Comparison of novel native probiotics and paraprobiotics in modulating oxidative stress and inflammation in DSS-induced colitis: implications for enhanced therapeutic strategies in high fat diet

Abstract

Aim

IBD is a condition that may result from the presence of oxidative stress. The objective of this research is to evaluate and compare the potency of probiotics and paraprobiotics to modulate oxidative stress and inflammation.

Methods and results

In the initial phase, the antioxidant capabilities of 88 strains from Lactobacillus and Bifidobacterium were evaluated. In the subsequent phase, during the in-vivo stage, four experimental groups were established, consisting of a high-fat diet (HFD) + PBS, HFD + DSS, HFD + DSS + 10^⁹ cfu/ml of 6 selected native probiotic, and HFD + DSS + 10^⁹ cfu/ml of paraprobiotic (from 6 selected strains), with male wild-type C57BL/6 mice being assigned to these groups. The phenotypical indices and pathological scores along with the evaluation of the expression of genes associated with the NF-kB and Nrf2 signaling pathways, as well as enzymes linked to oxidant/anti-oxidant activities, and proinflammatory/inflammatory cytokines were performed. A significant difference was noted among the groups exposed to DSS and groups that given our native agents. The mice receiving a blend of probiotics and paraprobiotics alongside DSS demonstrated a mitigation of the harmful impacts caused by DSS, both regarding phenotypic traits, including pathological scores and also the level of cytokines and antioxidant markers and also molecular indicators like the Nrf2 and NF-kB associated genes. Also, there was no notable difference between our native probiotic and paraprobiotic.

Conclusion

The study’s findings provide evidence that the expression of inflammation can be successfully alleviated by utilizing our native probiotics and paraprobiotics, with a greater emphasis on the latter due to its inherent safety.

Impact statements

This study highlighted the anti-inflammatory and antioxidant properties of probiotic and paraprobiotic that could be useful for patients with inflammatory status.

Introduction

Inflammatory Bowel Disease (IBD) is a disorder characterized by gastrointestinal symptoms, which encompasses two forms known as ulcerative colitis (UC) and Crohn’s disease (CD). Physician suspicion may arise from symptoms such as abdominal pain, diarrhea, rectal bleeding, and weight loss [1]. The precise etiology of IBD is remain unclear, however, genetic, immune system, and environmental factors, including smoking and stress have major role in causing IBD [2]. Besides, diet has the potential to impact the occurrence of IBD. It has been reported that a Western diet, which consists of excessive saturated fat, high sugar, and low fiber, as well as a high-fat diet (HFD) that contains significant amounts of fatty acids and lacks in fiber and vitamins, has recently been identified as a contributing factor to the development of IBD [3]. One of the most adverse effects of having high fat regimen is that long term use of HFD leads to increase the oxidative stress [4].

Oxidative stress is a common occurrence that could be seen when the normal balance of antioxidant and pro-oxidant is disrupted [5]. This phenomenon is another etiological reason for causing IBD. It could be said that in IBD, excessive immune response due to chronic inflammation and impaired tissue perfusion due to mucosal damage lead to excessive production of reactive oxygen and nitrogen species (ROS/RNS). Reactive oxygen species, produced by inflamed intestinal tissues and immune cells, elevate oxidative stress and play a role in the onset and advancement of chronic gastrointestinal infections. It has been reported that increased oxidized molecules due to excessive oxidative stress in IBD are correlated with severity of mucosal inflammation [6]. Given the potential deleterious consequences of oxidative stress and its potential involvement in the pathogenesis of IBD, it follows that any substance possessing antioxidant properties may exert a favorable influence on the regulation of this condition.

Probiotic strains, as affirmed by the World Health Organization (WHO) and the Food and Agriculture Organization (FAO), are advantageous microorganisms that must possess a minimum of two attributes, namely safety and the potential to promote well-being when administered in an appropriate dosage [7]. Paraprobiotics, referred to as inactive, non-living, or ghost probiotics, are microorganisms that possess similar advantageous properties to probiotics while being rendered inactive, thereby minimizing potential harm [8]. Both of these agents possess a multitude of beneficial properties, encompassing anti-inflammatory, immunomodulatory, anti-proliferative, antibacterial, and antioxidant actions [9]. One noteworthy observation is that peptidoglycans and lipopolysaccharides represent the predominant constituents of paraprobiotics, which retain their advantageous efficacy even after exposure to heat, thus encompassing antioxidant properties [10].

IBD is classified as a type of disease that, even with a variety of available medications and therapies, remains fundamentally incurable. The rising prevalence of IBD and the observable side effects resulting from the use of chemical medications indicate a relative deficiency in achieving desired outcomes [11]. Therefore, the recent focus has been primarily on the utilization of biological alternatives, encompassing agents that possess anti-inflammatory properties or any agents, such as probiotics or any derivatives derived from these agents, that may exert favorable effects against the pathophysiology of IBD, thus potentially serving as suitable means of managing IBD symptoms. Since, as previously mentioned, inflammation and oxidative stress are two primary phenomena influencing IBD, and given that probiotics and their derivatives appear to have positive effects on this condition, the main aim of the current study was to assess and analyze the antioxidant efficacy of our uniquely native probiotic cocktail, alongside a paraprobiotic cocktail, within a controlled group of mice that had undergone induced colitis, while concurrently exploring the potential presence of any noticeable differences or variations that could be recognized between these two advantageous agents thought to offer health benefits.

Materials and methods

In vitro experimental evaluation

Bacterial culture and preparation of paraprobiotic

A total of 88 probiotic strains that are native, were isolated from the breast milk and stool samples of healthy Iranian individuals. These strains include L. plantarum, L. reuteri, L. casei, L. rhamnosus, L. mucosae, L. fermentum, L. delbrueckii, and L. brevis, as well as B. bifidum, B. longum, and B. infantis [12, 13]. The procedure for culture of probiotic strains had been described, previously [14] and these previous studies were conducted according to the ARRIVE guidelines and were approved by the Experimentation Committee of the Pasteur Institute of Iran (IR.PII.REC.1398.060) and Iran University of Medical Science (IR.IUMS.REC 1395.9221133201) for the ethical care.

For preparation of paraprobiotics, after 18 h, bacterial suspension went through centrifugation at 12,000 rpm for 5 min at 4℃, followed by washing of each pellet twice with PBS. Each bacterium suspension, adjusted to a concentration of 10⁹ CFU/ml, was heated at 100 °C for 10 min. The inactive cells were sonicated every 10 min, with cycles lasting 1 min and frequencies set at 40 HZ, in a sonicated ice water bath. The suspension was then centrifuged at 10,000 rpm for 15 min at 4 °C, and the resulting precipitate was used as a paraprobiotic. To prepare a paraprobiotic mixture, all six selected strains (adjusted to 10⁹ CFU/mL) were combined and treated using the mentioned method.

The evaluation of antioxidant activity

The antioxidant efficacy of our native probiotic strains was evaluated by culturing the bacterial stocks in MRS broth (Ibresco, Life Science, Iran, LOT: MR241530819) at 37℃ for 18 h. Bacterial cell pellets were obtained by centrifugation at 8000 g for 10 min at 4 °C, and then washed twice with PBS. Antioxidant activity was assessed using biochemical assays in a three-step screening process. From 88 strains, finally six strains with the highest antioxidant activity, including Lactobacillus reuteri RP100, Lactobacillus plantarum RP42, Lactobacillus plantarum RP119, Lactobacillus plantarum RP155, Bifidobacterium bifidum RP1001, and Bifidobacterium longum RP1044) were selected through various biochemical tests, including DPPH, ABTS, superoxide anion and hydroxyl radical scavenging activity tests, reducing power and lipid peroxidation inhibition tests [15]. The antioxidant activity of these six strains was assessed using the same approach for paraprobiotic. MRS broth was used as the Blank in all experiments.

Animals and experimental design

A group of 24 male mice, aged 4–6 weeks and weighing 16 g, were obtained from the Pasteur Institute of Iran. The guidelines for the care and feeding of mice have been outlined before [16]. In brief, these mice lived in polycarbonate cages. They were in a controlled setting at 22 °C and 50% humidity, with a 12-hour light/dark cycle. The mice had access to water and food, and underwent a 2-week period of adjustment to a normal diet. After this period, the mice, now 8 weeks old, were put on a high-fat diet (HFD) for 28 days. The HFD provided 60% of total calories, with 35% from fat, 24% from protein, and 26% from carbohydrates, resulting in a caloric density of 52 kcal/g. In the third week of the HFD regimen, specific subgroups within the HFD category received different oral gavage treatments for two weeks. The treatments included a combination of HFD and PBS, a combination of HFD and 2% DSS (Dextran Sulfate Sodium), a combination of HFD and a mixture of 2% DSS and probiotics, and a combination of HFD and a mixture of 2% DSS and paraprobiotics.

Each mouse in every group was assessed daily for body weight, fecal consistency, and significant hemorrhaging. All evaluations were conducted in a blinded manner. All mice were sacrificed by cervical dislocation under anesthesia using ketamine and xylazine along with atropine sulfate at dose 200/15 mg/kg and atropine 0.05 mg/kg. The combinations were diluted in sterile saline and 0.1 mL per 10 g of body weight was injected intraperitoneally. This study was conducted according to the ARRIVE guidelines and all procedures involving animals were approved by the Animal Experimentation Committee of the Pasteur Institute of Iran (IR PII.REC1400.061) for the ethical care and use of laboratory mice.

The evaluation of the effects of our native agents on histopathological parameters

The evaluation of the Disease Activity Index (DAI) entails the analysis of a composite of variables, such as reduction in body mass, regularity of bowel movements, and occurrence of blood in the stool, as outlined by Kwon and colleagues [17]. The amalgamation of these three parameters is subsequently quantified to acquire a comprehensive clinical rating. The evaluation was conducted in a manner that guaranteed the evaluators were unaware of the particular details. The procedures of colon tissues preparation had been described, previously [16]. In summary, the colon was separated into three separate sections, and for histopathological analysis, the colon tissues were preserved using paraformaldehyde, embedded in paraffin, and sliced into 4 μm thick sections. The histological score was established by utilizing the criteria specified in a preceding study [18].

The evaluation of biomarkers of oxidative stress and cytokines

The concentrations of antioxidant/oxidant markers, including Malondialdehyde (MDA), glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) in both serum and sections of the distal intestinal tissue were assessed following the guidelines provided by the manufacturer, Navand Salamat, Iran, MDA (Cat No.: NS-15023), SOD (Cat No.: NS-15033, CAT (Lot: Cat No.: NS-15053), GSH (Cat No.: NS-15087, GPX (Cat No.: NS-15083). The concentrations of IL-1β and TNF-α, which are proinflammatory cytokines, as well as IL-4 and IL-10, which are anti-inflammatory cytokines, were evaluated in accordance with the instructions specified by the manufacturer (Karmania Pars Gene, Iran, Lot No.: MIL448231001).

Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) for Nrf2 and NF-kB pathways

The RNA extraction from the colon of mice was conducted using a kit made by Favorgen Biotech Corp in Taiwan (Cat No.: 4, FAPRK 000-Mini). For the reverse transcription of RNA into cDNA, a kit made by Yekta Tajhiz Azma Co in Iran (Cat No.: YT4500) was used. The quantitative PCR was carried out [19] using an instrument made by ABI Prism with the utilization of 2x SYBR-Green (Cat No.: 4344463), specifically a kit made by Amplicon A/S in Denmark. All primer sequences can be found in Table 1. The gene gapdh was used for normalization. The 2^−ΔΔCt method was used for gene expression quantification.

Table 1 The primers used in the current study

Statistical analysis

The statistical analysis was conducted employing the GraphPad Prism 8.0 software, devised by GraphPad Software Inc, located in CA, USA. The one-way test ANOVA was implemented, subsequently followed by Tukey’s post hoc test, when dealing with data that exhibited normal distribution. Conversely, the Kruskal-Wallis test was utilized for data that did not adhere to a normal distribution. The results were presented as the mean accompanied by the standard error. For the sake of statistical significance, a P-value lower than 0.05 was deemed noteworthy.

Results

The results of antioxidant activity among our native probiotic and paraprobiotic

The DPPH capabilities of six probiotic strains with high antioxidant capability varied from 52.33% (B. longum RP1044) to 72.67% (L. plantarum RP42), while the ABTS capabilities ranged from 50.33% (B. bifidum RP1001) to 66.67% (L. reuterri RP100). In the specific subset of the Hydroxyl Radical Scavenging (HRS) test, the activity level ranged from 50% for B. bifidum RP1001 to 70% for L. reuteri RP100. L. reuteri RP100 displayed a degree of activity equivalent to 68%, while B. longum 1044 exhibited an activity level of 50.2% in relation to the Superoxide Anion Assays. L. plantarum RP155 demonstrated the highest activity level with a value of 3.46, whereas L. plantarum RP42 showcased the lowest activity level with a value of 2.95 in the RP test. In the Lipid Peroxidation Inhibition test, B. longum RP1044 and L. reuteri RP100 displayed activity levels of 53% and 65%, respectively. Furthermore, it is important to note that the antioxidant capacity of our probiotic cocktail was significantly superior to that of each individual probiotic strain (Refer to Supplementary file 1, Table S1).

The levels of antioxidant activity observed in our native paraprobiotic were remarkably elevated. The strains displayed varying capabilities in terms of DPPH, ranging from 46.3% (B. bifidum RP1001) to 81% (L. reuteri RP100), and for ABTS, from 51.33% (B. longum RP1044) to 69.6% (L. plantarum RP155). Within this specific subset, the Hydroxyl Radical Scavenging (HRS) test exhibited a range of activity, spanning from 48% for B. bifidum RP1001 to 67.3% for L. reuteri RP100. L. plantarum RP155 displayed a level of activity reaching 51.3%, while L. reuteri RP100 demonstrated an activity level of 72.6% in relation to the Superoxide Anion Assays. Importantly, the RP test revealed that B. longum RP1044 and L. plantarum RP119 displayed the lowest and highest levels of activity, with values of 2.76 and 3.27, respectively. In the Lipid Peroxidation Inhibition test, L. plantarum RP155 and L. plantarum RP119 showcased activity levels of 50% and 61.3%, respectively. Furthermore, it is crucial to emphasize that the antioxidant capacity of our paraprobiotic cocktail was significantly superior to that of each individual paraprobiotic (See Supplementary file 1, Table S2). The examination of the outcomes of probiotic and paraprobiotic revealed that the antioxidative capacity of our native probiotic strains and paraprobiotic are nearly indistinguishable.

The effects of native probiotic and paraprobiotic on colitis-induced criteria

The results showed that our probiotic strains and paraprobiotic had remarkable anti-inflammatory effects. Using DSS in mice with HFD regimen caused weight loss and colon length decrease (p < 0.0001), as well as an increase in DAI and pathological scores (p > 0001). However, our probiotic and paraprobiotic significantly reduced DAI (p < 0.05) and pathological scores (p < 0.05) while increasing colon length (p < 0.001) compared to HFD + DSS group. Additionally, our probiotic and paraprobiotic resulted in less weight loss (p < 0.0001) compared to the HFD + DSS group. It is worth noting that the effects of our probiotic strains and paraprobiotic were similar, with no significant difference between them (See Fig. 1).

Fig. 1

Effects of probiotics and postbiotics mixture on disease severity in DSS-induced colitis mice. (A) Body weight changes, (B) DAI score, (C) Colon length, (D) H&E staining of colon section of mice (a: crypts architecture, b: inflammation, c: muscle thickness, d: goblet cells depletion, and e: crypts abscesses, The scale bar is 100 pixels. (E) histopathological score. Data are presented as the mean ± SD, N = 5 per group. Statistical significance was determined using the following symbols: a, p < 0.05; aa, p < 0.01; aaa, p < 0.001; aaaa, p < 0.0001 (HFD + PBS vs. Other groups), b, p < 0.05; bb, p < 0.01; bbb, p < 0.001; bbbb, p < 0.0001 (HFD + DSS vs. Other groups), The relatedness between HFD + DSS + probiotic and HFD + DSS + paraprobiotic groups. ND: no significant differences were found between the two supplements. HFD: 60%Kcal, Fat 35%, Protein 24%, Carbohydrate 26%, Calories 52 kcal/gr + PBS, DSS: DSS (2%) + PBS, HFD + Probiotic: HFD + probiotic cocktail (10⁹ CFU), HFD + paraprobiotic (HFD + paraprobiotic cocktail (10⁹ CFU)

The results of oxidative stress biomarkers and cytokines

The results of the analysis of antioxidant and oxidant enzymes in serum and digestive system are visually depicted in Figs. 2 and 3. Based on our observations of serum, the administration of DSS led to a decrease in the levels of markers such as SOD, CAT, GPX, and GSH, approaching negligible levels (p < 0.0001), while the levels of MDA increased subsequent to the utilization of DSS (p < 0.0001). Our native probiotic and paraprobiotic products were capable of significantly augmenting the levels of SOD, CAT, GPX, and GSH (p < 0.0001), while concurrently decreasing the levels of MDA (P < 0.0001). In the case of all markers, the effects of probiotic and paraprobiotic were similar, except for SOD, where our native probiotic strains exhibited a greater increase compared to paraprobiotic (p < 0.05). The same outcomes were replicated in regard to these markers within the digestive system, once again with no discernible distinction between probiotic and paraprobiotic derived from.

Fig. 2

The levels of SOD, CAT, GSH, GPX (antioxidant enzymes), and MDA oxidant enzyme in serum. Data are presented as the mean ± SD. Statistical significance was determined using the following symbols: a, p < 0.05; aa, p < 0.01; aaa, p < 0.001; aaaa, p < 0.0001 (HFD + PBS vs. Other groups), b, p < 0.05; bb, p < 0.01; bbb, p < 0.001; bbbb, p < 0.0001 (HFD + DSS vs. Other groups), *, p < 0.05; The relatedness between HFD + DSS + probiotic and HFD + DSS + paraprobiotic groups. ND: no significant differences were found between the two supplements. HFD: 60%Kcal, Fat 35%, Protein 24%, Carbohydrate 26%, Calories 52 kcal/gr + PBS, DSS: DSS (2%) + PBS, HFD + Probiotic: HFD + probiotic cocktail (10⁹ CFU), HFD + paraprobiotic (HFD + paraprobiotic cocktail (10⁹ CFU)

Fig. 3

The levels of SOD, CAT, GSH, GPX (antioxidant enzymes), and MDA oxidant enzyme in gut. Data are presented as the mean ± SD. Statistical significance was determined using the following symbols: a, p < 0.05; aa, p < 0.01; aaa, p < 0.001; aaaa, p < 0.0001 (HFD + PBS vs. Other groups), b, p < 0.05; bb, p < 0.01; bbb, p < 0.001; bbbb, p < 0.0001 (HFD + DSS vs. Other groups), The relatedness between HFD + DSS + probiotic and HFD + DSS + paraprobiotic groups. ND: no significant differences were found between the two supplements. HFD: 60%Kcal, Fat 35%, Protein 24%, Carbohydrate 26%, Calories 52 kcal/gr + PBS, DSS: DSS (2%) + PBS, HFD + Probiotic: HFD + probiotic cocktail (10⁹ CFU), HFD + paraprobiotic (HFD + paraprobiotic cocktail (10⁹ CFU)

The results of the effects of our native probiotic strains and paraprobiotic on the pro-inflammatory and anti-inflammatory cytokines could be seen in Fig. 4. According to the findings derived from our investigation, the utilization of DSS resulted in an escalation of pro-inflammatory cytokines, while concurrently causing a decline in anti-inflammatory cytokines (p < 0.0001). Nevertheless, our native probiotic and paraprobiotic agents yielded a considerable reduction in the levels of IL-1β and TNF-α, which are widely recognized as pro-inflammatory cytokines (p < 0.0001). Conversely, a noteworthy increase was observed in the levels of anti-inflammatory cytokines, icluding IL-4 and IL-10 (p < 0.0001). Once again, both of our agents produced similar outcomes in augmenting anti-inflammatory cytokines and diminishing pro-inflammatory cytokines, with no statistically significant difference.

Fig. 4

The Levels of IL-1β, TNF-α (Inflammatory cytokines) and, IL-4, IL-10 (anti-inflammatory cytokines), in serum. Data are presented as the mean ± SD. Statistical significance was determined using the following symbols: a, p < 0.05; aa, p < 0.01; aaa, p < 0.001; aaaa, p < 0.0001 (HFD + PBS vs. Other groups), b, p < 0.05; bb, p < 0.01; bbb, p < 0.001; bbbb, p < 0.0001 (HFD + DSS vs. Other groups),, The relatedness between HFD + DSS + probiotic and HFD + DSS + paraprobiotic groups. ND: no significant differences were found between the two supplements. HFD: 60%Kcal, Fat 35%, Protein 24%, Carbohydrate 26%, Calories 52 kcal/gr + PBS, DSS: DSS (2%) + PBS, HFD + Probiotic: HFD + probiotic cocktail (10⁹ CFU), HFD + paraprobiotic (HFD + paraprobiotic cocktail (10⁹ CFU)

The effects of native probiotic and paraprobiotic on the Nrf2 and NF-kB expression level

The native probiotic strains, along with with paraprobotic, demonstrated noteworthy antioxidant and anti-inflammatory effects by means of influencing the Nrf2 and NF-kB signaling pathway (see Figs. 5 and 6). With regards to the antioxidant activity of our native probiotic strains and paraprobiotic through affecting Nrf2 signaling pathway, employment of DSS resulted in a reduction in the level of expression of all genes (p < 0.0001). However, our native probiotic strains and paraprobiotic were capable of significantly augmenting the level of expression (p < 0.01) in comparison to the DSS group. In terms of the NF-kB genes that showed the anti-inflammatory effects of our agents, DSS instigated a substantial rise in the level of expression of genes (p < 0.0001). Nevertheless, our native probiotic strains and paraprobiotic were able to considerably diminish the level of expression (p < 0.0001). Over again, our native probiotic strains and paraprobiotics exhibited akin anti-inflammatory and antioxidant activities.

Fig. 5

Relative gene expression [mean fold change] of antioxidants and Nrf2 related pathway genes expression in the different groups of treatments. Data were normalized with gapdh. Data are presented as the mean ± SD. Statistical significance was determined using the following symbols: a, p < 0.05; aa, p < 0.01; aaa, p < 0.001; aaaa, p < 0.0001 (HFD + PBS vs. Other groups), b, p < 0.05; bb, p < 0.01; bbb, p < 0.001; bbbb, p < 0.0001 (HFD + DSS vs. Other groups),, The relatedness between HFD + DSS + probiotic and HFD + DSS + paraprobiotic groups. ND: no significant differences were found between the two supplements. HFD: 60%Kcal, Fat 35%, Protein 24%, Carbohydrate 26%, Calories 52 kcal/gr + PBS, DSS: DSS (2%) + PBS, HFD + Probiotic: HFD + probiotic cocktail (10⁹ CFU), HFD + paraprobiotic (HFD + paraprobiotic cocktail (10⁹ CFU)

Fig. 6

Relative gene expression [mean fold change] of NF-kB related pathway genes expression in the different groups of treatments. Data were normalized with gapdh. Data are presented as the mean ± SD. Statistical significance was determined using the following symbols: a, p < 0.05; aa, p < 0.01; aaa, p < 0.001; aaaa, p < 0.0001 (HFD + PBS vs. Other groups), b, p < 0.05; bb, p < 0.01; bbb, p < 0.001; bbbb, p < 0.0001 (HFD + DSS vs. Other groups), The relatedness between HFD + DSS + probiotic and HFD + DSS + paraprobiotic groups. ND: no significant differences were found between the two supplements. HFD: 60%Kcal, Fat 35%, Protein 24%, Carbohydrate 26%, Calories 52 kcal/gr + PBS, DSS: DSS (2%) + PBS, HFD + Probiotic: HFD + probiotic cocktail (10⁹ CFU), HFD + paraprobiotic (HFD + paraprobiotic cocktail (10⁹ CFU)

Discussion

IBD, a condition marked by periods of remission and relapse, encompasses a variety of factors that contribute to its development. Among these factors, nutrition and oxidative stress have emerged as significant determinants. Nutrition, for instance, can impact IBD by influencing the composition of the gut microbiome. The consumption of saturated fatty acids, for example, has been found to disrupt the balance of microorganisms in the gut, leading to an overgrowth of Bilophila wadsworthia. This, in turn, triggers an inflammatory response mediated by Th1 cells and ultimately results in colitis in mice model [20]. Besides, the consumption of a high-fat diet, which is commonly observed in the western lifestyle, may result in the induction of oxidative stress by disrupting mitochondrial regulation and promoting the overproduction of reactive oxygen species (ROS) [21] and the ROS could led to chronic inflammation that is seen in IBD [22]. Hence, employing substances such as probiotic and paraprobiotic agents which possess diverse advantageous attributes, encompassing the capability to influence dysbiosis and exhibit anti-oxidant efficacy [23]. Consequently, the aim of the current study was to exhibit the antioxidative and anti-inflammatory effectiveness of our native strains of probiotics and paraprobiotic in mice that were subjected to a high-fat diet. Additionally, the intention was to assess the efficiency of these substances in order to ascertain which one should be given precedence for application.

Our research revealed significant findings regarding the anti-inflammatory and anti-oxidant properties of our native probiotics and paraprobiotic in both in-vitro and in-vivo experiments. It is important to note that both of our agents in the cocktail format could demonstrate enhanced antioxidant potency. This could be attributed to their synergistic effects. Besides probiotic, according to the recent studies, paraprobiotics, which are inactivated or cellular fraction of probiotic microorganisms, can provide health benefits comparable to those of probiotic strains. Modulation of the immune system, enhanced adhesion to intestinal cells, inhibition of pathogens, secretion of metabolites, and balancing the gut microbiome are some recognized mechanisms, although the precise mode of action remains under investigation. Consequently, it appears that in the present study, the combination of these cell components may contribute to an increase in the overall biomass, thereby enhancing the anti-inflammatory and antioxidant efficacy [24,25,26]. In the in-vitro assay, the outcomes of the tests measuring antioxidant activity indicated a slight distinction between our agents, while both our native probiotic strains and paraprobiotic demonstrated similar results in terms of biochemical antioxidant assessments. These findings align with the findings presented by Kang et al. in Korea in 2021, as per their findings, the probiotic strains, such as Bifdobacterium bifidum MG731 and Bifdobacterium lactis MG741, exhibited the most notable DPPH and ABTS activities [27]. Jang et al. in Korea in 2018, also indicated that Lactobacillus plantarum Ln1 has the ability to demonstrate antioxidant activity by utilizing DPPH and ABTS assays, while the heat-killed cells of this probiotic strain exhibited antioxidant potential through β-carotene bleaching inhibition and reducing power assays [28]. Additionally, in accordance with the findings of İncili et al., in Turkey in 2023, the lyophilized or freeze-dried paraprobiotic derived from Pediococcus acidilactici exhibited significant levels of free radical scavenging capabilities during the evaluation of DPPH, ABTS, and FRAP [29]. According to the aforementioned findings, it can be argued that probiotic and paraprobiotic agents exhibit advantageous properties in counteracting oxidative processes that may arise due to certain phenomena, such as the consumption of a high-fat diet.

In the realm of in-vivo experimentation, our native probiotic and paraprobiotic agents yielded noteworthy findings. The results of our investigation pertaining to the markers associated with colitis indicate that our probiotic and paraprobiotic substances exhibited similar properties in terms of their ability to mitigate inflammation, as evidenced by alterations in body weight, colon length, DAI, and pathological scores. Song et al., in Korea in 2022, have presented findings that align with those of the present study. In their research, it was demonstrated that the probiotic and paraprobiotic properties of Lactiplantibacillus plantarum L67 had the capacity to restore normalcy to DAI and colon length in mice induced with DSS [30]. Sang et al., in China in 2015, also reported that VSL#3 (Lactobacillus plantarum, Lactobacillus Bulgaricus, Lactobacillus casei and Lactobacillus acidophilus, Bifidobacterium breve, Bifidobacterium longum and Bifidobacterium infantis and Streptococcus salivarius subspecies thermophilus in both format of live and heat-killed could show anti-inflammatory effects via affecting DAI, colon length, histological score and MPO activity [31]. Moreover, in a range of phenotypic assays encompassing the assessment of antioxidant markers in both the serum and gastrointestinal tract, as well as cytokine measurement, our indigenous agents once again demonstrated similar potent anti-inflammatory and antioxidant activities. Upon scrutinizing the molecular-level outcomes, particularly in relation to the assessment of the Nrf2 and NF-kB pathways, it becomes apparent that both probiotics and paraprobiotic agents exhibit a similar degree of effectiveness. Other studies also support the present inquiry. Liu and colleagues, in China in 2024, found that the paraprobiotic obtained from Lactiplantibacillus plantarum HF05 and Limosilactobacillus fermentum HF06 relieved inflammation, intestinal injury, and weight reduction in mice by inhibiting the activation of the NF-κB/NLRP3 pathway [32]. Another study, in Brazil in 2022, also demonstrated that the utilization of paraprobiotic derived from Bifidobacterium lactis, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus paracasei, and Streptococcus thermophilus exhibited a substantial decrease in inflammatory cytokines, notably IL-6, along with an augmentation of anti-inflammatory cytokines, such as IL-10 [33]. Another study conducted by Seong et al., in Korea in 2021, also reported that heat-killed Lactobacillus casei DKGF7 could decrease the levels of inflammatory cytokines in colonic tissue, including IL-1β, IL-12p70, and TNF-α [34]. Considering the antioxidant activities, El-Baz et al., in Egypt in 2020, could demonstrate that using Lactobacillus delbrueckii and Lactobacillus fermentum showed a noteworthy rise in levels of NrF2 and HO-1, as well as a substantial decline in TNF-α [35]. Consequently, it can be mentioned that the application of probiotic strains and their derivatives may influence oxidative stress and inflammatory conditions. Nevertheless, it is important to note that this investigation was performed on a colitic mouse model, and this in vivo approach may come with certain limitations regarding its applicability to human scenarios, owing to the pathophysiological diversity of the human condition; thus, conducting clinical trials with human participants appears essential to validate the beneficial potential of probiotic and paraprobiotic substances.

Conclusion

The study effectively demonstrates the antioxidant capabilities of our native probiotic strains of Lactobacillus and Bifidobacterium, highlighting their potential in mitigating the adverse effects of a high-fat diet. Notably, the administration of both native probiotics and paraprobiotics led to a reduction in oxidative stress markers and modulated key signaling pathways associated with inflammation, specifically NF-kB and Nrf2. The findings indicate that these microbial agents can counteract the detrimental effects of DSS, suggesting their potential as therapeutic interventions for conditions related to oxidative stress and inflammation. Importantly, the lack of significant differences between the effects of probiotics and paraprobiotics suggests that both forms may provide similar protective benefits, emphasizing the importance of further exploring their applications in clinical settings. However, it could be said that our native paraprobiotics take precedence as they are inactivated constituents derived from probiotic strains, and may serve as a safer alternative, especially for individuals who may experience undesirable side effects from probiotics. Overall, this research underscores the promising role of our native probiotics and paraprobiotics in enhancing gut health and reducing inflammation, paving the way for future studies aimed at optimizing their use in dietary interventions.