Revista Cienﬁca, FCV-LUZ / Vol. XXXV Received: 23/12/2025 Accepted: 23/02/2026 Published: 28/03/2026 hps://doi.org/10.52973/rcfcv-e35650 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 8 Revista Cienﬁca, FCV-LUZ / Vol. XXXVI hps://doi.org/10.52973/rcfcv-e362883 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Comparave evaluaon of bio-protecve eﬀects of alive and dead (heat- inacvated) Lacplanbacillus plantarum strains against Cadmium - induced toxicity in wistar rats Evaluación comparava de los efectos bioprotectores de cepas vivas y muertas (inacvadas por calor) de Lacplanbacillus plantarum frente a la toxicidad inducida por Cadmio en ratas Wistar Tevhide Elif Güner 1,* , Hasan Susar 2 , İhsan Kisadere 3 ¹ Balıkesir University, Faculty of Veterinary Medicine, Department Food Hygiene and Technology. Balıkesir, Türkiye ² Balıkesir University, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology. Balıkesir, Türkiye ³ Balıkesir University, Faculty of Veterinary Medicine, Department of Physiology.Balıkesir, Türkiye. *Corresponding author: elif.guner@balikesir.edu.tr ABSTRACT This study was conducted to evaluate the bio - protecve potenal of alive and dead (heat - inacvated) Lacplanbacillus plantarum strains against cadmium toxicity in Wistar rats. The aim was to determine whether alive and heat - inacvated Lacplanbacillus plantarum strains could alleviate cadmium - induced inﬂammatory and oxidave stress responses and reduce serum cadmium levels in rats. The study included forty-eight male albino Wistar rats, which were distributed equally among six groups: control (n = 8), cadmium (n = 8), alive Lacplanbacillus plantarum (n = 8), dead Lacplanbacillus plantarum (n = 8), cadmium + alive Lacplanbacillus plantarum, and cadmium + dead Lacplanbacillus plantarum. Rats in the cadmium, cadmium + alive Lacplanbacillus plantarum, and cadmium + dead Lacplanbacillus plantarum groups received cadmium chloride (2 mg / kg) orally by gastric gavage three mes a week for 4 weeks. Alive and dead Lacplanbacillus plantarum (10 9 – 10 10 cfu / mL) suspensions were administered to alive Lacplanbacillus plantarum, dead Lacplanbacillus plantarum, cadmium + alive Lacplanbacillus plantarum, and cadmium + dead Lacplanbacillus plantarum groups with the same frequency and duraon. The control group received physiological saline. Aﬅer experimental compleon, decapitaon was performed, and serum samples were collected for cadmium concentraons, cytokines (TNF - α, IL - 6, IL - 10, IL - 1β), and anoxidant (Malondialdehyde, superoxide dismutase, glutathione peroxidase, catalase) levels. cadmium administraon caused a signiﬁcant increase in serum TNF - α and IL - 6 levels but decreased IL - 1β levels (P < 0.05). Superoxide dismutase and glutathione peroxidase levels were reduced, but Malondialdehyde concentraons elevated in only the cadmium - exposed group (P < 0.05). Lacplanbacillus plantarum administraons alleviated the inﬂammatory response by providing a signiﬁcant decrease in IL - 6 levels, especially in the heat - inacvated form (cadmium + dead Lacplanbacillus plantarum) (P < 0.05). Both alive and dead forms showed paral recovery trends in superoxide dismutase, glutathione peroxidase, and catalase. Lacplanbacillus plantarum exhibited parally an-inﬂammatory, anoxidant, and metal-binding properes against cadmium toxicity in both forms and presented a usable bioprotecve model in terms of food safety and veterinary toxicology when applied with a milk matrix. Key words: Cadmium, inﬂammaon, L. plantarum, oxidave stress, paraprobioc RESUMEN Este estudio se llevó a cabo para evaluar el potencial bioprotector de cepas vivas y muertas (inacvadas por calor) de Lacplanbacillus plantarumfrente a la toxicidad por cadmio en ratas Wistar. El objevo fue determinar si las cepas vivas y termoinacvadas de Lacplanbacillus plantarum podían atenuar las respuestas inﬂamatorias y de estrés oxidavo inducidas por cadmio y reducir los niveles séricos de cadmio en las ratas. Cuarenta y ocho ratas macho albinas Wistar se dividieron en seis grupos iguales : control (n = 8), cadmio (n = 8), Lacplanbacillus plantarum viva (n = 8), Lacplanbacillus plantarum muerta (n = 8), cadmio + Lacplanbacillus plantarum viva y cadmio + Lacplanbacillus plantarum muerta. Las ratas de los grupos cadmio, cadmio + Lacplanbacillus plantarum viva y cadmio + Lacplanbacillus plantarum muerta recibieron cloruro de cadmio (2 mg / kg) por vía oral mediante gavage gástrico, tres veces por semana durante 4 semanas. Las suspensiones de Lacplanbacillus plantarum viva y muerta (10 9 – 10 10 UFC / mL) se administraron a los grupos Lacplanbacillus plantarum viva, Lacplanbacillus plantarum muerta, cadmio + Lacplanbacillus plantarum viva y cadmio + Lacplanbacillus plantarum muerta con la misma frecuencia y duración. El grupo control recibió solución salina ﬁsiológica. Posteriormente, los animales fueron decapitados y se recolectaron muestras de suero para la determinación de las concentraciones séricas de cadmio, citocinas (TNF - α, IL - 6, IL - 10, IL - 1β) y niveles de anoxidantes (malondialdehído, superóxido dismutasa, glutaon peroxidasa, catalasa). La administración de cadmio provocó un aumento signiﬁcavo de los niveles séricos de TNF - α e IL - 6, pero una disminución de los niveles de IL - 1β (P < 0,05). Los niveles de superóxido dismutasa y glutaon peroxidasa se redujeron, mientras que las concentraciones de malondialdehído se elevaron únicamente en el grupo expuesto a cadmio (P < 0,05). Las administraciones de Lacplanbacillus plantarum atenuaron la respuesta inﬂamatoria al provocar una disminución signiﬁcava de los niveles de IL - 6, especialmente en su forma inacvada por calor (cadmio + Lacplanbacillus plantarum muerta) (P < 0,05). Tanto las formas vivas como las muertas mostraron tendencias de recuperación parcial en los niveles de superóxido dismutasa, glutaon peroxidasa y catalasa. Lacplanbacillus plantarum presentó propiedades parcialmente aninﬂamatorias, anoxidantes y de unión a metales frente a la toxicidad por cadmio en ambas formas, y mostró un modelo bioprotector ulizable en términos de seguridad alimentaria y toxicología veterinaria cuando se aplicó junto con una matriz láctea. Palabras clave: Cadmio, inﬂamación, L. plantarum, estrés oxidavo, paraprobióco

L. plantarum Protecon in Cd Exposure / Güner et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Cadmium (Cd) is an environmentally prevalent metal that exerts pronounced toxic eﬀects on biological systems even at relavely low exposure levels. It enters the ecosystem through industrial processes, phosphate ferlizers, plasc manufacturing, baeries, and wastewater. Due to its lengthy biological half - life, Cd accumulates parcularly in the liver, kidneys, lungs, brain, and cardiovascular system and leads to deterious eﬀects [1 , 2] Environmental or food - borne exposure to Cd results in disrupon of the redox balance, oxidave stress, and triggering of the inﬂammatory response in the organism. Cd ions bind to sulydryl groups and inhibit the acon of anoxidant enzymes such as glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD). This disturbance promotes oxidave stress, manifested by increased lipid peroxidaon (LPO), genomic damage, and intracellular accumulaon of reacve oxygen species (ROS) [3 , 4]. In addion to oxidave stress, Cd is also known to increase the synthesis of proinﬂammatory cytokines such as tumor necrosis factor alpha (TNF - α), interleukin - 6 (IL - 6), and IL - 1beta (β) by acvang the nuclear factor kappa – light – chain - enhancer of acvated B cells (NF - κB) signaling pathway. These mechanisms trigger systemic inflammation, leading to disruption of tissue integrity and loss of metabolic homeostasis [5 , 6]. Transmission of Cd to humans and animals through the food chain is one of the most signiﬁcant aspects of exposure. It accumulates in soil and water, then enters the food chain through plant products, feeds, and therefore animal foods. In terms of food safety and environmental toxicology, this condion presents a major public health concern [7]. For this reason, interest in biological detox techniques that will reduce the bioavailability of Cd and facilitate its eliminaon from the body is increasing day by day (d). Certain probioc bacteria, parcularly Lacplanbacillus plantarum (Lp), have been shown in recent studies to have a protecve eﬀect against heavy metal poisoning [8 ,9]. It has been suggested that the protecve eﬀect of Lp is mulfaceted. The cell surface carboxyl and phosphate groups of Lp bind Cd²⁺ ions and restrict the metal’s absorpon from the gastrointesnal system by biosorpon [10 , 11]. Furthermore, the producon of short - chain fay acids and anoxidant metabolites supports epithelial barrier integrity by lowering intesnal pH and reduces the transfer of toxic ions into the circulaon. These mechanisms suggest that Lp funcons as an anoxidant and immunomodulatory bioprotecve agent not only at the local intesnal level but also at the systemic level [12 , 13]. Lacplanbacillus plantarum is a probioc microorganism that is commonly found in fermented products of plant origin and can also adapt to a milk environment and is therefore frequently used in funconal food formulaons. Probiocs play a crucial role in bridging the gap between veterinary toxicology and food safety by maintaining their viability in biological environments such as milk and enhancing their bioavailability against toxic chemicals [14 , 15]. In previous research, no comprehensive study was found to comparavely evaluate the live and heat-inacvated (paraprobioc) forms of Lp under heavy metal toxicity condions and to reveal their bioprotecve mechanisms at the cellular level in a holisc manner. Addionally, Zhai et al. [16] invesgated the short-term protecve eﬀects of heat-inacvated and live Lp CCFM8610 against acute Cd poisoning; however, the study had limited parameters and a 48 - hour experimental duraon. The present study aimed to overcome this limitaon and evaluate the eﬀects of alive and inacve forms of Lp on some serum anoxidant and inﬂammatory (cytokine) parameters in a holisc manner under longer - term Cd exposure. Thus, probiocs’ paraprobioc potenal, which does not depend on viability, is being examined from a fresh angle in the context of toxicity and food safety. The study’s novel experimental model ﬁlls a major gap in the literature. The informaon gathered is expected to oﬀer a scienﬁc foundaon for lowering the risks of foodborne illness and creang probioc - based bioprotecve products. MATERIAL AND METHODS Ethics statement This research was conducted with the approval of the Balıkesir University Animal Experimentaon Local Ethics Commiee (Ethics Commiee Approval No : 2024 / 11 - 10). All experimental procedures were conducted in accordance with the “European Council Direcve 2010 / 63 / EU on the Welfare and Experimental Use of Animals” and the “Balıkesir University Experimental Animal Ethics Commiee Working Procedures and Principles.” Throughout the experiment, unnecessary stress was avoided in the animals, the number of animals used was minimized, and the 3R (Replacement, Reducon, Reﬁnement) principles were observed. Animal material In this study, 48 three – week - old male Wistar albino rats (Raus norvegecis) with an average body weight (Kern, EW 620-3NM, Germany) of 200 ± 30 g were used. Male Wistar rats were supplied by the Experimental Animal Producon, Care, Applicaon, and Research Center of Balıkesir University. Following acclimazaon, the animals were allocated into six groups of equal size using a randomizaon procedure. Throughout the experimental period, rats were maintained in plasc cages under controlled environmental condions with a 12 hours (h) light/12 h dark cycle. The room temperature and a relave humidity were 23 ± 2 °C, 50 ± 10 %, respecvely. Standard rat chow and fresh water were provided to the animals ad libitum during the study. Experimental groups Cadmium Group (Cd) : Cadmium chloride (CdCl₂) was administered by oral gavage at a dose of 2 mg/kg, three mes per week for four weeks [17]. Alive Lp Group (ALP) : Suspension containing approximately 10 9 – 10 10 cfu / mL of live bacteria was given by oral gavage three mes per week for four weeks [18]. Cd + ALP Group : CdCl 2 (2 mg / kg) and 10 9 – 10 10 cfu / mL live Lp suspension were administered by oral gavage three mes per week for four weeks [17 , 18]. 2 of 8

Revista Cienﬁca, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Dead Lp Group (DLP) : Approximately 10 9 – 10 10 cfu / mL of heat - inacvated bacterial suspension was applied (by oral gavage) three mes a week for four weeks [18]. Cd + DLP Group : CdCl 2 (2 mg / kg) and 10 9 – 10 10 cfu / mL of inacvated Lp suspension were administered orally (by gavage) three mes per week for four weeks [17 , 18]. Control Group (C) : Physiological saline was administered orally via gavage for the same periods of me as stated above. Analysis of cadmium - resistant strain Four Lp strains were screened for Cd tolerance by assessing minimum inhibitory and lethal concentraon thresholds (MIC and MLC), and the strain exhibing the highest resistance was chosen for subsequent experiments. Each strain was grown in de Man, Rogosa, and Sharpe (MRS) broth containing diﬀerent Cd concentraons with some adaptaons to the method of Zhai et al. [19]. In this study, Cd concentraons ranging from 0.1 to 200 mg / L were used, and all required concentraons were prepared by making appropriate diluons from a 1.000 mg / L CdCl 2 stock soluon. Lp strains inoculated into each tube were incubated (Memmert, model E412.0361, Schwabach, Germany) under microaerophilic condions at 37 °C for 24 – 48 hours (h). Bacterial growth was monitored by measuring the opcal density (OD 600 ) at 600 nm and by a visual assessment of turbidity (SPECTROstar Nano, BMG LABTECH GmbH, Ortenberg, Germany). MIC was determined as the lowest concentraon of cadmium that inhibited observable bacterial growth, while MLC was idenﬁed as the lowest concentraon resulng in the absence of growth aﬅer transfer to fresh, Cd-free MRS agar plates. Each experiment was independently repeated three mes. At the end of the incubaon period, the Lp Biofen (DSMZ 16627) strain, which was able to grow at the highest Cd concentraon, was selected as the most Cd - resistant strain for use in the rest of the study. Preparaon of bacterial suspensions The selected Lp strain was culvated in 10 mL of de MRS broth (Merck, Darmstadt, Germany) and incubated at 30 °C for 18 – 20 h in a temperature-controlled incubator (Memmert, model E412.0361, Schwabach, Germany). At the end of the incubaon, the cultures were centrifuged (Universal 320 R, Hech Zentrifugen, Germany) at 4200 × G for 5 minutes (min) in a centrifuge cooled to 4 °C. After removing the supernatants, the bacterial pellets were resuspended in 10 mL of sterile skim milk powder soluon (Merck, 115363, Darmstadt, Germany). The ﬁnal bacterial suspension was prepared to contain approximately 10 9 – 10 10 cfu/mL, as described previously with minor modiﬁcaons [8 , 18]. Inacvaon process of Lp Bacterial suspensions were heat-inacvated by autoclaving (Hirayama, Hiclave HV85, Japan) at 121 °C for 15 min, following previously described procedures for paraprobioc preparaons [14 , 20]. Loss of viability was conﬁrmed by the absence of growth aﬅer incubaon on MRS agar. Detecng of serum cadmium concentraons The wet ash digeson process was used to mineralize whole blood samples at atmospheric pressure. Each sample’s 1 mL of blood was combined with 5 mL of concentrated nitric acid (HNO 3 ) and 2 mL of hydrogen peroxide (H 2 O 2 ), then heated to 90 °C. Until a definitive solution was found, the procedure was repeated. After, the solutions were put into volumetric flasks and filled with distilled water to a volume of 10 mL. Prior to analysis, prepared samples were kept at 4 °C in a refrigerator (Siemens, model KD56NNW22N, Germany). Cd concentraons were determined using inducvely coupled plasma–opcal emission spectrometry (ICP-OES; Opma 7300, PerkinElmer, USA) following the method of Saglam et al. [21]. Detecon of serum Malondialdehyde concentraons and some anoxidant levels Serum samples were analyzed using commercially obtained Enzyme-Linked Immunosorbent Assay (ELISA) kits, following the protocols provided by the manufacturer, and measurements were performed with an ELISA microplate reader (SPECTROstar Nano, BMG LABTECH GmbH, Ortenberg, Germany), the concentraons of Malondialdehyde (MDA) (BT Lab, E0156Ra, China), GPx values (BT Lab, E1172Ra, China), SOD (BT Lab, E1444Ra, China), and CAT (BT Lab, E0869Ra, China) enzyme acvies were determined [8]. Detecon of some serum cytokine levels Serum TNF - α [(Bioassay Technology Laboratory (BT Lab), E0764Ra, China)], IL – 1 - β [(BT Lab, E0119Ra, China)], IL - 6 [(BT Lab, E0135Ra, China)], and IL - 10 [(BT Lab, E0108Ra, China)] were determined by ELISA employing commercial kits, and absorbance readings were obtained using an ELISA reader (SPECTROstar Nano, BMG LABTECH GmbH, Ortenberg, Germany) [8]. Experimental workﬂow The overall experimental workﬂow, including Cd exposure, probioc administraon, sample collecon and analysis steps, is illustrated in FIG 1. Stascal analysis Stascal analyses were carried out using the Stascal Package for the Social Sciences soﬅware (SPSS version 25.0; SPSS Inc., Chicago, IL, USA). Mean ± standard error (SEM) is used to express the results. The Shapiro - Wilk test was used to evaluate data distribuon and homogeneity of variance. The Duncan mulple comparison test and one-way analysis of variance were used to invesgate group diﬀerences. Stascally signiﬁcant diﬀerences were deﬁned as P < 0.05. 3 of 8

L. plantarum Protecon in Cd Exposure / Güner et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico FIGURE 1. Experimental design and overview of the study. Cadmium (Cd) and Lacplan- bacillus plantarum (Lp) suspensions were prepared and administered to rats by oral gavage for 4 weeks. Experimental groups received Cd alone or Cd in combinaon with alive (ALP) or dead (DLP) L. plantarum. At the end of the experimental period, animals were anesthezed, blood samples were collected, serum was separated, and Cd concentraons, anoxidant parameters, and cytokine levels were analyzed. The bar graph illustrates changes in serum Cd concentraons following ALP and DLP treatments. Values are expressed as mean ± SEM. Diﬀerent leers (a - d)above bars indicate stascally signiﬁcant diﬀerences among groups (P < 0.05) RESULTS AND DISCUSSION Serum cadmium concentraons FIGURE 2. Serum cadmium (Cd) concentraons (µg/L) in the experimental groups aﬅer the 4-week treatment period. Control animals received no treatment. Dead Lp represents rats administered heat-inacvated Lacplanbacillus plantarum, while Alive Lp indicates rats receiving viable L. plantarum. Cd + Dead Lp and Cd + Alive Lp groups received cadmium in combinaon with dead or alive L. plantarum, respecvely. The Cd group received cadmium alone. Values are presented as mean ± 95 % conﬁdence interval (CI). Diﬀerent leers (a–d) above bars indicate stascally signiﬁcant diﬀerences among groups (P < 0.05) Serum Cd concentraon was found to be higher in the Cd group than in other experimental groups in the present study (P < 0.05) (FIG. 2). Besides, its concentraon was detected lower in the Cd + ALP and Cd + DLP (especially) groups compared to the Cd group in this study (P < 0.05). In addion, it was not determined any change among the groups (C, ALP, and DLP) regarding serum Cd levels (P > 0.05), which were likely increased only due to fress drinking water and free dietary intake. It was reported that chronic Cd exposure led to an increase in blood Cd concentraons and altered some anoxidant and cytokine levels in living beings [8]. In this study, Cd administraon increased the serum Cd concentraons in the Cd group when compared to the C group. These ﬁndings were similar to previous studies [8 , 22 , 23]. On the other hand, the Lp - administrated groups (Cd + ALP and Cd + DLP) had lower serum Cd concentraons than the Cd group in this invesgaon. The systemic bioavailability of Cd was considerably decreased by both treatments, while the death strain’s (DLP) decline was somewhat more than the alive strain’s (ALP). This result is consistent with studies showing that Lp inhibits metal absorpon by binding to Cd 2 ⁺ ions in the intesnal environment, thus reducing serum levels and systemic bioavailability via the gut-liver axis [8 ,9, 16 , 24 , 25 , 26]. Moreover, following heat treatment, the bacterial cell wall’s enhanced exposure of funconal groups, including phosphate, carboxyl, and teichoic acids, may have expanded the number of binding sites for Cd ions. Some of these locaons may have been masked by surface proteins and metabolic processes in living bacteria. As a result, the ion exchange sites on the surface of dead Lp would be more accessible compared to the living form, and their biosorpon capability might have risen [10]. Serum malondialdehyde concentraons and some anoxidant levels Cadmium treatment resulted in increased serum MDA concentraons relave to the C group (P < 0.05) (TABLE I). Alive or dead (heat - inacvated) Lp administraons could not ameliorate the MDA concentraons in the Cd + ALP and Cd + DLP groups when compared to the Cd group (P > 0.05). On the other hand, the serum MDA levels of the Cd + ALP and Cd + DLP (especially) groups parally decreased following Lp administraon; however, this decline was not stascally signiﬁcant (P > 0.05). Compared with the control group, the Cd group exhibited signiﬁcantly lower serum GPx levels (p < 0.05). Administraon of alive or dead (heat - inacvated) Lp administraons did not aﬀect the serum GPx levels in the Cd + ALP and Cd + DLP groups compared to the Cd group (P > 0.05). All administraons (ALP, DLP, and Cd) had no eﬀect on serum CAT enzyme acvies, although all of the experimental groups’ SOD enzyme acvies were lower than those of the C group (P < 0.05). On the other hand, the alive Lp strain administraon showed a tendency toward higher SOD and GPx acvity in the Cd + ALP group compared to the Cd group. In comparison to the acve strain (ALP), the mean GPx level seemed to be marginally greater in the dead strain administered group (Cd + DLP). In addion, the heat - inacvated (dead) strain showed a tendency toward higher CAT acvity compared to the Cd group (P > 0.05). 4 of 8

Revista Cienﬁca, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico TABLE I Malondialdehyde concentraons and some anoxidant levels in the experimental groups (mean ± SEM) Groups n MDA (mmol / mL) GPx (U / mL) SOD (ng / mL) CAT (ng / mL) Control 8 0.68 ± 0.09 b 1042.50 ± 27.17 a 22.56 ± 3.37 a 5.21 ± 0.89 n.s ALP 8 0.95 ± 0.16 b 940.62 ± 4.27 ab 12.65 ± 1.77 b 4.14 ± 1.29 n.s DLP 8 0.81 ± 0.17 b 988.12 ± 90.03 ab 14.19 ± 4.43 b 5.10 ± 0.85 n.s Cd 8 1.80 ± 0.27 a 892.50 ± 40.91 b 6.55 ± 2.45 b 2.98 ± 0.24 n.s Cd + ALP 8 1.60 ± 0.07 a 897.50 ± 15.08 b 6.64 ± 1.79 b 3.35 ± 0.31 n.s Cd + DLP 8 1.48 ± 0.02 a 906.25 ± 10.72 b 6.76 ± 0.43 b 4.08 ± 0.37 n.s MDA: Malondialdehyde; GPx: Glutathione peroxidase; SOD: Superoxide dismutase; CAT: Catalase; ALP: Alive Lacplanbacillus plantarum; DLP: Dead Lacplanbacillus plantarum; Cd: Cadmium; Cd + Alive Lp: Cadmium and Alive Lacplanbacillus plantarum; CD + DLP: Cadmium and Dead Lacplanbacillus plantarum. n: number of animals in each experimental group; mmol: milimol; mL: mililiter; U: Unit; ng: nanogram; The data is represented as the mean ± standard error; n.s: nonsigniﬁcant. *Diﬀerent leers (a – c) in the same column indicate a stascally signiﬁcant diﬀerence between groups (P < 0.05). Previously, many studies have demonstrated that Cd smulates the formaon of ROS and disrupts intracellular redox balance in living organisms [1 , 4]. In the present study, Cd administraon increased the serum MDA (the principal product of polyunsaturated fay acid peroxidaon) concentraons in the Cd group compared to the C group. The present results are in agreement with earlier reports [27 , 28 , 29]. In contrast, administraon of Lp was associated with a paral reducon in serum MDA concentraons in the Cd + ALP and parcularly the Cd + DLP groups; however, this decrease did not reach stascal signiﬁcance in the current study. These ﬁndings were compable with previous research [8 ,9]. Conversely, it was reported that neither Cd nor the other experimental groups (Bacillus coagulans, L. plantarum, and inulin) showed any discernible variaons in MDA concentraons throughout the course of 42 d in rats [30]. This discrepancy is thought to be due to signiﬁcant diﬀerences in the experimental condions of the two studies. The signiﬁcantly lower Cd dose (200 μg / rat / d) used by Jafarpour et al. [30] compared to the dose used in the present experiment, the administraon of probiocs in synbioc form (L. plantarum + inulin), the diﬀerent Lp strains used, and the longer meframe (21 and 42 d) for assessing oxidave stress parameters may have led to the diﬀerent observed MDA responses. Because these variables can directly aﬀect the severity of oxidave stress, the observaon that Cd administraon signiﬁcantly increased MDA levels, while this increase was parally limited in the Lp - treated groups, although not stascally signiﬁcant, can be explained by these methodological diﬀerences. It has been reported that the ability of defense enzymes (such as SOD, GPx, and CAT) to funcon is limited when Cd ions interact with cellular sulydryl groups and bind to the acve sites of anoxidant enzymes [31 , 32]. In the present study, exposure to cadmium resulted in reduced of GPx levels in the Cd group compared to the C group. Although serum CAT enzyme acvies were not aﬀected, serum SOD levels were reduced by Cd administraon in the Cd group compared to the C group in this study. Xue et al. [33] reported a reducon in serum CAT and GPx levels following cadmium exposure (CdCl 2 , 6 mg / kg, b.w.), whereas SOD acvity in serum remained unchanged in Sprague Dawley female rats. Conversely, it has been shown that cadmium exposure at a dose of 2.04 mg/mL administered orally for 28 days had no measurable eﬀect on serum GSH concentraons or SOD and CAT acvies in female rats [8]. These diﬀerences may have varied depending on diﬀerent doses or gender. On the other hand, the alive Lp strain administraon showed a nonsigniﬁcant tendency toward higher SOD and GPx acvity in the Cd + ALP group compared to the Cd group. In comparison to the acve strain (ALP), the mean SOD and GPx level seemed to be marginally greater (not meaningful) in the dead strain administared group (Cd + DLP). In addion, the heat-inacvated (dead) strain showed a tendency toward higher CAT acvity compared to the Cd group. This phenomenon could be due to funconal groups on the cell surface sll being able to bind Cd ions even aﬅer the cell is no longer viable. However, there was no discernible diﬀerence among the groups regarding serum CAT levels in rats. These results were in line with research in the literature that showed heat-inacvated Lp’s ability to bind Cd and how it regulated oxidave stress [16]. The administraon of Lp decreased the oxidave load brought on by exposure to Cd in both situaons. This paral recovery may be indicave of Cd accumulaon within the ssues, which keeps the pressure on anoxidant enzyme systems parally intact. In addion, these results demonstrate that Lp exerts a protecve eﬀect against Cd toxicity through both physicochemical (adsorpon) and biological (acvaon of anoxidant enzymes) mechanisms. Likewise, numerous probioc strains have been documented in the literature to migate oxidave stress resulng from heavy metal exposure and maintain anoxidant enzyme acvies [9 , 34]. Some serum cytokine levels In this invesgaon, serum TNF - α levels were shown to be greater in the Cd group than in the C group (P < 0.05) (TABLE II). Besides, IL - 6 levels were observed to be higher in the Cd group than in the C group (P < 0.05), although serum IL - 10 levels were unaﬀected by any administraon (P > 0.05). 5 of 8

L. plantarum Protecon in Cd Exposure / Güner et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico TABLE II Some serum cytokine levels in the diﬀerent experimental groups (mean ± SEM) Groups n TNF - α (ng / L) IL - 6 (ng / L) IL - 10 (pg / mL) IL - 1β (pg / mL) Control 8 2.25 ± 0.05 c 0.85 ± 0.01 c 158.41 ± 52.31 n.s 32.96 ± 9.45 a ALP 8 2.32 ± 0.16 bc 1.08 ± 0.06 bc 117.56 ± 15.47 n.s 14.97 ± 2.54 b DLP 8 2.18 ± 0.16 c 1.19 ± 0.10 bc 96.46 ± 11.25 n.s 19.91 ± 8.60 ab Cd 8 2.69 ± 0.11 a 1.76 ± 0.30 a 82.98 ± 2.10 n.s 3.28 ± 0.59 b Cd + ALP 8 2.63 ± 0.11 ab 1.34 ± 0.11 ab 89.79 ± 8.84 n.s 8.98 ± 1.27 b Cd + DLP 8 2.42 ± 0.07 ab 1.14 ± 0.13 bc 88.17 ± 12.42 n.s 4.23 ± 2.08 b TNF – α: Tumor necrosis factor alpha; IL – 6: Interleukin - 6; IL – 10: Interleukin – 10 ; IL - 1β: Interleukin - 1 beta ; ALP: Alive Lacplanbacillus plantarum; DLP: Dead Lacplanbacillus plantarum; Cd: Cadmium; Cd + Alive Lp: Cadmium and Alive Lacplanbacillus plantarum; CD + DLP: Cadmium and Dead Lacplanbacillus plantarum. n: number of animals in each experimental group; ng : nanogram; L: Liter; pg: pictogram; mL: mililiter; The data is represented as the mean ± standard error; n.s: nonsigniﬁcant. *Diﬀerent leers (a – c) in the same column indicate a stascally signiﬁcant diﬀerence between groups (P < 0.05) An important decrease in IL - 6 levels was observed only in the heat-inacvated strain (Cd + DLP) (P < 0.05), while there was a decreasing trend in the alive strain (Cd + ALP) (P > 0.05) in the present study. Serum IL - 1ß levels were detected to be lower in the Cd, ALP, Cd + ALP, and also Cd + DLP groups compared to the C group (P < 0.05), interesngly. However, serum IL-1β levels in the Cd + ALP group showed a paral restoraon when compared with the Cd-only group. Cadmium is a potent pro - oxidant and inﬂammatory agent due to its tendency to accumulate in living ssues, long half - life, and capacity to disrupt redox balance. Prior research indicates that Cd - induced oxidave stress acvates the NF - κB complex and promotes the transcription of proinflammatory mediators such as TNF - α, IL - 6, IL - 1β, and cyclooxygenase - 2 [5 , 6]. Consistent with this mechanism, Cd exposure was associated with higher serum levels of TNF-α and IL-6, whereas IL-10 concentraons showed a non-signiﬁcant downward trend. According to Choudhury et al. [35], the immune response switches to a proinﬂammatory axis when IL - 10 levels fall, prevenng the resoluon of inﬂammaon. On the other hand, it was reported that Cd administraon did not cause any signiﬁcant changes regarding serum TNF - α and IL - 1β levels in female rats [8]. In a previous study, it was also suggested that Cd administered orally at a dose of 2 mg/kg over a 4-week period failed to signiﬁcantly aﬀect serum IL-1β, but led to increased TNF-α levels in male Wistar rats [36]. In agreement with previous ﬁndings, similar results were reported by Han et al. [37] in mice. Addionally, a decline in serum IL-1β levels was observed following Cd administraon. According to previous studies, the producon of this cytokine is suppressed at high or prolonged exposure and increased at low levels [36 , 38]. This could be explained by Cd blocking the caspase - 1 enzyme, which stops IL - 1β from maturing. Lp administraon resulted in a paral improvement in cytokine proﬁles in the Cd + ALP and Cd + DLP groups compared to the Cd group. An important decrease in IL - 6 levels was observed only in the heat-inacvated strain (Cd + DLP), while a decreasing trend was observed in the alive strain (Cd + ALP). Furthermore, no signiﬁcant diﬀerence was found among the groups (Cd, Cd + ALP, and Cd + DLP) regarding TNF - α levels. However, a partial recovery was observed in the Cd + ALP group’s serum IL - 1β levels compared to the Cd group. Despite Cd exposure, the administraon of alive Lp resulted in a nonsigniﬁcant paral decrease in TNF - α and IL - 6 levels, a nonsigniﬁcantly improvement in IL - 1β, and a not meaningful recovery in IL - 10. These ﬁndings demonstrate that Lp has a limited immunomodulatory capacity, and it can regulate cytokine balance. Similarly, it has been reported in the literature that this probioc suppresses the Cd - induced inﬂammatory response and improves the cytokine proﬁle [8 , 39 , 40]. The aenuaon of the systemic inﬂammatory response associated with Cd toxicity aﬅer Lp administraon suggests that this bacterium both directly interacts with immune cells and creates an indirect regulatory eﬀect through the intesnal barrier [9 ,[16]. CONCLUSIONS When serum Cd levels and anoxidant enzyme acvies of rats were evaluated, it was determined that DLP administraon was more eﬀecve than ALP administraon in eﬀecvely reducing serum Cd levels (as a metal binding agent), thereby a parally decreasing lipid peroxidaon and oxidave stress. Moreover, it was also observed that DLP administraon had a slightly beer eﬀect (nonsigniﬁcant) on serum pro-inﬂammatory cytokine acvity, while ALP had a paraly posive eﬀect (nonsigniﬁcant) on an-inﬂammatory cytokine acvity. These ﬁndings suggest that diﬀerent forms of probioc bacteria (live or heat - inacvated) may be eﬀecve as bioprotecve agents in reducing the bioavailability of foodborne contaminants and controlling oxidave stress and inﬂammaon. Furthermore, Lp, naturally occurring in plant - based fermented foods and adaptable to milk matrices, is thought to contribute to the development of innovave approaches for food safety and veterinary public health. In this context, probioc - containing dairy products can be considered funconal foods that help reduce systemic oxidave damage and inﬂammaon, parcularly due to toxic metals such as Cd. The applicability of Lp in food matrices oﬀers a praccal advantage for use in ﬁeld condions. Furthermore, the ability of paraprobioc forms to exhibit similar protecve eﬀects without requiring viability signiﬁcantly contributes to product stability and safety. This allows the use of both live and inacvated probiocs in funconal dairy products. 6 of 8

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