Application method of ginsenoside Rg3 for relieving immune stress of broiler chickens

Abstract

The invention discloses an application method of ginsenoside Rg3 for relieving broiler chicken immune stress, and relates to the technical field of broiler chicken immune stress intervention. The invention reveals that ginsenoside Rg3 drives the polarization conversion of macrophages from M1 type to M2 type by regulating MAPK and mTOR / PI3K / AKT signal channels for the first time, and alleviates the immune stress of broiler chickens.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of broiler immune stress intervention, in particular to an application method of ginsenoside Rg3 for relieving broiler immune stress. BACKGROUND

[0002] Ginsenosides Rg1 and Re are known to have certain anti-inflammatory and immune stress relieving effects, but research shows that their effects are weaker than g3 (effect Rg3 > Rg1 > Re), and the specific mechanism of their action through regulating macrophage polarization has not been clearly defined.

[0003] Macrophage polarization imbalance (M1 pro-inflammatory / M2 anti-inflammatory) is the core mechanism of broiler immune stress, but existing technologies lack targeted regulation strategies for this key node.

[0004] The deficiencies of the prior art are as follows:

[0005] Unclear mechanism: existing research has not revealed the specific signal pathway (such as MAPK, mTOR / PI3K / AKT, etc.) through which ginsenoside Rg3 regulates the balance of macrophage M1 / M2 polarization to relieve broiler immune stress.

[0006] Limited application: other ginsenosides (such as Rg1) have weaker binding ability with related targets, and limited inhibitory effect on inflammation and immune stress relief.

[0007] Species difference: existing research focuses on mammalian macrophages (such as mouse RAW264.7), while the avian immune system has evolutionary differences (such as differences in TLR4 signaling pathway). This patent first confirms that Rg3 regulates M1 / M2 polarization to relieve immune stress in avian macrophages (HD11 cell line) and broiler hypothalamic tissue, filling the gap in avian-specific mechanisms.

[0008] Lack of targeting: existing intervention programs do not precisely intervene at the key node of immune regulation, macrophage polarization.

[0009] Therefore, a new solution is needed to address the above problems. SUMMARY

[0010] The purpose of the present application is to provide an application method of ginsenoside Rg3 for relieving broiler immune stress to solve the technical problems raised in the background art.

[0011] To achieve the above purpose, the present application provides the following technical solution: an application method of ginsenoside Rg3 for relieving broiler immune stress, wherein ginsenoside Rg3 is applied in the preparation of a drug or feed additive for relieving broiler immune stress, and the ginsenoside Rg3 relieves broiler immune stress by regulating the balance of macrophage M1 / M2 polarization.

[0012] Further, the ginsenoside Rg3 alleviates the immune stress of broilers by regulating the M1 / M2 polarization balance of macrophages, and at least includes the following steps:

[0013] Using ginsenoside Rg3 as an active ingredient;

[0014] By regulating the MAPK (such as p38MAPK, JNK) and mTOR / PI3K / AKT signaling pathways, the macrophages are driven to transform from M1 type (pro-inflammatory) to M2 type (anti-inflammatory);

[0015] The down-regulation of M1 markers (iNOS, IL-1β, CD86, etc.) and the up-regulation of M2 markers (Arg-1, CD206, IL-10, etc.) are used as the efficacy determination standard;

[0016] By regulating the polarization balance of macrophages, Rg3 alleviates the immune stress response of broilers, especially in avian macrophages and hypothalamic tissues of broilers.

[0017] Compared with the prior art, the beneficial effects of the present application are:

[0018] The present application first discloses that ginsenoside Rg3 alleviates the immune stress of broilers by regulating the MAPK and mTOR / PI3K / AKT signaling pathways, and driving the macrophages to transform from M1 type to M2 type. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiment description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0020] Figure 1 Schematic diagram for the influence of in vitro LPS stimulation on inflammatory factors of HD11 cells;

[0021] Figure 2 Schematic diagram for qPCR detection of M1 type macrophage polarization related cytokines;

[0022] Figure 3 Schematic diagram for qPCR detection of M2 type macrophage polarization related cytokines;

[0023] Figure 4 Schematic diagram for transcriptomic analysis of M1 and M2 polarization related genes;

[0024] Figure 5 Schematic diagram for the influence of ginsenoside Rg3 on avian macrophage polarization related indicators;

[0025] Figure 6 Schematic diagram of local structure of transmission electron microscope photos X of each group of HD11 cells;

[0026] Figure 7 Schematic diagram of ELISA detection of M1 and M2 polarization related cytokine content in cell supernatant;

[0027] Figure 8 Schematic diagram of gene expression level of pathway;

[0028] Figure 9 Schematic diagram of qPCR detection of M1 / M2 type macrophage related factors in peripheral blood of immune stress broilers;

[0029] Figure 10 Schematic diagram of HE pathological section of broiler hypothalamus;

[0030] Figure 11 Schematic diagram of Nissl staining section of broiler hypothalamus;

[0031] Figure 12 Schematic diagram of transmission electron microscope photos of hypothalamic neuron cells and microglial cells in each group;

[0032] Figure 13 Schematic diagram of qPCR detection of M1 type microglial cell polarization related cytokines;

[0033] Figure 14 Schematic diagram of qPCR detection of M2 type microglial cell polarization related cytokines;

[0034] Figure 15 Schematic diagram of gene expression level of hypothalamic cell pathway;

[0035] Figure 16 Schematic diagram of molecular docking of target points of MAPK signaling pathway and ginsenoside Rg3. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments.

[0037] The application method of ginsenoside Rg3 for relieving immune stress of broilers is that the ginsenoside Rg3 is applied in the preparation of a drug or feed additive for relieving immune stress of broilers, and the ginsenoside Rg3 relieves immune stress of broilers by regulating the M1 / M2 polarization balance of macrophages.

[0038] Further, the process of the ginsenoside Rg3 for relieving immune stress of broilers by regulating the M1 / M2 polarization balance of macrophages at least includes the following steps:

[0039] Ginsenoside Rg3 is used as the active ingredient;

[0040] By regulating MAPK (such as p38MAPK, JNK) and the mTOR / PI3K / AKT signaling pathway, macrophages are driven to transform from M1 (pro-inflammatory) to M2 (anti-inflammatory).

[0041] The efficacy was determined by downregulation of M1 markers (iNOS, IL-1β, CD86, etc.) and upregulation of M2 markers (Arg-1, CD206, IL-10, etc.).

[0042] It is applied to broilers, especially targeting avian macrophages (such as the HD11 cell line) and broiler hypothalamic tissue.

[0043] The principle is explained as follows:

[0044] 1. Rg3 binds to core targets of the MAPK pathway (such as MAPK14 / p38, MAPK8 / JNK) and targets of the mTOR / PI3K / AKT pathway, activating these pathways.

[0045] 2. The activated MAPK and mTOR / PI3K / AKT pathways work synergistically to inhibit the release of pro-inflammatory factors (such as IL-1β and TNF-α) while promoting the expression of anti-inflammatory factors (such as IL-10 and TGF-β).

[0046] 3. This regulatory effect drives the transformation of macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type, which is manifested by a decrease in the expression of M1 markers (such as iNOS, IL-1β, CD86) and an increase in the expression of M2 markers (such as Arg-1, CD206, IL-10).

[0047] 4. Applied to broilers, especially avian macrophages (such as the HD11 cell line) and broiler hypothalamic tissue.

[0048] The specific mechanism is explained as follows:

[0049] 1) Predicting potential mechanisms of action:

[0050] We used RNA-seq to screen differentially expressed genes (DEGs) and combined this with GO and KEGG enrichment analysis to explore the key biological pathways by which Rg3 regulates M1 / M2 polarization. Based on network pharmacology, we predicted the potential mechanism by which Rg3 alleviates immune stress in broilers and verified its binding activity to core targets (such as AKT2, MAPK14, TGFBR1, and VEGFA) using molecular docking technology.

[0051] 2) In vitro cell experiments:

[0052] This study used the avian bone marrow macrophage cell line (HD11) for in vitro experiments. During cell resuscitation, pre-warmed culture medium and liquid nitrogen-frozen cells were rapidly thawed in a 37°C water bath. After centrifugation (1000 rpm, 5 min), the supernatant was discarded, and the cells were resuspended in complete culture medium and cultured overnight. For passage, the cell layer was washed with PBS, and trypsin digested until slight cell detachment occurred. Digestion was stopped with serum-containing culture medium, and the cells were centrifuged, resuspended, and counted at a ratio of 1.5 × 10⁻⁶ cells / cells. 5 Inoculate new culture flasks with cells at a density of 1 × 10⁶ cells / mL. Resuspend cells in PBS before cryopreservation and adjust the density to 1 × 10⁶ cells / mL. 6 -1.5×10 6 Quantity / mL, add cryopreservation solution and store at -80℃.

[0053] Constructing the M1 type polarization model:

[0054] HD11 cells were treated with LPS (0, 0.05, 0.1, 0.2, 0.4, 0.5, 1, 2 μg / mL) for 24 hours. IL-1β mRNA expression was detected by qPCR, and the optimal LPS concentration was determined to be 0.05 μg / mL. Cells were further treated with this concentration for 0, 2, 6, and 12 hours, and the optimal stimulation time was determined to be 2 hours. For screening the optimal concentration of ginsenoside Rg3, cells were pretreated with 0-200 μg / mL of Rg3 for 1 hour, and IL-1β levels were detected by qPCR, determining 100 μg / mL as the optimal intervention concentration. Figure 1 ).

[0055] Experimental Groups:

[0056] Control group (CON): cultured in basal medium;

[0057] LPS group: treated with 0.05 μg / mL LPS for 2 hours;

[0058] LPS+Rg3 group: After 1 hour of pretreatment with 100 μg / mL Rg3, stimulation with LPS (0.05 μg / mL) was performed for 2 hours.

[0059] Rg3 group: 100 μg / mRg3 was used for treatment alone for 1 hour.

[0060] Detection method:

[0061] qPCR: RNA was extracted using the Trizol method, precipitated with isopropanol after chloroform separation, washed with 75% ethanol, and reverse transcribed using a TAKARA kit. The reaction system contained 5 μL of TBGreen Premix, 0.4 μL of each primer, and 1 μL of cDNA template. β-actin was used as an internal control, and gene expression was analyzed using the 2-ΔΔCt method.

[0062] ELISA: Collect cell supernatant and detect cytokines such as IL-1β and IL-10 according to the kit instructions.

[0063] Transmission electron microscopy (TEM): Cells were double-fixed with 2.5% glutaraldehyde and 1% osmium tetroxide, dehydrated with a gradient of acetone (30%→100%), infiltrated with a gradient of acetone-embedding solution, and ultra-thin sections (60-90nm) were stained with uranium acetate and lead citrate to observe the ultrastructure.

[0064] 3) Animal experiments with broilers:

[0065] Seventy-two one-day-old healthy white-feathered broiler chickens (Sichuan Lihua Poultry Co., Ltd.) were selected and approved by the Animal Ethics Committee of Southwest University (IACUC-20240508-02). After three days of acclimatization, they were randomly divided into four groups (18 chickens per group):

[0066] Control group (CON): Intraperitoneal injection of normal saline;

[0067] LPS group: LPS (0.25 mg / kg BW) was injected intraperitoneally;

[0068] LPS+Rg3 group: Rg3 (1 mg / kg BW) was injected intraperitoneally 2 hours before LPS injection;

[0069] Rg3 group: Rg3 (1 mg / kg BW) was injected alone.

[0070] The experiment started at 15 days old and lasted for 15 days. The temperature in the chicken house was initially 33-35℃, then decreased by 2-3℃ every 2 days until it stabilized at 23℃. The house was cleaned twice a day, and chickens had free access to feed and water.

[0071] Samples were collected at the end of the experiment:

[0072] Serum: Blood was collected from the jugular vein, incubated at 37°C for 1 hour, then centrifuged (3500 r / min, 10 min). The separated serum was stored at -80°C and used for ELISA detection of IL-1β, IL-6, Arg1, and IL-10.

[0073] Hypothalamic tissue processing:

[0074] 4% paraformaldehyde fixation: used for paraffin sections (HE staining, Nissl staining);

[0075] 2.5% glutaraldehyde fixation: used for TEM ultrastructure observation;

[0076] Flash-freeze in liquid nitrogen: Store at -80℃ for qPCR detection of gene expression.

[0077] Detection method:

[0078] Histopathology:

[0079] HE staining: After dehydration by gradient ethanol, clearing with xylene, embedding in paraffin, sectioning (5μm), staining with hematoxylin for 5 minutes, differentiating with hydrochloric acid alcohol, counterstaining with eosin for 30 seconds, and mounting with neutral resin.

[0080] Nissl staining: After dewaxing the sections, treat them with Nissl staining solution for 10 minutes, dehydrate and clear them with graded ethanol, and then mount them.

[0081] TEM: Fixed tissues were dehydrated with acetone, embedded in epoxy resin, and then double-stained after ultrathin sectioning (uranium acetate for 20 minutes + lead citrate for 12 minutes) to observe the ultrastructure of neurons and microglia.

[0082] qPCR: New primers include CD11b, MHCII, and mTOR, and the steps are the same as for cell experiments.

[0083] This mechanism solves the problem of the lack of precise regulation strategies for macrophage polarization in existing technologies, and also fills the research gap in avian-specific immune regulation mechanisms.

[0084] Experiments were conducted to verify the content of the examples:

[0085] 1. Ginsenoside Rg3 inhibits M1 polarization of macrophages (cell experiment)

[0086] Model construction: LPS (0.05 μg / mL, 2 h) successfully induced HD11 cells to polarize towards the M1 type, significantly upregulating pro-inflammatory factors (IL-1β, iNOS, IL-6, CD86) and downregulating anti-inflammatory factors (VEGFA, TGF-β). Figures 2-3 ).

[0087] Rg3 mechanism of action: Pretreatment (100 μg / mL, 1 h) significantly inhibited the expression of M1 marker genes (IL-1β↓, IL-6↓, CD86↓) and promoted the expression of M2 marker genes (VEGFA↑, TGF-β↑). Figure 5 , (A) IL-1β; (B) IL-6; (C) Arg1; (D) IL-10 (n=8)).

[0088] Transmission electron microscopy showed that Rg3 alleviated LPS-induced nuclear shrinkage, endoplasmic reticulum expansion, and mitochondrial cristae breakage. Figure 6 (Black arrows represent mitochondria, and white arrows represent endoplasmic reticulum).

[0089] ELISA assay: Decreased IL-1β protein in cell supernatant (↓P<0.001), increased IL-10 (↑P<0.01) and TGF-β1 (↑P<0.001).

[0090] 0001)( Figure 7(A is M1, B is M2).

[0091] Conclusion: Rg3 exerts its anti-inflammatory effect by inhibiting M1 polarization and promoting M2 phenotypic transformation.

[0092] 2. Rg3 regulates the inflammatory response through the MAPK / mTOR pathway.

[0093] Transcriptomics analysis: LPS group vs CON group: 5282 differentially expressed genes (DEGs) were identified, of which 1862 showed reversed expression trends by Rg3.

[0094] GO / KEGG enrichment: Target genes were significantly enriched in the mTOR signaling pathway and MAPK signaling pathway (such as Toll-like receptor and NOD-like receptor pathway).

[0095] Pathway validation: qPCR confirmed that Rg3 regulates the expression of key genes in the MAPK (p-38MAPK, ERK, JNK) and mTOR / PI3K / Akt pathways. Figure 8 (A) mTOR / PI3K / AKT signal path; (B) MAPK signal path (n=6)).

[0096] Conclusion: Rg3 reverses LPS-induced inflammatory gene expression by intervening in the MAPK / mTOR signaling axis.

[0097] 3. Rg3 alleviates immune stress and hypothalamic damage in broilers (animal experiments)

[0098] Serum markers: Rg3 reduces LPS-induced pro-inflammatory factors (IL-1β↓, IL-6↓) and increases anti-inflammatory factors (IL-10↑). Figure 9 (A) CD11b; M1 polarization related: (B) IL-1β; (C) TNF-α; (D) iNOS; (E) IL-6; (F) MHCII; (G) CD86; M2 polarization related: (H) IL-10; (I) TGF-β (n=4)).

[0099] Hypothalamic protection: HE staining / Nissl staining: Rg3 reduces neuronal vacuolar degeneration and restores Nissl body structure. Figures 10-11 ).

[0100] Transmission electron microscopy: Improved neuronal mitochondrial swelling and microglial endoplasmic reticulum dilatation ( Figure 12 (A) Neurons; (B) Microglia.

[0101] qPCR detection: Inhibition of microglial M1 polarization (CD86↓, IL-1β↓), (see...) Figure 13(A) CD86; (B) CD80; (C) IL-12; (D) IL-1β (n=3)), promote M2 polarization (CD206↑, VEGFA↑) Figure 14 , (A) CD206; (B) VEGFA (n=3)).

[0102] Pathway activation: Rg3 significantly upregulated the mRNA expression of mTOR, p-38MAPK, PI3K, and AKT in the hypothalamus. Figure 15 , (A) mTOR; (B) p-38MAPK; (C) PI3K; (D) AKT (n=3)).

[0103] Conclusion: Rg3 alleviates immune stress damage by regulating hypothalamic microglia polarization and the MAPK / mTOR pathway.

[0104] In summary, the present invention possesses several undeniable advantages:

[0105] 1) Core Applications:

[0106] The application of ginsenoside Rg3 in the preparation of drugs or feed additives to alleviate immune stress in broilers is achieved by regulating the polarization balance of macrophages M1 / M2.

[0107] 2) Target mechanism:

[0108] Ginsenoside Rg3 targets the core targets of the MAPK pathway (such as MAPK14 / p38, MAPK8 / JNK) and the mTOR / PI3K / AKT pathway (molecular docking binding energy ≤ -7.0 kcal / mol) (Table 1). Figure 16 ), which regulates macrophage polarization by activating the above pathways.

[0109] Table 1. Binding energy of ginsenoside Rg3 and its core target

[0110]

[0111] 3) Combination of markers:

[0112] The efficacy was determined by downregulation of M1 markers (iNOS, IL-1β, CD86, etc.) and upregulation of M2 markers (Arg-1, CD206, IL-10, etc.).

[0113] 4) Pathway synergistic effect:

[0114] Rg3 inhibits the release of pro-inflammatory factors (downregulation of IL-1β and TNF-α) and promotes the expression of anti-inflammatory factors (upregulation of IL-10 and TGF-β) by simultaneously activating the MAPK (p38 / JNK) and mTOR / PI3K / AKT pathways.

[0115] 5) Application scenario extension:

[0116] A veterinary preparation containing ginsenoside Rg3, used to prevent immune stress-related damage or inflammation-related functional disorders in poultry, such as those affecting the intestines and hypothalamus.

[0117] Transcriptome sequencing:

[0118] In the LPS group, the overall expression levels of multiple genes related to M1 polarization were significantly upregulated, including iNOS, CD86, CD40, and IL-6, with IL-1β showing a change of more than 45-fold, indicating that LPS effectively induced the M1 phenotype. In the LPS+Rg3 group, the expression of these M1 phenotype marker genes was significantly lower than that in the LPS group, suggesting that ginsenoside Rg3 has a certain inhibitory effect on LPS-induced M1 polarization. Figure 4 A).

[0119] like Figure 4 As shown in Figure B, multiple genes related to M2 polarization showed a downregulation trend in the LPS group, while their expression was generally restored in the LPS+Rg3 group. For example, the expression levels of Arg2 and VEGFA increased by 1-fold and 2-fold, respectively, suggesting that ginsenoside Rg3 can promote the transcriptional expression of M2 phenotype-related genes to a certain extent, thereby counteracting the LPS-induced inflammatory state.

[0120] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A method for relieving immune stress in broilers using ginsenoside Rg3, characterized by: Ginsenoside Rg3 is used in the preparation of drugs or feed additives to alleviate immune stress in broilers. Ginsenoside Rg3 alleviates immune stress in broilers by regulating the M1 / M2 polarization balance of macrophages.

2. The method for applying ginsenoside Rg3 to alleviate immune stress in broilers according to claim 1, characterized in that: The process by which ginsenoside Rg3 alleviates immune stress in broilers by regulating the M1 / M2 polarization balance of macrophages includes at least the following steps: Ginsenoside Rg3 is used as the active ingredient; By regulating the MAPK and mTOR / PI3K / AKT signaling pathways, macrophages are driven to transform from M1 to M2. The efficacy was determined by upregulation of M1 markers and downregulation of M2 markers. By regulating macrophage polarization balance, Rg3 alleviates immune stress in broilers, particularly in avian macrophages and broiler hypothalamic tissue.

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