Application of lysolecithin in preparation of product for protecting immune stress of pigs

Abstract

The invention discloses an application of lysolecithin in preparation of a product for protecting immune stress of pigs, the body health of the pigs in an immune stress scene can be protected by feeding the pigs with the product containing the lysolecithin, and the damage of intestinal tracts and livers of the pigs caused by lipopolysaccharide stimulation can be relieved, so that the immune stress of the pigs can be protected. The purpose of preventing the pig intestinal injury and / or liver injury can be achieved, the purpose of treating the pig intestinal injury and / or liver injury can also be achieved, and the pig intestinal injury and / or liver can be protected.

Description

Technical Field

[0001] This invention relates to the field of pig feed products technology, specifically to the application of lysophosphatidylcholine in the preparation of products that protect against immune stress in pigs. Background Technology

[0002] Immune stress in pigs is a common problem in pig production. It activates the immune system to synthesize and secrete large amounts of cytokines (such as IL-6 and TNF-α), which leads to severe damage to pig tissues and organs and a decline in growth performance, causing significant economic losses to pig production.

[0003] For example, in large-scale intensive pig farming, vaccination and stimulation from various pathogens can trigger immune stress in pigs, thereby activating the immune system and causing immune cells to release excessive amounts of inflammatory factors such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. Studies have reported that the excessive release of inflammatory factors can cause damage and dysfunction in pigs' liver and intestines, and the immune stress in pigs reduces the economic benefits of pig farming.

[0004] Lysophosphatidylcholine (LPC) is a monoacylphosphatidylcholine derivative produced by the hydrolysis of fatty acyl groups in phospholipid molecules catalyzed by phospholipase. Studies have shown that lysophosphatidylcholine not only emulsifies fats and promotes the body's absorption of fats, but also plays a variety of biological functions. However, research on the protective effect of lysophosphatidylcholine against immune stress in pigs is relatively limited. Summary of the Invention

[0005] The purpose of this invention is to provide the application of lysophosphatidylcholine in the preparation of products that protect against porcine immune stress.

[0006] Feeding pigs products containing lysophosphatidylcholine can protect their health during immune stress.

[0007] Furthermore, the present invention aims to provide the use of lysophosphatidylcholine in the preparation of products that protect the porcine intestine and / or liver.

[0008] Furthermore, the present invention aims to provide the use of lysophosphatidylcholine in the preparation of products for the prevention or treatment of intestinal and / or liver damage in pigs.

[0009] As a feed nutrition product, lysophosphatidylcholine can be used in the daily life of pig farming to prevent and treat intestinal and / or liver damage in pigs, thereby protecting the pig's intestines and / or liver.

[0010] Furthermore, the intestinal and / or liver damage is induced by immune stress.

[0011] Furthermore, the intestinal and / or liver damage is induced by lipopolysaccharide.

[0012] Furthermore, in any of the above applications, the product is any one of a drug, feed additive, or feed.

[0013] The drugs, feed additives, and feeds contain lysophosphatidylcholine. Lysophosphatidylcholine can be prepared into swine drug products for feeding pigs, or it can be combined with other pharmaceutical excipients to prepare swine drug products for feeding pigs; it can also be prepared into swine feed additive products for feeding pigs, or it can be combined with other feed excipients to prepare swine feed additive products for feeding pigs; it can also be prepared into swine feed products for feeding pigs, or it can be combined with other feed excipients to prepare swine feed products for feeding pigs.

[0014] Another objective of this invention is to provide the application of lysophosphatidylcholine in the preparation of products that regulate the Teff / Treg balance in the porcine intestine via the JAK / STAT signaling pathway.

[0015] In this invention, lysophosphatidylcholine can alleviate intestinal damage caused by lipopolysaccharide by regulating the Teff / Treg balance in the porcine intestine through the JAK / STAT signaling pathway, and can also alleviate intestinal damage in porcine intestine by regulating the secretion of Teff / Treg cell-related factors.

[0016] Another object of the present invention is to provide the application of lysophosphatidylcholine in the preparation of products that inhibit the polarization of porcine liver macrophages through glycolysis and the citric acid cycle signaling pathway.

[0017] Currently, there are few research reports on whether lysophosphatidylcholine can regulate porcine liver macrophage polarization and maintain porcine liver function through glycolysis and the citric acid cycle signaling pathway.

[0018] This invention revealed that lipopolysaccharide (LPS) stimulation significantly increased the mRNA expression levels of polarization-related factors in M1 and M2 macrophages in porcine liver. Lysolecithin significantly alleviated the increase in LPS-induced M1 macrophage polarization-related factor mRNA expression in porcine liver. LPS stimulation significantly increased the mRNA expression levels of hexokinase 2 (HK2) during glycolysis and the citrate cycle, and significantly decreased the mRNA expression levels of pyruvate kinase (PK), pyruvate dehydrogenase (PDH), citrate synthase (CS), isocitrate dehydrogenase (IDH), and dihydrolipoamide succinyl (DLST). Lysolecithin alleviated the LPS-induced increase in HK2 mRNA expression in the liver.

[0019] This invention reveals that lysophosphatidylcholine alleviates liver inflammation by regulating glycolysis and the citric acid cycle in porcine liver macrophages. Lysophosphatidylcholine can alleviate lipopolysaccharide (LPS)-induced liver macrophage polarization through glycolysis and the citric acid cycle signaling pathway. Lysophosphatidylcholine alleviates the decrease in mRNA and protein levels of LPS-related factors in the liver mTOR pathway, and it can inhibit the polarization of porcine liver macrophages towards the M1 direction. Lysophosphatidylcholine has a regulatory effect on LPS-induced liver macrophage polarization and can reduce liver damage by decreasing the expression of pro-inflammatory cytokine mRNA in M1 macrophages.

[0020] Another object of the present invention is to provide the use of lysophosphatidylcholine in the preparation of regulators having any one or more of the following functions:

[0021] (1) Increase villus height and villus height / crypt depth in the jejunum and ileum of pigs;

[0022] (2) Increase the activity of maltase in the jejunum, sucrase and maltase in the ileum of pigs;

[0023] (3) It alleviates the decrease in the mRNA expression level of porcine jejunum ZO-1 caused by lipopolysaccharide stimulation and increases the expression level of porcine jejunum Occludin protein;

[0024] (4) It can alleviate the decrease in mRNA expression levels of TNF-α, IFN-γ, IL-4, IL-17A and GATA3 in the jejunum caused by lipopolysaccharide stimulation, and alleviate the increase in mRNA expression levels of IL-17F, TGF-β and FOXP3 in the jejunum caused by lipopolysaccharide stimulation.

[0025] (5) It alleviates the decrease in mRNA expression of IL-4, GATA3 and FOXP3 in the ileum caused by lipopolysaccharide stimulation, and alleviates the increase in mRNA expression of IL-17A and IL-17F in the ileum caused by lipopolysaccharide stimulation.

[0026] (6) Increase the mRNA expression levels of JAK3, STAT4, STAT5A in the jejunum and STAT4 in the ileum of pigs;

[0027] (7) Relieves the increase in serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) in pigs caused by lipopolysaccharide stimulation;

[0028] (8) Alleviate the increase in mRNA expression of polarization-related factors in porcine liver M1 macrophages caused by lipopolysaccharide stimulation;

[0029] (9) Alleviates the increase in HK2 mRNA expression in pig liver caused by lipopolysaccharide stimulation;

[0030] (10) Alleviate the decrease in mRNA expression levels of S6K1, hypoxia-inducible factor (HIF)-1α and silencing information regulator (SIRT1) in porcine liver caused by lipopolysaccharide stimulation;

[0031] (11) Relieves the decrease in expression levels of mTOR and p-mTOR proteins in pig liver caused by lipopolysaccharide stimulation.

[0032] The regulator is any one of drugs, feed additives, or feed, and the regulator contains lysophosphatidylcholine.

[0033] Furthermore, the application method of any of the above-mentioned applications is as follows: feeding pigs a basal diet supplemented with lysophosphatidylcholine, wherein the amount of lysophosphatidylcholine added to the basal diet is 0.01%.

[0034] The basic diet can be formulated by the pigs themselves according to their nutritional needs, or commercially available basic diets can be purchased directly.

[0035] The beneficial effects of this invention are:

[0036] This invention provides the application of lysophosphatidylcholine in the preparation of products that protect against immune stress in pigs. By feeding pigs products containing lysophosphatidylcholine, the health of pigs under immune stress can be protected, and the damage to the pig intestines and liver caused by lipopolysaccharide stimulation can be alleviated. This invention can achieve the purpose of preventing and / or treating intestinal and / or liver damage in pigs, thus achieving the protection of the pig intestines and / or liver.

[0037] This invention provides an innovative technical solution for improving the health of pigs by alleviating immune stress through nutritional regulation. In the face of the era's development requirements of "antibiotic ban" in feed products and "antibiotic reduction and restriction" in the breeding process, the technical solution of this invention is of great significance. Attached Figure Description

[0038] Figure 1 Morphological observation of the effect of lysophosphatidylcholine on the morphology of the intestines (jejunum and ileum) of piglets stimulated by LPS (100×).

[0039] Figure 2 This is a data analysis graph showing the effect of lysophosphatidylcholine on the expression of tight junction proteins in the intestinal jejunum of piglets stimulated by LPS.

[0040] Figure 3 This is a data analysis graph showing the effect of lysophosphatidylcholine on the expression of tight junction proteins in the intestinal (ileum) of piglets stimulated by LPS.

[0041] Figure 4Morphological observation of the effect of lysophosphatidylcholine on the liver morphology of piglets stimulated by LPS (400×). (In this figure: a) disappearance of hepatocyte nuclei; b) inflammatory cell infiltration; c) nuclear pyknosis.

[0042] Figure 5 This is a data analysis figure showing the effect of lysophosphatidylcholine on the expression of mTOR and LKB1 pathway-related factor proteins in piglet liver macrophages stimulated by LPS. Detailed Implementation

[0043] The following are specific embodiments of the present invention, which further describe the technical solution of the present invention. However, the scope of protection of the present invention is not limited to these embodiments. All changes or equivalent substitutions that do not depart from the concept of the present invention are included within the scope of protection of the present invention.

[0044] Lipopolysaccharide (LPS), a major component of the cell wall of Gram-negative bacteria, is a typical endotoxin. In piglet studies, intraperitoneal injection of LPS effectively induces systemic inflammatory responses, leading to intestinal damage and dysfunction. It can decrease intestinal barrier function, impair liver function and structure, and significantly increase the expression of pro-inflammatory cytokine mRNA in the liver. LPS-induced immune stress results in tissue damage.

[0045] In this embodiment of the invention, an immune stress model was established by injecting LPS into the peritoneum of pigs to investigate the protective effect of lysophosphatidylcholine (hereinafter referred to as LPC) on immune stress-induced intestinal and liver damage in pigs, and to investigate the regulatory effect of lysophosphatidylcholine on LPS-induced intestinal and liver damage in pigs.

[0046] Experiment 1: The regulatory effect of lysophosphatidylcholine on LPS-induced intestinal injury in piglets

[0047] The gut is not only the main site for the digestion and absorption of nutrients in pigs, but also the largest immune organ in their bodies. In intensive farming, piglets are exposed to vaccinations and various pathogens, which can cause immune stress, thereby activating the immune system, releasing excessive inflammatory factors, leading to intestinal homeostasis disorder, and affecting the health of piglets.

[0048] This experiment established an intestinal immune stress model by injecting LPS into the peritoneum of pigs, and explored whether lysophosphatidylcholine (LPC) could alleviate the intestinal Teff / Treg imbalance caused by LPS through the JAK / STAT signaling pathway, providing an innovative technical solution for studying the protective effect of lysophosphatidylcholine (LPC) on the pig intestine.

[0049] 1. Materials and Methods

[0050] 1.1 Test Materials

[0051] The experiment used a 2×2 factorial design, resulting in the following four treatment groups:

[0052] (1) Control group (group code Ctrl): fed with basal diet;

[0053] (2) Lysolecithin group (group code LPC): fed with the experimental diet;

[0054] (3) LPS group (group code LPS): fed with basal diet;

[0055] (4) Lysophosphatidylcholine + LPS group (group code LPC+LPS): fed with experimental diet.

[0056] The basal diet is a complete diet for pigs. The basal diet can be formulated according to the nutritional needs of the experimental animals, or commercially available basal diets can be purchased directly.

[0057] The experimental diet was prepared by adding 0.01% lysophosphatidylcholine by mass to the basic diet and mixing them evenly; that is, for example, adding 0.01 kg of lysophosphatidylcholine to 100 kg of the basic diet and mixing them evenly to prepare the experimental diet.

[0058] Twenty-four Duroc × Landrace × Large White piglets (28±1 days old, average initial weight 8.62±0.63 kg) were randomly divided into four treatment groups, corresponding to the control group (Ctrl), lysophosphatidylcholine group (LPC), LPS group (LPS), and lysophosphatidylcholine + LPS group (LPC+LPS)). Each treatment group was fed according to the feeding protocol of the corresponding four treatment groups. There were six replicates in each treatment group, with one pig per replicate.

[0059] The experiment lasted 28 days. After feeding the piglets in all four treatment groups for 28 days, the following treatments were administered:

[0060] (1) Control group (group code Ctrl): 100 μg / kg body weight of physiological saline was injected into the peritoneum of pigs;

[0061] (2) Lysophosphatidylcholine group (group code LPC): 100 μg / kg body weight of physiological saline was injected into the peritoneum of pigs;

[0062] (3) LPS group (group code LPS): 100 μg / kg body weight of LPS was injected into the peritoneum of pigs;

[0063] (4) Lysophosphatidylcholine + LPS group (group code LPC+LPS): 100 μg / kg body weight of LPS was injected into the peritoneum of pigs.

[0064] Blood was collected 4 hours after the above treatment was completed, and intestinal samples were taken from anesthetized and slaughtered piglets for testing.

[0065] LPC was purchased from Shanchuan Biotechnology (Wuhan) Co., Ltd.; LPS, Escherichia coli serotype O55:B5, with an active ingredient content of >99%, was purchased from Sigma.

[0066] 1.2 Feeding and Management

[0067] The experiment was conducted at the pig farm of Auden Agricultural and Animal Husbandry Technology Co., Ltd. in Tianmen City, Hubei Province. Piglets had free access to feed and water.

[0068] 1.3 Sample Collection

[0069] Four hours after injection of LPS or saline, blood samples were collected and placed in heparin sodium vacuum blood collection tubes. The blood samples were then centrifuged and stored at -80°C for subsequent analysis. Four hours after blood collection, piglets were injected with sodium pentobarbital and then slaughtered. The jejunum and ileum were dissected and immediately placed on ice, then rinsed with cold phosphate-buffered saline (PBS). A 3 cm segment of the intestine was cut from the middle and fixed with 4% paraformaldehyde for intestinal morphology observation. The remaining portion was cut open with scissors, rinsed with cold saline to remove contents, and the inner surface of the intestinal mucosa was blotted dry with filter paper. The intestinal mucosa was then carefully scraped off with a clean glass slide, mixed thoroughly, and quickly placed into 1.5 mL sterile EP tubes. The tubes were immediately placed in liquid nitrogen for rapid freezing and then transferred to a -80°C freezer for later analysis.

[0070] 1.4 Detection Indicators

[0071] 1.4.1 Morphological observation of intestinal tissue

[0072] The jejunum and ileum samples were first dehydrated and embedded, then prepared into 5 μm paraffin sections. After HE staining and resin mounting, the sections were finally observed morphologically using the HPIAS-1000 high-resolution color pathology image and text report analysis system. The villus height and crypt depth were measured, and the ratio of villus height to crypt depth was calculated.

[0073] 1.4.2 Determination of intestinal mucosal protein, DNA and RNA content

[0074] The intestinal mucosa was placed in a mortar and ground into powder under liquid nitrogen conditions. A certain amount was accurately weighed and added to physiological saline at a ratio of weight (g):volume (L) = 1:9 for homogenization. The supernatant was separated by low-temperature centrifugation at 3000 rpm / min. Protein content was determined by the Coomassie brilliant blue method, and DNA and RNA content were determined by spectrophotometry.

[0075] 1.4.3 Determination of intestinal mucosal disaccharidase activity

[0076] The method for preparing 10% intestinal mucosal homogenate is the same as above. The intestinal disaccharidase activity was measured using a disaccharidase kit (lactase, sucrase, and maltase) purchased from Nanjing Jiancheng Bioengineering Institute. The measurement method was strictly performed according to the instructions. The enzyme activity in the intestine is expressed as U / mg protein.

[0077] 1.4.4 mRNA expression level determination

[0078] The study measured the secretion of cytokines (IL-10 and TGF-β) in Treg cells from the jejunum and ileum of piglets, the secretion of cytokines (IL-6, IFN-γ, IL-1β, and TNF-α) in TH1 cells, the secretion of cytokines (IL-4) in TH2 cells, and the secretion of cytokines (IL-17A and IL-17F) in TH1, TH2, TH17, and Treg cells. The mRNA expression levels of nuclear transcription factors (T-bet, GATA3, RORγt, and FOXP3) in TH1, TH2, TH17, and Treg cells were also measured. The mRNA expression levels of JAK / STAT signaling pathway-related factors (JAK1, JAK2, JAK3, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6) were also determined. Primer sequences are shown in Table 1. Real-time PCR data analysis used β-actin as an internal reference gene.

[0079] Table 1 Primer sequence information

[0080]

[0081] Continued from Table 1

[0082]

[0083] 1.4.5 Protein Expression Level Measurement

[0084] Wesernt blot was used to determine the tight junction proteins (Occludin and ZO-1) in the piglet intestine, with β-actin as an internal control. Information on primary and secondary antibodies is shown in Table 2. Protein expression levels were expressed as the ratio of the band intensity of the target protein to the band intensity of the corresponding internal control.

[0085] Table 2. Description of primary and secondary antibodies used in Western blot analysis.

[0086]

[0087] 1.5 Statistical Analysis

[0088] All the experimental data were analyzed by two-way ANOVA using SPSS 20.0 statistical software. The main effects of the model included the addition of lysophosphatidylcholine, LPS treatment, and the interaction between the two. Duncan's multiple comparison was used. The results were expressed as mean ± standard error. A significant difference was indicated by P < 0.05, and a trend of significance was indicated by 0.05 < P < 0.10.

[0089] 2 Experimental results

[0090] 2.1 Effects of lysophosphatidylcholine on intestinal morphology of LPS-stimulated piglets

[0091] As Figure 1 can be seen, the villus structures of the jejunum and ileum in the Ctrl group and the LPC group were intact and arranged closely. LPS stimulation caused sparse and disordered arrangement of villi in the jejunum and ileum, with a large number of villi broken and shed. Compared with the LPS group, the villi in the LPC + LPS group were arranged more closely, the tight junctions were clearer, and a small amount of villi were shed.

[0092] As shown in Table 3, LPS stimulation significantly decreased the villus height and villus height / crypt depth of the jejunum and ileum (P < 0.05), and there was a downward trend in the crypt depth of the jejunum (0.05 < P < 0.10). There was an interaction between the effect of LPC on the crypt depth of the ileum and LPS stimulation (P < 0.05), that is, LPC alleviated the decrease in the crypt depth of the ileum caused by LPS stimulation. LPC increased the villus height and villus height / crypt depth of the jejunum and ileum of piglets (P < 0.05).

[0093] 2.2 Effects of lysophosphatidylcholine on intestinal protein, DNA, and RNA contents of LPS-stimulated piglets

[0094] As shown in Table 4, LPS stimulation significantly decreased the RNA / DNA of the ileum and significantly increased the protein / DNA (P < 0.05).

[0095] 2.3 Effects of lysophosphatidylcholine on intestinal disaccharidase activities of LPS-stimulated piglets

[0096] As shown in Table 5, LPS stimulation significantly decreased the activity of sucrase in the ileum (P < 0.05). LPC significantly increased the activities of maltase in the jejunum and sucrase and maltase in the ileum of piglets (P < 0.05).

[0097] 2.4 Effects of lysophosphatidylcholine on intestinal tight junction protein expression of LPS-stimulated piglets

[0098] As Figure 2-3It was found that LPS stimulation significantly reduced the mRNA expression levels of jejunal Occludin and ZO-1 (P<0.05), while LPC increased the mRNA expression levels of jejunal Occludin and ileal ZO-1 (P<0.05). The effect of LPC on jejunal ZO-1 mRNA expression was interactive with that of LPS (P=0.068), meaning that LPC alleviated the decrease in jejunal ZO-1 mRNA expression caused by LPS stimulation (P<0.05).

[0099] 2.5 Effects of lysophosphatidylcholine on mRNA expression levels of LPS-stimulated intestinal Teff / Treg balance-related indicators in piglets

[0100] Table 6 shows that LPS stimulation significantly increased the mRNA expression levels of IL-6, FOXP3, and TGF-β in the jejunum of piglets (P<0.05) and significantly decreased the mRNA expression level of TNF-α (P<0.05). LPC interacted with LPS in reducing the mRNA expression levels of TNF-α, IL-4, IL-17A, IL-17F, TGF-β, GATA3, and FOXP3 in the jejunum (P<0.05). Specifically, LPC alleviated the decrease in TNF-α, IL-4, IL-17A, and GATA3 mRNA expression caused by LPS stimulation (P<0.05) and the increase in IL-17F, TGF-β, and FOXP3 mRNA expression caused by LPS stimulation (P<0.05), while no such effect was observed in pigs injected with saline.

[0101] Table 6 shows that LPS stimulation significantly increased the mRNA expression level of FOXP3 in the ileum (P<0.05) and significantly decreased the mRNA expression levels of TNF-α, IL-4, and IL-10 in the ileum (P<0.05). LPC interacted with LPS to reduce the mRNA expression levels of IL-4, IL-17A, IL-17F, GATA3, and FOXP3 in the ileum (P<0.05), meaning that LPC could alleviate the decrease in IL-4, GATA3, and FOXP3 mRNA expression levels caused by LPS stimulation (P<0.05) and the increase in IL-17A and IL-17F mRNA expression levels caused by LPS stimulation (P<0.05), while no such effect was observed in pigs injected with saline.

[0102] 2.6 Effects of lysophosphatidylcholine on the expression levels of JAK / STAT signaling pathway-related molecules in the intestine of piglets stimulated by LPS

[0103] As shown in Table 7, LPS stimulation significantly decreased the mRNA expression level of JAK2 in the jejunum of piglets (P<0.05), and the mRNA expression level of STAT2 showed a downward trend (0.05<P<0.10). LPC tended to increase the mRNA expression levels of JAK3, STAT4, and STAT5A in the jejunum (0.05<P<0.10). There was no interaction between LPC and LPS stimulation on the mRNA expression of factors related to the JAK / STAT signaling pathway in the jejunum.

[0104] As shown in Table 7, LPC tended to increase the mRNA expression level of STAT4 in the ileum (0.05<P<0.10). There was no interaction between LPC and LPS stimulation on the mRNA expression of factors related to the JAK / STAT signaling pathway in the ileum.

[0105] Table 3 Effects of Lysophosphatidylcholine on the Intestinal Morphology of LPS-Stimulated Piglets

[0106]

[0107] Note: ab Different letters in the same row indicate significant differences (P<0.05). The P value in the diet column represents the difference between the basal diet and the experimental diet, and the P value in the LPS column represents the difference between saline injection and LPS injection.

[0108] Table 4 Effects of Lysophosphatidylcholine on the Contents of Intestinal Protein, DNA, and RNA in LPS-Stimulated Piglets

[0109]

[0110] Note: The P value in the diet column represents the difference between the basal diet and the experimental diet, and the P value in the LPS column represents the difference between saline injection and LPS injection.

[0111] Table 5 Effects of Lysophosphatidylcholine on the Activities of Intestinal Disaccharidases in LPS-Stimulated Piglets

[0112]

[0113] Note: The P value in the diet column represents the difference between the basal diet and the experimental diet, and the P value in the LPS column represents the difference between saline injection and LPS injection.

[0114] Table 6 Effects of Lysophosphatidylcholine on the mRNA Expression Levels of Intestinal Teff / Treg Balance-Related Indicators in LPS-Stimulated Piglets

[0115]

[0116] Note: abDifferent letters in the same row indicate significant differences (P<0.05). In the diet column, the P value represents the difference between the basal diet and the experimental diet, and in the LPS column, the P value represents the difference between the injection of physiological saline and the injection of LPS.

[0117] Continued from Table 6

[0118]

[0119] Note: ab Different letters in the same row indicate significant differences (P<0.05). In the diet column, the P value represents the difference between the basal diet and the experimental diet, and in the LPS column, the P value represents the difference between the injection of physiological saline and the injection of LPS.

[0120] Table 7. Effects of lysophosphatidylcholine on the mRNA expression levels of JAK / STAT signaling pathway-related molecules in the intestinal tract of piglets stimulated by LPS.

[0121]

[0122] Note: In the diet column, the P-value represents the difference between the basal diet and the experimental diet, and in the LPS column, the P-value represents the difference between the injection of physiological saline and the injection of LPS.

[0123] Continued from Table 7

[0124]

[0125] Note: In the diet column, the P-value represents the difference between the basal diet and the experimental diet, and in the LPS column, the P-value represents the difference between the injection of physiological saline and the injection of LPS.

[0126] 3. Conclusion

[0127] The intestines, as vital digestive and absorptive organs in pigs, not only play a crucial role in the breakdown and absorption of nutrients but also form an important barrier against the invasion of pathogens and toxins. When the intestines are stimulated by external factors such as pathogenic microorganisms and toxins, the tight junctions between intestinal epithelial cells can be disrupted, leading to impaired intestinal barrier function and ultimately affecting the overall health of the pig.

[0128] Intestinal villus height and crypt depth are key morphological indicators for assessing intestinal physiological status, and the villus height / crypt depth ratio comprehensively reflects intestinal mucosal structure and function. Studies have shown that LPS stimulation significantly reduces villus height and the villus height / crypt depth ratio in the jejunum of piglets, indicating impaired intestinal morphology. In the embodiments of this invention, LPS stimulation caused villus arrangement disorder and breakage in the jejunum and ileum of piglets, resulting in a decrease in intestinal villus height and villus height / crypt depth. LPC alleviated the villus breakage and crypt depth decrease caused by LPS stimulation in piglets. LPC significantly increased villus height and the villus height / crypt depth ratio in the jejunum and ileum. The results indicate that adding LPC to the pig diet has a protective effect on the intestinal morphology and structure of pigs.

[0129] Protein, DNA, and RNA are important indicators reflecting intestinal growth, development, and damage repair. Changes in their relative contents can reflect the physiological state of intestinal cells. The RNA / DNA ratio assesses cellular protein synthesis activity, while the protein / DNA ratio reflects the level of intracellular protein deposition. Studies have shown that LPS stimulation significantly decreases the protein / DNA ratio in the ileum of piglets, weakening protein synthesis and carbohydrate breakdown and utilization. In the experiments of the embodiments of this invention, LPS stimulation significantly reduced the ileal RNA / DNA ratio and significantly increased the protein / DNA ratio.

[0130] Disaccharides (such as sucrose, maltose, and lactose), as important carbohydrates, must be hydrolyzed into monosaccharides by corresponding disaccharidases before they can be effectively absorbed by intestinal epithelial cells. Therefore, the activity level of intestinal disaccharidases is an important indicator reflecting the digestive and absorptive capacity of the intestine. In the experiments of this invention, LPS stimulation reduced the activity of sucrase in the ileum. LPC significantly increased the activities of maltase in the jejunum, sucrase in the ileum, and maltase. The results indicate that LPC can increase the disaccharidase activity in the intestine of piglets.

[0131] Tight junction proteins between intestinal epithelial cells are a crucial physical basis for the intestinal physical barrier. These proteins effectively seal the gaps between intestinal epithelial cells by forming continuous intercellular junction complexes, constituting the first line of defense against the invasion of pathogenic microorganisms and toxins. In the experiments of this invention, LPS stimulation reduced the mRNA expression levels of Occludin and ZO-1 in the jejunum, while LPC increased the expression level of Occludin protein in the jejunum. LPC alleviated the decrease in ZO-1 mRNA expression in the jejunum caused by LPS stimulation. The results indicate that LPC can improve the intestinal barrier function of piglets.

[0132] The intestine is the largest immune organ in pigs, rich in immune cells. The balance of Teff / Treg cells plays a crucial role in maintaining intestinal homeostasis and health; an imbalance can lead to related diseases. In the experiments of this invention, LPS stimulation increased the mRNA expression of the nuclear transcription factor FOXP3 in jejunal and ileal Treg cells and decreased the mRNA expression of TNF-α in Teff cells. LPC interacted with LPS to reduce the mRNA expression of IL-4, IL-17A, IL-17F, GATA3, and FOXP3 in the jejunum and ileum. LPC alleviated the LPS-induced decrease in IL-4, IL-17A, and GATA3 mRNA expression in the jejunum and ileum, and also alleviated the LPS-induced increase in IL-17F and FOXP3 mRNA expression. These results suggest that LPC may alleviate intestinal damage by regulating the secretion of Teff / Treg cell-related factors.

[0133] The JAK / STAT signaling pathway plays a crucial role in cell growth, differentiation, proliferation, and immune function regulation. In livestock production, JAK / STAT signaling is considered to have a significant regulatory effect on animal growth and development. In the experiments of this invention, LPS stimulation significantly reduced the mRNA expression level of JAK2 in the jejunum of piglets, and the mRNA expression of STAT2 showed a decreasing trend. LPC increased the mRNA expression levels of JAK3, STAT4, and STAT5A in the jejunum, while the mRNA expression level of STAT4 in the ileum showed an increasing trend. The results indicate that the JAK / STAT signaling pathway can regulate the intestinal Teff / Treg cell homeostasis.

[0134] In conclusion, adding lysophosphatidylcholine (LPC) to the basal diet of pigs can alleviate intestinal damage caused by immune stress, protect the pig's intestines, and safeguard the pig's health under immune stress.

[0135] Experiment 2: Regulatory effect of lysophosphatidylcholine on LPS-induced liver injury in piglets

[0136] The liver is a vital metabolic organ and detoxification site. When the liver is attacked by pathogens, toxins, or under stress, it triggers a massive release of inflammatory cytokines, leading to an inflammatory response and impaired liver function. LPS, a major component of the outer membrane of Gram-negative bacteria, can cause a large release of pro-inflammatory cytokines, resulting in liver inflammation and dysfunction.

[0137] Macrophages are the most abundant immune cells in the liver. They play a crucial role in clearing cellular debris, eliminating pathogens, regulating inflammatory responses, and maintaining liver homeostasis. M1 macrophages secrete large amounts of pro-inflammatory cytokines to eliminate host pathogens. M2 macrophages secrete anti-inflammatory factors to suppress inflammatory responses. Macrophage polarization is closely related to liver inflammation.

[0138] Macrophage polarization is closely related to glycolysis and the citric acid cycle. Glycolysis and the citric acid cycle are regulated by signaling pathways such as mammalian target of rapamycin (mTOR) and hepatic kinase B1 (LKB1). Under conditions of insufficient cellular energy, LKB1 and its downstream silencing signaling regulator (SIRT1) can inhibit cellular glycolysis by suppressing mTOR activity and the expression of related glycolytic genes, thereby promoting the citric acid cycle and oxidative phosphorylation.

[0139] Currently, there are few research reports on whether lysophosphatidylcholine can regulate macrophage polarization and maintain liver function through glycolysis and the citric acid cycle signaling pathway.

[0140] This experiment established a liver immune stress model by injecting LPS into the peritoneum of pigs to explore whether lysophosphatidylcholine (LPC) can alleviate liver macrophage polarization caused by LPS stimulation through glycolysis and the citric acid cycle signaling pathway, providing an innovative technical solution for studying the protective effect of lysophosphatidylcholine (LPC) on pig liver.

[0141] 1. Materials and Methods

[0142] 1.1 Test Materials

[0143] Same as Experiment 1: Regulatory effect of lysophosphatidylcholine on LPS-induced intestinal injury in piglets, "1.1 Experimental materials".

[0144] 1.2 Feeding and Management

[0145] Same as Experiment 1: The regulatory effect of lysophosphatidylcholine on LPS-induced intestinal damage in piglets, "1.2 Feeding and management".

[0146] 1.3 Sample Collection

[0147] Four hours after injection of LPS or saline, blood samples were collected and placed in heparin sodium vacuum blood collection tubes. The blood samples were then centrifuged and stored at -80°C for subsequent analysis. Four hours after blood collection, piglets were injected with sodium pentobarbital and then slaughtered. The livers were dissected, immediately placed on ice, rinsed with cold phosphate-buffered saline (PBS), and a 0.5 cm section was cut off. 3Small pieces were fixed with 4% paraformaldehyde for liver observation. Another portion of the liver was wrapped in aluminum foil and rapidly frozen in liquid nitrogen, then stored at -80°C until further analysis.

[0148] 1.4 Detection Indicators

[0149] 1.4.1 Observation of liver morphology

[0150] Liver tissue morphology sections were stained with hematoxylin and eosin (HE), and the main steps included dewaxing, staining, dehydration and clearing, and blocking.

[0151] 1.4.2 Serum Biochemical Indicators Measurement

[0152] The serum total protein (TP), albumin (ALB), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma-glutamyl transferase (GGT) of piglets were measured using a fully automated serum biochemical analyzer.

[0153] 1.4.3 mRNA expression level determination

[0154] The mRNA expression levels of key genes (IL-6, IL-1β, TNF-α, iNOS, CD80, and CD86) in liver M1 macrophages, key genes (IL-10, TGF-β, Arg-1, and CD206) in liver M2 macrophages, key glycolytic enzymes (HK2, PFK, and PK), key citrate cycle enzymes (PDH, CS, IDH, and DLST), and key factors in the mTOR and LKB1 signaling pathways (mTOR, S6K1, 4EBP1, HIF-1α, LKB1, and SIRT1) were detected using real-time quantitative PCR. Primer sequences are shown in Table 1. β-actin was used as an internal reference gene for Real-time PCR data analysis.

[0155] 1.4.4 Protein Expression Level Measurement

[0156] The expression levels of mTOR, p-mTOR, 4EBP1, p-4EBP1, and HIF-1α proteins in piglet liver were determined using Western blot, with β-actin as an internal control. Information on primary and secondary antibodies is provided in Table 2. Protein expression levels are expressed as the ratio of the band intensity of the target protein to the band intensity of the corresponding internal control.

[0157] 1.5 Statistical Analysis

[0158] Two-way ANOVA was performed on all experimental data using SPSS 20.0 statistical software. The main effects of the model included the addition of LPC, LPS treatment, and the interaction between the two. Duncan's multiple comparison was used. The results were expressed as mean ± standard error. A significant difference was indicated by P < 0.05, and a trend of significance was indicated by 0.05 < P < 0.10.

[0159] 2 Experimental results

[0160] 2.1 Effects of lysophosphatidylcholine on the liver morphology of LPS-stimulated piglets

[0161] As Figure 4 shown, the hepatic cell structure of pigs injected with normal saline was clear, and the hepatic cords were regular. However, the liver of piglets in the LPS injection group was significantly damaged, manifested as the disappearance of hepatic nuclei, infiltration of inflammatory cells, and karyopyknosis. The addition of LPC in the diet significantly alleviated the liver damage caused by LPS stimulation.

[0162] 2.2 Effects of lysophosphatidylcholine on the serum biochemical indexes of LPS-stimulated piglets

[0163] As shown in Table 8, LPS stimulation significantly decreased the contents of total protein (TP) and albumin (ALB) in the serum of piglets (P < 0.05), significantly increased the contents of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) (P < 0.05), and there was an upward trend in the content of serum glutamyl transpeptidase (GGT) (0.05 < P < 0.10). The effect of LPC on the contents of serum AST, ALT, and ALP interacted with LPS stimulation (P < 0.05), and LPC alleviated the increase in the contents of serum AST, ALT, and ALP caused by LPS stimulation (P < 0.05).

[0164] 2.3 Effects of lysophosphatidylcholine on the mRNA expression levels of molecules related to macrophage polarization in the livers of LPS-stimulated piglets

[0165] As shown in Table 9, LPS stimulation significantly increased the mRNA expression levels of IL-1β, IL-6, TNF-α, IFN-γ, iNOS, and CD80, which are related to M1 macrophage polarization in the liver (P<0.05). The mRNA expression level of CD86 showed an upward trend (0.05<P<0.10). The mRNA expression levels of IL-10, TGF-β, and Arg-1, which are related to M2 macrophage polarization, were significantly increased (P<0.05). There was an interaction between LPC and LPS stimulation on the mRNA expression levels of IL-1β, IL-6, TNF-α, IFN-γ, iNOS, CD80, and CD86 in the liver (P<0.05). LPC alleviated the increase in the mRNA expression levels of IL-1β, IL-6, TNF-α, IFN-γ, and iNOS in the liver caused by LPS stimulation (P<0.05), and alleviated the decrease in the mRNA expression levels of CD80 and CD86 in the liver caused by LPS stimulation (P<0.05), while this effect was not observed in pigs injected with saline.

[0166] 2.4 Effects of lysophosphatidylcholine on the mRNA expression levels of glycolysis- and citric acid cycle-related molecules in macrophages of the livers of LPS-stimulated piglets

[0167] As shown in Table 10, LPS stimulation significantly increased the mRNA expression level of HK2 during glycolysis and the citric acid cycle (P<0.05), while the mRNA expression levels of PK, PDH, CS, IDH, and DLST were significantly decreased (P<0.05).

[0168] There was an interaction between LPC and LPS stimulation on the mRNA expression level of HK2 in the liver (P<0.05), that is, LPC alleviated the increase in the mRNA expression level of HK2 in the liver caused by LPS stimulation (P<0.05).

[0169] 2.5 Effects of lysophosphatidylcholine on the mRNA expression levels of mTOR- and LKB1-pathway-related molecules in macrophages of the livers of LPS-stimulated piglets

[0170] As shown in Table 11, LPS stimulation significantly decreased the mRNA expression levels of mTOR, S6K1, 4EBP1, and LKB1 in the liver (P<0.05). There was an interaction between LPC and LPS stimulation on the mRNA expression levels of S6K1, HIF-1α, and SIRT1 in the liver (P<0.05), that is, LPC alleviated the decrease in the mRNA expression levels of S6K1, HIF-1α, and SIRT1 in the liver caused by LPS stimulation (P<0.05).

[0171] 2.6 Effects of lysophosphatidylcholine on the protein expression of mTOR- and LKB1-pathway-related molecules in macrophages of the livers of LPS-stimulated piglets

[0172] Depend on Figure 5 It was found that LPS stimulation significantly reduced the expression levels of hepatic mTOR and p-mTOR proteins (P<0.05). LPC interacted with LPS stimulation on the expression of hepatic mTOR and p-mTOR proteins (P<0.05), meaning that LPC alleviated the decrease in hepatic mTOR and p-mTOR protein expression caused by LPS stimulation (P<0.05).

[0173] Table 8 Effects of lysophosphatidylcholine on serum biochemical parameters of LPS-stimulated piglets

[0174]

[0175] Note: abc Different letters in the same row indicate significant differences (P<0.05). In the diet column, the P value represents the difference between the basal diet and the experimental diet, and in the LPS column, the P value represents the difference between the injection of physiological saline and the injection of LPS.

[0176] In Table 8: TP is total protein, ALB is albumin, AST is aspartate aminotransferase, ALT is alanine aminotransferase, ALP is alkaline phosphatase, and GGT is gamma-glutamyl transferase.

[0177] Table 9. Effects of lysophosphatidylcholine on the mRNA expression levels of LPS-stimulated macrophage polarization-related molecules in piglet livers.

[0178]

[0179] Note: abc Different letters in the same row indicate significant differences (P<0.05). In the diet column, the P value represents the difference between the basal diet and the experimental diet, and in the LPS column, the P value represents the difference between the injection of physiological saline and the injection of LPS.

[0180] Table 10. Effects of lysophosphatidylcholine on the mRNA expression levels of molecules related to glycolysis and citric acid cycling in LPS-stimulated piglet liver macrophages.

[0181]

[0182] Note: ab Different letters in the same row indicate significant differences (P<0.05). In the diet column, the P value represents the difference between the basal diet and the experimental diet, and in the LPS column, the P value represents the difference between the injection of physiological saline and the injection of LPS.

[0183] Table 11 Effects of lysophosphatidylcholine on the expression levels of mTOR and LKB1 pathway-related molecules in piglet liver macrophages stimulated by LPS.

[0184]

[0185] Note: ab Different letters in the same row indicate significant differences (P<0.05). In the diet column, the P value represents the difference between the basal diet and the experimental diet, and in the LPS column, the P value represents the difference between the injection of physiological saline and the injection of LPS.

[0186] 3. Conclusion

[0187] The liver, as a crucial detoxification organ in pigs, is highly sensitive to pathogenic infections and immune stress. In the embodiments of this invention, the protective effect of dietary lysophosphatidylcholine (LPC) on lipopolysaccharide-induced liver damage in piglets was first investigated, followed by a further study of the molecular mechanisms of liver inflammation. The changes in liver morphology and structure were analyzed, revealing that LPS stimulation led to the disappearance of hepatocyte nuclei, inflammatory cell infiltration, and nuclear pyknosis in piglets. Dietary LPC supplementation alleviated LPS-induced liver morphological damage.

[0188] The chemical composition of blood plasma mainly comes from nutrients digested and absorbed by the gastrointestinal tract. Therefore, plasma biochemical indicators can reflect the health status and nutritional metabolism level of an animal. Albumin (ALB) and total protein (TB) can indirectly reflect the body's immunity.

[0189] In the experiments of this invention, LPS stimulation led to a decrease in TP and ALB levels in piglet serum. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma-glutamyl transferase (GGT) are important biochemical indicators for assessing liver health. When hepatocytes are damaged or inflamed, these enzymes are released from the damaged hepatocytes into the bloodstream, resulting in a significant increase in their concentration in serum. Therefore, detecting the levels of these enzymes can serve as an important basis for diagnosing abnormal liver function.

[0190] In the experimental embodiments of this invention, LPS stimulation caused an increase in serum AST, ALT, ALP, and GGT levels, while LPC alleviated the increase in serum AST, ALT, and ALP caused by LPS stimulation. LPC can alleviate liver function damage caused by LPS stimulation.

[0191] Macrophages are the most abundant immune cells in the liver. M1 macrophages mainly secrete pro-inflammatory factors such as IL-1β, IL-6, TNF-α, and iNOS, participating in the inflammatory response. M2 macrophages can secrete anti-inflammatory factors such as IL-10, TGF-β, and Arg-1 to inhibit inflammation. Under normal circumstances, M1 and M2 macrophages are in dynamic equilibrium. When stimulated by external factors, the number and function of M1 and M2 macrophages change, leading to inflammation and disease.

[0192] In the experimental embodiments of this invention, LPS stimulation increased the mRNA expression levels of polarization-related factors in piglet liver M1 macrophages, and also increased the mRNA expression levels of polarization-related factors in M2 macrophages. LPC could alleviate the increase in the mRNA expression levels of liver M1 macrophage polarization-related factors induced by LPS stimulation. LPC has a regulatory effect on the polarization of liver macrophages induced by LPS stimulation and can alleviate liver damage by reducing the expression of pro-inflammatory factor mRNAs in M1 macrophages.

[0193] Studies have found that glycolysis and the citric acid cycle play crucial roles in regulating macrophage polarization. Inhibiting HK2 gene expression can suppress LPS-induced M1 macrophage polarization. A fusion protein containing a lysogenic death receptor 5-Fc antibody reduces the mRNA level of HK2 in M1 macrophages via a concentration gradient, thereby inhibiting M1 macrophage polarization by suppressing glycolysis and reducing the secretion of inflammatory cytokines IL-1β and TNF-α.

[0194] In the experiments of this invention, LPS stimulation increased the mRNA expression of HK2, a key enzyme in macrophage glycolysis, and decreased the mRNA expression of other key enzymes in glycolysis and the citrate cycle. LPC alleviated the increase in macrophage HK2 mRNA expression induced by LPS stimulation. LPC also alleviated the increase in liver HK2 mRNA expression induced by LPS stimulation, inhibited glycolysis, and reduced the mRNA expression of macrophage M1 polarization-related factors, such as pro-inflammatory factors like IL-1β, IL-6, and TNF-α, thereby alleviating liver inflammation. These results suggest that LPC may alleviate liver inflammation by regulating macrophage glycolysis and the citrate cycle.

[0195] Studies have shown that the mTOR and LKB1 pathways regulate macrophage glycolysis and citric acid cycling. To further explore the molecular mechanism by which LPC regulates glycolysis and citric acid cycling, we measured the mRNA and protein expression levels of key genes in the mTOR and LKB1 signaling pathways. In the experiments of this invention, LPS stimulation significantly reduced the expression levels of hepatic mTOR, S6K1, 4EBP1, and LKB1 mRNA, while LPC alleviated the decrease in hepatic S6K1, HIF-1α, and SIRT1 mRNA expression caused by LPS stimulation. In the experiments of this invention, LPS stimulation reduced the expression levels of hepatic mTOR and p-mTOR proteins, while LPC alleviated the decrease in hepatic mTOR and p-mTOR protein expression caused by LPS stimulation. The fact that LPC alleviated the decrease in mRNA and protein levels of hepatic mTOR pathway-related factors caused by LPS stimulation indicates that LPC can inhibit macrophage polarization towards the M1 direction.

[0196] In conclusion, adding lysophosphatidylcholine (LPC) to the basal diet of pigs can alleviate liver damage caused by immune stress, protect the pig's liver, and safeguard the pig's health under immune stress.

Claims

1. Application of lysophosphatidylcholine in the preparation of products that protect against porcine immune stress.

2. Use according to claim 1, characterized in that, Application of lysophosphatidylcholine in the preparation of products that protect the intestines and / or liver of pigs.

3. Use according to claim 2, characterized in that, Application of lysophosphatidylcholine in the preparation of products for the prevention or treatment of intestinal and / or liver damage in pigs.

4. The application according to claim 3, characterized in that, The intestinal and / or liver damage is induced by immune stress.

5. The application according to claim 3 or 4, characterized in that, The intestinal and / or liver damage was induced by lipopolysaccharide.

6. The application according to any one of claims 1-3, characterized in that, The product can be any one of the following: medicine, feed additive, or feed.

7. Application of lysophosphatidylcholine in the preparation of products that regulate the Teff / Treg balance in the porcine intestine through the JAK / STAT signaling pathway.

8. Application of lysophosphatidylcholine in the preparation of products that inhibit porcine liver macrophage polarization through glycolysis and citric acid cycle signaling pathways.

9. Application of lysophosphatidylcholine in the preparation of regulators possessing one or more of the following functions: (1) Increase villus height and villus height / crypt depth in the jejunum and ileum of pigs; (2) Increase the activity of maltase in the jejunum, sucrase and maltase in the ileum of pigs; (3) It alleviates the decrease in the mRNA expression level of porcine jejunum ZO-1 caused by lipopolysaccharide stimulation and increases the expression level of porcine jejunum Occludin protein; (4) It can alleviate the decrease in mRNA expression levels of TNF-α, IFN-γ, IL-4, IL-17A and GATA3 in the jejunum caused by lipopolysaccharide stimulation, and alleviate the increase in mRNA expression levels of IL-17F, TGF-β and FOXP3 in the jejunum caused by lipopolysaccharide stimulation. (5) It alleviates the decrease in mRNA expression of IL-4, GATA3 and FOXP3 in the ileum caused by lipopolysaccharide stimulation, and alleviates the increase in mRNA expression of IL-17A and IL-17F in the ileum caused by lipopolysaccharide stimulation. (6) Increase the mRNA expression levels of JAK3, STAT4, STAT5A in the jejunum and STAT4 in the ileum of pigs; (7) Relieves the increase in serum aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase in pigs caused by lipopolysaccharide stimulation; (8) Alleviate the increase in mRNA expression of polarization-related factors in porcine liver M1 macrophages caused by lipopolysaccharide stimulation; (9) Alleviates the increase in HK2 mRNA expression in pig liver caused by lipopolysaccharide stimulation; (10) Alleviate the decrease in mRNA expression levels of S6K1, hypoxia-inducible factor (HIF)-1α and silencing information regulator SIRT1 in porcine liver caused by lipopolysaccharide stimulation; (11) Relieves the decrease in expression levels of mTOR and p-mTOR proteins in pig liver caused by lipopolysaccharide stimulation.

10. The application according to claims 1, 2, 3, 7, 8, and 9, characterized in that, The application method is as follows: feed pigs a basal diet supplemented with lysophosphatidylcholine, wherein the amount of lysophosphatidylcholine added to the basal diet is 0.01%.

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