Application of glucuronolactone in the preparation of feed to alleviate spleen damage caused by combined PRRSV and vomitoxin

Abstract

This invention belongs to the field of animal feed technology and discloses the application of glucuronolactone in the preparation of feed that alleviates spleen damage caused by the combined effects of PRRSV and DON. This invention discovers that glucuronolactone can effectively alleviate spleen damage in piglets caused by co-exposure to PRRSV and DON. It exerts its protective effect by activating the Nrf2 antioxidant signaling pathway, improving spleen tissue structure, alleviating oxidative stress, inhibiting inflammatory responses, and inhibiting apoptosis. This invention also provides a feed containing the aforementioned effective amount of glucuronolactone, with the basic feed tailored to the nutritional needs of piglets. This invention fills the technological gap in effective intervention methods for spleen damage under PRRSV and DON co-exposure. The glucuronolactone addition process is simple, the effect is significant, and it is suitable for large-scale feed production. It can effectively solve the problem of immunosuppression in pig herds caused by the combined harm of PRRSV and DON in the pig industry, and has important practical value.

Description

Technical Field

[0001] This invention relates to the field of animal feed technology, specifically to the application of glucuronolactone in the preparation of feed that alleviates spleen damage in piglets caused by the combined effects of porcine reproductive and respiratory syndrome virus (PRRSV) and vomitoxin (DON). Background Technology

[0002] DON (vomiting toxin) is a typical mycotoxin produced by Fusarium. It easily contaminates feed ingredients such as corn and soybean meal. When ingested by pigs, it can lead to decreased feed intake, reduced growth performance, and even immune dysfunction in pigs. Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen causing immunosuppression in pig herds. It targets and attacks the alveolar macrophages, spleen, and other lymphoid tissues in pigs, causing severe immune damage.

[0003] Studies have confirmed that piglets are susceptible to simultaneous PRRSV infection and DON feed contamination during weaning, with a synergistic effect between the two, resulting in an additive effect on pig health. The spleen, as the largest peripheral immune organ, is the core site for immune response. Co-exposure to PRRSV and DON severely damages spleen tissue structure, leading to lymphocyte depletion and functional loss. This is a key reason for vaccine failure and a significantly increased rate of secondary infections in piglets. Currently, there are no effective interventions for spleen damage caused by co-exposure to PRRSV and DON in pigs.

[0004] Glucuronolactone is a classic hepatoprotective and detoxifying agent that has been proven to enhance liver detoxification function. Patent CN119969507A reported its application in improving lung damage in pigs caused by co-exposure to PRRSV and DON. However, there are no publicly available reports on whether glucuronolactone has a protective function on the spleen, a core immune organ, under co-exposure to PRRSV and DON. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the primary objective of this invention is to provide the application of glucuronolactone in the preparation of feed that alleviates spleen damage caused by the combined effects of PRRSV and vomitoxin, overcoming the technical deficiencies of existing technologies that lack effective protection against spleen damage in pigs caused by co-exposure to PRRSV and DON; simultaneously, it provides a feed containing glucuronolactone to alleviate the aforementioned spleen damage, offering a practical solution for ensuring the immune health of pig herds.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides the use of glucuronolactone in the preparation of feed that alleviates spleen damage caused by the combined effects of PRRSV and vomitoxin, wherein the spleen damage includes damage to spleen tissue structure, oxidative stress, inflammatory response, and / or apoptosis:

[0008] (1) Damage to the spleen’s tissue structure manifests as blurred boundaries between red and white pulp, dilated lymphatic sinuses, lymphocyte necrosis and inflammatory cell infiltration;

[0009] (2) Oxidative stress is manifested as a decrease in catalase (CAT) activity;

[0010] (3) The inflammatory response is characterized by increased expression of pro-inflammatory factors IL-1β, IL-18, and IL-6 and / or decreased expression of anti-inflammatory factor IL-10;

[0011] (4) Apoptosis is manifested by increased expression of pro-apoptotic proteins Bax and Caspase-3 and / or decreased expression of anti-apoptotic protein Bcl-2.

[0012] Furthermore, the glucuronolactone exerts a protective effect on the spleen by activating the Nrf2 antioxidant signaling pathway. This activation manifests as an increase in the P-Nrf2 / Nrf2 ratio, an increase in HO-1 protein expression, and an increase in the expression of GCLM, HO-1, and NQO-1 genes.

[0013] Furthermore, based on the feed weight, the effective amount of glucuronolactone added to the feed to alleviate spleen damage caused by the combined effects of PRRSV and vomitoxin is 50–600 mg / kg, preferably 200 mg / kg.

[0014] Secondly, the present invention provides a feed for alleviating spleen damage caused by the combined effects of PRRSV and vomitoxin, comprising a basal feed and an effective amount of glucuronolactone; the amount of glucuronolactone added is 50-600 mg / kg, preferably 200 mg / kg, based on the total weight of the feed.

[0015] Furthermore, the formulation of the basic feed is based on conventional piglet feed formulations in the art, and includes the following components: corn, protein substances, low-protein whey powder, oils, sugars, amino acids, feed additives, enzyme preparations, and premixes.

[0016] Furthermore, in one specific implementation, the basic feed comprises, by weight parts, the following components: 450-650 parts corn, 250-350 parts protein, 40-80 parts low-protein whey powder, 15-35 parts oil, 10-30 parts carbohydrates, 3.2-7.6 parts amino acids, 5.5-10 parts feed additives, 1-3 parts enzyme preparations, and 10-30 parts premix.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) Filling the technological gap and expanding the application scenarios of glucuronolactone: This invention found that glucuronolactone can effectively alleviate spleen damage in piglets caused by co-exposure to PRRSV and DON. Compared with the existing technology that only focuses on the protection of lung damage, this invention focuses on the spleen, a core immune organ, and provides a more comprehensive strategy for immune protection of pigs co-exposure to PRRSV and DON.

[0019] (2) Multidimensional protective effect with clear mechanism: This invention systematically reveals the protective effect of glucuronolactone on the spleen at the histopathological, cellular and molecular levels. It can not only improve the spleen tissue structure, but also systematically enhance the antioxidant capacity by activating the Nrf2 pathway, while inhibiting excessive inflammatory response and cell apoptosis, and maintaining the homeostasis and function of spleen immune cells. In particular, the inhibitory effect of glucuronolactone on spleen cell apoptosis is a first discovery and is an important mechanism innovation that distinguishes it from the existing technology.

[0020] (3) Significant effect and strong practicality: The present invention has been proven by rigorous animal experiments that adding an effective amount of glucuronolactone to feed can significantly reverse the abnormalities of various spleen damage indicators caused by co-exposure to PRRSV and DON. Moreover, glucuronolactone is a mature chemical raw material that is easy to obtain and has a simple addition process, making it suitable for large-scale feed production. It can provide a practical and effective means for the pig industry to solve the problem of combined harm caused by PRRSV and DON. Attached Figure Description

[0021] Figure 1 Effects of dietary supplementation with glucuronolactone on spleen pathological changes in piglets co-exposed to PRRSV and DON (HE staining, ×200); A: PRRSV group; B: PRRSV+DON group; C: PRRSV+DON+GLU group; Yellow arrow: lymphocyte aggregation; Blue arrow: lymphocyte necrosis; Orange arrow: neutrophils; Brown arrow: splenic sinusoidal dilation; Green arrow: plasma cells.

[0022] Figure 2 Effects of dietary supplementation with glucuronolactone on antioxidant indices of spleen in piglets co-exposed to PRRSV and DON.

[0023] Figure 3 Effects of dietary supplementation with glucuronolactone on mRNA expression of antioxidant-related genes in the spleen of piglets co-exposed to PRRSV and DON.

[0024] Figure 4 Effects of dietary supplementation with glucuronolactone on the expression of Nrf2 pathway-related proteins in the spleen of piglets co-exposed to PRRSV+DON.

[0025] Figure 5 Effects of dietary supplementation with glucuronolactone on mRNA expression of spleen inflammation-related genes in piglets co-exposed to PRRSV-DON.

[0026] Figure 6 Effects of dietary supplementation with glucuronolactone on the expression of inflammation-related proteins in the spleen of piglets co-exposed to PRRSV and DON.

[0027] Figure 7 Effects of dietary supplementation with glucuronolactone on mRNA expression of apoptosis-related genes in the spleen of piglets co-exposed to PRRSV and DON.

[0028] Figure 8 Effects of dietary supplementation with glucuronolactone on the expression of apoptosis-related proteins in the spleen of piglets co-exposed to PRRSV and DON. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0031] Experimental Example 1: Experimental Design and Animal Grouping

[0032] 1. Experimental animals:

[0033] Thirty 21-day-old weaned piglets (Duroc × Landrace × Large White) with similar weight (7.41±0.15 kg) and parity (PRRSV double negative, castrated boars) were selected and randomly divided into 3 groups of 10 piglets each.

[0034] 2. Experimental grouping and treatment:

[0035] The first group was the PRRSV infection group (PRRSV group), which was fed a basal diet;

[0036] The second group was the PRRSV and DON co-infection group (PRRSV+DON group), which was fed a basal diet supplemented with 2 mg / kg DON;

[0037] The third group was the glucuronolactone treatment group (PRRSV+DON+GLU group), which was fed a basal diet supplemented with 2 mg / kg DON and 200 mg / kg glucuronolactone.

[0038] All piglets were fed the appropriate diet from day 1 of the experiment, four times a day, with free access to feed. On day 15 of the experiment, all piglets were inoculated with 2 mL of PRRSV (10) via intranasal drip. 5 TCID 50 / mL). The experiment lasted for 35 days. After the experiment, all piglets were slaughtered and spleen tissue was collected for subsequent testing.

[0039] The basic feed formula was designed with reference to the NRC (2012) recommended nutritional requirements for 7-25 kg piglets. The specific components and contents are shown in Table 1.

[0040] Table 1. Composition and nutrient levels of the experimental diets (% of basal feeding)

[0041] Experimental Example 2: Effects of glucuronolactone on spleen pathological damage in piglets co-infected with PRRSV and DON

[0042] 1. Detection Method

[0043] Spleen tissues from piglets in each group were collected, fixed with 4% paraformaldehyde for 24 h, then dehydrated in a gradient manner, cleared, embedded in paraffin, and 5 μm serial sections were prepared. The sections were stained with hematoxylin and eosin (hematoxylin staining for 5 min, eosin staining for 3 min) and scanned with a digital bright-field microscope to observe the pathological structural changes of the spleen tissues.

[0044] 2. Test Results

[0045] The results are as follows Figure 1 As shown, the white and red pulp layers of the spleen in piglets in the PRRSV group were relatively clear, with uniform distribution of lymphatic sinuses and tightly arranged lymphocytes. Compared with the PRRSV group, the spleen of piglets in the PRRSV+DON group showed severe pathological damage, characterized by blurred boundaries between the red and white pulp, dilated lymphatic sinuses, reduced lymphocyte count with necrosis, and significant neutrophil infiltration. Compared with the PRRSV+DON group, the pathological damage of the spleen in piglets in the PRRSV+DON+GLU group was significantly reduced, with the red pulp congestion disappearing, lymphocytes returning to a tighter arrangement, and reduced inflammatory cell infiltration.

[0046] The above results indicate that co-exposure to PRRSV and DON exacerbates spleen tissue damage in piglets, while the addition of glucuronolactone to the diet can significantly improve the pathological structure of the spleen and alleviate spleen damage caused by co-exposure to both.

[0047] Experimental Example 3: Effect of glucuronolactone on the antioxidant capacity of spleen in piglets co-infected with PRRSV and DON

[0048] 1. Detection Method

[0049] Frozen spleen tissue was collected and homogenized with physiological saline at a weight-to-volume ratio of 1:10. The homogenate was then ground under ice bath conditions and centrifuged at 12000 r / min for 15 min at 4℃. The supernatant was collected. Total antioxidant capacity (T-AOC), malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activities were determined using kits provided by Nanjing Jiancheng Bioengineering Research Institute, strictly following the instructions. Results are expressed as per milligram of protein.

[0050] 2. Test Results

[0051] The results are as follows Figure 2 As shown, compared with the PRRSV group, the CAT activity in the spleen of piglets in the PRRSV+DON group was significantly decreased (P < 0.05), while the MDA content showed an increasing trend. Compared with the PRRSV+DON group, the CAT activity in the spleen of piglets in the PRRSV+DON+GLU group was significantly increased (P < 0.05).

[0052] The above results indicate that co-exposure to PRRSV and DON induces oxidative stress in the spleen of piglets, while the addition of glucuronolactone to the diet can significantly increase the activity of antioxidant enzymes in the spleen and alleviate oxidative damage.

[0053] Experiment 4: Effect of glucuronolactone on mRNA expression of antioxidant-related genes in the spleen of piglets co-infected with PRRSV and DON.

[0054] 1. Detection Method

[0055] Total RNA was extracted from piglet spleen samples using the Trizol method, and RNA concentration and purity (OD) were detected using a nucleic acid protein analyzer. 260 / 280 Between 1.8 and 2.0, OD 260 / 230 (between versions 1.7 and 2.0). Uses PrimeScript. TM cDNA was synthesized via reverse transcription using the RT Reagent kit and detected by real-time quantitative PCR using the SYBR Green Master Mix PCR kit. The relative mRNA expression level was calculated using the 2⁻ΔΔCt method. Primer sequences are shown in Table 2 (Table 2 in the paper can be cited here).

[0056] 2. Test Results

[0057] The results are as follows Figure 3As shown, compared with the PRRSV group, the mRNA expression levels of the glutamate-cysteine ​​ligase catalytic subunit (GCLC), glutamate-cysteine ​​ligase modified subunit (GCLM), heme oxygenase 1 (HO-1), and NAD(P)H quinone oxidoreductase 1 (NQO1) genes in the spleen of piglets in the PRRSV+DON group showed a decreasing trend. Compared with the PRRSV+DON group, the mRNA expression levels of the GCLM, HO-1, and NQO1 genes in the spleen of piglets in the PRRSV+DON+GLU group were significantly increased (P < 0.05).

[0058] These results indicate that glucuronolactone can upregulate the expression of antioxidant-related genes, thereby enhancing the spleen's antioxidant defense capabilities at the transcriptional level.

[0059] Experimental Example 5: Effects of glucuronolactone on the expression of Nrf2 pathway-related proteins in the spleen of piglets co-infected with PRRSV and DON.

[0060] 1. Detection Method

[0061] Proteins were extracted from spleen tissue using RIPA lysis buffer, and protein concentrations were determined using the BCA method. Protein samples were separated by 10% SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. The membrane was blocked with rapid blocking buffer at room temperature for 30 min. Primary antibodies Nrf2, P-Nrf2, HO-1, and Keap1 (all diluted 1:1000) were added, and the membrane was incubated overnight at 4°C. After washing, HRP-labeled secondary antibody (1:5000) was added, and the membrane was incubated at room temperature for 1 h. The membrane was then developed using ECL chemiluminescence buffer, and images were taken using a chemiluminescent gel imaging system. The grayscale values ​​of the bands were analyzed using ImageJ software.

[0062] 2. Test Results

[0063] The results are as follows Figure 4 As shown, compared with the PRRSV group, the P-Nrf2 / Nrf2 ratio and HO-1 protein expression level in the spleen of piglets in the PRRSV+DON group were significantly decreased (P < 0.05). Compared with the PRRSV+DON group, the P-Nrf2 / Nrf2 ratio and HO-1 protein expression level in the spleen of piglets in the PRRSV+DON+GLU group were significantly increased (P < 0.05).

[0064] The above results indicate that co-exposure to PRRSV and DON inhibits the activation of the Nrf2 pathway, while glucuronolactone can significantly activate the Nrf2 signaling pathway, systematically enhance the spleen's antioxidant defense capacity, and effectively alleviate oxidative stress damage.

[0065] Experimental Example 6: Effects of glucuronolactone on the expression of inflammation-related genes and proteins in the spleen of piglets co-infected with PRRSV and DON.

[0066] 1. Detection Method

[0067] The mRNA expression detection method for inflammation-related genes (IL-1β, IL-6, IL-18, IL-10, ASC) was the same as in test example 4. The Western blot detection method for inflammation-related proteins (IL-1β, IL-6) was the same as in test example 5.

[0068] 2. Test Results

[0069] mRNA levels: Results are as follows Figure 5 As shown, compared with the PRRSV group, the mRNA expression levels of pro-inflammatory factors IL-1β and IL-18 in the spleen of piglets in the PRRSV+DON group were significantly increased (P < 0.05), while the mRNA expression levels of anti-inflammatory factors IL-10 and the inflammasome-related gene ASC were significantly decreased (P < 0.05). Compared with the PRRSV+DON group, the mRNA expression levels of IL-1β and IL-18 in the spleen of piglets in the PRRSV+DON+GLU group were significantly decreased (P < 0.05), while the mRNA expression levels of IL-10 and ASC were significantly increased (P < 0.05).

[0070] Protein levels: Results as follows Figure 6 As shown, compared with the PRRSV group, the expression level of IL-6 protein in the spleen of piglets in the PRRSV+DON group was significantly increased (P < 0.05). Compared with the PRRSV+DON group, the expression level of IL-6 protein in the spleen of piglets in the PRRSV+DON+GLU group was significantly decreased (P < 0.05).

[0071] The above results indicate that glucuronolactone can inhibit the expression of pro-inflammatory factors and promote the expression of anti-inflammatory factors, effectively alleviating the splenic inflammatory response caused by co-exposure to PRRSV and DON, and reducing splenic inflammatory damage.

[0072] Experimental Example 7: Effects of glucuronolactone on the expression of apoptosis-related genes and proteins in the spleen of piglets co-infected with PRRSV and DON.

[0073] 1. Detection Method

[0074] The mRNA expression detection method for apoptosis-related genes (ATG5, LC3BI, LC3BI, PINK1, FUNDC1, BCL-2) was the same as in test example 4. The Western blot detection method for apoptosis-related proteins (Bax, Bcl-2, Caspase-3) was the same as in test example 5.

[0075] 2. Test Results

[0076] mRNA levels: Results are as follows Figure 7As shown, compared with the PRRSV group, the mRNA expression levels of autophagy and apoptosis-related genes ATG5, LC3BI, LC3BI, and PINK1 in the spleen of piglets in the PRRSV+DON group were significantly increased (P < 0.05). Compared with the PRRSV+DON group, the mRNA expression levels of ATG5, LC3BI, LC3BI, PINK1, and FUNDC1 in the spleen of piglets in the PRRSV+DON+GLU group were significantly decreased (P < 0.05).

[0077] Protein levels: Results as follows Figure 8 As shown, compared with the PRRSV group, the expression levels of pro-apoptotic proteins Bax and Caspase-3 in the spleen of piglets in the PRRSV+DON group were significantly increased (P < 0.05). Compared with the PRRSV+DON group, the expression level of anti-apoptotic protein Bcl-2 in the spleen of piglets in the PRRSV+DON+GLU group was significantly increased (P < 0.05).

[0078] The above results indicate that co-exposure to PRRSV and DON induces excessive apoptosis of spleen cells, while glucuronolactone can inhibit the expression of pro-apoptotic proteins and promote the expression of anti-apoptotic proteins, effectively alleviating the apoptosis process of spleen cells and maintaining the homeostasis of spleen immune cells.

[0079] In summary, this invention, by adding an effective amount of glucuronolactone to the feed, synergistically alleviates spleen damage in piglets caused by co-exposure to PRRSV and DON from four dimensions: improving spleen tissue structure, alleviating oxidative stress, inhibiting inflammatory response, and inhibiting cell apoptosis. This effectively solves the technical problem of the lack of effective intervention methods for this type of damage in the prior art.

[0080] Obviously, the specific implementation schemes described above are merely a further detailed explanation of the purpose, technical solution and beneficial effects of the present invention. It should be understood that the above descriptions are only specific examples of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of glucuronolactone in the preparation of feed to alleviate spleen damage caused by the combined effects of PRRSV and vomitoxin.

2. The application according to claim 1, characterized in that, The spleen injury includes damage to spleen tissue structure, oxidative stress, inflammatory response and / or apoptosis.

3. The application according to claim 1 or 2, characterized in that, The effective amount of glucuronolactone added to the feed is 50–600 mg / kg based on the total feed mass.

4. The application according to claim 3, characterized in that, The effective amount of glucuronolactone added to the feed is 200 mg / kg based on the total mass of the feed.

5. A feed for alleviating spleen damage caused by the combined effects of PRRSV and vomitoxin, characterized in that, It consists of a basic feed and an effective amount of glucuronolactone; the amount of glucuronolactone added is 50-600 mg / kg based on the total feed mass.

6. The feed according to claim 5, characterized in that, The amount of glucuronolactone added is 200 mg / kg based on the total weight of the feed.

7. The feed according to claim 5 or 6, characterized in that, The basic feed includes the following components: corn, protein substances, low-protein whey powder, oils, sugars, amino acids, feed additives, enzyme preparations, and premixes.

8. The feed according to claim 7, characterized in that, By weight, the basic feed comprises the following components: 450-650 parts corn, 250-350 parts protein, 40-80 parts low-protein whey powder, 15-35 parts oil, 10-30 parts carbohydrates, 3.2-7.6 parts amino acids, 5.5-10 parts feed additives, 1-3 parts enzyme preparations, and 10-30 parts premix.

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