Application of rhizoma atractylodis macrocephalae in preparation of feed for increasing antibody titer of weaned piglets, improving immune balance of intestinal mucosa of weaned piglets and regulating intestinal oxidative stress and cell apoptosis of weaned piglets

Abstract

The invention provides application of rhizoma atractylodis macrocephalae in preparation of feed for increasing antibody titer of weaned piglets, improving immune balance of intestinal mucosa of the weaned piglets and adjusting intestinal oxidative stress and cell apoptosis of the weaned piglets, and belongs to the technical field of livestock breeding. According to the invention, the bighead atractylodes rhizome is added into the daily ration, so that the immunity of the weaned piglets can be improved by adjusting the titer of an antibody, and the immune balance of intestinal mucosa of the weaned piglets can be adjusted by adjusting the mRNA expression level of interferon and defensin, promoting the secretion of SIgA and increasing the positive area of CD4 + T cells; the antioxidant capacity of the weaned piglets is improved by regulating and controlling a Keap1 / Nrf2 oxidative stress signal channel; excessive cell apoptosis in intestinal tracts of weaned piglets can be relieved, so that the effect of regulating intestinal health is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of animal husbandry, and particularly relates to an application of Atractylodes macrocephala in preparing a feed for increasing antibody titer of weaned piglets, improving intestinal mucosal immune balance of weaned piglets, and regulating intestinal oxidative stress and cell apoptosis. Background Art

[0002] In recent years, the pig farming industry has developed rapidly, from family farms to large-scale and intensive farming, and domestic and foreign capital has entered the pig farming industry. Animal nutrition technology and feed industry have also developed vigorously, and veterinary drugs and various additives have developed rapidly. However, there are problems to varying degrees in feeding management, environmental cleanliness, and biosafety, which have led to a high incidence of piglet weaning stress syndrome, increased pig production costs, and reduced production efficiency, which has brought great development resistance to the pig farming industry. Piglet weaning stress syndrome refers to the phenomenon that after piglets are weaned at 20-28 days of age, due to various stress factors (food nutrition, feeding environment, pathogen infection, maternal or autoimmune conditions), piglets suffer from severe diarrhea, fat loss, poor feed utilization, slow growth, stagnant growth and development, and even stiff pigs. At the same time, diarrhea leads to reduced digestion and absorption capacity, decreased immunity, weakened resistance to diseases, and easy secondary infection of other diseases.

[0003] The decline in immunity caused by weaning stress in piglets is mainly manifested in the decrease in the number and imbalance of T lymphocytes and B lymphocytes in the blood of weaned piglets, and the decrease in the secretion of cytokines, immunoglobulins, and specific antibodies. In addition, weaning stress can cause damage to the intestinal barrier of piglets, which is specifically manifested as follows: ① The mechanical barrier is destroyed, the intestinal villi are atrophied, the crypts are hyperplastic, the intestinal tight junction proteins are destroyed, and the secretion of intestinal digestive enzymes is reduced; ② The intestinal chemical barrier is destroyed, the intestinal mucus layer is damaged, the number of goblet cells is reduced, the arrangement is disordered, and the secretion of mucin is reduced; ③ The intestinal immune barrier is destroyed, the intestinal inflammatory cytokines are upregulated, and in severe cases, it may also cause intestinal oxidative stress, damage the intestine, and ultimately lead to apoptosis and cell cycle arrest of small intestinal epithelial cells in weaned piglets; ④ The microbial flora changes, and the proportion of dominant flora decreases. Therefore, the above factors together lead to reduced digestion and absorption capacity, decreased feed intake, diarrhea, aggravated immunity decline, increased susceptibility to pathogens, and increased mortality in weaned piglets.

[0004] Chinese (veterinary) medicine believes that the spleen is responsible for transportation and transformation, is the source of qi and blood biochemical transformation, and provides nutrition and energy for various tissues of the body. The physiological characteristics of young animals are summarized as "vigorous vitality, rapid development, delicate organs, incomplete physical form, weak spleen and stomach, loose pores, and insufficient kidney qi". Therefore, young animals are often congenitally deficient in spleen and stomach, and their spleen and stomach are not fully developed. In addition, they do not know how to control their food. If they are not properly fed, or if they are sick or have improper medicine and food, their spleen and stomach will be over-consumed, which can easily cause abnormalities in the spleen and stomach's function of receiving and fermenting, resulting in spleen and stomach diseases. Over time, the spleen is weak and unable to transport and transform, causing indigestion and diarrhea in young animals. From the cause of the disease, whether it is food damage and milk damage (nutrition), exogenous cold, dampness, heat, wind and dryness (environment), or spleen and stomach disharmony, and kidney yang is not warm, the root cause of indigestion and diarrhea can be attributed to spleen and stomach weakness. Weaned piglets have delicate organs, incomplete physical form, weak spleen and stomach, and food damage and heat accumulation are the main causes of diarrhea in early weaned piglets. Therefore, piglets often show symptoms of spleen deficiency, which can lead to insufficient qi and blood, malnutrition of the five internal organs and various physiological dysfunctions. During the piglet care process, piglets have weak spleen and stomach, are prone to weaning stress, and have low disease resistance. They are easily attacked by pathogens and then suffer from diarrhea, cough and asthma, which increases mortality and reduces production performance, causing great harm to the pig farming industry.

[0005] In view of the current development trend of "green, efficient and safe" in the breeding industry and the position of "banning antibiotics" (Gadde, U., Kim, WH, Oh, ST, Lillehoj, HS, 2017. Alternatives to antibiotics for maximizing growth performance and feed efficiency in poultry: a review. Animal health research reviews 18, 26-45.). The additives we choose to replace antibiotics should not only improve the efficiency of breeding production, but also meet the needs of health, effectiveness and sustainability. At present, 118 natural plants with medicinal and edible properties have been included in China's "Catalogue of Feed Ingredients". Some of these natural plants and their extracts have been proven to relieve animal stress and improve feed conversion rate. Therefore, this type of natural plants with medicinal and food homology are a good choice for antibiotic substitutes.

[0006] The traditional Chinese medicine Atractylodes macrocephala Koidz.AM belongs to the Asteraceae family and plays a role in antioxidant, immunomodulatory, and antiviral processes. The biological activities of Atractylodes macrocephala are mainly attributed to its essential oils, sesquiterpenes, sesquiterpenoids, polysaccharides, and polyacetylenes (Gu, S., Li, L., Huang, H., Wang, B., Zhang, T., 2019. Antitumor, Antiviral, and Anti-Inflammatory Efficacy of Essential Oils from Atractylodesmacrocephala Koidz. Produced with Different Processing Methods. Molecules (Basel, Switzerland) 24. / Lv, WJ, Ma, YM, Huang, JY, He, SQ, Li, SP, Lin, J., Chen, R., Lun, JC, Liu, J., Guo, SN, 2022. Polysaccharides derived from ShenlingBaizhu San improve colitis via modulating tryptophan metabolisminmice.International journal of biological macromolecules 222,1127-1136. / Zhu,B.,Zhang,QL,Hua,JW,Cheng,WL,Qin,LP,2018.The traditional uses,phytochemistry,and pharmacology of Atractylodes macrocephala Koidz.:Areview.Journal of ethnopharmacology 226,143-167. / Zhu,Q.,Lin,M.,Zhuo,W.,Li,Y.,2021.Chemical Constituents from the Wild Atractylodes macrocephala Koidzand Acetylcholinesterase Inhibitory Activity Evaluation as Well as MolecularDocking Study.Molecules (Basel, Switzerland)26.), these chemical components of Atractylodes macrocephala are believed to be responsible for the ability of traditional Chinese medicine to promote intestinal motility, increase gastric mucosal healing and other beneficial effects through various mechanisms (Yang, L., Yu, H., Hou, A., Man, W., Wang, S., Zhang, J., Wang, X., Zheng, S., Jiang, H., Kuang, H., 2021. A Review of the Ethnopharmacology, Phytochemistry, Pharmacology, Application, Quality Control, Processing, Toxicology, and Pharmacokinetics of the Dried Rhizome of Atractylodes macrocephala. Frontiers in pharmacology 12, 727154.). The anhydrous ethanol extract of Atractylodes macrocephala can also change the structure of human intestinal flora, increase the abundance of Bifidobacterium, and reduce the Bacteroidetes, which are particularly related to gastrointestinal harm (Lemons, JMS, Narrowe, AB, Liu, L., Firrman, J., Mahalak, KK, Van den Abbeele, P., Baudot, A., Deyaert, S., Li, Y., Yu, LL, 2023. Impact of Baizhu, Daqingye, and Hehuanhua extracts on the human gut microbiome. Frontiers in cellular and infection microbiology 13, 1298392.).

[0007] There are some studies on the positive effects of the traditional Chinese medicine Atractylodes macrocephala on gastrointestinal function, but whether Atractylodes macrocephala can improve the intestinal health of weaned piglets and improve weaning stress syndrome in weaned piglets needs further exploration. Summary of the invention

[0008] In view of this, the object of the present invention is to provide an application of Atractylodes macrocephala in preparing feed for increasing antibody titer of weaned piglets, improving intestinal mucosal immune balance of weaned piglets, and regulating intestinal oxidative stress and cell apoptosis.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The invention provides application of Atractylodes macrocephala in preparing feed for improving intestinal health of weaned piglets.

[0011] The invention also provides application of Atractylodes macrocephala in preparing feed for improving immunity of weaned piglets.

[0012] The invention also provides the use of Atractylodes macrocephala in preparing feed for improving the antioxidant capacity of weaned piglets.

[0013] The invention also provides application of Atractylodes macrocephala in preparing feed for regulating the titer of serum antibodies of weaned piglets, wherein the antibodies include PRV-Ab and CSFV-Ab.

[0014] The present invention also provides the use of Atractylodes macrocephala in preparing feed for regulating the mRNA expression level of intestinal interferon and defensin in weaned piglets. The interferon includes IFN-α, IFN-β and IFN-γ, and the defensin includes pBD2 and pBD129.

[0015] The present invention also provides the use of Atractylodes macrocephala in preparing feed for promoting SIgA secretion in the intestine of weaned piglets.

[0016] The invention also provides a method for preparing a method for increasing intestinal CD4 in weaned piglets. + Application of feed to the T cell positive area.

[0017] The present invention also provides the use of Atractylodes macrocephala in preparing feed for regulating the Keap1 / Nrf2 oxidative stress signal pathway in the intestine of weaned piglets, wherein the expression level of Keap1 is reduced, and the expression level of Nrf2 is increased; the expression levels of HO-1 and antioxidant enzymes in the Keap1 / Nrf2 oxidative stress signal pathway are increased, and the antioxidant enzymes include Cat, SOD, Nqo1 and Nos2.

[0018] The present invention also provides the use of Atractylodes macrocephala in preparing feed for alleviating intestinal cell apoptosis in weaned piglets, wherein the apoptosis-related proteins include anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax, the expression level of the anti-apoptotic protein Bcl-2 is increased, and the expression level of the pro-apoptotic protein Bax is decreased;

[0019] The expression level of the anti-apoptotic protein Bcl-2 is increased to inhibit the expression of Caspase-3, and the protein expression level of Caspase-3 is reduced.

[0020] Preferably, the amount of Atractylodes macrocephala added is 200-800 mg / kg -1 .

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The invention adds Atractylodes macrocephala to the diet, can improve the immunity of weaned piglets by regulating antibody titer; can regulate the intestinal mucosal immune balance of weaned piglets by regulating the mRNA expression levels of interferon and defensin, promoting the secretion of SIgA, and increasing the positive area of ​​CD4+T cells; can improve the antioxidant capacity of weaned piglets by regulating the Keap1 / Nrf2 oxidative stress signaling pathway; can alleviate excessive cell apoptosis in the intestine of weaned piglets, thereby achieving the effect of regulating intestinal health. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the effect of adding AM to the diet on the antibody titer of weaned piglets (where A is the effect of adding AM to the diet on CSFV-Ab in the serum of weaned piglets 14 and 28 days after the addition of AM to the diet, and B is the effect of adding AM to the diet on PRV-Ab in the serum of weaned piglets 14 and 28 days after the addition of AM to the diet);

[0024] Figure 2 The effects of AM on intestinal mucosal immunity of weaned piglets (A is the effect of AM added to the diet on the mRNA expression level of intestinal related cytokines detected by RT-qPCR; B is the effect of AM added to the diet on the expression level of intestinal SIgA detected by Western Blot; C is the effect of AM added to the diet on the expression level of intestinal CD4 + T cell positive area; D is the effect of adding AM to the diet on the expression level of intestinal SIgA by immunohistochemical staining);

[0025] Figure 3 The effect of AM on the antioxidant function of weaned piglets (A and B are Western Blot tests of the effect of AM added to the diet on the expression level of related proteins in the Keap1 / Nrf2 signaling pathway in the intestine of weaned piglets, and C is RT-qPCR tests of the effect of AM added to the diet on the mRNA expression level of antioxidant enzymes related to oxidative stress in the intestine of weaned piglets);

[0026] Figure 4 The effects of AM on intestinal cell apoptosis (A and B are Western Blot tests of the effects of AM added to the diet on the expression levels of intestinal cell apoptosis-related proteins in weaned piglets, and C and D are immunohistochemical staining tests of the effects of AM added to the diet on the expression levels of Bax in the intestine). DETAILED DESCRIPTION

[0027] The invention provides application of Atractylodes macrocephala in preparing feed for improving intestinal health of weaned piglets.

[0028] The invention also provides application of Atractylodes macrocephala in preparing feed for improving immunity of weaned piglets.

[0029] The invention also provides the use of Atractylodes macrocephala in preparing feed for improving the antioxidant capacity of weaned piglets.

[0030] The invention also provides application of Atractylodes macrocephala in preparing feed for regulating the titer of serum antibodies of weaned piglets, wherein the antibodies include PRV-Ab and CSFV-Ab.

[0031] The present invention also provides the use of Atractylodes macrocephala in preparing feed for regulating the mRNA expression level of intestinal interferon and defensin in weaned piglets. The interferon includes IFN-α, IFN-β and IFN-γ, and the defensin includes pBD2 and pBD129.

[0032] The present invention also provides the use of Atractylodes macrocephala in preparing feed for promoting SIgA secretion in the intestine of weaned piglets.

[0033] The invention also provides a method for preparing a method for increasing intestinal CD4 in weaned piglets. + Application of feed to the T cell positive area.

[0034] The present invention also provides the use of Atractylodes macrocephala in preparing feed for regulating the Keap1 / Nrf2 oxidative stress signal pathway in the intestine of weaned piglets, wherein the expression level of Keap1 is reduced, and the expression level of Nrf2 is increased; the expression levels of HO-1 and antioxidant enzymes in the Keap1 / Nrf2 oxidative stress signal pathway are increased, and the antioxidant enzymes include Cat, SOD, Nqo1 and Nos2.

[0035] The present invention also provides the use of Atractylodes macrocephala in preparing feed for alleviating intestinal cell apoptosis in weaned piglets. The apoptosis-related proteins include anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax. The expression level of the anti-apoptotic protein Bcl-2 is increased, and the expression level of the pro-apoptotic protein Bax is decreased. The expression level of the anti-apoptotic protein Bcl-2 is increased to inhibit the expression of Caspase-3, and the protein expression level of Caspase-3 is decreased.

[0036] In the present invention, the amount of Atractylodes macrocephala added is preferably 200 to 800 mg kg -1 , more preferably 400 mg·kg -1 .

[0037] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0038] Example 1

[0039] (1) Experimental design

[0040] A total of 64 35-day-old weaned piglets (initial weight 11.34±0.43kg) of three-way crossbreed (Durham × Long Beach × Long Beach) with similar body weight and good health were randomly divided into four treatment groups, with 16 piglets in each group, namely the basal diet group (fed with the basal diet, Control group), 200mg·kg -1 Atractylodes macrocephala powder group (200 mg kg -1 Atractylodes macrocephala powder, AML group), 400 mg·kg -1 Atractylodes macrocephala powder group (400 mg kg -1 Atractylodes macrocephala powder, AMM group), 800 mg·kg -1 Atractylodes macrocephala powder group (800 mg kg -1 Atractylodes macrocephala powder (AMH group), the experimental period was 28 days, and the Atractylodes macrocephala powder (AM) used in the experiment was purchased from Anning Zhengdetang Medicinal Materials Store (Gansu, China).

[0041] (2) Basic diet and feeding management

[0042] Piglets were fed a basal diet prepared with corn-soybean meal. The nutrients and feed composition of the basal diet are shown in Table 1. Each group of pigs was housed individually in a pen with a stainless steel feeder and waterer. The room temperature was between 26 and 28 °C. In addition, the relative humidity level was maintained in the range of 65% to 75%. Feeding was done three times a day at 8:00, 14:00, and 20:00. During the 28-day trial, piglets had unlimited access to food and water. The animal research and related protocols used in this experiment were approved by the Animal Ethics Committee of the Lanzhou Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences (License No.: SCXK Gansu 2023-021). The basal diet used in the experiment met the nutritional requirements recommended by the National Research Council of the United States (Table 1).

[0043] Table 1 Basic feed composition and nutritional level

[0044]

[0045]

[0046] 1 Premix provides: 9555IU vitamin A, 3000IU vitamin C per kg of basic diet

[0047] Vitamin D3, 81IU Vitamin E, 3mg Vitamin K3, 3mg Vitamin B, 10.2

[0048] mg vitamin B2, 6 mg vitamin B6, 0.036 mg vitamin B12, 0.24 mg

[0049] D-biotin, 30mg D-pantothenic acid, 36mg niacinamide, 300.06mg choline chloride,

[0050] 135mg iron, 115mg copper, 60mg manganese, 1144.5mg zinc, 118.2mg methionine

[0051] Acid, 1638mg L-lysine.

[0052] 2 Calculated values ​​of each nutrient level.

[0053] (3) Sample collection

[0054] After 28 days of feeding, 15 mL of blood was collected from the anterior vena cava using a vacuum blood collection tube (without anticoagulant) and then allowed to rest for 10 min at 3500 r / min. -1 Centrifuge for 10 minutes and take the supernatant for testing. After slaughter, 10 cm tissue segments were taken from the duodenum, jejunum, and mid-ileum, and then the intestinal cavity was thoroughly washed with ice-cold phosphate-buffered saline (PBS) solution, and 2-3 cm intestinal segments were taken and fixed in paraformaldehyde. The remaining tissue was placed in a 2 mL cryovial, immediately immersed in liquid nitrogen, and then stored at -80 °C for subsequent analysis.

[0055] Example 2

[0056] Adding Atractylodes macrocephala to diet can improve the immunity of weaned piglets by regulating antibody titer

[0057] After weaning, piglets are exposed to various stress factors, which lead to a decrease in immunity and an increased infection rate for various pathogens. As swine fever virus and pseudorabies virus can infect pigs of all ages, especially piglets, are more susceptible to infection and the symptoms are more severe, causing significant losses to the pig industry. Therefore, the prevention and control of these two diseases is particularly important in breeding.

[0058] (1) ELISA kit detection method

[0059] According to the test instructions of the ELISA kit (Jianglai Biotechnology Co., Ltd.), the levels of porcine pseudorabies virus antibodies (PRV-Ab) and classical swine fever virus antibodies (CSFV-Ab) in serum were determined.

[0060] (2) Data processing method

[0061] All data were expressed as "Mean±SEM". The data were first tested for normal distribution and homogeneity of variance. For those that conformed to normal distribution and had homogeneous variance, the One-Way ANOVA method was used for comparison between groups. For those that did not conform to normal distribution, non-parametric tests were used. A trend of significant difference was considered when 0.05 < P < 0.10, and a significant difference was considered when P < 0.05. SPSS 27 was used for data statistical analysis, and GraphPad Prism 9 was used for graphing.

[0062] (3) Test results

[0063] As Figure 1 shown. On the 14th day of the experiment, compared with the Control group, the PRV-Ab level in the AMM group was significantly increased (P < 0.05, Figure 1 B in), and on the 28th day of the experiment, although there were no significant changes in PRV-Ab and CSFV-Ab (P > 0.05), the PRV-Ab and CSFV-Ab in both the AML group and the AMM group remained at a relatively high level ( Figure 1 A and B in). Therefore, adding AM to the diet can improve the immunity of weaned piglets by regulating antibody titers.

[0064] Example 3

[0065] Adding Atractylodes macrocephala to the diet can regulate the mRNA expression levels of interferon and defensin, promote the secretion of SIgA, and increase the positive area of CD4 + T cells, thereby regulating the intestinal mucosal immune balance of weaned piglets

[0066] Interferons (IFNs) have antiviral activity, promote T cells to phagocytize pathogens, and are closely related to mucosal immunity. Porcine β-defensin (pBD), as a natural antimicrobial peptide in the body, has dual antibacterial and antiviral effects and is also an integral part of the innate immunity of the intestine. IFN-α, IFN-β, and IFN-γ can be produced by most immune cells and have the effects of restricting the spread of infectious agents (especially viral pathogens), regulating the balance of innate immune responses, and activating adaptive immunity to promote the antigen-specific responses of T cells and B cells. Intestinal mucosal immunity is an important part of the body's immune system. SIgA is considered the main product of intestinal mucosal immunity and can effectively inhibit the adhesion, colonization, or invasion of pathogens on the mucosal surface. Together with mucus and antimicrobial peptides, it forms the first line of defense of intestinal epithelial cells and protects them from pathogens in the intestine. In addition, CD4 + T cells play an important role in intestinal mucosal immunity and can promote the secretion of SIgA. SIgA is mainly produced by CD4 +After T cells differentiate into follicular helper T cells, the plasma cells in the lamina propria secrete SIgA after receiving stimulation from follicular helper T cells. However, due to the underdeveloped intestinal immune system of piglets, the maternal immune antibody channel is interrupted after weaning, making it impossible for the intestine to secrete enough SIgA, resulting in decreased immunity of weaned piglets.

[0067] (1) Detection of gene expression levels

[0068] Trizol was added to the intestinal tissue to extract total RNA. The RNA concentration was detected by ultra-micro spectrophotometer according to the instrument instructions and the reverse transcription system was prepared: 2 μL of 5×gDNA, 4 μL of 5×Evo M-mLV RT Reaction MIX, and 14 μL of RNA+DEPC water were used to prepare 20 μL of reverse transcription system for reverse transcription. Green Pro Tap HS 5μL, upstream and downstream primers 0.2μL each, DEPC water 4.1μL, cDNA 0.5μL to prepare 10μL PCR system, use QuantStudio TM Design & Analysis real-time fluorescence quantitative PCR instrument was used for amplification, and β-actin was used as the internal reference. 95℃ pre-denaturation for 5 min, 95℃ denaturation for 10 s, 60℃ annealing for 20 s, 72℃ extension for 20 s, 40 cycles. The △CT method (2 -△△CT ) Calculate the change of CT value to judge the expression change of target gene. The primer sequences are shown in Table 2.

[0069] (2) Protein expression level detection

[0070] The intestinal tissue was lysed with PMSF lysis buffer, and the supernatant was collected as a protein extract by centrifugation at 4°C and the concentration of the protein sample was measured according to the bicinchoninic acid assay (BCA). Equal amounts of protein samples were separated by 8-12% SDS-PAGE and then electrotransferred to a polyvinylidene difluoride membrane (Millipore, MA, USA). The membrane was sealed with 5% skim milk at room temperature for 1.5 h, incubated with SIgA (1:1000), and HRP-conjugated goat anti-mouse IgG secondary antibody (1:5000) was added. Then, 1 mL of each of A and B solutions were mixed using ultrasensitive ECL chemiluminescent solution (Apply.gen, Beijing, China), and dropped onto the NC membrane. The detection was performed using a Biosciences Imager, and β-actin (1:1000) was used as a reference protein for comparison. The grayscale value of the band was analyzed and calculated by Image J software.

[0071] (3) Immunohistochemical staining

[0072] The intestinal tissues of piglets were collected, fixed in 4% neutral formaldehyde for 24 h, embedded in paraffin after dehydration, sectioned, dewaxed, washed, retrieved by high-pressure antigen retrieval, blocked for endogenous peroxidase, incubated with 3% hydrogen peroxide for 30 min, blocked with goat serum for 30 min, and then incubated with the primary antibody SIgA overnight at 4°C. The next day, incubated with the secondary antibody biotinylated goat anti-rabbit IgG polymer for 30 min, and then added with the working solution of streptavidin labeled with horseradish peroxidase for 30 min. After coloring, counterstaining with hematoxylin, fractionation with hydrochloric acid ethanol, and sealing, microscopic examination was performed.

[0073] (4) Immunofluorescence analysis

[0074] After dewaxing the sections to water, they were immersed in EDTA buffer for microwave antigen repair. After natural cooling, blocked with 3% BSA at room temperature for 30 min, and then incubated with the primary antibody CD4 + T cells overnight in a wet box at 4°C. After washing the slides with PBS, added with the secondary antibody biotinylated goat anti-rabbit IgG polymer and incubated in the dark at room temperature for 50 min. Then counterstained the cell nuclei with DAPI in the dark. After washing and centrifuging the slides dry, added with an anti-fluorescence quenching agent to seal the slides. An IF laser scanning confocal microscope (NIKON ECLIPSE C1, Nikon, Japan) was used to observe the fluorescence images at the excitation wavelengths of 488 nm (green) and 405 nm (blue).

[0075] (5) Data processing method

[0076] All data were expressed as "mean ± standard deviation (Mean ± SEM)". The data were first tested for normal distribution and homogeneity of variance. If they conformed to normal distribution and had homogeneous variance, the One-Way ANOVA method was used for comparison between groups. If they did not conform to normal distribution, non-parametric tests were used. A trend of significant difference was considered when 0.05 < P < 0.10, and a significant difference was considered when P < 0.05. SPSS 27 was used for data statistical analysis, and GraphPad Prism 9 was used for graphing.

[0077] Table 2 Primer sequences

[0078]

[0079]

[0080] (6) Test results

[0081] As Figure 2As shown. Compared with the control group, the mRNA expression levels of IFN-α, IFN-β and IFN-γ in the duodenum, jejunum and ileum were significantly increased (P<0.05). Compared with the control group, the mRNA expression levels of pBD2 and pBD129 in the duodenum were significantly increased after adding low and medium doses of AM (P<0.05). The mRNA expression level of pBD2 in the AMH group was also significantly increased (P<0.05). The jejunum and ileum also showed a similar trend ( Figure 2 A in the figure). It can be seen that the addition of AM in the diet can regulate the intestinal mucosal immune health of weaned piglets by regulating the expression levels of related cytokines.

[0082] Compared with the control group, the AML group significantly increased the expression levels of SIgA in the jejunum and ileum (P<0.05), and the AMM group significantly increased the expression level of SIgA in the ileum (P<0.05, Figure 2 B).

[0083] In addition, immunohistochemical results also showed that medium-dose AM significantly increased the expression of SIgA in the duodenum, jejunum, and ileum (P<0.05, Figure 2 C).

[0084] The results of immunofluorescence detection showed that the addition of medium-dose AM to the diet could significantly increase the number of CD4 + T cell positive area (P<0.05, Figure 2 D).

[0085] The above results show that the addition of AM to the diet can promote SIgA secretion and increase CD4 + T cell positive expression, thereby enhancing the intestinal mucosal immunity of weaned piglets.

[0086] Example 4

[0087] Supplementation of Atractylodes macrocephala in diet can improve the antioxidant capacity of weaned piglets by regulating the Keap1 / Nrf2 oxidative stress signaling pathway

[0088] Intestinal oxidative stress in piglets can lead to intestinal damage, which in turn affects the immune function of the intestinal mucosa. Keap1 / Nrf2 is an important endogenous oxidative stress pathway in the body. Under normal circumstances, Nrf2 and Keap-1 are combined and exist in the cytoplasm. However, in the oxidative stress response, the activity of the Keap-1 complex is inhibited, which promotes the aggregation of Nrf2 in the nucleus, induces the expression of downstream HO-1, and increases the expression of antioxidant enzymes such as Cat and SOD.

[0089] (1) Western blot analysis

[0090] The intestinal tissues were lysed with PMSF lysis buffer, and the supernatant was collected by centrifugation at 4°C as the protein extract. The concentration of the protein sample was measured according to the bicinchoninic acid assay (BCA). Equal amounts of protein samples were separated by 8-12% SDS-PAGE and then electrotransferred onto a polyvinylidene difluoride membrane (Millipore, MA, USA). The membrane was blocked with 5% non-fat milk at room temperature for 1.5 h and incubated with Nrf2 (1:1000), Keap-1 (1:1000), and HO-1 (1:1000) respectively. After adding the HRP-conjugated goat anti-mouse IgG secondary antibody (1:5000) or HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000), 1 mL each of the hypersensitive ECL chemiluminescent solution (Apply.gen, Beijing, China) A and B solutions were mixed and dropped onto the NC membrane, and detection was performed using a Biosciences Imager. β-actin (1:1000) was used as a reference protein for comparison. In addition, phosphorylated proteins were compared against their respective total proteins. The gray values of the bands were analyzed and calculated using Image J software.

[0091] (2) Detection of gene expression levels:

[0092] Total RNA of intestinal tissues in each group was extracted using TRIzol reagent, and the total RNA was reverse transcribed into cDNA according to the PrimeScript RT reverse transcription kit. Using SYBR Premix Ex Taq II reagent and an Applied Biosystems real-time fluorescence quantitative PCR system, the expression of target gene mRNA was quantitatively analyzed using the 2-ΔΔCT method with reference to the β-actin internal reference gene. The primer sequences of the target genes are shown in Table 2.

[0093] (3) Data processing and analysis:

[0094] Image J was used to process the Western blot images. Each data was expressed as "mean ± standard deviation (Mean ± SEM)". The data were first tested for normal distribution and homogeneity of variance. If they conformed to normal distribution and had homogeneous variance, the One-Way ANOVA method was used for comparison between groups. If they did not conform to normal distribution, non-parametric tests were used. A trend of significant difference was considered when 0.05 < P < 0.10, and a significant difference was considered when P < 0.05. SPSS 27 was used for data statistical analysis, and GraphPad Prism 9 was used for graphing.

[0095] (4) Experimental results:

[0096] As Figure 3As shown. In the duodenum of weaned piglets, compared with the Control group, the expression level of HO-1 in the three AM supplementation groups was significantly increased (P < 0.05). The expression level of Keap-1 was significantly decreased (P < 0.05); in the jejunum, compared with the Control group, the protein expression levels of Nrf2, HO-1 and in the AML group and AMM group were significantly increased (P < 0.05), and the protein level of Keap-1 was significantly decreased (P < 0.05, Figure 3 A or B in the figure); similar results were found in the ileum. In addition, we detected key factors in the intestinal Keap1 / Nrf2 signaling pathway. Compared with the Control group, the mRNA expression levels of antioxidant enzymes Cat, SOD, Nqo1 and Nos2 in the AML and AMM groups were significantly increased (P < 0.05, Figure 3 C). Therefore, dietary AM can improve the antioxidant capacity of weaned piglets by regulating the Keap1 / Nrf2 oxidative stress signaling pathway.

[0097] Example 5

[0098] Supplementation of Atractylodes macrocephala in diet can alleviate excessive cell apoptosis in the intestine of weaned piglets, thereby regulating intestinal health

[0099] Cell apoptosis is closely related to oxidative stress. Excessive production of ROS can cause protein dysfunction, lipid peroxidation and DNA damage, leading to excessive cell apoptosis and then damaging intestinal tissue. Bcl-2 and Bax are common apoptosis-related proteins. Decreased Bcl-2 activity can open mitochondrial membrane channels, promote the release of cytochrome C, and further activate Caspase-3. Activated Caspase-3 can lead to a large amount of cellular energy consumption, and apoptosis plays a key role in mitochondrial outer membrane permeability and subsequent cell apoptosis.

[0100] (1) Western blot analysis

[0101] The intestinal tissue was lysed with PMSF lysis buffer, and the supernatant was collected by centrifugation at 4°C as the protein extract. The concentration of the protein sample was measured according to the bicinchoninic acid assay (BCA). Equal amounts of protein samples were separated by 8-12% SDS-PAGE and then electrotransferred onto a polyvinylidene difluoride membrane (Millipore, MA, USA). The membrane was blocked with 5% skim milk at room temperature for 1.5 h and incubated with Bax (1:1000), Bcl-2 (1:1000), pro-Caspase 3 (1:1000), and Cleaved-Caspase 3 (1:1000) respectively. After adding the HRP-conjugated goat anti-mouse IgG secondary antibody (1:5000) or HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000), 1 mL each of the hypersensitive ECL chemiluminescence solution A and B (Apply.gen, Beijing, China) was mixed well and dropped onto the NC membrane. Detection was performed using a Biosciences Imager, and β-actin (1:1000) was used as a reference protein for comparison. In addition, phosphorylated proteins were compared against their respective total proteins. The gray values of the bands were analyzed and calculated using Image J software.

[0102] (2) Immunohistochemical analysis

[0103] The intestinal tissue of piglets was collected, fixed in 4% neutral formaldehyde for 24 h, dehydrated, paraffin-embedded, sectioned, dewaxed, washed, retrieved by high-pressure antigen retrieval, blocked for endogenous peroxidase, incubated with 3% hydrogen peroxide for 30 min, blocked with goat serum for 30 min, and then incubated with the primary antibody Bax overnight at 4°C. The next day, it was incubated with the biotin-labeled goat anti-rabbit IgG polymer secondary antibody for 30 min, and then the working solution of streptavidin labeled with horseradish peroxidase was added for 30 min. After coloring, counterstaining with hematoxylin, fractionation with hydrochloric acid ethanol, and sealing, microscopic examination was performed.

[0104] (3) Data processing and analysis

[0105] Image J was used to process the Western blot pictures. Each data was expressed as "mean ± standard deviation (Mean ± SEM)". The data was first tested for normal distribution and homogeneity of variance. If it conformed to the normal distribution and had homogeneous variance, the One-Way ANOVA method was used for comparison between groups; if it did not conform to the normal distribution, non-parametric tests were used. A trend of significant difference was considered when 0.05 < P < 0.10, and a significant difference was considered when P < 0.05. SPSS 27 was used for data statistical analysis, and GraphPad Prism 9 was used for graphing.

[0106] (5) Experimental results

[0107] As Figure 4Compared with the Control group, the three dietary AM supplementation groups could increase the expression level of anti-apoptotic protein Bcl-2 and reduce the protein expression levels of pro-apoptotic proteins Bax and Caspase-3 in the intestine of weaned piglets (P<0.05, Figure 4 At the same time, the immunohistochemical results also showed that the expression level of intestinal Bax protein in the AMM group was significantly reduced compared with the Control group (P < 0.05, Figure 4 C and D in Figure 3). Therefore, dietary AM can alleviate excessive cell apoptosis in the intestine of weaned piglets, thereby regulating intestinal health.

[0108] It can be seen from the above embodiments that the present invention can improve the immunity of weaned piglets by adding Atractylodes macrocephala to the diet by adjusting the antibody titer; regulate the mRNA expression level of interferon and defensin, promote the secretion of SIgA, increase the positive area of ​​CD4+T cells, and thus regulate the intestinal mucosal immune balance of weaned piglets; improve the antioxidant capacity of weaned piglets by regulating the Keap1 / Nrf2 oxidative stress signaling pathway; and alleviate excessive cell apoptosis in the intestine of weaned piglets, thereby achieving the effect of regulating intestinal health.

[0109] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of Atractylodes macrocephala in preparing feed for improving intestinal health of weaned piglets.

2. Application of Atractylodes macrocephala in preparing feed for improving immunity of weaned piglets.

3. Application of Atractylodes macrocephala in preparing feed for improving the antioxidant capacity of weaned piglets.

4. The use of Atractylodes macrocephala in preparing feed for regulating serum antibody titer of weaned piglets, characterized in that: The antibodies include PRV-Ab and CSFV-Ab.

5. The use of Atractylodes macrocephala in preparing a feed for regulating the mRNA expression level of intestinal interferon and defensin in weaned piglets, characterized in that: The interferons include IFN-α, IFN-β and IFN-γ, and the defensins include pBD2 and pBD129.

6. Application of Atractylodes macrocephala in preparing feed for promoting intestinal SIgA secretion in weaned piglets.

7. Atractylodes macrocephala preparation increases intestinal CD4 in weaned piglets + Application of feed to the T cell positive area.

8. The use of Atractylodes macrocephala in preparing feed for regulating Keap1 / Nrf2 oxidative stress signaling pathway in the intestine of weaned piglets, characterized in that: The expression level of Keap1 is reduced, and the expression level of Nrf2 is increased; the expression levels of HO-1 and antioxidant enzymes in the Keap1 / Nrf2 oxidative stress signaling pathway are increased, and the antioxidant enzymes include Cat, SOD, Nqo1 and Nos2.

9. The use of Atractylodes macrocephala in preparing feed for alleviating intestinal cell apoptosis in weaned piglets, characterized in that: The apoptosis-related proteins include anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax, the expression level of the anti-apoptotic protein Bcl-2 is increased, and the expression level of the pro-apoptotic protein Bax is decreased; The expression level of the anti-apoptotic protein Bcl-2 is increased to inhibit the expression of Caspase-3, and the protein expression level of Caspase-3 is reduced.

10. The use according to any one of claims 1 to 9, characterized in that: The amount of Atractylodes macrocephala added is 200-800 mg kg -1 .

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