; doi: 10.1080/10408398.2024.2361299;CTYPE:STRING:JOURNAL. Caetano-Silva ME, Netto FM, Bertoldo-Pacheco MT, et al. Peptide-metal complexes: obtention and role in increasing bioavailability and decreasing the pro-oxidant effect of minerals. Crit Rev Food Sci Nutr [Internet]. 2021 [cited 2025 May 21];61(9):1470–1489. Kontny E, Maśliński W. Interleukin 6 – biological activities and role in rheumatoid arthritis pathogenesis. Reumatologia [Internet]. [cited 2025 May 21];47(1):24–33. Al-Qahtani AA, Alhamlan FS, Al-Qahtani AA. Pro-Inflammatory and Anti-Inflammatory Interleukins in Infectious Diseases: A Comprehensive Review. Trop Med Infect Dis [Internet]. 2024 [cited 2025 May 21];9(1):13. Souza RF, Caetano MAF, Magalhães HIR, et al. Study of tumor necrosis factor receptor in the inflammatory bowel disease. World J Gastroenterol [Internet]. 2023 [cited 2025 May 21];29(18):2733–2746. Theiss AL, Simmons JG, Jobin C, et al. Tumor necrosis factor (TNF) α increases collagen accumulation and proliferation in intestinal myofibroblasts via TNF receptor 2. Journal of Biological Chemistry [Internet]. 2005 [cited 2025 May 21];280(43):36099–36109. Ye H, Wang Y, Bennett Jenson A, et al. Identification of inflammatory factor TNFα inhibitor from medicinal herbs. Exp Mol Pathol [Internet]. 2016 [cited 2025 May 21];100(2):307–311. Parameswaran N, Patial S. Tumor necrosis factor-a signaling in macrophages. Crit Rev Eukaryot Gene Expr [Internet]. 2010 [cited 2025 May 21];20(2):87–103. Tessema TS, Schwamb B, Lochner M, et al. Dynamics of gut mucosal and systemic Th1/Th2 cytokine responses in interferon-gamma and interleukin-12p40 knock out mice during primary and challenge Cryptosporidium parvum infection. Immunobiology [Internet]. 2009 [cited 2025 May 21];214(6):454–466. Mordue DG, Monroy F, La Regina M, et al. Acute Toxoplasmosis Leads to Lethal Overproduction of Th1 Cytokines. The Journal of Immunology [Internet]. 2001 [cited 2025 May 21];167(8):4574–4584. Mocchegiani E, Romeo J, Malavolta M, et al. Zinc: Dietary intake and impact of supplementation on immune function in elderly. Age (Omaha). 2013;35(3):839–860. Saraiva M, O’Garra A. The regulation of IL-10 production by immune cells. Nature Reviews Immunology 2010 10:3 [Internet]. 2010 [cited 2025 May 23];10(3):170–181. Bao B, Prasad AS, Beck FWJ, et al. Zinc supplementation decreases oxidative stress, incidence of infection, and generation of inflammatory cytokines in sickle cell disease patients. Translational Research [Internet]. 2008 [cited 2025 May 23];152(2):67–80. Prasad AS. Zinc is an Antioxidant and Anti-Inflammatory Agent: Its Role in Human Health. Front Nutr [Internet]. 2014 [cited 2025 May 23];1:100515. Ibs KH, Rink L. Zinc-altered immune function. Journal of Nutrition [Internet]. 2003 [cited 2025 May 23];133(5 SUPPL. 2). Cereda G, Ciappolino V, Boscutti A, et al. Zinc as a Neuroprotective Nutrient for COVID-19–Related Neuropsychiatric Manifestations: A Literature Review. Advances in Nutrition [Internet]. 2021 [cited 2025 May 23];13(1):66. Perestiuk V, Kosovska T, Volianska L, et al. Association of zinc deficiency and clinical symptoms, inflammatory markers, severity of COVID-19 in hospitalized children. Front Nutr. 2025;12:1566505. Shankar AH, Prasad AS. Zinc and immune function: the biological basis of altered resistance to infection. Am J Clin Nutr [Internet]. 1998 [cited 2025 May 23];68(2):447S-463S. Hojyo S, Fukada T. Roles of Zinc Signaling in the Immune System. J Immunol Res [Internet]. 2016 [cited 2025 May 23];2016. Fraker PJ, King LE. Reprogramming of the immune system during zinc deficiency. Annu Rev Nutr [Internet]. 2004 [cited 2025 May 23];24:277–298. Weström B, Arévalo Sureda E, Pierzynowska K, et al. The Immature Gut Barrier and Its Importance in Establishing Immunity in Newborn Mammals. Front Immunol. 2020;11. Additional Declarations No competing interests reported. Supplementary Files Supplementarydata1abundance.tsv Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7478928","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":507198057,"identity":"f429beee-7bb5-467a-ba0d-ed508a39645f","order_by":0,"name":"Gita 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Agency","correspondingAuthor":false,"prefix":"","firstName":"Suci","middleName":"Zulaikha","lastName":"Hildayani","suffix":""},{"id":507198069,"identity":"75ba5fef-e676-438d-8f47-dc6e7035abb4","order_by":8,"name":"Amanah Amanah","email":"","orcid":"","institution":"Universitas Swadaya Gunung Jati","correspondingAuthor":false,"prefix":"","firstName":"Amanah","middleName":"","lastName":"Amanah","suffix":""}],"badges":[],"createdAt":"2025-08-28 09:53:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7478928/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7478928/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90498637,"identity":"3f8d7589-78e7-41fd-a691-9e23a400b7e0","added_by":"auto","created_at":"2025-09-03 11:11:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":326743,"visible":true,"origin":"","legend":"\u003cp\u003eResearch flowchart of animal experimental for zinc-chelating peptides therapy for ZD.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7478928/v1/151cdb196341061e210122a8.png"},{"id":90498640,"identity":"608cda95-d4c0-4a71-b19f-1933ebb41fe9","added_by":"auto","created_at":"2025-09-03 11:11:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":188968,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of top 100 microbial species across sample.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7478928/v1/9bc4de3b32607189b5572390.png"},{"id":90498638,"identity":"6790470b-3e81-4be4-9c22-d0362c3bb2b5","added_by":"auto","created_at":"2025-09-03 11:11:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":72745,"visible":true,"origin":"","legend":"\u003cp\u003eBarplot of the 19 most abundant taxa at the species rank in all the samples. Any remaining taxa have been collapsed under the 'Other' category to facilitate the visualization. The y-axis indicates the relative abundance of each taxon in percentages for each sample.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7478928/v1/c1ed19ee2bf26b4f52e92f45.png"},{"id":90498967,"identity":"0d4112c4-8f71-48a1-a6d3-de1f300a46c9","added_by":"auto","created_at":"2025-09-03 11:19:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":109843,"visible":true,"origin":"","legend":"\u003cp\u003eThe concentration of cytokines secretion consisted of six groups: healthy rat pups (HG), healthy rat pups + ZCP 4 mg/kg BW (HG40), ZD rat pups + ZCP 4 mg/kg BW (ZD4), ZD rat pups + ZCP 40 mg/kg BW (ZD40), ZD rat pups + ZnSO\u003csub\u003e4\u003c/sub\u003e 40 mg/kg BW (ZDZn), and ZD rat pups without any therapies (ZD)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7478928/v1/3985c422a08637f7416bb779.png"},{"id":90498642,"identity":"fac717b7-4b51-4339-9b5e-23141ba03af6","added_by":"auto","created_at":"2025-09-03 11:11:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":165563,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of correlation of cytocine markers in ZD model (A) and ZD which received the supplement model (B)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7478928/v1/5007aee6a95275a048483243.png"},{"id":91901709,"identity":"ceb5d070-b679-4edd-a4c8-4cb6955ab964","added_by":"auto","created_at":"2025-09-22 21:46:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1818283,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7478928/v1/da031f2e-e80c-4c99-9181-3091ab91b653.pdf"},{"id":90498646,"identity":"0500ec30-c92b-467d-ab3c-f40e9ed576a4","added_by":"auto","created_at":"2025-09-03 11:11:16","extension":"tsv","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":90874,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata1abundance.tsv","url":"https://assets-eu.researchsquare.com/files/rs-7478928/v1/cfb6480f5b7d9ab346d48deb.tsv"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eEvaluation of Zn-Peptides from Holothuria scabra: Gut Microbiota Profile and Cytokine Signalling Pathways in Rat Pups Born to Zinc-Deficient Parent\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGrowth retardation is a well-documented and significant consequence of zinc deficiency (ZD) in young animals [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This impairment in growth may arise from a combination of factors, including a reduction in the body's ability to synthesize proteins, which are the building blocks of tissues, and an increase in catabolic processes that break down existing tissues [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Zinc plays a crucial role in the processes of bone growth and development, influencing the proliferation and differentiation of chondrocytes, the cells responsible for cartilage formation in the growth plates [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Consequently, insufficient zinc can directly impede the longitudinal growth of bones, contributing to overall growth stunting. The intricate relationship between zinc and fundamental anabolic processes highlights its essential role in achieving normal growth and development in young mammals [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn young mammals, including rat pups which serve as a valuable model for studying early development, ZD can lead to a spectrum of serious health consequences. These include impaired growth and overall development, hindering the achievement of full physical potential[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Furthermore, ZD severely compromises the function of the immune system, leading to an increased susceptibility to a wide range of infections. Behavioural patterns can also be negatively affected, manifesting as impairments in activity, attention, and motor development [\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Notably, maternal ZD during pregnancy can also have detrimental effects on offspring, influencing their growth, neurobehavioral development, and immune function [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The multifaceted negative impacts of ZD during this sensitive life stage underscore the critical need for effective preventative and therapeutic strategies.\u003c/p\u003e\u003cp\u003eThe immune system exhibits a particular sensitivity to fluctuations in zinc status. Zinc is vital for the proper development, differentiation, and function of a wide array of immune cells, including T cells, B cells, natural killer (NK) cells, and neutrophils.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] Furthermore, zinc is involved in the production and regulation of cytokines, which are signaling molecules that coordinate immune responses [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. A deficiency in zinc can disrupt the delicate balance of these immune components, leading to impaired immune function and an increased vulnerability to infections. The impact of zinc on the immune system underscores its critical role in maintaining the body's defenses against pathogens, particularly in young, developing individuals whose immune systems are still maturing [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Studies show that ZD in humans reduces the production of interferon-gamma (IFN-γ) and have a negative effect on cell-mediated immunity [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The IFN-γ increase is associated with epigenetic and redox-mediated mechanisms [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eZD has been demonstrated to elevate reactive oxygen species (ROS) production, which in turn leads to increased activation of p38 mitogen-activated protein kinase (MAPK). This increased activation is necessary for the synthesis of interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Many of the adverse effects of ZD on cytokine production can be reversed through zinc supplementation. Supplementation corrects the production of both interleukin-2 (IL-2) and interferon-gamma (IFN-γ) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Nonetheless, there is a paucity of research on the topic, but studies on zinc chelates have not yet been published in relation to cytokine secretion resulting from the supplementation with this Zn-peptides type.\u003c/p\u003e\u003cp\u003eZinc and the microbiota of the gastrointestinal tract have been demonstrated to be interrelated. Research findings indicate that zinc levels can influence the composition and function of the gastrointestinal microbiota, and vice versa. The gut microbiota, a complex ecosystem present within the gastrointestinal tract, plays a pivotal role in various physiological processes, including nutrient absorption, intestinal permeability regulation, host metabolism, and modulation of mucosal and systemic immunity [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Physiological and nutritional doses of zinc have been demonstrated to enhance gut wall integrity, thereby mitigating the translocation of bacteria and gut microbiome metabolites into the systemic circulation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSpecific bacteria caused of high zinc diets can enrich \u003cem\u003eAkkermansia\u003c/em\u003e, \u003cem\u003eFaecalibaculum\u003c/em\u003e, \u003cem\u003eHelicobacter, Dubosiella, Caulobacter\u003c/em\u003e, and \u003cem\u003eBradyrhizobium\u003c/em\u003e. High-zinc diets are associated with a lower abundance of \u003cem\u003eSphingomonas Romboutsia, Bacteroides, Lactobacillus, and Bifidobacterium\u003c/em\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Zinc supplementation has been shown to improve the integrity of the gut wall, reduce the translocation of bacteria from the gut into the bloodstream, and influence the diversity and composition of the gut microbiota [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Furthermore, it can play a role in modulating the production of cytokines, often leading to a reduction in the levels of pro-inflammatory cytokines [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. These findings indicate that addressing ZD through supplementation can have direct benefits for gut health by strengthening the intestinal barrier and promoting a more balanced inflammatory state within the gut [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis study employs zinc-chelating peptides (ZCPs) derived from \u003cem\u003eHolothuria scabra\u003c/em\u003e, the characteristics of which have been established in prior investigations [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The evaluation of Zn-Peptides sourced from \u003cem\u003eHolothuria scabra\u003c/em\u003e with respect to cytokine secretion and gut microbiota profiles in zinc-deficient rat offspring has not been previously documented. The present research is proposed to elucidate the comprehensive potential of third-generation zinc supplementation through a metagenomic approach and its impact on the immune system of zinc-deficient rats offspring.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cp\u003eThis study employed an in vivo methodology to ascertain the capacity of ZCPs to normalize Zn absorption in ZD rat offspring from the maternal uterine environment. The methodology employed to develop zinc-deficient animals during the first 1000 days of life (conversion with human age) and the composition of the zinc-deficient feed utilized to obtain the experimental animal model have been registered with Indonesian patent number P00202410265. The research flowchart is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Research Ethics\u003c/h2\u003e\u003cp\u003e This study has been granted ethical approval by the Health Research Ethics Committee of the Faculty of Medicine, University of Indonesia (FKUI) - RSUPN Dr. Cipto Mangunkusumo (certificate number: Ket-309/UN2.F1/ETIK/PPM/00.02/2022; protocol number: 22-03-0342).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Animals Subject\u003c/h2\u003e\u003cp\u003eThis investigation employed an adult cohort of male and female Sprague-Dawley rats, comprising a balanced population with a weight of 150\u0026ndash;200 grams at the age of 8 weeks. The animal subjects were obtained from the Animal Research Facilities at Faculty of Medicine, University of Indonesia and maintained in a room with a constant temperature of 25\u0026ndash;27\u0026deg;C and constant humidity (45\u0026ndash;65 RH). The lighting schedule was maintained at an adequate level, with 12 hours of light and 12 hours of dark. The rats were provided with feed in the form of pellets and had access to drinking water at all times. Subsequent to this, the male and female rats were bred with previously categorized healthy rats and ZD rats, which were provided with feed supplementation. The rat pups from healthy and ZD rats were separated into six groups with males and females randomly allocated in three-week-old. The facility for animal subject experiments certified (SNI ISO/IEC 17025:2017) and accredited by the National Accreditation Committee, Republic of Indonesia No. LP-1855-IDN.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Zinc deficiency model\u003c/h2\u003e\u003cp\u003eThe present study commenced with the preparation of zinc-deficient rats by feeding a zinc-deficit diet. The composition of the feed used to develop the ZD rat model was as follows (per kilo of feed): pollard 13.5%; corn 40%; soybean meal 15%; corn gluten meal 22%; tapioca starch 2%; morin mix 2.5%; vegetable oil 2%. The composition of the feed was as follows: calcium carbonate (1.3%), mineral premix (without Zn) (0.2%), and dicalcium phosphate (1.5%). The mothers were fed a zinc deficit diet during both gestation and lactation. The rat pups were weaned at 3 weeks of age and continued with zinc deficit feeding until seven weeks of age (registered Indonesian patent number P00202410265).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Zinc-chelating peptides therapy design\u003c/h2\u003e\u003cp\u003eAs referenced in previous studies, therapeutic ZCPs derived from \u003cem\u003eHolothuria scabra\u003c/em\u003e will be utilized in this investigation as a therapeutic supplement to ZD [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In the therapeutic intervention of ZD rat pups, ZCPs therapy are employed by dividing the variation of group doses of 4 and 40 mg/kg BW. The other group serving as the control group, which is administered with ZnSO4 at a dose of 40 mg/kg BW [\u003cspan additionalcitationids=\"CR29 CR30 CR31 CR32\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe study consisted of six groups: healthy rat pups (HG), healthy rat pups\u0026thinsp;+\u0026thinsp;ZCP 4 mg/kg BW (HG40), ZD rat pups\u0026thinsp;+\u0026thinsp;ZCP 4 mg/kg BW (ZD4), ZD rat pups\u0026thinsp;+\u0026thinsp;ZCP 40 mg/kg BW (ZD40), ZD rat pups\u0026thinsp;+\u0026thinsp;ZnSO\u003csub\u003e4\u003c/sub\u003e 40 mg/kg BW (ZDZn), and ZD rat pups without any therapies (ZD). These abbreviations will be employed subsequently in the results and discussion section.\u003c/p\u003e\u003cp\u003eZCPs therapy were administered to 3-week-old rat pups along with a low-zinc diet for four weeks. Following the conclusion of the study, all rats were euthanized with a ketamine and xylazine ratio of 5:1 (mg/kg BW) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Blood samples were obtained from the orbital plexus and from the heart, then stored in EDTA vacutainers. Samples were kept at cold temperature before being separated by 15 min centrifuge at 4000 rpm with 4\u003csup\u003e0\u003c/sup\u003eC to collect the serum. The serum was then stored at -80\u0026deg;C for the analysis of cytocine secretion.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 DNA microbiota sequencing and data analysis\u003c/h2\u003e\u003cp\u003eAll of groups were colected the segment of the rat pups sigmoid colon under sterile conditions and stored in temperature \u0026minus;\u0026thinsp;80\u003csup\u003e0\u003c/sup\u003eC until analysis. Total microbial DNA was extracted using the the Zymo Quick-DNA Fecal/Soil Microbe Miniprep Kit following the manufacturer\u0026rsquo;s protocol. DNA concentration and purity were assessed using a Nanodrop spectrophotometer (Thermo Fisher Scientific, USA).\u003c/p\u003e\u003cp\u003eMicrobial DNA was amplified targeting the 16S rRNA gene using universal primers 27F (5\u0026prime;-AGAGTTTGATCMTGGCTCAG-3\u0026prime;) and 1492R (5\u0026prime;-CGGTTACCTTGTTACGACTT-3\u0026prime;) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. PCR amplification was performed using the (16S barcoding (SQK-16SO24) Kit on a Applied Biosystems Thermal Cyclers for PCR Thermo Fisher PCR machine. Amplicon concentration was confirmed using a Qubit Fluorometer (Invitrogen, USA) Qubit 3 Fluorometer dengan KIT Qubit\u0026trade; dsDNA HS Assay Kit, Invitrogen by Thermo Fisher to ensure sufficient template quantity for sequencing. Amplicon size and specificity were further verified by agarose gel electrophoresis using a 1% (w/v) agarose gel with Gel Doc\u0026trade; EZ Imager.\u003c/p\u003e\u003cp\u003eFor next-generation sequencing (NGS) library preparation, the 16S amplicons were barcoded using the 16S Barcoding Kit 1\u0026ndash;24 (SQK-16S024, Oxford Nanopore Technologies, UK) according to the manufacturer\u0026rsquo;s instructions. Libraries were pooled in equimolar concentrations and prepared for sequencing using the Ligation Sequencing Kit (SQK-LSK114, Oxford Nanopore Technologies, UK). Sequencing was conducted on a MinION Mk1C device (Oxford Nanopore Technologies, UK) equipped with a FLO-MIN106 (R9.4.1) flow cell. The sequencing run was managed using MinKNOW software (version 24.02.16) with real-time basecalling performed using Dorado (version 7.3.11), configured with the fast model optimized for 450 bp reads [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The sequencing workflow was carried out at the Sequencing Laboratory of the National Research and Innovation Agency (BRIN).\u003c/p\u003e\u003cp\u003eRaw FASTQ files were processed. First. adapter sequences were trimmed using Porechop (version 0.2.4) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and reads were quality-filtered using NanoFilt (version 2.8.0), applying a minimum Phred quality score threshold of Q10. Reads were further filtered based on length, retaining those between 1,300 bp and 1,700 bp to match targeted amplicon of 27F and 1492R. Read quality, both pre- and post-filtering, was assessed using NanoPlot (version 1.20.0) (De Coster et al., 2018). Filtered reads were classified using Kraken2 (version 2.1.3) within the 16S pipeline (version 1.4.0) implemented in EPI2ME (Oxford Nanopore Technologies, UK) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Taxonomic classification was performed against the NCBI 16S_18S reference database with default parameters, including a minimum percent identity of 95% and a minimum reference coverage of 90%. Data visualization and statistical analyses were conducted in R (version 4.3.1)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Concentration of cytokine secretion assay\u003c/h2\u003e\u003cp\u003eCytokine secretion was quantitatively analysed in serum samples obtained from all groups of rat pups. The cytokine parameters examined included IL6, IL10, TNFα, and IFNγ levels, utilizing the ElabScience ELISA Kit for Rat. The experimental procedures were conducted in accordance with the protocols outlined in the kit's assay protocol for each parameter and previous study results [\u003cspan additionalcitationids=\"CR40 CR41 CR42 CR43 CR44\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn briefly, The ELISA procedure begins with preparing reagents by bringing them to room temperature and diluting the concentrated wash buffer. The standard is reconstituted, and both the biotinylated detection Ab working solution and the HRP conjugate working solution are made. Next, diluted standard, blank, and sample are added to designated wells, and the plate is incubated. Following incubation, the liquid is decanted, biotinylated detection Ab working solution is added, and the plate is incubated again. A wash step is performed, and then HRP conjugate working solution is added with another incubation. Another wash is performed before adding Substrate Reagent and incubating in the dark. The reaction is stopped with Stop Solution, and the optical density is read using a micro-plate reader.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Statistical analysis\u003c/h2\u003e\u003cp\u003eThe presentation of the data was as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) or median (range). The statistical analysis used a t-test and a one-way ANOVA to compare differences in treatment between groups. Tukey's multiple comparisons test was then used for post hoc analysis. A significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was set. Correlation analysis was conducted using Pearson\u0026rsquo;s test. The analysis and calculation of the data was performed using GraphPad Prism 9.0 for Windows.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Sequencing data and microbial diversity\u003c/h2\u003e\u003cp\u003eA total of 866,465 sequence reads were successfully assigned to taxonomic groups, spanning 667 species, 301 genera, 71 families, 33 orders, 16 classes, and 10 phyla (\u003cb\u003eSupplementary Data 1\u003c/b\u003e \u0026ndash; Abundance TSV).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eReads data before and after filtering across sample.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFiltering\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTotal reads\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTotal bases\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMean read length\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMean Q\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eHG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBefore\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e199,868\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e310,889,655\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1,555.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e11.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAfter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e176,462\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e261,778,874\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1,483.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e12.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eHG40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBefore\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e13,076\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19,661,907\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1,503.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e11.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAfter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e11,005\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16,088,090\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1,461.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e11.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eZD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBefore\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e153,965\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e239,060,313\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1,552.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e11.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAfter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e134,188\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e199,980,948\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1,490.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e12.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eZD4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBefore\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e145,632\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e223,901,584\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1,537.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e11.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAfter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e127,177\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e187,484,810\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1,474.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e12.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eZD40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBefore\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e155,101\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e242,950,714\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1,566.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e11.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAfter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e136,079\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e204,091,741\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1,499.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e12.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eZDZn\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBefore\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e365,872\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e535,680,079\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1,464.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e11.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAfter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e281,554\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e414,641,295\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1,472.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e11.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAmong the samples, the HG group harbored 266 microbial species, including 24 species unique to this sample. The HG40 group contained 55 microbial species, 7 of which were exclusive to this environment. The ZD group exhibited 220 microbial species, with 16 unique species identified. In the ZD4 group, 275 microbial species were identified, including 39 species exclusive to this group. The ZD40 group demonstrated greater diversity with 346 microbial species, of which 63 were unique. Notably, the ZDZn group exhibited the highest microbial diversity, with 446 species identified, including 99 unique species absent from all other samples. Reads data for all sample summarize in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSample diversity indices\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRichness\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eShannon diversity index\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSimpson's index\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eInverse Simpson's index\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e266\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.06\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHG40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.39\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eZD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e220\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.08\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eZD4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e275\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eZD40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e346\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eZDZn\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e446\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.04\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe ZDZn group demonstrated the highest microbial richness, with 446 species identified, and had the greatest Shannon diversity index (4.16), indicating both a high number of species and an even distribution among them. Its Simpson's index (0.96) highlighted minimal dominance by individual species, further supported by an inverse Simpson's index of 1.04. The ZD40 group followed, with a richness of 346 species and a Shannon diversity index of 3.28, reflecting substantial diversity. Its Simpson's index (0.87) and inverse Simpson's index (1.16) indicated slightly higher species dominance compared to ZDZn (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe ZD4 and ZD groups displayed moderate diversity, with 275 and 220 species, respectively. The Shannon diversity indices were 3.37 for ZD4 and 3.22 for ZD, suggesting relatively even species distributions. Both groups exhibited Simpson's indices above 0.9, indicating low species dominance, with inverse Simpson's indices of 1.10 for ZD4 and 1.08 for ZD. The HG group, containing 266 species, had a Shannon diversity index of 3.6, reflecting higher species evenness compared to ZD4 and ZD. Its Simpson's index (0.94) and inverse Simpson's index (1.06) supported this observation. In contrast, the HG40 group exhibited the lowest richness (55 species) and the lowest Shannon diversity index (2.23), indicating limited diversity. Its Simpson's index (0.72) and inverse Simpson's index (1.39) suggested greater dominance by a few species compared to the other groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe composition of microbial communities varied significantly among the samples, as visualized in a heatmap of the top 100 microbial species (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In the HG group, Enterococcus faecalis was the most abundant species (28,537 reads), followed by \u003cem\u003eLigilactobacillus murinus\u003c/em\u003e (10,880 reads) and \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e (10,784 reads). The HG40 group was dominated by \u003cem\u003eCollinsella aerofaciens\u003c/em\u003e (5,285 reads), with lower abundances of \u003cem\u003eEnterococcus faecalis\u003c/em\u003e (1,260 reads) and \u003cem\u003eLactobacillus hominis\u003c/em\u003e (483 reads). In the ZD group, \u003cem\u003eLigilactobacillus murinus\u003c/em\u003e (19,668 reads), \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e (18,582 reads), and \u003cem\u003eL. hominis\u003c/em\u003e (15,019 reads) were the dominant taxa, collectively accounting for the majority of reads. The ZD4 group was characterized by a high abundance of \u003cem\u003eRomboutsia timonensis\u003c/em\u003e (28,954 reads), \u003cem\u003eRomboutsia ilealis\u003c/em\u003e (16,201 reads), and \u003cem\u003eL. johnsonii\u003c/em\u003e (12,408 reads). The ZD40 group exhibited a significant presence of \u003cem\u003eL. johnsonii\u003c/em\u003e (44,535 reads), \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e (13,716 reads), and \u003cem\u003eAnaerobiospirillum succiniciproducens\u003c/em\u003e (8,473 reads). Finally, the ZDZn group, with the highest diversity, was enriched with \u003cem\u003eRomboutsia timonensis\u003c/em\u003e (34,050 reads), \u003cem\u003eL. hominis\u003c/em\u003e (23,369 reads), and \u003cem\u003eR. ilealis\u003c/em\u003e (16,201 reads).\u003c/p\u003e\u003cp\u003eAs depicted in ZD barplot (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cb\u003eSupplementary Data 2 -\u003c/b\u003e Sankey diagram) Bacillota (98%) and Pseudomonadota (2%) are the two identified bacterial phyla that make up ZD. Bacilli (64,18%) and Lactobacillales (64.24%) prevailed at the class and ordo-levels. The most prevalent family among all six families found was Lactobacillales (64.24%), which included three dominant genera from the same family: \u003cem\u003eLimosilactobacillus\u003c/em\u003e (22.66%), \u003cem\u003eLactobacillus\u003c/em\u003e (21.9%), and \u003cem\u003eLigilactobacillus\u003c/em\u003e (21.89%). \u003cem\u003eStreptococcus, Romboutsia, Clostridium\u003c/em\u003e, and \u003cem\u003eAnaerobiospirillum\u003c/em\u003e are the additional genera that have been identified. Finally, at the species-level profilling showed the \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e (28.63%), \u003cem\u003eLactobacillus hominis\u003c/em\u003e (24.68%), \u003cem\u003eLigilactobacillus murinus\u003c/em\u003e (30.3%), \u003cem\u003eRomboutsia timonensis\u003c/em\u003e (19.59%), and \u003cem\u003eClostridium disporicum\u003c/em\u003e (13.98%) dominated.\u003c/p\u003e\u003cp\u003eBased on the diagram for ZD40 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), it shown that ZD40 was composed of three bacterial phyla, which are Bacillota, Bacteroidota, and Pseudomonadota. Bacillota (85.58%) was identified as the most abundant phylum followed by Pseudomonadota (12.67%), and Bacteroidota (1.25%). At the class level, Bacilli (69.50%) was the dominant and Lactobacillales (69.48) was the dominant for ordo level. Lactobacillaceae (65.37%) was identified to be the most dominant family among all the bacterial families (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8). Genus level microbial resolution revealed the predominance of \u003cem\u003eLactobacillus\u003c/em\u003e (49.01%) among all identified genera (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;11). Finally, species-level profiling revealed the abundance of \u003cem\u003eL. johnsonii\u003c/em\u003e (33.38%), \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e (10.28%), \u003cem\u003eAnaerobiospirillum succiniciproducens\u003c/em\u003e (6.35%), and \u003cem\u003eL. hominis\u003c/em\u003e (5.10%) among other specises (n\u0026thinsp;=\u0026thinsp;14).\u003c/p\u003e\u003cp\u003eFor ZDZn (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) reveals that ZDZn contains two bacterial phyla, which are Bacillota as the most abundant (93.99%) and Bacteroidota (6.01%). The dominant class of ZDZn was Clostridia (73.41%), followed by Bacilli (24.90%) and Bacteroidia (1.69%). Lachnospirales was the most abundant ordo (37.19%) while Lachnospiraceae was the most abundant family (38.47%) among all the bacterial families (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8). For the genus level, Blautia was the most dominant (23.73%) followed by Romboutsia (23.33%) among all genera (n\u0026thinsp;=\u0026thinsp;16). At the species level, \u003cem\u003eR. timonenses\u003c/em\u003e revealed as the most abundant species (18.22%) followed by \u003cem\u003eL. hominis\u003c/em\u003e (12.50%) among all identified species (n\u0026thinsp;=\u0026thinsp;21).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e3.2 Concentration of cytokines secretion\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e showed that, Interleukin-10 (IL10) typically considered an anti-inflammatory cytokine. The lowest level is seen in the healthy control group (HG, approx. 80 pg/mL). The zinc-deficient group (ZD) shows a significantly higher level (approx. 130 pg/mL) compared to HG (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). All treatment groups (ZD4, ZD40, ZDZn) and the healthy treated group (HG40) also show elevated IL10 levels compared to HG (ranging approx. 110\u0026ndash;120 pg/mL), but these levels appear slightly lower than the untreated ZD group. ZD significantly elevates IL10 levels. Treatments, including ZCP and ZnSO₄ in deficient rats and ZCP in healthy rats, also result in IL10 levels higher than healthy controls, though potentially slightly reduced compared to untreated ZD.\u003c/p\u003e\u003cp\u003eMeanwhile, Interleukin-6 (IL6) generally considered a pro-inflammatory cytokine. The HG group has the lowest level (approx. 125 pg/mL). The ZD group shows a significantly increased IL6 level (approx. 210 pg/mL) compared to HG. The ZCP-treated ZD groups (ZD4 and ZD40) also have high IL6 levels (approx. 190\u0026ndash;205 pg/mL). The ZnSO₄-treated group (ZDZn, approx. 155 pg/mL) and the healthy treated group (HG40, approx. 140 pg/mL) show IL6 levels lower than the ZD group but still elevated compared to the HG group. ZD significantly increases IL6 levels. ZCP treatment at both doses maintains high IL6 levels in deficient rats. ZnSO₄ treatment in deficient rats and ZCP treatment in healthy rats appear to partially moderate this increase compared to ZD, but levels remain above healthy controls.\u003c/p\u003e\u003cp\u003eThe ratio of the pro-inflammatory IL6 to the anti-inflammatory IL10. A higher ratio might suggest a more pro-inflammatory state. The ratios across all groups range from about 1.3 to 1.7. HG is around 1.5. ZD, ZD4, and ZD40 appear slightly higher (around 1.6\u0026ndash;1.7). ZDZn and HG40 appear slightly lower (around 1.3\u0026ndash;1.4). There are no significance markers shown on this graph. Based on the visual representation and lack of significance markers, there may not be statistically significant differences in the IL6/IL10 ratio between the groups. However, there's a slight trend towards a higher ratio in ZD, ZD4, and ZD40 groups compared to HG, ZDZn, and HG40.\u003c/p\u003e\u003cp\u003eResult shows, Tumor Necrosis Factor-alpha (TNFα), another pro-inflammatory cytokine. All groups show similar mean levels of TNFα (around 70\u0026ndash;75 pg/mL) and no significance markers are present. The treatments and/or ZD state did not appear to cause significant changes in TNFα levels among the different groups in this study. Interferon-gamma, a cytokine involved in immune response, often associated with Th1-type inflammation. The ZD group shows a significantly higher level (approx. 440 pg/mL) compared to the HG group (approx. 380 pg/mL) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Interestingly, all treatment groups (ZD4, ZD40, ZDZn) and the healthy treated group (HG40) show markedly lower IFNγ levels (around 300\u0026ndash;330 pg/mL) compared to both the ZD and the HG groups. ZD significantly increases IFNγ levels compared to healthy controls. All tested treatments (ZCP in deficient and healthy rats, ZnSO₄ in deficient rats) appear to reduce IFNγ levels below those seen in both untreated deficient and healthy control groups.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Correlation of cytokine release in ZD model and ZD which received the supplement model\u003c/h2\u003e\u003cp\u003eThe provided heatmap illustrates (Figure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) the relationships between key cytokines in zinc-deficient rat pups. Each cell in the matrix represents the Pearson correlation coefficient, indicating the strength and direction of the linear association between two cytokines. Heatmap of Figure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA showed, strong negative correlation is observed between IL-10 and both IL-6 (r = -0.49) and TNFα (r = -0.52), suggesting an inverse relationship where increased levels of the anti-inflammatory cytokine IL-10 are associated with decreased levels of these pro-inflammatory cytokines. IL-6 and TNFα exhibit a weak positive correlation (r = -0.12), indicating a minimal linear relationship. The correlation between IL-10 and IFNγ is weakly positive (r = -0.23). IL-6 shows a moderate negative correlation with IFNγ (r = -0.32), while TNFα and IFNγ have a weak positive correlation (r = -0.11). Overall, the data suggests a complex interplay between these cytokines in ZD, with IL-10 potentially playing a role in modulating the levels of pro-inflammatory cytokines.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMeanwhile heatmap in 5B analyzing the inter-cytokine correlations reveals several relationships in ZD which received the supplement (ZD4 model). A moderate positive correlation exists between TNFα and IL6 (r\u0026thinsp;=\u0026thinsp;0.40). Conversely, a notable strong negative correlation is observed between IL6 and IL10 (r = -0.63). TNFα also shows a moderate negative correlation with IL10 (r = -0.40). IFNγ exhibits weaker correlations with the other cytokines: a weak negative correlation with TNFα (r = -0.22), a weak to moderate negative correlation with IL6 (r = -0.32), and a weak positive correlation with IL10 (r\u0026thinsp;=\u0026thinsp;0.23). These findings highlight potential coordination or opposition in the expression patterns of these cytokines under the conditions studied, with the IL6-IL10 relationship being the most strongly antagonistic.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Zinc deficiency significantly alters the gut microbiome composition\u003c/h2\u003e\u003cp\u003eThe study definitively identified microbial diversity in the intestinal organs of the colon segment through 16S rRNA sequencing in six groups of rats. HG, HG40, ZD, ZD4, ZD40, and ZDZn. The objective was straightforward: to identify the microbial diversity and associated effects in each group, with a particular focus on zinc metabolism. The comparison between the healthy (HG) and zinc-deficient (ZD) groups indicates that ZD impacts the gut microbiome primarily by causing shifts in its composition rather than drastically reducing overall diversity.\u003c/p\u003e\u003cp\u003eThe ZD group showed slightly lower species richness and Shannon diversity compared to the HG group, their Simpson's diversity index remained similar [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Zinc is indispensable for bacteria, acting as a cofactor for hundreds of enzymes crucial for growth and virulence. However, the host actively combats bacterial infections by limiting zinc availability at infection sites \u0026ndash; a process called \"nutritional immunity.\" Proteins like S100A8/A9 (calprotectin) are deployed by immune cells to sequester zinc [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhile systemic ZD (low zinc throughout the body) isn't the same as targeted zinc withholding by the immune system, it does mean less zinc is available overall. This can directly inhibit the growth of bacteria that cannot efficiently scavenge scarce zinc. Pathogenic bacteria often possess high-affinity zinc uptake systems (like the ZnuABC transporter) specifically to overcome host zinc restriction [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Zinc is critical for the development and function of immune cells (T-cells, B-cells, macrophages).\u003c/p\u003e\u003cp\u003eDeficiency impairs immune responses, leading to increased susceptibility to infections. However, it doesn't simply weaken all defences; it can dysregulate the immune system, sometimes leading to inappropriate inflammation or altered cytokine profiles, which changes the environment bacteria experience. An altered immune landscape due to ZD can shift the competitive balance between different microbial species[\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Zinc is vital for maintaining the intestinal epithelial barrier. It supports the function of tight junctions, the structures that seal the space between gut cells.\u003c/p\u003e\u003cp\u003eDeficiency can increase intestinal permeability (\"leaky gut\"). A compromised barrier allows bacterial products (like LPS) to cross into tissues, potentially triggering inflammation. It also changes the conditions within the gut lumen, affecting microbial composition (dysbiosis) [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Due to varying zinc requirements and acquisition capabilities (e.g., presence of high-affinity transporters), different bacterial species and strains exhibit different sensitivities to zinc limitation [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Therefore, ZD doesn't simply inhibit all bacteria equally. It creates a complex, altered environment where the interplay between impaired bacterial metabolism, altered host immunity, compromised barrier function, and inter-species competition for limited zinc resources can lead to significant shifts in the microbiota composition, potentially favouring certain (sometimes pathogenic) species over others.\u003c/p\u003e\u003cp\u003eThe microbial composition clearly differs substantially across the groups (HG, HG40, ZD, ZD4, ZD40, ZDZn), as expected based on distinct treatments or conditions. The stark differences between the HG (control) and ZD (zinc deficient) groups highlight the significant impact of ZD on gut microbial composition, changing the dominant species entirely. The various treatment groups (ZD4, ZD40, ZDZn) applied to the zinc-deficient state each resulted in unique microbial signatures, suggesting dose-dependent effects or different mechanisms of action if the treatments varied beyond zinc levels.\u003c/p\u003e\u003cp\u003eZinc is an essential micronutrient for both the host and gut bacteria. It plays critical catalytic, structural, and regulatory roles in cellular physiology [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. The microbiota composition changes, particularly the increase in certain \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eRomboutsia\u003c/em\u003e species with presumed zinc supplementation, and the differing profiles in zinc-deficient states, align with broader research indicating that zinc is a crucial modulator of gut microbiota [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The microbial shifts observed in this study, particularly in the zinc-related groups (ZD, ZD4, ZD40, ZDZn), can be contextualized with existing research on zinc's impact on gut microbiota.\u003c/p\u003e\u003cp\u003eIn ZD group, dominance of \u003cem\u003eLigilactobacillus murinus\u003c/em\u003e, \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e, and \u003cem\u003eLactobacillus hominis\u003c/em\u003e findings in zinc-deficient models, but shifts in \u003cem\u003eLactobacillus\u003c/em\u003e species are commonly reported. \u003cem\u003eL. murinus\u003c/em\u003e has been shown to modulate intestinal barrier damage and gut microbiota in immunosuppressed models, often increasing short-chain fatty acid-producing bacteria [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. \u003cem\u003eL. reuteri\u003c/em\u003e is a well-studied probiotic with anti-inflammatory properties and the ability to modulate gut microbiota. Some studies have linked \u003cem\u003eLactobacillaceae\u003c/em\u003e family members abundance to metal availability, including zinc [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. The presence of these species in the ZD group is interesting and might reflect a complex interplay where some lactobacilli might thrive or be less affected by moderate zinc scarcity compared to other bacteria. Their relative abundance increases as other zinc-dependent microbes decrease. Their persistence or increase in ZD40 (for \u003cem\u003eL. reuteri\u003c/em\u003e) suggests a positive response to or role during zinc therapy.\u003c/p\u003e\u003cp\u003eIn treatment groups (ZD4, ZD40, ZDZn), zinc supplementation can modulate the gut microbiota, often reversing some of the changes caused by deficiency and sometimes promoting the growth of beneficial bacteria. A systematic review highlighted that high dietary zinc could alter bacterial taxa, sometimes increasing metal resistance genes [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. The shifts towards \u003cem\u003eRomboutsia timonensis\u003c/em\u003e, \u003cem\u003eRomboutsia ilealis\u003c/em\u003e, and \u003cem\u003eLactobacillus johnsonii\u003c/em\u003e (ZD4), increased \u003cem\u003eLactobacillus johnsonii\u003c/em\u003e and \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e (ZD40), and enrichment of \u003cem\u003eRomboutsia timonensis\u003c/em\u003e and \u003cem\u003eLactobacillus hominis\u003c/em\u003e (ZDZn) in this study are specific changes that would be interesting to compare to targeted supplementation studies.\u003c/p\u003e\u003cp\u003e\u003cem\u003eRomboutsia timonensis\u003c/em\u003e and \u003cem\u003eRomboutsia ilealis\u003c/em\u003e (dominant in ZD4 and ZDZn groups). \u003cem\u003eRomboutsia\u003c/em\u003e is a genus within the \u003cem\u003eClostridiales\u003c/em\u003e order. While less studied than \u003cem\u003eLactobacillus\u003c/em\u003e or \u003cem\u003eBifidobacterium\u003c/em\u003e, emerging research is exploring its role. The significant increase in these species in the ZD4 and ZDZn groups is a key finding. This suggests that these \u003cem\u003eRomboutsia\u003c/em\u003e species might be favored by or play a role in the gut environment shaped by zinc therapy. General reviews on dietary zinc mention that zinc status alters bacterial taxa, but specific effects on \u003cem\u003eRomboutsia\u003c/em\u003e would require more targeted literature [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. The varied profiles in ZD4, ZD40, and ZDZn groups highlight that zinc therapy can selectively promote certain bacterial taxa, such as specific \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eRomboutsia\u003c/em\u003e species. The differences between these supplemented groups underscore that the \u003cem\u003edose or form\u003c/em\u003e of zinc may fine-tune these microbial shifts [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e4.2 Zinc deficiency significantly alters inflammatory cytokine profile\u003c/h2\u003e\u003cp\u003eThe significant increase in the anti-inflammatory cytokine IL-10 in the zinc-deficient (ZD) group (approx. 130 pg/mL vs. 80 pg/mL in HG, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and its persistence at elevated levels in all treatment groups (ZD4, ZD40, ZDZn, HG40) compared to healthy controls, is a pivotal observation. While IL-10 is a potent anti-inflammatory agent, its chronic elevation can sometimes be indicative of an immune system struggling to resolve ongoing inflammation or an attempt to counteract a strong pro-inflammatory drive [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. In the context of ZD, which generally promotes inflammation (as seen with IL-6 and IFN-γ), this IL-10 surge could indeed be a compensatory mechanism [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. This elevation may result from disrupted zinc homeostasis in immune cells, particularly affecting regulatory T cells (Treg), which are primary producers of IL-10. Zinc deficiency impairs Treg differentiation by downregulating Foxp3 expression, leading to compensatory IL-10 overproduction as the immune system attempts to counterbalance inflammation [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. The fact that even zinc supplementation (both ZCP and ZnSO₄) did not normalize IL-10 levels, and ZCP in healthy rats (HG40) also increased it, suggests a more complex interaction than simple deficiency correction. It might imply that, the underlying inflammatory insult caused by the initial deficiency has long-lasting effects on immune programming. The forms of zinc used, or the act of supplementation itself, might directly stimulate IL-10 producing cells (e.g., regulatory T cells, certain macrophage subsets) [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. This finding links to the concept of immune homeostasis and how the body attempts to restore balance. Persistent IL-10 elevation, while seemingly beneficial, warrants scrutiny as it can sometimes be associated with immune exhaustion or even contribute to pathogen persistence or tumor immune evasion in other contexts if not properly regulated [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eZinc deficiency robustly increased pro-inflammatory IL-6 (approx. 210 pg/mL in ZD vs. 125 pg/mL in HG). Notably, ZCP treatment in deficient rats (ZD4, ZD40) did little to reduce these high levels, whereas ZnSO₄ (ZDZn) showed a more pronounced (though incomplete) reduction. Even ZCP in healthy rats (HG40) slightly elevated IL-6. This elevation could be due to a disruption in zinc homeostasis in immune cells, particularly affecting regulatory T cells (Treg), which produce IL-10. Zinc deficiency impairs Treg differentiation by downregulating Foxp3 expression, prompting the immune system to produce more IL-10. This increase in IL-6 is driven by macrophages and intestinal epithelial cells, which are triggered by increased gut permeability (leaky gut) and NF-κB signaling. The strong IL-6 induction in ZD is well-documented and often linked to the activation of the NF-κB pathway, a critical transcription factor for many pro-inflammatory genes [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eZinc is known to inhibit NF-κB activation; thus, its deficiency unleashes this pathway [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. The differential effect of ZCP versus ZnSO₄ is significant. While ZnSO₄ is a readily ionizable form of zinc, zinc-peptide chelates (like ZCP) are proposed to have different absorption pathways and bioavailability characteristics. It is suggested that ZCP may depend on peptide-mediated uptake via transporters such as ZIP4, which has a higher expression in ZD. However, this may not effectively deliver zinc to the intracellular compartments that are essential for NF-κB inhibition, constraining its anti-inflammatory effectiveness in comparison to ZnSO₄ [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. The inability of ZCP to effectively lower IL-6 in ZD rats, possible because poorer dissociation of zinc from the peptide at relevant cellular sites, this indicates that the release of zinc from its peptides may be inefficient, potentially resulting in insufficient zinc availability for its anti-inflammatory functions within the cell. Besides that, the peptide moiety itself having some mild pro-inflammatory activity or interfering with zinc's anti-inflammatory action against IL-6 specifically. Meanwhile, ZCP need for higher or more prolonged dosing because capability to insufficient to achieve a significant decrease in IL-6 [\u003cspan additionalcitationids=\"CR76\" citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe balance between pro-inflammatory (IL-6) and anti-inflammatory (IL-10) cytokines is critical in determining the net immune status. A sustained high IL-6/IL-10 ratio is often implicated in the pathogenesis of chronic inflammatory diseases [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. While not statistically significant in the provided data, the slight trend towards a higher IL-6/IL-10 ratio in ZD, ZD4, and ZD40 (more pro-inflammatory) versus a lower ratio in ZDZn and HG40 is suggestive. ZnSO₄ more effective at tipping the balance away from a pro-inflammatory state in deficient animals compared to ZCP. For ZCP supplementation, in healthy animals (HG40) promote a relatively more anti-inflammatory balance, primarily driven by its IL-10 elevating and IFN-γ suppressing effects, despite slightly raising IL-6. In essence, these trends suggest a differential role for ZnSO₄ and ZCP depending on the physiological state (deficient vs. healthy) of the animals. ZnSO₄ better for correcting deficiency-induced inflammation, while ZCP more subtle, immunomodulatory benefit in healthy states, potentially by promoting a regulatory (IL-10) and dampening an effector (IFN-γ) immune response. It can potentially be attributed to the slower zinc release kinetics of ZCP, which may offer preferential support to Treg function in contrast to the rapid correction of pro-inflammatory signalling. These are critical distinctions for understanding the potential therapeutic applications of each zinc form [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan additionalcitationids=\"CR78\" citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eZD is often reported to increase TNF-α (as a primary pro-inflammatory cytokine), this result is somewhat unexpected. According to previous studies, the specific model of ZD used (duration, severity) did not reach the threshold for systemic TNF-α elevation. TNF-α responses are more localized to specific tissues rather than being reflected systemically at the measured time point. Other cytokines (IL-6, IFN-γ) are the predominant drivers of inflammation in this particular context. The observed IL-10 elevation effectively dampened potential TNF-α increases [\u003cspan additionalcitationids=\"CR81 CR82\" citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eZinc deficiency significantly increased IFN-γ (approx. 440 pg/mL in ZD vs. 380 pg/mL in HG, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Strikingly, all treatments (ZD4, ZD40, ZDZn, HG40) not only reversed this increase but suppressed IFN-γ to levels markedly below those of healthy controls (around 300\u0026ndash;330 pg/mL). IFN-γ is crucial for Th1-mediated immunity, vital for controlling intracellular pathogens. While its elevation in ZD suggests immune activation/dysregulation, the profound suppression by all zinc treatments below healthy baseline is a critical point [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. The phenomenon of suppression may be attributable to zinc's modulation of T-bet and STAT1 signalling. These are pivotal for Th1 cell differentiation and IFN-γ production. Zinc overcorrection of these pathways, particularly with ZCP, may impair Th1 responses. This could be beneficial in Th1-dominant autoimmune or inflammatory conditions [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. However, it raises concerns about potentially impairing necessary Th1 responses and increasing susceptibility to certain infections or hampering anti-tumor immunity [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. Literature indicates zinc is essential for IFN-γ production, but excessive zinc or particular supplementation forms might lead to over-suppression. The extent of IFN-γ suppression observed here suggests a powerful modulatory effect of the administered zinc forms, perhaps impacting transcription factors like T-bet (for Th1 differentiation) or STATs involved in IFN-γ signalling [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe study findings collectively demonstrate that ZD induces a complex inflammatory state characterized by elevated IL-6, IFN-γ, and a possibly compensatory rise in IL-10, while TNF-α remains stable. Zinc supplementation, regardless of the form (ZCP or ZnSO₄), appears to exert potent immunomodulatory effects that go beyond simple repletion, most notably the profound suppression of IFN-γ below healthy levels. The differential impact of ZCP and ZnSO₄ on IL-6 levels further highlights that the choice of zinc supplement can lead to distinct immunological outcomes. Future studies could incorporate flow cytometry analysis to confirm changes in immune cell populations, such as Th1, Treg, and macrophages, thereby providing deeper insights into these cellular mechanisms.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Zinc deficiency leads to a dysregulated cytokine network\u003c/h2\u003e\u003cp\u003eThe shifts in cytokine correlations between your zinc-deficient (ZD) rat pups and those receiving zinc supplementation (ZD4 model) provide critical insights into how zinc modulates the immune system inflammatory response. In both ZD and ZD4 groups, IL-10 (a key anti-inflammatory cytokine) showed strong to moderate negative correlations with the major pro-inflammatory cytokines IL-6 and TNF-α. IL-10 is critical for limiting host immune responses to pathogens and preventing excessive tissue damage by inhibiting the production of pro-inflammatory cytokines like TNF-α and IL-6 by macrophages and other cells [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIL-6 and TNF-α are pivotal pro-inflammatory cytokines often produced in concert during an inflammatory response, driven by common signalling pathways such as NF-ĸB. Their coordinated action is essential for effective pathogen clearance and initiation of adaptive immunity, but dysregulation contributes to chronic inflammatory diseases. In the ZD group, the weak negative (or essentially uncoordinated) relationship between IL-6 and TNF-α a might reflect a dysregulated and inefficient inflammatory signalling cascade characteristic of ZD.\u003c/p\u003e\u003cp\u003eThe inflammatory response may be chronic and disorganized. The shift to a positive correlation in the ZD4 group suggests that zinc supplementation helps restore a more coordinated and potentially more effective pro-inflammatory response. This could mean that when inflammation is required, these cytokines are produced in a more synchronized and controlled manner, potentially leading to more efficient resolution rather than chronic, low-grade inflammation [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e, \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn the ZD group, the weak negative correlation between IFN-γ and IL-10 could indicate a generally suppressed or imbalanced Th1/Treg axis. With impaired Th1 function due to zinc lack, any IFN-γ production might occur in an environment where IL-10 is also variably present, leading to a slight inverse trend as part of overall immune dysregulation. The shift to a weak positive IFN-γ /IL-10 correlation in the ZD4 group is intriguing. It doesn't suggest strong synergy but might imply a more balanced immune state where both cell-mediated immunity (potentially enhanced IFN-γ capacity due to zinc repletion) and its regulation (IL-10) can coexist and be modulated more appropriately. For instance, an appropriate immune response involves an initial IFN-γ surge followed by IL-10 to control it. This weak positive correlation might capture moments of this dynamic balance across different animals. Zinc supplementation has been shown to restore Th1/Th2 balance, which involves IFN-γ and IL-10 [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e, \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eZD leads to a dysregulated cytokine network. While anti-inflammatory mechanisms (IL-10) are active, their interplay with pro-inflammatory cytokines (IL-6, TNF-α) and other immune modulators (IFN-γ) appears disorganized. This aligns with human and animal studies showing ZD is associated with increased susceptibility to infections, chronic inflammation, and impaired immune cell function [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e, \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e]. The lack of positive coordination between IL-6 and TNF-α in ZD rats might reflect an inefficient and prolonged inflammatory state [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eZinc supplementation (therapy) appears to restore a more coordinated and balanced cytokine network. This is evident in the strengthened IL-10 regulatory axis, the establishment of a positive correlation between key pro-inflammatory cytokines (suggesting more controlled responses when needed), and subtle shifts in IFN-γ interactions indicative of better immune homeostasis. Effective zinc therapy aims to normalize immune cell function, leading to appropriate cytokine production and better control over inflammatory processes [\u003cspan additionalcitationids=\"CR94\" citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e4.4 Zinc deficiency leads to a dysbiosis gut microbiome and a disorganized systemic inflammatory response\u003c/h2\u003e\u003cp\u003eThe interplay between gut microbiota and host immunity is a critical determinant of health, particularly under conditions of nutritional stress such as ZD. Our findings reveal a significant modulation of both the gut microbial composition and the systemic cytokine network in a zinc-deficient rat model following zinc supplementation (ZD4 group). In the zinc-deficient (ZD) state, the gut microbiome was primarily dominated by species such as \u003cem\u003eLigilactobacillus murinus\u003c/em\u003e, \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e, and \u003cem\u003eLactobacillus hominis\u003c/em\u003e. This microbial profile was associated with a distinct cytokine correlation pattern characterized by strong negative correlations between the anti-inflammatory cytokine IL-10 and the pro-inflammatory cytokines IL-6 (r = -0.49) and TNF-α (r = -0.52), alongside weak, largely uncoordinated relationships between the pro-inflammatory markers IL-6 and TNF-α (r = -0.12). This suggests an immune environment under ZD where regulatory mechanisms (IL-10) are active but struggle against a backdrop of potentially disorganized inflammation. Dysbiosis increases gut permeability and allows bacterial components like lipopolysaccharides (LPS) to activate Toll-like receptor 4 (TLR4) on immune cells. This triggers the production of IL-6 and other pro-inflammatory cytokines.\u003c/p\u003e\u003cp\u003eUpon zinc supplementation (ZD4 model), a marked shift occurred in both microbial and immune landscapes. The microbiota transitioned towards a community dominated by \u003cem\u003eRomboutsia timonensis\u003c/em\u003e, \u003cem\u003eRomboutsia ilealis\u003c/em\u003e, and \u003cem\u003eLactobacillus johnsonii\u003c/em\u003e. Concurrently, the cytokine network demonstrated evidence of enhanced coordination and regulation. Notably, the correlation between IL-6 and TNF-α shifted to moderate positive (r\u0026thinsp;=\u0026thinsp;0.40), suggesting a more synchronized pro-inflammatory response. Furthermore, the negative correlation between IL-6 and IL-10 intensified (r = -0.63), indicating a more robust anti-inflammatory control loop. Additionally, the relationship between IL-10 and IFN-γ inverted from weak negative (r = -0.23) to weak positive (r\u0026thinsp;=\u0026thinsp;0.23), potentially reflecting a rebalancing of Th1-type and regulatory responses. Short-chain fatty acids (SCFAs) like butyrate are produced by Lactobacillus johnsonii and Romboutsia species and may contribute to immune modulation. SCFAs bind to G protein-coupled receptor 43 (GPR43) on immune cells, promoting IL-10 production by regulatory T cells and inhibiting pro-inflammatory cytokine expression via histone deacetylase (HDAC) inhibition [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Zinc supplementation strengthens gut barrier integrity, reducing LPS translocation and subsequent TLR4 activation, thereby limiting systemic inflammation [\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThese parallel shifts strongly suggest a functional link between the gut microbiota and host immune regulation, mediated by zinc status. The emergence of \u003cem\u003eLactobacillus johnsonii\u003c/em\u003e, a species with documented immunomodulatory properties including the capacity to promote IL-10 production, likely contributes to the enhanced regulatory control observed in the ZD4 group. Similarly, the significant increase in \u003cem\u003eRomboutsia\u003c/em\u003e species, members of the \u003cem\u003eClostridiales\u003c/em\u003e order, may indicate an altered production of short-chain fatty acids (SCFAs). SCFAs, particularly butyrate, are known to exert potent immunomodulatory effects, including the promotion of regulatory T cells and IL-10, which aligns with the observed cytokine changes. The restoration of gut barrier function by zinc is hypothesised to reduce dysbiosis-induced TLR signalling, thereby supporting a balanced immune response[\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Future studies could confirm the role of microbial metabolites in modulating the cytokine network.\u003c/p\u003e\u003cp\u003eThese findings corroborate previous studies demonstrating that zinc is essential for both maintaining a healthy gut microbiome and ensuring balanced immune function. Zinc supplementation appears to not only directly support immune cell activity, improving cytokine signaling and regulation (e.g., via NF-ĸB modulation), but also indirectly reshape the gut microbial community towards a profile more conducive to immune homeostasis. Thus, the correction of ZD facilitates a transition from a dysregulated, pro-inflammatory state towards a more coordinated and effectively regulated immune environment, highlighting the therapeutic potential of zinc in modulating the gut-immune axis.\u003c/p\u003e\u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe impact of zinc deficiency (ZD) in rat offspring on the gut-immune axis is significant, with the resultant consequences including gut microbiota dysbiosis and a disorganized cytokine network. The hallmark of this condition is the presence of elevated levels of pro-inflammatory cytokines, such as IL-6 and IFN-γ, accompanied by a compensatory rise in the anti-inflammatory cytokine IL-10. The correlation between pro-inflammatory markers is weak and uncoordinated in ZD rats. The supplementation of zinc, in the form of zinc sulphate (ZnSO₄) or zinc-chelating peptides (ZCP), has been demonstrated to exert an influence on both the composition of the gut microbiome and the immune response. ZnSO₄ therapy has been demonstrated to result in the most significant restoration of microbial diversity and the most effective reduction of IL-6 levels in comparison with ZCP. This finding indicates that ZnSO₄, as a readily ionizable zinc source, may be a more effective means of rapidly correcting the inflammatory state by targeting intracellular pathways such as NF-κB inhibition. Conversely, ZCP therapy, while less effective at reducing IL-6, has been shown to initiate a significant shift in the gut microbial community towards species like \u003cem\u003eRomboutsia\u003c/em\u003e and \u003cem\u003eLactobacillus johnsonii\u003c/em\u003e. This microbial change has been shown to be associated with a restructuring of the cytokine network, leading to a more coordinated pro-inflammatory response and a strengthened IL-10 regulatory axis. The shift in microbial profile may contribute to immune regulation through the production of short-chain fatty acids (SCFAs). In essence, ZnSO₄ is effective in rapidly reducing inflammation in acute ZD, while ZCP more effective in the long-term modulation of the gut-immune axis and the promotion of a balanced, well-regulated immune state. The selection of supplement is contingent upon the therapeutic objectives: the expeditious management of inflammation with ZnSO₄, while the promotion of long-term immune homeostasis through gut microbiome support with ZCP.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis research was supported the Vaccine and Drug Research Program of the BRIN Health Research Organization. Grant number 134/BR/VIII/2024\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThe conceptualisation of the study was a collaborative effort between G. Syahputra, F. Shidiq, and Y. Maladan. The methodology was designed by G. Syahputra, N. Gustini, Y. Hapsari and Y. Maladan, who also performed the experiments. The responsibility for the analysis and visualisation of the data was undertaken by F. Shidiq, N. Gustini, W. Susmayanti, and A. Rosyidah. The initial manuscript was drafted by G. Syahputra. A. Amanah, S. Hidayani, Y. Maladan and F. Shidqi were responsible for the review and editing of the manuscript. All authors contributed to the interpretation of results, provided critical feedback, and approved the final version of the manuscript for submission.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis research was supported by the BRIN Genomics Laboratory through its sequencing facilities and funded by the Vaccine and Drug Research Program of the BRIN Health Research Organization.Conflicts of Interest: The authors declare no conflict of interest.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYu X, Chen W, Wei Z, et al. Effects of maternal mild zinc deficiency and different ways of zinc supplementation for offspring on learning and memory. Food Nutr Res [Internet]. 2016 [cited 2025 Mar 29];60.\u003c/li\u003e\n\u003cli\u003eFosmire GJ, Sandstead HH. 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Front Immunol. 2020;11.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"immunity, infection, microbes, sea cucumber, supplementation","lastPublishedDoi":"10.21203/rs.3.rs-7478928/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7478928/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eZinc is essential for skeletal development and maintaining immune homeostasis, with zinc deficiency (ZD) leading to compromised immunity and heightened infection risk. This study explores the potential of Zinc-chelating Peptides (ZCP), a novel third-generation zinc supplement sourced from \u003cem\u003eHolothuria scabra\u003c/em\u003e, to ameliorate ZD-induced immune dysregulation and gut dysbiosis in rat offspring. Through a metagenomic approach, we demonstrate that ZD markedly increases pro-inflammatory cytokines (IL-6, IL-10, IFN-γ) and disrupts gut microbial diversity. ZCP treatment effectively modulated these cytokine profiles, with ZnSO\u003csub\u003e4\u003c/sub\u003e supplementation (ZDZn group) leading to the greatest restoration of microbial diversity. These results underscore the critical role of the gut microbiome in mediating zinc's immunomodulatory effects and suggest that ZCP holds promise for restoring both gut health and immune balance in ZD states, necessitating further exploration of its underlying mechanisms and long-term implications.\u003c/p\u003e","manuscriptTitle":"Evaluation of Zn-Peptides from Holothuria scabra: Gut Microbiota Profile and Cytokine Signalling Pathways in Rat Pups Born to Zinc-Deficient Parent","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-03 11:11:11","doi":"10.21203/rs.3.rs-7478928/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"33578b51-03ad-4792-8c9e-06701b4dc0ad","owner":[],"postedDate":"September 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-22T21:38:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-03 11:11:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7478928","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7478928","identity":"rs-7478928","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}