Inhibitor for weaned piglet intestinal ferroptosis caused by vomitoxin and preparation method thereof

Abstract

The invention relates to the technical field of animal husbandry, in particular to an inhibitor for weaned piglet intestinal ferroptosis caused by vomitoxin and a preparation method, an iron chelating agent (deferoxamine and DFO), a GPX4 activator (selenomethionine and SeMet) and anti-inflammatory components (curcumin, astragalus polysaccharide and dihydroartemisinin) are integrated, and three major ferroptosis core pathways such as iron accumulation, GPX4 inactivation and lipid peroxidation are covered; meanwhile, the components are coated with pH-sensitive chitosan-sodium alginate nano-particles, so that the components can pass through the stomach and are prevented from being damaged by gastric acid, it is ensured that the medicine is accurately released in the alkaline environment of the intestinal tract, and therefore the utilization rate of the medicine is increased; moreover, the selenomethionine, the curcumin, the astragalus polysaccharide and the dihydroartemisinin are all natural sources, so that the risk of chemical drug residues can be avoided, and the selenium methionine, the curcumin, the astragalus polysaccharide and the dihydroartemisinin can be used as a feed additive to be applied to weaned piglets.

Description

Technical Field

[0001] The present invention relates to the field of animal husbandry technology, and in particular to an inhibitor of intestinal ferroptosis of weaned piglets induced by vomitoxin and a preparation method thereof. Background Art

[0002] Deoxynivalenol (DON) is a common contaminant in grain feed that can cause intestinal barrier damage, diarrhea, and decreased growth performance in weaned piglets. Ferroptosis is an iron-dependent, novel form of cell death characterized by glutathione (GSH) depletion and lipid peroxidation. GPX4, as the only key enzyme that reduces lipid peroxides to non-toxic alcohols, plays an important role in the process of ferroptosis. Studies have found that exposure to DON reduces GPX4 activity and GSH content in weaned piglets, leading to intestinal ferroptosis. Existing prevention and treatment methods for ferroptosis mainly include antioxidants and probiotics, which mainly target oxidative stress and inflammatory responses, but cannot simultaneously regulate iron metabolism imbalance and GPX4 inactivation. There is a lack of targeted and targeted intervention for the role of ferroptosis in DON-induced intestinal damage.

[0003] To address the above issues, existing selenium supplements alleviate ferroptosis by upregulating GPX4 expression in the intestines of weaned piglets, but do not integrate the synergistic effects of iron metabolism regulation and anti-inflammatory pathways; compound antimicrobial peptides can repair intestinal damage in weaned piglets, but do not involve the core mechanism of ferroptosis.

[0004] In summary, the current nutritional regulation methods for intestinal ferroptosis in piglets mainly include antioxidants, probiotics, etc., but there is a lack of multi-target synergistic intervention strategies, which cannot simultaneously inhibit iron accumulation, lipid peroxidation and GPX4 inactivation; and ordinary preparations are easily degraded by gastric acid, making it difficult to release active ingredients in the intestine, resulting in low utilization of ferroptosis inhibitors. Summary of the Invention

[0005] The purpose of the present invention is to provide an inhibitor of intestinal ferroptosis caused by vomitoxin in weaned piglets and a preparation method, aiming to solve the technical problems that the nutritional regulation methods for intestinal ferroptosis in piglets in the existing technology lack a multi-target synergistic intervention strategy and cannot simultaneously inhibit iron accumulation, lipid peroxidation and GPX4 inactivation; and ordinary preparations are easily degraded by gastric acid and it is difficult to release active ingredients in the intestine in a targeted manner, thereby resulting in low utilization rate of ferroptosis inhibitors.

[0006] To achieve the above objectives, the present invention uses an inhibitor of intestinal ferroptosis of weaned piglets caused by vomitoxin, comprising an active ingredient and a carrier system, wherein the active ingredient comprises 15g of an iron chelator, 8g of a GPX4 activator, and an anti-inflammatory compound, wherein the anti-inflammatory compound comprises 3g of curcumin, 3g of astragalus polysaccharide, and 1.5g of dihydroartemisinin;

[0007] The carrier system is a chitosan-sodium alginate double-layer nanoparticle coating, wherein the outer layer is pH-sensitive chitosan and the inner layer is a sodium alginate sustained-release core.

[0008] The chitosan-sodium alginate double-layer nanoparticles have a particle size of 80-120 nm. In the carrier system, the pH-sensitive chitosan serving as the outer shell degrades when the pH is greater than 7.

[0009] Wherein, the iron chelating agent is deferoxamine, and the concentration of the deferoxamine solution is 10-20%;

[0010] The GPX4 activator is selenomethionine, and the concentration of the selenomethionine is 5-10%.

[0011] The present invention also provides a method for preparing an inhibitor of intestinal ferroptosis caused by vomitoxin in weaned piglets, which is used to prepare the above-mentioned inhibitor of intestinal ferroptosis caused by vomitoxin in weaned piglets.

[0012] The steps include:

[0013] Step 1: Pretreatment of active ingredients: 3 g of curcumin powder was mixed with polyethylene glycol-400, and stirred in a 60°C water bath for 30 min to form a transparent micellar solution to obtain a curcumin dispersion; 15 g of deferoxamine, 8 g of selenomethionine, 3 g of astragalus polysaccharide, and 1.5 g of dihydroartemisinin were dissolved in 50 mL of deionized water and sonicated for 15 min until completely dissolved to obtain an aqueous phase mixture;

[0014] Step 2: Prepare the sodium alginate core carrier: Slowly add the curcumin dispersion to the aqueous phase mixture, magnetically stir at 800 rpm to form an emulsified solution, add 100 mL of sodium alginate solution with a pH value of 6.5 and a solution concentration of 2%, and continue stirring for 20 minutes; then add 10 mL of 0.5% calcium chloride solution dropwise to form sodium alginate microspheres with uniform particle size through ionic crosslinking;

[0015] Step 3: Construct a pH-sensitive chitosan coating layer: Dissolve 1.5 g of chitosan in a 1% acetic acid solution, add 10 mL of a 0.5% sodium tripolyphosphate solution, and stir to form a pH-sensitive chitosan-TPP colloid; then add the sodium alginate microsphere suspension dropwise to the chitosan colloid, magnetically stir at 500 rpm for 30 minutes, centrifuge at 8000 rpm for 10 minutes, and collect the coated particles; then wash three times with phosphate buffer to remove unbound components;

[0016] Step 4: Mix the nanoparticle suspension with 5% mannitol, pre-freeze to -80°C, and freeze-dry under vacuum for 24 hours to obtain a white loose powder, which is then stored at 4°C away from light.

[0017] The particle size of the sodium alginate microspheres is 100-150 nm.

[0018] The present invention discloses an inhibitor of intestinal ferroptosis caused by vomitoxin in weaned piglets and a preparation method thereof. The present invention integrates an iron chelator (deferoxamine, DFO), a GPX4 activator (selenomethionine, SeMet) and anti-inflammatory ingredients (curcumin, astragalus polysaccharide, dihydroartemisinin), covering the three core pathways of ferroptosis, namely iron accumulation, GPX4 inactivation, and lipid peroxidation.

[0019] At the same time, pH-sensitive chitosan-sodium alginate nanoparticles are used to coat the above ingredients, allowing them to pass through the stomach and avoid damage by gastric acid, ensuring the precise release of the drugs in the alkaline environment of the intestine, thereby improving the utilization rate of the drugs.

[0020] In addition, selenomethionine, curcumin, astragalus polysaccharide, and dihydroartemisinin are all of natural origin, which can avoid the risk of chemical drug residues and can be used as feed additives in weaned piglets.

[0021] The experiment showed that after intervention with inhibitors, the ADFI of weaned piglets increased by 18%, ADG increased by 39%, F / G decreased by 15%, and the diarrhea rate decreased by 73%. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 It is a schematic diagram of the inhibitor of vomitoxin-induced intestinal ferroptosis in weaned piglets of the present invention. DETAILED DESCRIPTION

[0024] The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0025] See also Figure 1 , Figure 1 It is a schematic diagram of the inhibitor of vomitoxin-induced intestinal ferroptosis in weaned piglets of the present invention.

[0026] The present invention provides an inhibitor of intestinal ferroptosis in weaned piglets induced by vomitoxin, comprising an active ingredient and a carrier system, wherein the active ingredient comprises 15g of an iron chelator, 8g of a GPX4 activator, and an anti-inflammatory compound, wherein the anti-inflammatory compound comprises 3g of curcumin, 3g of astragalus polysaccharide, and 1.5g of dihydroartemisinin;

[0027] The carrier system is a chitosan-sodium alginate double-layer nanoparticle coating, wherein the outer layer is pH-sensitive chitosan and the inner layer is a sodium alginate sustained-release core.

[0028] The chitosan-sodium alginate double-layer nanoparticles have a particle size of 80-120 nm. In the carrier system, the pH-sensitive chitosan serving as the outer shell degrades when the pH is greater than 7.

[0029] Wherein, the iron chelating agent is deferoxamine, and the concentration of the deferoxamine solution is 10-20%;

[0030] The GPX4 activator is selenomethionine, and the concentration of the selenomethionine is 5-10%.

[0031] The present invention also provides a method for preparing an inhibitor of intestinal ferroptosis caused by vomitoxin in weaned piglets, which is used to prepare the above-mentioned inhibitor of intestinal ferroptosis caused by vomitoxin in weaned piglets.

[0032] The steps include:

[0033] Step 1: Pretreatment of active ingredients: 3 g of curcumin powder was mixed with polyethylene glycol-400, and stirred in a 60°C water bath for 30 min to form a transparent micellar solution to obtain a curcumin dispersion; 15 g of deferoxamine, 8 g of selenomethionine, 3 g of astragalus polysaccharide, and 1.5 g of dihydroartemisinin were dissolved in 50 mL of deionized water and sonicated for 15 min until completely dissolved to obtain an aqueous phase mixture;

[0034] Step 2: Prepare the sodium alginate core carrier: Slowly add the curcumin dispersion to the aqueous phase mixture, magnetically stir at 800 rpm to form an emulsified solution, add 100 mL of sodium alginate solution with a pH value of 6.5 and a solution concentration of 2%, and continue stirring for 20 minutes; then add 10 mL of 0.5% calcium chloride solution dropwise to form sodium alginate microspheres with uniform particle size through ionic crosslinking;

[0035] Step 3: Construct a pH-sensitive chitosan coating layer: Dissolve 1.5 g of chitosan in a 1% acetic acid solution, add 10 mL of a 0.5% sodium tripolyphosphate solution, and stir to form a pH-sensitive chitosan-TPP colloid; then add the sodium alginate microsphere suspension dropwise to the chitosan colloid, magnetically stir at 500 rpm for 30 minutes, centrifuge at 8000 rpm for 10 minutes, and collect the coated particles; then wash three times with phosphate buffer to remove unbound components;

[0036] Step 4: Mix the nanoparticle suspension with 5% mannitol, pre-freeze to -80°C, and freeze-dry under vacuum for 24 hours to obtain a white loose powder, which is then stored at 4°C away from light.

[0037] The particle size of sodium alginate microspheres is 100-150nm.

[0038] The experiment on the inhibitor of vomitoxin-induced intestinal ferroptosis in weaned piglets is as follows:

[0039] 1.1 Experimental design and sample collection

[0040] A total of 180 21-day-old weaned piglets (Du × Chang × Da) of similar body weight were randomly divided into three groups, with six replicates per group and ten piglets per replicate. The CON group was fed a basal diet, the DON group was fed a basal diet supplemented with 1 mg / kg DON, and the intervention group was fed a basal diet supplemented with 2 mg / kg inhibitor and 1 mg / kg DON. The experimental period lasted 28 days. Growth performance was analyzed during the experimental period. At 49 days of age, blood was collected from the anterior vena cava of one piglet of similar body weight in each replicate. Half of each piglet was anesthetized and slaughtered. The abdominal cavity was opened, and the jejunum was harvested. A 1 cm segment of intestinal material was cut, rinsed with PBS, and fixed in 4% paraformaldehyde. The intestinal segments were dissected, and the jejunal mucosa was scraped and stored at -80°C for subsequent measurement.

[0041] 1.2 Measurement indicators

[0042] (1) Growth performance: ADFI, ADG, F / G, diarrhea rate

[0043] (2) Intestinal morphology: villus height, crypt depth, villus height / crypt depth

[0044] (3) Intestinal inflammatory response: IL-1β, IL-6, TNF-α

[0045] (4) Antioxidant function: T-AOC, T-SOD, GSH-Px, CAT, MDA

[0046] (5) Ferroptosis indicators: GPX4, GSH, Fe 2+

[0047] (6) Expression of genes related to ferroptosis pathway: DMT1, TFR1, GPX4, SCL7A11, ALOX15, ACSL4, NF-κB

[0048] 1.3 Test results

[0049] As shown in Table 1, DON significantly reduced (P<0.05) the final weight, ADFI and ADG of weaned piglets, and increased (P<0.05) the F / G ratio and diarrhea rate; inhibitor intervention significantly increased (P<0.05) the final weight, ADFI and ADG of weaned piglets induced by DON, and reduced (P<0.05) the F / G ratio and diarrhea rate.

[0050] Table 1 Effects of inhibitor intervention on the growth performance of weaned piglets induced by vomitoxin

[0051]

[0052] Note: The numbers with different letters in the same column indicate significant differences (P<0.05).

[0053] As shown in Table 2, DON reduced (P<0.05) the villus height and villus height / crypt depth of the jejunum of weaned piglets, and increased (P<0.05) the crypt depth; inhibitor intervention increased (P<0.05) the DON-induced villus height and villus height / crypt depth of the jejunum of weaned piglets, and reduced (P<0.05) the crypt depth.

[0054] Table 2 Effects of inhibitor intervention on intestinal morphology of weaned piglets induced by vomitoxin

[0055]

[0056]

[0057] Note: The numbers with different letters in the same column indicate significant differences (P<0.05).

[0058] As shown in Table 3, DON increased (P<0.05) the levels of IL-1β, IL-6, and TNF-α in the jejunum of weaned piglets, and inhibitor intervention decreased (P<0.05) the DON-induced jejunal levels of IL-1β, IL-6, and TNF-α in weaned piglets.

[0059] Table 3 Effects of inhibitor intervention on intestinal inflammatory response induced by vomitoxin in weaned piglets

[0060]

[0061] Note: The numbers with different letters in the same column indicate significant differences (P<0.05).

[0062] As shown in Table 4, DON reduced (P<0.05) the level of T-AOC, the activities of T-SOD, T-SOD and GSH-Px in the jejunum of weaned piglets, and increased (P<0.05)(P<0.05) the content of MDA; the addition of inhibitors significantly increased the level of T-AOC, the activities of T-SOD, T-SOD and GSH-Px in the jejunum of weaned piglets induced by DON, and reduced (P<0.05) the content of MDA.

[0063] Table 4 Effects of inhibitor intervention on intestinal antioxidant capacity of weaned piglets induced by vomitoxin

[0064]

[0065] Note: The numbers with different letters in the same column indicate significant differences (P<0.05).

[0066] As shown in Table 5, DON decreased (P<0.05) the GPX4 activity and GSH content in the jejunum of weaned piglets, and increased (P<0.05) the Fe 2+ The addition of inhibitors significantly increased (P<0.05) DON-induced GPX4 activity and GSH content in the jejunum of weaned piglets, and decreased (P<0.05) Fe 2+ content.

[0067]

[0068] Note: The numbers with different letters in the same column indicate significant differences (P<0.05).

[0069] As shown in Table 6, DON increased (P<0.05) the relative expression levels of DMT1, TFR1, ALOX15 and NF-κB mRNA in the jejunum of weaned piglets, and decreased (P<0.05) the relative expression levels of GPX4 and SCL7A11 mRNA. The addition of inhibitors decreased (P<0.05) the DON-induced relative expression levels of DMT1, TFR1, ALOX15 and NF-κB mRNA in the jejunum of weaned piglets, and increased (P<0.05) the relative expression levels of GPX4 and SCL7A11 mRNA.

[0070] Table 6 Effects of inhibitor intervention on the expression of genes related to the intestinal ferroptosis pathway induced by deoxynivalenol in weaned piglets

[0071]

[0072] Note: The numbers with different letters in the same column indicate significant differences (P<0.05).

[0073] An inhibitor of intestinal ferroptosis caused by vomitoxin in weaned piglets and a preparation method thereof are used in the present embodiment. First, 3 g of curcumin powder is mixed with polyethylene glycol-400, and stirred in a water bath at 60° C. for 30 min to form a transparent micellar solution to obtain a curcumin dispersion; 15 g of deferoxamine, 8 g of selenomethionine, 3 g of astragalus polysaccharide and 1.5 g of dihydroartemisinin are dissolved in 50 mL of deionized water, and ultrasonically treated for 15 min until completely dissolved to obtain an aqueous phase mixture; then, a sodium alginate core carrier is prepared: the curcumin dispersion is slowly added to the aqueous phase mixture, magnetically stirred at 800 rpm to form an emulsified solution, 100 mL of sodium alginate solution with a pH value of 6.5 and a solution concentration of 2% is added, and stirring is continued for 20 min; then 10 mL of 0.5% calcium chloride solution is added to allow ionic cross-linking to form sodium alginate microspheres with consistent particle size; then a pH-sensitive chitosan coating layer is constructed: 1.5 g of chitosan is dissolved in a 1% acetic acid solution, 10 mL of a 0.5% sodium tripolyphosphate solution is added, and the mixture is stirred to form a pH-sensitive chitosan-TPP colloid; then the sodium alginate microsphere suspension is added dropwise to the chitosan colloid, magnetically stirred at 500 rpm for 30 minutes, and centrifuged at 8000 rpm for 10 minutes to collect the coated particles; then washed three times with phosphate buffer to remove unbound components; finally, the nanoparticle suspension is mixed with 5% mannitol and pre-frozen to -80°C; vacuum freeze-dried for 24 hours to obtain a white loose powder, which can be stored at 4°C away from light.

[0074] The present invention integrates iron chelators (deferoxamine, DFO), GPX4 activators (selenomethionine, SeMet) and anti-inflammatory ingredients (curcumin, astragalus polysaccharide, dihydroartemisinin), covering the three core pathways of ferroptosis, including iron accumulation, GPX4 inactivation, and lipid peroxidation.

[0075] At the same time, pH-sensitive chitosan-sodium alginate nanoparticles are used to coat the above ingredients, allowing them to pass through the stomach and avoid damage by gastric acid, ensuring the precise release of the drugs in the alkaline environment of the intestine, thereby improving the utilization rate of the drugs.

[0076] In addition, selenomethionine, curcumin, astragalus polysaccharide, and dihydroartemisinin are all of natural origin, which can avoid the risk of chemical drug residues and can be used as feed additives in weaned piglets.

[0077] The experiment showed that after intervention with inhibitors, the ADFI of weaned piglets increased by 18%, ADG increased by 39%, F / G decreased by 15%, and the diarrhea rate decreased by 73%.

[0078] The above disclosure is only a preferred embodiment of the present invention, and certainly cannot be used to limit the scope of the rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiment and equivalent changes made in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. An inhibitor of intestinal ferroptosis caused by deoxynivalenol in weaned piglets, characterized in that: The invention comprises an active ingredient and a carrier system, wherein the active ingredient comprises 15g of an iron chelator, 8g of a GPX4 activator, and an anti-inflammatory compound, wherein the anti-inflammatory compound comprises 3g of curcumin, 3g of astragalus polysaccharide, and 1.5g of dihydroartemisinin; The carrier system is a chitosan-sodium alginate double-layer nanoparticle coating, wherein the outer layer is pH-sensitive chitosan and the inner layer is a sodium alginate sustained-release core.

2. The inhibitor of vomitoxin-induced intestinal ferroptosis in weaned piglets according to claim 1, characterized in that The chitosan-sodium alginate double-layer nanoparticles have a particle size of 80-120 nm. In the carrier system, the pH-sensitive chitosan serving as the outer shell degrades when the pH is greater than 7.

3. The inhibitor of vomitoxin-induced intestinal ferroptosis in weaned piglets according to claim 2, characterized in that: The iron chelator is deferoxamine, and the concentration of the deferoxamine solution is 10-20%; The GPX4 activator is selenomethionine, and the concentration of the selenomethionine is 5-10%.

4. A method for preparing an inhibitor of intestinal ferroptosis caused by vomitoxin in weaned piglets, which is used to prepare the inhibitor of intestinal ferroptosis caused by vomitoxin in weaned piglets according to claim 3, characterized in that: The steps include: Step 1: Pretreatment of active ingredients: 3 g of curcumin powder was mixed with polyethylene glycol-400, and stirred in a 60°C water bath for 30 min to form a transparent micellar solution to obtain a curcumin dispersion; 15 g of deferoxamine, 8 g of selenomethionine, 3 g of astragalus polysaccharide, and 1.5 g of dihydroartemisinin were dissolved in 50 mL of deionized water and sonicated for 15 min until completely dissolved to obtain an aqueous phase mixture; Step 2: Prepare the sodium alginate core carrier: Slowly add the curcumin dispersion to the aqueous phase mixture, magnetically stir at 800 rpm to form an emulsified solution, add 100 mL of sodium alginate solution with a pH value of 6.5 and a solution concentration of 2%, and continue stirring for 20 minutes; then add 10 mL of 0.5% calcium chloride solution dropwise to form sodium alginate microspheres with uniform particle size through ionic crosslinking; Step 3: Construct a pH-sensitive chitosan coating layer: Dissolve 1.5 g of chitosan in a 1% acetic acid solution, add 10 mL of a 0.5% sodium tripolyphosphate solution, and stir to form a pH-sensitive chitosan-TPP colloid; then add the sodium alginate microsphere suspension dropwise to the chitosan colloid, magnetically stir at 500 rpm for 30 minutes, centrifuge at 8000 rpm for 10 minutes, and collect the coated particles; then wash three times with phosphate buffer to remove unbound components; Step 4: Mix the nanoparticle suspension with 5% mannitol, pre-freeze to -80°C, and freeze-dry under vacuum for 24 hours to obtain a white loose powder, which is then stored at 4°C away from light.

5. The method for preparing an inhibitor of vomitoxin-induced intestinal ferroptosis in weaned piglets according to claim 4, characterized in that: The particle size of sodium alginate microspheres is 100-150nm.

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