Anti-oxidative stress composition and application thereof in improving neonatal necrotizing enterocolitis

Abstract

The invention relates to the field of infant food, in particular to an anti-oxidative stress composition and application thereof to improvement of neonatal necrotizing enterocolitis. The composition is prepared from the following components: neutral oligosaccharide and sialylated oligosaccharide. The neutral oligosaccharide is 3-fucosyllactose, and the sialylated oligosaccharide is 3 '-sialic acid lactose. The invention provides a composition capable of relieving neonatal necrotizing enterocolitis. Through combination of the 3-fucosyllactose and the 3 '-sialic acid lactose, related symptoms of the neonatal necrotizing enterocolitis can be improved by inhibiting a TLR4 / NF-kappa B inflammation pathway, improving cell oxidative stress, protecting cell barriers and reducing cell apoptosis, and the 3-fucosyllactose and the 3'-sialic acid lactose have important application value in the field of functional foods or formula foods.

Description

Technical Field

[0001] This invention relates to the field of infant food, and more particularly to an antioxidant stress composition and its application in improving neonatal necrotizing enterocolitis. Background Technology

[0002] Necrotizing enterocolitis (NEC) is a common and serious inflammatory bowel disease that primarily affects premature infants. Its pathological features include intestinal barrier disruption, bacterial invasion, and localized intestinal tissue necrosis. Despite significant advancements in neonatal intensive care technology, NEC remains a major threat to neonatal health. Currently, clinical treatment for NEC mainly relies on the use of broad-spectrum antibiotics, interruption of enteral nutrition, and surgical resection of necrotic bowel segments. However, these conventional treatments have significant limitations and are often accompanied by serious long-term complications, such as short bowel syndrome, growth retardation, and neurodevelopmental disorders. Therefore, a deeper understanding of the pathogenesis of NEC and the search for safer and more effective prevention and treatment strategies are urgent clinical challenges.

[0003] Existing research indicates that the pathogenesis of NEC is complex, involving multiple factors such as immature intestinal development, excessive inflammatory response, and abnormal immune regulation. Among these, gut microbiota dysbiosis is considered a key contributing factor. Compared to healthy infants, children with NEC often exhibit significantly altered gut microbiota structure, characterized by a decrease in the relative abundance of Firmicutes and Bacteroidetes, an abnormally high abundance of Proteobacteria, and a decline in overall microbiota diversity. This dysbiosis further exacerbates intestinal barrier damage and induces cytokine storms. Therefore, restoring gut microbiota balance and enhancing intestinal barrier function through microecological regulation (such as probiotic supplementation) has become a research hotspot. For example, probiotics such as Bifidobacteria and Lactobacillus have been shown to reduce the risk of NEC to some extent through mechanisms such as competitively inhibiting pathogenic bacterial colonization and upregulating tight junction protein expression.

[0004] Besides probiotics themselves, human milk oligosaccharides (HMOs), as important bioactive components of breast milk, play an irreplaceable role in maintaining the gut health of infants and young children. HMOs are composed of monomers such as glucose, galactose, N-acetylglucosamine, fucose, and sialic acid, and possess various biological activities, including regulating immunity, inhibiting pathogen adhesion, and promoting neural development. In particular, 3-fucosylated lactose (3-FL) and 3'-sialylated lactose (3'-SL) not only act as prebiotics, promoting the proliferation of beneficial bacteria such as Bifidobacteria and metabolizing them into short-chain fatty acids, but also directly act on intestinal epithelial cells, exerting anti-inflammatory and barrier-protective functions. Further research is needed on strategies for improving NEC (Neurotic Endocrine Disorder) related to HMOs. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides an antioxidant stress composition and its application in improving neonatal necrotizing enterocolitis. This aims to resolve the shortcomings and unclear mechanisms of existing solutions for neonatal necrotizing enterocolitis.

[0006] In a first aspect, the present invention provides an antioxidant stress composition comprising: neutral oligosaccharides and sialylated oligosaccharides.

[0007] Furthermore, the neutral oligosaccharide is fucoidyl lactose, preferably 3-fucosyl lactose.

[0008] Furthermore, the sialylated oligosaccharide is 3'-sialyl lactose.

[0009] Furthermore, the weight ratio of the neutral oligosaccharide to the sialylated oligosaccharide is (0.5-50):1.

[0010] Preferably, the weight ratio of the neutral oligosaccharide to the sialylated oligosaccharide is (1-20):1; More preferably, the weight ratio of the neutral oligosaccharide to the sialylated oligosaccharide is (1-7):1; Furthermore, the neutral oligosaccharide is 0.1-3 mg / mL.

[0011] Furthermore, the composition is a liquid or a solid, preferably a powdered solid.

[0012] Secondly, the present invention provides a formulated food product comprising the aforementioned composition.

[0013] Furthermore, the formulated food includes pasta, beverages, instant foods, baked goods, sauces, or functional nutritional supplements.

[0014] Furthermore, the reconstituteable food includes milk powder.

[0015] Further, wherein the formulated food is an infant formula or an infant supplement; and in solid form, the amount of the composition added is: 0.3-1.75g / 100g of 3-fucosylated lactose and 0.1-0.28g / 100g of 3'-sialic acid lactose.

[0016] Thirdly, the present invention provides the use of the aforementioned composition in the preparation of a product for improving neonatal necrotizing enterocolitis; said product being a food, feed additive, or health product.

[0017] Furthermore, the improvement of neonatal necrotizing enterocolitis includes the following: (1) It alleviates the weakened degradation ability of lipid peroxides; (2) Relieves or restores one or more of the following: intestinal edema, pneumocystis, villus loss, intestinal barrier damage, and tissue necrosis; (3) Inhibit the release of inflammatory factors promoted by the TLR4 / NF-κB pathway; (4) Restore the content of short-chain fatty acids in the intestine.

[0018] Furthermore, the lipid peroxide may be MDA.

[0019] Furthermore, the ability to mitigate the weakening of lipid peroxide degradation includes the following: (1) Restore the activity and content of antioxidant enzymes in the intestine; (2) Reduces lipid oxidation product MDA; (3) Inhibit the development of oxidative stress through the Nrf2 / NQO1 pathway.

[0020] "Oxidative stress" refers to an imbalance between oxidation and antioxidation in the body, which can damage various cellular components and tissues, including DNA, leading to oxidative stress injury and ultimately oxidative stress-mediated diseases. For example, oxidative stress may cause cell damage, impaired cell function, and / or cell death ("apoptosis").

[0021] The difference between neonatal necrotizing enterocolitis (NEC) and ordinary enteritis lies in the fact that NEC involves more pathological targets. Due to the incomplete development of the neonatal intestine, the pathological process in NEC presents a "catastrophic" response. A large number of pathogens invade the intestinal wall, producing cytotoxins and gas, causing intestinal gas accumulation, oxidative ischemia, and structural abnormalities in the intestinal wall. At the same time, severe oxidative stress occurs in the intestine, further aggravating oxidative damage, ultimately leading to intestinal necrosis, and even more serious intestinal perforation and portal venous gas accumulation. Ordinary enteritis, on the other hand, is only a local immune and inflammatory response triggered by pathogens, and is more reversible.

[0022] The present invention has the following beneficial effects: This invention reveals that the combined use of neutral oligosaccharides and sialylated oligosaccharides can synergistically prevent, improve, and alleviate neonatal necrotizing enterocolitis (NEC) through a multi-target, integrative mechanism that inhibits the TLR4 / NF-κB inflammatory pathway, improves cellular oxidative stress, protects the cell barrier, regulates gut microbiota, and reduces apoptosis. This has significant application value in the field of neonatal necrotizing enterocolitis management. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 This invention relates to the effects of 3-FL, 3'-SL, and their combination on ROS production in LPS-induced IEC-6 cells, as provided in Example 1 of this invention.

[0025] Figure 2 This invention relates to the effects of 3-FL, 3'-SL, and their combination on the mRNA and protein expression of TJ and Muc2 proteins in LPS-induced IEC-6 cells, as provided in Example 1 of this invention.

[0026] Figure 3 This invention relates to the effects of 3-FL, 3'-SL, and their combination on LPS-induced expression of NF-κB pathway mRNA and related proteins in IEC-6 cells, as provided in Example 1 of this invention.

[0027] Figure 4 This is a diagram showing the effects of 3-FL, 3'-SL and their combination on the expression of Nrf2 / HO-1 / NQO1 pathway-related mRNAs and proteins in LPS-induced IEC-6 cells, as provided in Example 1 of this invention.

[0028] Figure 5 This is a diagram showing the expression of 3-FL, 3'-SL and their combination in LPS-induced IEC-6 cells of eNOS mRNA and protein, as provided in Example 1 of this invention.

[0029] Figure 6 This is a graph showing the effects of 3-FL and 3'-SL, and their combination, provided in Example 1 of this invention, on body weight and clinical disease index in rats with necrotizing enterocolitis.

[0030] Figure 7 This is a diagram showing the effects of 3-FL and 3'-SL, and their combination, provided in Example 1 of this invention, on the macroscopic state of the intestine and tissue damage in rats with necrotizing enterocolitis.

[0031] Figure 8 This is a diagram showing the effects of 3-FL and 3'-SL, and their combination, provided in Example 1 of this invention, on cytokines in the intestinal tissue of rats with necrotizing enterocolitis.

[0032] Figure 9 This is a graph showing the effects of 3-FL and 3'-SL, and their combination, provided in Example 1 of this invention, on oxidative stress indicators in the intestinal tissue of rats with necrotizing enterocolitis.

[0033] Figure 10 This is a diagram showing the effect of 3-FL and 3'-SL and their combination on the intestinal barrier integrity in rats with necrotizing enterocolitis, as provided in Example 1 of this invention.

[0034] Figure 11 This is a diagram showing the effect of 3-FL and 3'-SL and their combination on the expression of TLR4 / NF-κB pathway-related proteins in the intestine of rats with necrotizing enterocolitis, as provided in Example 1 of this invention.

[0035] Figure 12 This is a diagram showing the effect of 3-FL and 3'-SL, and their combination, provided in Example 1 of this invention, on the expression of Nrf2 / NQO1 pathway-related proteins in the intestine of rats with necrotizing enterocolitis.

[0036] Figure 13 This is a graph showing the effect of 3-FL and 3'-SL and their combination on short-chain fatty acids in the intestine of rats with necrotizing enterocolitis, as provided in Example 1 of this invention.

[0037] Figure 14 This is a graph showing the effect of 3-FL and 3'-SL and their combination on the alpha diversity of the rat gut microbiota, as provided in Example 1 of this invention. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0039] This invention discovers that the combined action of neutral oligosaccharides (such as fucose-based lactose) and sialylated oligosaccharides can improve intestinal inflammation, especially for neonatal necrotizing enterocolitis.

[0040] Fucosyllactose is an oligosaccharide containing fucose residues. The following are examples of different types of fucosyllactose: 2'-Fucosyllactose (2'-FL), a neutral trisaccharide composed of L-fucose, D-galactose, and D-glucose units, where the monosaccharide L-fucose is linked to the disaccharide D-lactose via an α (1→2) bond. 3-Fucosyllactose (3-FL), a neutral trisaccharide composed of L-fucose, D-galactose, and D-glucose units, where the monosaccharide L-fucose is linked to D-glucose via an α (1→3) bond. Lactose-N-fucopentose I (LNFP I), a neutral pentasaccharide composed of L-fucose, D-glucose, two molecules of D-galactose, and N-acetylglucosamine units, where the monosaccharide L-fucose is linked to D-glucose via an α (1→3) bond. Lactose-N-difucohexose I (LNDFH I) is a neutral hexasaccharide composed of two molecules of L-fucose, D-glucose, D-galactose, and N-acetylglucosamine, wherein the monosaccharide L-fucose is linked to D-galactose and N-acetylglucosamine via α (1→2) and α (1→4) bonds, respectively. Lactose-N-difucohexose II (LNDFH II) is also a neutral hexasaccharide composed of two molecules of L-fucose, D-glucose, D-galactose, and N-acetylglucosamine, wherein the monosaccharide L-fucose is linked to D-glucose and N-acetylglucosamine via α (1→3) and α (1→4) bonds, respectively. The human milk oligosaccharides (HMOs) described in this invention include at least the aforementioned neutral fucoidan-lactose; in some preferred embodiments, the fucoidan-lactose is preferably 3-fucosyllactose (3-FL).

[0041] The sialylated oligosaccharides of this invention include any one or more of 3'-sialylated lactose, 6'-sialylated lactose, sialylated-lactose-N-tetrasaccharide a, sialylated-lactose-N-tetrasaccharide b, sialylated-lactose-N-tetrasaccharide c, and disialiacid-lactose-N-tetrasaccharide. In some preferred embodiments, the sialylated oligosaccharide of this invention is 3'-sialylated lactose.

[0042] This invention does not impose any particular limitation on the source of 3-FL and 3'-SL. Typically, they can be obtained by conventional chemical synthesis methods, microbial fermentation, or other means in the art. Furthermore, 3-FL and 3'-SL can also be derived from animal milk, such as cow's milk.

[0043] There are no particular limitations on the formulated foods involved in this invention, which can generally be pasta, beverages, instant foods, baked goods, sauces, or functional nutritional supplements. In the case of instant foods, typical examples include instant milk powder products, such as infant formula, adult milk powder, and milk powder for the elderly. In particular, the compositions of this invention are very suitable for the preparation of infant formula. Furthermore, there are no particular limitations on functional nutritional supplements, which can be used as nutritional supplements or meal replacements. Such foods, in addition to including the two functional components of this invention, may also include one or more of the following components: protein, fat, essential carbohydrates, dietary fiber, supplementary elements, vitamins, plant or dietary ingredients, etc.

[0044] Unless otherwise specified, all methods used in the examples were conventional or performed according to techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents and instruments used without specified manufacturers were all conventional products that can be purchased through legitimate channels.

[0045] Example 1 1. Experimental materials The IEC-6 cell line of rat small intestinal crypt epithelial cells was purchased from Shangen Biotechnology Co., Ltd.

[0046] 2. Experimental Grouping (1) ctrl group: No LPS induction treatment is performed.

[0047] (2) LPS group: LPS induction treatment was performed only. The treatment method was to treat IEC-6 cells with 10 μg / mL LPS for 24 hours.

[0048] (3) 3-FL: Based on the LPS group, the patients were first treated with 1 mg / mL of 3-FL (which has been verified to be cytotoxic) for 24 hours, and then LPS induction treatment was performed.

[0049] (4) 3'-SL: Based on the LPS group, the patients were first treated with 0.01 mg / mL of 3'-SL (which has been verified to be cytotoxic) for 24 hours, and then LPS induction treatment was performed.

[0050] (5) 3-FL+3'-SL: Based on the LPS group, the patient was first treated with 0.5 mg / mL 3-FL + 0.05 mg / mL 3'-SL (which has been verified to be cytotoxic) for 24 hours, and then LPS induction treatment was performed.

[0051] All of the above treatments were performed in culture medium.

[0052] 3. Analytical Methods The data in the following methods were analyzed using SPSS, with multiple comparisons performed using the Tukey method. A p-value < 0.05 was considered statistically significant. Experimental data are expressed as mean ± standard deviation, and each group was repeated at least three times.

[0053] (1) IEC-6 cell culture Place the cell culture consumables in the laminar flow hood beforehand and sterilize them with UV light for 30 minutes. After sterilization, wipe the prepared DMEM complete culture medium and trypsin with 75% ethanol and then place them in the laminar flow hood.

[0054] (1.1) Cell resuscitation Remove the cryovials containing IEC-6 cells from liquid nitrogen and quickly place them in a 37 ℃ constant temperature water bath. Gently agitate to thaw them rapidly. Wipe with 75% ethanol and transfer to a clean bench. Transfer the cell suspension from the cryovials to a 15 mL centrifuge tube containing an appropriate amount of complete culture medium. Gently pipette to mix and centrifuge (1000×g, 5 min). Discard the supernatant and resuspend the cells in 1 mL of complete culture medium. Mix well and transfer to a T25 culture flask containing 4 mL of complete culture medium. Incubate at 37 ℃ with 5% CO2.

[0055] (1.2) Cell passage Under an inverted microscope, cells were passaged when they reached 80%-90% confluence. First, the original culture medium was discarded, and the cells were washed with sterile PBS, the waste liquid aspirated, and this process was repeated twice. Then, 1 mL of trypsin was added for digestion, and the cells were gently agitated to distribute the trypsin evenly. The cells were then placed in an incubator at 37 ℃ and 5% CO2. When the cells became rounded and the intercellular spaces increased under the microscope, 3 mL of LDM complete culture medium was immediately added to stop the digestion. The adherent cells were gently detached by pipetting, and the cell suspension was transferred to a 15 mL centrifuge tube. The tubes were centrifuged (1000×g, 5 min), the supernatant was discarded, and the cells were resuspended in 3 mL of complete culture medium. The cells were then seeded into new culture flasks at a 1:3 ratio and incubated at 37 ℃ and 5% CO2.

[0056] (1.3) Cell cryopreservation Following cell passage methods, cells were digested with trypsin, resuspended in culture medium, and centrifuged (1000×g, 5 min) to obtain a cell suspension. The supernatant was discarded, and the cells were resuspended in 1 mL of cryopreservation buffer and transferred to cryovials. The cell type and cryopreservation time were labeled on the cryovials, and then a gradient cryopreservation process was performed. The cryovials were first placed in a 4 ℃ freezer for 30 min, then in a -20 ℃ freezer for 2 h, then in a -80 ℃ freezer overnight, and finally stored in a liquid nitrogen tank for long-term preservation.

[0057] (2) Determination of lipopolysaccharide (LPS) concentration IEC-6 cells were used at a rate of 6 × 10⁶ cells per well.3 Cells were seeded at a density of [number] cells / well in 96-well plates and incubated for 24 h until complete adherence. Cells were divided into a control group and different LPS treatment groups (0.1, 1, 5, 10, 25, 50, and 100 μg / mL), with three replicates per group. After 24 hours of treatment, the original culture medium was discarded. Medium containing 10% CCK-8 was added to each well, and the cells were incubated for 2 h. The absorbance (OD) was measured at 450 nm using a microplate reader, and cell viability was calculated to determine the optimal LPS induction concentration. A 10 μg / mL LPS treatment regimen was ultimately determined.

[0058] (3) Morphological observation of cell damage IEC-6 cells in the logarithmic growth phase and in good condition were collected. When the cells reached 60% confluence, they were subjected to the aforementioned treatments. Cell morphological changes were observed and photographed under a fluorescence inverted microscope (MF53, Guangzhou Mingmei Optoelectronic Technology Co., Ltd.). The image resolution was 1920×1200, and the magnification was 100×.

[0059] The control group (ctrl group, the same below) showed monolayer adherent growth, resembling cobblestones, with a tight arrangement and clear boundaries. After stimulation with 10 μg / ml LPS for 24 h, IEC-6 cells underwent significant morphological changes, with a marked decrease in cell density, a disordered and irregular overall arrangement, and numerous cells exhibiting shrinkage and detachment. Pretreatment with 1 mg / mL 3-FL and 0.01 mg / mL 3'-SL significantly increased cell number and reduced cell fragmentation. The combined effect of 3-FL and 3'-SL had a more pronounced effect on the morphological changes of IEC-6 cells induced by LPS stimulation, significantly increasing cell density, improving shrinkage and detachment, and restoring cell morphology to a more normal state.

[0060] (4) Measurement of reactive oxygen species (ROS) generation After cell culture, discard the culture medium, wash twice with 2 mL PBS, digest with trypsin to stop digestion, collect cells into centrifuge tubes, centrifuge (200×g, 4 ℃, 5 min), discard the supernatant, wash twice more with PBS, centrifuge (200×g, 4 ℃, 5 min), discard the supernatant, add 1 mL of 10 μM DCFH-DA staining solution, mix well, and incubate in a CO2 cell culture incubator (5% CO2, 37 ℃) for 30 min. During incubation, gently invert the tube every 5 min to ensure thorough contact between the probe and cells. After incubation, wash twice with serum-free DMEM to remove DCFH-DA, resuspend cells in an appropriate amount of DMEM, and analyze using flow cytometry.

[0061] The results are as follows Figure 1 As shown: Compared with the control group, the intracellular ROS production was significantly increased after LPS induction. P <0.05%. Compared with the model group, the amount of ROS produced in cells pretreated with 1 mg / mL 3-FL was significantly reduced ( P <0.05), the amount of ROS generated in the 3'-SL treatment group was not significantly different from that in the model group ( P >0.05), while the reduction in ROS production was most significant in cells treated with a combination of 3-FL and 3'-SL. P <0.01). ROS can serve as signaling molecules in inflammatory responses, and high levels of ROS production are associated with increased inflammation. Experimental results show that LPS treatment can activate intracellular oxidative stress, promote ROS production, and thus exacerbate the inflammatory response. Combined treatment with 3-FL and 3'-SL can significantly reduce ROS production, thereby maintaining intracellular redox stability and alleviating the inflammatory response.

[0062] (5) mRNA and protein expression analysis of tight junction proteins and mucins in IEC-6 cells This invention analyzed the expression levels of ZO-1, Occludin, Claudin-1, and Muc2 proteins in IEC-6 cells using q-PCR and Western blot. The results are as follows: Figure 2 As shown: Compared with the control group, the mRNA expression levels of TJ and Muc2 proteins in LPS-stimulated IEC-6 cells were significantly reduced. P <0.01), while compared with the model group, the mRNA expression levels of ZO-1 and occludin in each HMO treatment group were significantly increased ( P <0.05), the mRNA expression levels of claudin-1 and Muc2 were significantly increased in the 3-FL and 3-FL+3'-SL groups, while the increase in the mRNA expression levels of claudin-1 and Muc2 in the 3'-SL group was not significant. P >0.05). Furthermore, Western blot results showed that the expression levels of TJ and Muc2 proteins in the model group were significantly lower than those in the control group ( P <0.05), while compared with the corresponding proteins induced by LPS in cells, the expression of TJ protein and Muc2 protein in the three groups of cells after 3-FL and 3'-SL and combined administration was significantly increased ( P <0.05%. The effect was most pronounced in the 3-FL+3'-SL group ( P<0.01). This indicates that 3-FL, 3'-SL, and their combined action can regulate the expression of TJ protein and mucin Muc to maintain the integrity of the intestinal barrier, thereby playing a regulatory role in the development of cellular inflammation.

[0063] (6) Effects on the TLR4 / NF-κB inflammatory pathway To further investigate the alleviating effect of HMO on lipopolysaccharide (LPS)-induced inflammation in IEC-6 cells, this invention used q-PCR and Western blot to detect the expression levels of related mRNAs and proteins in the inflammatory pathway, respectively. The results are as follows: Figure 3 As shown: Compared with the control group, the mRNA expression levels of TLR4 and MyD88 in LPS-stimulated IEC-6 cells were significantly increased. P <0.05), which increased by 3.31-fold and 3.50-fold, respectively; among them, compared with the model group, the mRNA expression level of TLR4 in each HMO treatment group decreased by 33.64%, 17.29% and 50.13%, respectively; since TLR4 and MyD88 are key proteins in the NF-κB signaling pathway, it indicates that 3-FL, 3'-SL and the combined action of the two can inhibit the expression of key protein mRNA induced by LPS, thereby inhibiting the NF-κB signaling pathway and ultimately inhibiting the expression and release of inflammatory factors.

[0064] In addition, Western blot results showed that LPS treatment significantly increased the expression levels of TLR4, MyD88, and iNOS proteins. P <0.05), and the p-P65 to P65 ratio also increased significantly. This indicates that a large amount of P65 is phosphorylated and participates in the reaction, exacerbating cellular inflammation. Compared with the corresponding proteins induced by LPS in cells, the expression of TLR4, MyD88, and iNOS in the three groups of cells after 3-FL, 3'-SL, and combined administration was significantly reduced. P <0.05), p-P65 / P65 was also significantly reduced ( P <0.05), with the 3-FL+3'-SL group showing the most significant effect. This indicates that 3-FL, 3'-SL, and their combined treatment inhibited the signaling factors TLR4, MyD88, and iNOS in the NF-κB pathway, and also inhibited the phosphorylation of p65. Therefore, these results suggest that 3-FL, 3'-SL, and their combined action may suppress inflammation by reducing the expression of the NF-κB signaling pathway, thereby alleviating NEC.

[0065] (7) Effects on the Nrf2 / HO-1 / NQO1 pathway This invention analyzed the Nrf2 expression level in IEC-6 cells using q-PCP. The results are as follows: Figure 4As shown: Compared with the control group, the mRNA expression level of Nrf2 in LPS-stimulated IEC-6 cells was significantly reduced ( P <0.05, compared with the model group, the mRNA expression levels of Nrf2 in the 3-FL, 3'-SL and combined treatment groups were significantly increased ( P <0.05). Furthermore, Western blot results showed that, compared to LPS-induced corresponding proteins in cells, the expression of Nrf2 and NQO1 was significantly increased in all three groups of cells after 3-FL and 3'-SL administration, as well as after combined administration. P <0.05%. The effect was particularly pronounced in the 3-FL+3'-SL group. P <0.05%. HO-1 protein expression was also significantly increased in the 3-FL and combined groups. P <0.05), while the expression level of HO-1 protein also increased in the 3-SL group, but the difference from the model group was not significant ( P >0.05). The results showed that 3-FL, 3'-SL, and their combined treatment promoted the dissociation of Nrf2 and Keap1 into the nucleus, increasing the expression of downstream antioxidant proteins HO-1 and NQO1. Previous studies have found that HMOs can promote the production of indole-3-lactic acid, a major metabolite of Bifidobacterium infantis, and protect cultured intestinal epithelial cells by activating the Nrf2 pathway. Therefore, these results indicate that 3-FL, 3'-SL, and their combined action can alleviate LPS-induced NEC by inhibiting the development of oxidative stress through the Nrf2 / HO-1 / NQO1 pathway.

[0066] (8) Effects on eNOS (endothelial nitric oxide synthase) Experimental results are as follows Figure 5 As shown: 3-FL and 3'-SL, and their combined treatment, can increase the intracellular eNOS content, promote NO production, thereby regulating cell metabolism and reducing the negative effects of LPS on cells. The mRNA expression level of eNOS in LPS-stimulated IEC-6 cells was significantly reduced. P <0.05. Compared with the model group, the mRNA expression level of eNOS was significantly increased in both the 3-FL and 3-FL+3'-SL treatment groups. P <0.05). This indicates that LPS reduced the level of eNOS mRNA in cells through a series of reactions, consistent with findings in primary cultured endothelial cells. Western blot results showed that, compared with the corresponding protein induced by LPS, the expression of eNOS in the three groups of cells, including those treated with 3-FL and 3'-SL, and those treated with combination therapy, was significantly increased. P <0.05%. The effect was most pronounced in the 3-FL+3'-SL group ( P<0.05%, or even returned to normal levels, with no significant difference from the control group ( P <0.05). This indicates that 3-FL, 3-SL, and especially their combined action can promote the expression of eNOS protein, thereby maintaining the normal function of intestinal cells and alleviating the LPS-induced inflammatory response.

[0067] Example 2 I. Experimental Methods 1. Animal grouping (1) Con group: No NEC procedure induction.

[0068] (2) Mod group: NEC program induction was performed: rats were placed in a hypoxic environment (95% N2 + 5% O2) for 10 minutes, then placed in a 4℃ environment for 10 minutes, 3 times / day for 3 consecutive days. During this period, rats were fed with hypertonic formula milk (15 g PreNAN milk powder dissolved in 75 mL LESbilac canine milk substitute) 4 times / day, with an increase of 0.1 mL every 24 h. The mice were weighed every morning. After the last gavage, the rats were sacrificed 12 h after fasting. The spleen and the 1 cm segment of the terminal ileum were fixed with paraformaldehyde fixative. The segment of the lower duodenum to the ileocecal junction and the intestinal contents were stored at -80 ℃.

[0069] (3) 3-FL group: In addition to the Mod group, 3-FL was fed daily at a rate of 1500 mg / kg / day.

[0070] (4) 3'-SL group: In addition to the Mod group, 3'-SL was fed daily at a rate of 300 mg / kgL / day.

[0071] (5) 3-FL+3'-SL group: In addition to the Mod group, 750mg / kg / day of 3-FL+150mg / kg / day of 3'-SL was fed daily.

[0072] 2. NEC Severity Assessment Before euthanizing the rats, their condition was observed in terms of appearance, tactile response, natural activity, and body color. The overall activity of the rats was assessed according to the scoring criteria of the Clinical Disease Index (Table 1). After euthanasia, the appearance of the intestines was observed, and the macroscopic condition of the intestines was scored according to the scoring criteria (Table 2) in terms of intestinal continuity, intestinal color, and intestinal distension.

[0073] Table 1 Clinical Disease Index Scores

[0074] Table 2. Intestinal macroscopic condition score

[0075] 3. Histopathological analysis Spleen and terminal ileum segments fixed in paraformaldehyde were dehydrated and embedded. Embedded tissue samples were cut into 4 μm pieces, dewaxed, stained with hematoxylin and eosin (H&E), and mounted. Sections were observed using an inverted fluorescence microscope, and images were scored. NEC was assessed in terminal ileum tissue sections according to the following criteria: 0 points, intact structure; 1 point, upper part of villi destroyed and mild submucosal separation; 2 points, mild villi separation and mild submucosal edema; 3 points, villi detached and submucosal edema; 4 points, most villi detached or complete loss of structure. Mice scoring 2 points or higher were considered to have NEC.

[0076] 4. Inflammatory cytokine assay A 0.1 g rat ileum tissue sample was mixed with physiological saline to prepare a tissue homogenate. After centrifugation at 4 ℃ (3000 g, 10 min), the supernatant was collected and stored at -80 ℃ for later analysis. Following the manufacturer's instructions, the levels of inflammatory cytokines (TNF-α), (IL-1β), (IL-6), (IL-10), and immunoglobulins (IgA) and (IgG) in the tissue supernatant were determined using an enzyme-linked immunosorbent assay (ELISA) kit.

[0077] The contents of malondialdehyde (MDA), (GSH), (SOD), and (CAT) in the supernatant were determined using a kit developed in Nanjing (Nanjing, China). All procedures were performed in accordance with the instructions.

[0078] 5. Immunofluorescence staining The paraffin-embedded block of the terminal ileum was cut into 4 μm thin sections, dewaxed, and rehydrated. The sections were then blocked with 5% BSA (room temperature, 20 min). After washing with PBS to remove BSA, the sections were incubated overnight at 4°C with ZO-1, Occludin, and Claudin-1 antibodies, respectively, followed by washing three times with PBS. Secondary antibody was then added to cover the tissue sections, and the sections were incubated at room temperature for 50 min. The sections were then washed three times with PBS in the dark. Finally, 4,6-diamino-2-phenylindole (DAPI) was added and the sections were incubated at room temperature for 10 min to stain the cell nuclei, followed by washing with PBS. Images were observed under a microscope and quantitative analysis was performed.

[0079] 6. Real-time quantitative polymerase chain reaction (RT-qPCR) Total RNA was extracted from small intestinal tissue samples using the Trizol kit, and cDNA was obtained according to the reverse transcription kit instructions. Finally, the expression level of the target gene was determined using a quantitative real-time PCR kit. Data were analyzed using 2... -ΔΔCtThe method is used for computational analysis, and normalization is performed using GAPDH as an internal reference.

[0080] 7. Protein blotting Small intestinal tissue samples were homogenized and centrifuged (10000 g, 5 min) after being added to a lysis buffer containing protease and phosphatase inhibitors, and the supernatant was collected. Protein content was determined using a BCA kit. Protein samples were separated by 10% polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride (PVDF) membrane, and blocked with 5% BSA at room temperature for 1 h. The PVDF membrane was then incubated overnight at 4 °C with antibodies (TLP4, MyD88, NF-kB p65, p-NF-kB p65), followed by incubation with secondary antibody at room temperature for 1 h. After washing three times, the samples were developed and exposed using an ECL chemiluminescence detection kit (Meilunbio, Dalian, China). Finally, the gray values ​​of the target bands were analyzed.

[0081] 8. Gut microbiota analysis based on 16S rRNA sequencing The 16S rRNA sequencing process followed established procedures: Genomic DNA was extracted from fresh fecal samples using the OMEGA Soil DNA Kit (Omega Bio-Tek), DNA concentration was quantified using a Nanodrop spectrophotometer (NC2000, ThermoScientific), and DNA integrity was checked by 0.8% agarose gel electrophoresis. Subsequently, the V3-V4 hypervariable region of the 16S rRNA gene was amplified using a PCR amplification instrument (ABI2720) with primers 338F (5'-ACTCCTACGGAGGCAGCA-3') and 806R (5'-GGACTACCAGGGTATCTAAT-3'). The concentration of PCR products was measured using a microplate reader (BioTek, FLx800) with the Quant-iT PicoGreends DNA assay kit. Sequencing libraries were constructed using the TruSeq Nano DNALT library preparation kit, and sequencing was finally completed using the Illumina NovaSeq platform.

[0082] 9. Quantitative analysis of short-chain fatty acids Small intestinal tissue was homogenized in PBS, the supernatant was mixed, and then combined with phosphate, internal standard, and ether. After standing for 2 minutes, the mixture was centrifuged, and the supernatant was passed through a 0.22 μm organic membrane for measurement. Detection was performed using a Thermo Trace 1310 (Thermo Fisher Scientific, USA) gas chromatography system.

[0083] 10. Statistical analysis.

[0084] Experimental data were statistically analyzed using one-way ANOVA, supplemented by Tukey's multiple comparison test. Results are expressed as mean ± standard deviation (SD). P < 0.05 was considered statistically significant.

[0085] II. Experimental Results 1. Weight and Clinical Disease Index Analysis There was no difference in the weight of newborn rats among the treatment groups before modeling. P >0.05). During the modeling period, the body weight of rats in the blank group maintained a steady increase, and the weight gain was significant by the time the modeling was completed. 24 h after modeling, the body weight gain of rats in the NEC model group was slow, and the body weight change was not significant compared with that 24 h before, with no statistically significant difference. P >0.05). At 48 h and 72 h after modeling, the body weight of rats in the NEC model group even decreased to varying degrees. The body weight of rats in each NEC treatment group was significantly different from that in the control group at the same time point, and all were statistically significant. P <0.05). Except for the blank group, there was no significant difference in rat body weight among the groups.

[0086] Assess disease status in young mice using clinical disease indices. Figure 6 The control group rats had rosy skin, sensitive tactile responses, and could move freely and normally within their cages. However, after three days of hypoxia and cold stimulation, the model group rats had pale, loose skin and sluggish responses. On the third day, the model group rats achieved a clinical disease score of 7.67. Treatment with 3-FL and 3'-SL, alone or in combination, effectively reduced NEC-related clinical disease scores. The clinical disease scores of rats in the 3-FL group, 3'-SL group, and 3-FL+3'-SL group were 4, 4.82, and 3.03, respectively.

[0087] The results indicate that 3-FL, 3'-SL, and 3-FL+3'-SL can all reduce mortality and clinical disease scores in NEC rats, macroscopically alleviating the severity of NEC and demonstrating good preventive and therapeutic effects. The combined treatment group showed better results than the single treatment group.

[0088] 2. Macroscopic condition and histopathological changes of the intestines Macroscopic condition of the intestines, such as Figure 7As shown. Gross morphological observation of rat intestinal tissue revealed that in the control group, the rat intestines exhibited normal color, good elasticity, and no dilation. In the model group, the rat intestines showed a yellow-red discoloration, were easily broken, had significantly reduced elasticity, and some segments showed dilation, sometimes exhibiting a beaded appearance. Rats fed diets of 3-FL, 3'-SL, and 3-FL+3'-SL showed significant improvements in intestinal continuity, color, and dilation. The macroscopic evaluation score of the model group rat intestines was 4.24, significantly higher than that of the normal group of young rat intestines. P <0.05). However, treatment with 3-FL, 3'-SL, and 3-FL+3'-SL reduced the macroscopic score of the rat intestine to 2.70, 3.23, and 2.08, respectively, with the combined group showing the most significant decrease, reducing it to 50.94% of the model group ( P <0.05).

[0089] From the tissue sections of the terminal ileum, the intestinal tissue structure of the control group was normal, and the intestinal villi were neatly and tightly arranged. The intestinal tissue of the model group was severely damaged, with severe villus breakage and even necrosis, and tissue sloughing or even disappearance. The intestinal tissue condition of the young mice in the 3-FL group, 3'-SL group, and 3-FL+3'-SL group was significantly improved, and the intestinal histological score decreased from 3.26 in the model group to 2.46, 2.62, and 1.70, respectively. P <0.05).

[0090] The results above indicate that 3-FL, 3'-SL, and 3-FL+3'-SL treatments all have a protective effect on the intestines, with the combined treatment group showing the best improvement in intestinal damage in NEC pups.

[0091] 3. Cytokine analysis in intestinal tissue Results of cytokines in rat intestinal tissue as follows Figure 8 As shown. Compared with the blank group, the levels of TNF-α, IL-1β, and IL-6 in the intestinal tissue of rats in the model group were significantly increased ( P <0.05%, IL-10 levels decreased significantly ( P <0.05 indicates that the NEC program induced inflammation in rats. In the 3-FL group, 3'-SL group, and 3-FL+3'-SL group, the levels of TNF-α, IL-1β, and IL-6 were significantly decreased ( P <0.05), while IL-10 levels increased significantly, indicating that treatment with 3-FL, 3'-SL, and 3-FL+3'-SL alleviated NEC-induced intestinal inflammation.

[0092] The above results indicate that 3-FL and 3'-SL may alleviate the severity of NEC in newborn mice by inhibiting pro-inflammatory cytokines and promoting anti-inflammatory cytokines, and the combined use of the two has the best effect in relieving intestinal inflammation.

[0093] 4. Analysis of oxidative stress indicators in intestinal tissue Results of cytokines in rat intestinal tissue as follows Figure 9 As shown. Compared with the blank group, the MDA content in the model group was significantly increased ( P <0.05%, the levels of SOD, CAT and GSH were significantly reduced ( P <0.05). However, compared with the corresponding indicators in the model groups, the MDA content in the 3-FL group, 3'-SL group, and 3-FL+3'-SL group was significantly decreased ( P <0.05, SOD, CAT and GSH levels increased significantly ( P <0.05).

[0094] These results suggest that 3-FL and 3'-SL may exert their antioxidant effects by regulating the levels of lipid peroxidation products and antioxidant enzymes, thereby alleviating changes in oxidative stress in NEC rats and reducing intestinal damage in NEC rats.

[0095] 5. Impact on the integrity of the intestinal barrier The expression levels of TJ proteins (ZO-1, Occludin, Claudin-1) and mucin MUC-related mRNAs were detected by q-PCR. Figure 10 The results showed that feeding with 3-FL, 3'-SL, and 3-FL+3'-SL significantly reversed the decrease in mRNA expression levels of two TJ proteins and MUC2 in the rat intestine compared to the model group. P <0.05%, with the combined group showing the most significant increase, where the mRNA levels of ZO-1, Occludin, and MUC increased by 3.45-fold, 4.01-fold, and 3.23-fold, respectively, compared to the model group. While Claudin-1 protein expression was increased in the intestines of rats in the 3'-SL group, the difference from the model group was not significant. P >0.05). Even so, the expression of Claudin-1 protein mRNA in the rat intestine was significantly higher after the combination of 3-FL and 3'-SL than that in the single treatment group.

[0096] The above results indicate that 3-FL and 3'-SL may act on TJ and MUC2 proteins, thereby maintaining the integrity of the rat intestinal barrier structure and alleviating NEC symptoms by increasing the expression levels of TJ and MUC2 proteins.

[0097] 6. Effects on the expression of TLR4 / NF-κB pathway proteins The results are as follows Figure 11 As shown, Western blot results revealed significantly increased expression levels of TLR4 and MyD88 proteins in the intestines of NEC rats. P The p-NF-κB / NF-κB ratio was significantly increased (p < 0.05). This indicates that a large amount of NF-κB is phosphorylated, participating in the inflammatory response process and exacerbating cellular inflammation. Compared with the model group, the expression of TLR4 and MyD88 in the intestines of rats in the three groups after 3-FL, 3'-SL, and combined feeding was significantly reduced (P < 0.05), and the p-NF-κB / NF-κB ratio was also significantly reduced (P < 0.05). P <0.05), especially the 3-FL+3'-SL group, which showed the most significant effect, with the p-NF-κB to NF-κB ratio decreasing by 52.19% compared to the model group. Since TLR4 and MyD88 are key proteins in the NF-κB signaling pathway, this indicates that 3-FL, 3'-SL, and their combined treatment can inhibit the signaling factors TLR4 and MyD88 in the NF-κB pathway and suppress NF-κB phosphorylation.

[0098] The above results suggest that 3-FL, 3'-SL, and their combined action may inhibit inflammation by reducing the expression of the NF-κB signaling pathway, thereby alleviating NEC.

[0099] 7. Effects on protein expression in the Nrf2 / NQO1 pathway The results are as follows Figure 12 As shown, compared with the blank group, the expression levels of Nrf2 and NQO1 in the intestine of rats in the model group were significantly reduced ( P <0.05), while the expression levels of Nrf2 and NQO1 in the rat intestine were significantly increased in the 3-FL, 3'-SL and combined treatment groups. P <0.05), with the combined group showing the most significant increase, increasing by 6.85-fold and 8.12-fold respectively, indicating that 3-FL, 3'-SL and their combined treatment promoted Nrf2 nuclear translocation and increased the expression of the downstream antioxidant protein NQO1.

[0100] The above results indicate that 3-FL, 3'-SL, and their combined action can inhibit the development of oxidative stress through the Nrf2 / NQO1 pathway, thereby reducing NEC damage.

[0101] 8. Effects on short-chain fatty acids The results of the determination of acetic acid, propionic acid, butyric acid and isobutyric acid in the intestinal contents of rats in each group are as follows: Figure 13 As shown, the NEC procedure significantly reduced the levels of acetic acid, propionic acid, and butyric acid in the intestinal contents of mice. P<0.05), decreased by 37.48%, 53.18%, and 56.53%, respectively, with no significant change in isobutyric acid levels. Interventions with 3-FL, 3'-SL, and 3-FL+3'-SL significantly restored the levels of acetic acid, propionic acid, and butyric acid ( ). P <0.05), especially the 3-FL+3'-SL intervention, which increased its content to 1.30 times, 1.49 times and 1.58 times that of the model group, respectively. P <0.05). The results indicate that the combined intervention of 3-FL and 3'-SL has the function of restoring the level of SCFAs in the intestine of NEC rats, which may be one of the ways to alleviate NEC symptoms in rats.

[0102] 9. Analysis of the gut microbiota ALpha diversity results show ( Figure 14 Compared with the control group, the Chao1 index of the gut microbiota in the model group rats was significantly decreased (P<0.05). After intervention with 3-FL, 3'-SL, and 3-FL+3'-SL, the Chao1 index of each group increased to varying degrees, indicating that 3-FL and 3'-SL intervention helped increase the number of species in the gut microbiota of NEC rats. In addition, the NEC induction program significantly reduced the Simpson index of the gut microbiota in rats (P<0.05), however, after intervention with 3-FL, 3'-SL, and 3-FL+3'-SL, the Simpson index of the gut microbiota in rats was significantly increased (P<0.05). The results show that 3-FL and 3'-SL, alone or in combination, can restore the richness and evenness of species in the gut microbiota of NEC rats, improve the gut microbiota dysbiosis in NEC rats, and thus alleviate NEC symptoms.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antioxidant stress composition, characterized in that, The composition consists of the following components: neutral oligosaccharides and sialylated oligosaccharides.

2. The composition according to claim 1, characterized in that, The neutral oligosaccharide is fucose-based lactose, preferably 3-fucosyllactose.

3. The composition according to claim 1 or 2, characterized in that, The sialylated oligosaccharide is 3'-sialyl lactose.

4. The composition according to any one of claims 1-3, characterized in that, The weight ratio of the neutral oligosaccharide to the sialylated oligosaccharide is (0.5-50):

1.

5. A formulated food product, characterized in that, Includes the composition according to any one of claims 1-4.

6. The formulated food according to claim 5, characterized in that, The formulated food is an infant formula or an infant supplement; in solid form, the amount of the composition added is: 0.3-1.75g / 100g of 3'-fucosylated lactose and 0.1-0.28g / 100g of 3'-sialic acid lactose.

7. The formulated food according to claim 5 or 6, characterized in that, The formulated foods include pasta, beverages, instant foods, baked goods, sauces, or functional nutritional supplements.

8. The use of the composition according to any one of claims 1-4 in the preparation of a product for improving necrotizing enterocolitis in newborns; said product being a food, feed additive, drug, or health product.

9. The application according to claim 8, characterized in that, The improvement of neonatal necrotizing enterocolitis includes the following: (1) The ability to improve the degradation of lipid peroxides is weakened; (2) Relieves or restores one or more of the following: intestinal edema, pneumocystis, villus loss, intestinal barrier damage, and tissue necrosis; (3) Inhibit the release of inflammatory factors promoted by the TLR4 / NF-κB pathway; (4) Restore the content of short-chain fatty acids in the intestine.

10. The application according to claim 9, characterized in that, The reduced ability to improve lipid peroxide degradation includes the following: (1) Restore the activity and content of antioxidant enzymes in the intestine; (2) Reduce the content of lipid oxidation product MDA; (3) Inhibit the development of oxidative stress through the Nrf2 / NQO1 pathway.

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