Application of ginsenoside Rg1 in preparation of product for relieving or preventing intestinal barrier injury

Abstract

The invention discloses application of ginsenoside Rg1 in preparation of a product for relieving or preventing intestinal barrier injury, and belongs to the technical field of biological medicine and agricultural feed. Experiments prove that ginsenoside Rg1 can regulate polarization balance of macrophages M1 / M2, inhibit activity of a TLR4-NF-kappa B pathway and an MAPK pathway, up-regulate expression of intestinal tight junction protein and reduce permeability of intestinal mucosa, so that the protective effect on intestinal barriers is achieved. The application and action mechanism of the ginsenoside Rg1 in intestinal barrier injury protection are defined for the first time, a new effective scheme is provided for prevention and treatment of intestinal diseases, and the ginsenoside Rg1 has important clinical and agricultural application values.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and agricultural feed technology, and in particular to the application of ginsenoside Rg1 in the preparation of products for alleviating or preventing intestinal barrier damage. Background Technology

[0002] Intestinal barrier damage is a common pathological feature of many intestinal diseases, such as intestinal damage caused by weaning stress in piglets and inflammatory bowel disease. In the agricultural breeding sector, weaning stress in piglets often leads to intestinal barrier dysfunction, causing problems such as diarrhea and growth retardation, which seriously affects breeding efficiency. In the medical field, intestinal barrier damage is closely related to the occurrence and development of diseases such as inflammatory bowel disease.

[0003] Existing research indicates that immune cell dysfunction is a key contributing factor to intestinal barrier damage. Macrophages, as important immune cells, play a crucial role in regulating intestinal inflammation and maintaining barrier function through the balance of their polarization state (the balance between pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages). When macrophage polarization is imbalanced, with overactivation of M1 macrophages and suppression of M2 macrophage function, intestinal inflammation can persist and worsen, ultimately damaging the integrity of the intestinal barrier. Therefore, regulating macrophage polarization has become a recognized potential strategy for treating intestinal barrier damage. Summary of the Invention

[0004] The purpose of this invention is to provide the application of ginsenoside Rg1 in the preparation of products for alleviating or preventing intestinal barrier damage, so as to solve the problem of insufficient existing intestinal barrier damage prevention and treatment methods, especially to provide a new and effective solution for weaning stress-related intestinal damage and LPS-induced intestinal damage in piglets.

[0005] To achieve the above objectives, on the one hand, the present invention provides the application of ginsenoside Rg1 in the preparation of products for alleviating or preventing intestinal barrier damage, wherein the intestinal barrier damage is lipopolysaccharide-induced intestinal barrier damage, and the ginsenoside Rg1 exerts its effect by regulating the polarization balance of macrophages M1 / M2.

[0006] Preferably, the intestinal barrier damage is intestinal barrier damage related to weaning stress in piglets, and the ginsenoside Rg1 exerts its effect by inhibiting the activity of the TLR4-NF-κB and MAPK pathways.

[0007] Preferably, the purity of the ginsenoside Rg1 is ≥98%.

[0008] Preferably, the dosage of ginsenoside Rg1 in the drug is 10 mg / kg body weight, and the administration method is intraperitoneal injection, and the drug is administered continuously for 6 days before the intestinal barrier injury model is established.

[0009] Preferably, the ginsenoside Rg1 regulates the polarization balance of macrophages M1 / M2 by downregulating the expression of M1 macrophage markers IL-1β, TNF-α, IL-6, iNOS, and MHC II, and upregulating the expression of M2 macrophage markers IL-10, TGF-β, Arg-1, and CD163.

[0010] Preferably, the ginsenoside Rg1 inhibits the activity of the TLR4-NF-κB pathway by reducing the expression of TLR4, MyD88, and NF-κB p65 proteins and inhibiting the nuclear translocation of NF-κB p65; and inhibits the activity of the MAPK pathway by downregulating the phosphorylation levels of p38 and JNK.

[0011] Preferably, the protective effect of ginsenoside Rg1 on the intestinal barrier is also manifested in: upregulating the mRNA expression levels of intestinal tight junction proteins ZO-1, Claudin-1, and Occludin, and restoring the activity of DAO enzyme, an indicator of intestinal mucosal permeability.

[0012] Preferably, the feed additive is used to prevent weaning stress diarrhea or antibiotic-associated enteritis in piglets.

[0013] On the other hand, the present invention provides a veterinary preparation for alleviating or preventing intestinal barrier damage, the active ingredient being ginsenoside Rg1, wherein the purity of ginsenoside Rg1 is ≥98%, and the veterinary preparation is used for piglets at a dosage of 10 mg / kg body weight.

[0014] Preferably, the veterinary preparation alleviates or prevents LPS-induced intestinal barrier damage in piglets by regulating the M1 / M2 polarization balance of macrophages, the activity of the TLR4-NF-κB pathway and the MAPK pathway.

[0015] Therefore, the application of ginsenoside Rg1 in the preparation of products for alleviating or preventing intestinal barrier damage has the following beneficial effects: (1) The application of ginsenoside Rg1 in the preparation of drugs or feed additives for alleviating or preventing intestinal barrier damage was proposed for the first time, especially for weaning stress and LPS-induced intestinal damage in piglets, filling the gap in existing technology.

[0016] (2) This invention clarifies that ginsenoside Rg1 exerts its intestinal barrier protective effect by regulating the polarization balance of macrophages M1 / M2 and inhibiting the activity of the TLR4-NF-κB and MAPK pathways; (3) Through histological, molecular biological and statistical analysis, in-depth and reliable data support was provided to confirm the effectiveness of ginsenoside Rg1; the application potential of ginsenoside Rg1 in the fields of veterinary drugs and feed additives was clarified, and it can be used to prevent weaning stress diarrhea or antibiotic-associated enteritis in piglets, which has important agricultural application value; at the same time, it also provides a new research direction for the prevention and treatment of diseases related to human intestinal barrier damage, which has potential clinical application value.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 HE staining and scoring of piglet jejunum; where A is the control group, B is the LPS group, C is the Rg1 group, and D is the pathological section score. Figure 2 The results of ELISA and qPCR for polarization-related cytokines and genes in M1 macrophages are shown below; where A represents IL-1β content, B represents IL-1β mRNA expression level, C represents iNOS mRNA expression level, D represents MHCII mRNA expression level, E represents IL-6 mRNA expression level, and F represents TNF-α mRNA expression level. Figure 3 The results are qPCR of polarization-related cytokines and genes in M2 macrophages; where A represents the mRNA expression level of Arg-1; B represents the mRNA expression level of TGF-β; C represents the mRNA expression level of CD163; and D represents the mRNA expression level of IL-10. Figure 4 The results are qPCR of TLR4-NF-κB pathway-related genes; where A is the mRNA expression level of TLR4; B is the mRNA expression level of MyD88; C is the mRNA expression level of NF-κB; D is the mRNA expression level of IKBα; and E is the mRNA expression level of p65. Figure 5 The results are qPCR values ​​for genes related to the MAPK pathway; where A represents the mRNA expression level of MAPK1; B represents the mRNA expression level of p38; C represents the mRNA expression level of JNK1; D represents the mRNA expression level of STAT1; and E represents the mRNA expression level of SOCS1. Figure 6 The values ​​represent the expression levels of tight junction proteins in the jejunum of piglets; where A represents the mRNA expression level of ZO-1; B represents the mRNA expression level of Claudin-1; C represents the mRNA expression level of Occludin; and D represents the mRNA expression level of DAO. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0022] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0023] Example 1 The construction of an animal model of intestinal barrier injury includes the following procedures: (1) Experimental animals and feeding: Healthy weaned (21-28 days old) Taihu piglets were selected and acclimatized in a standard experimental animal room for 7 days, with free access to food and water.

[0024] All animal handling procedures strictly adhere to the ethical guidelines for laboratory animal welfare and have been reviewed and approved by the Laboratory Animal Ethics Committee of Southwest University (License No.: IACUC-20250107-01).

[0025] (2) Drugs and dosing regimen: The drug is ginsenoside Rg1, with a purity ≥98%, synthesized in a yeast cell factory and dissolved in sterile physiological saline. The dosage is 10 mg / kg body weight, administered via intraperitoneal injection, for 6 consecutive days prior to intestinal barrier injury modeling.

[0026] (3) Establishment of intestinal barrier injury model: After 6 consecutive days of administration, an intestinal barrier injury model was established by intraperitoneal injection of lipopolysaccharide (LPS). The LPS injection dose was 100 μg / kg body weight, dissolved in sterile physiological saline. Animals were fasted for 12 hours before injection (water was not restricted).

[0027] (4) Sample collection: Piglets were euthanized 6 hours after modeling, weighed and intestinal tissue and serum samples were collected for subsequent testing.

[0028] The efficacy of ginsenoside Rg1 was verified using multiple standardization methods. All data are expressed as mean ± standard deviation. One-way ANOVA was used for inter-group comparisons. P A value <0.05 was considered statistically significant; sample size n=8 / group; statistical power >0.9. The specific testing method is as follows: (1) Histomorphological analysis: Hematoxylin-eosin (H&E) staining was used to observe the pathological changes in intestinal tissue (such as villus height, crypt depth, inflammatory infiltration, etc.) and histological damage score was performed.

[0029] The results are as follows Figure 1 As shown, the control group (injected with the same volume of sterile saline as the Rg1 group) had intact intestinal mucosal structure, neatly arranged villi, and normal crypt morphology. Compared with the control group, the LPS group showed severe intestinal mucosal damage, villi atrophy and necrosis, and intestinal mucosal sloughing. Compared with the LPS group, the Rg1 group showed gradual recovery of intestinal mucosal structure, reduced villi damage, and significant improvement in intestinal damage. The histological damage score of the Rg1 group was significantly lower ( P <0.01).

[0030] (2) Detection of inflammatory factor levels: The protein concentrations of cytokines IL-1β, TNF-α, IL-6 and IL-10 in serum were detected by enzyme-linked immunosorbent assay (ELISA).

[0031] (3) Gene expression analysis: Transcriptomics sequencing and real-time quantitative polymerase chain reaction (qRT-PCR) were used to verify the mRNA expression level of inflammation-related genes, with GAPDH as the internal reference gene.

[0032] ELISA and qPCR results of M1 macrophage polarization-related cytokines and genes are as follows: Figure 2 As shown, compared with the control group, the LPS group upregulated the level of IL-1β in piglet serum, while the level in the Rg1 group showed a decreasing trend. qPCR results showed that, compared with the control group, the mRNA expression of M1 macrophage polarization-related genes IL-1β, iNOS, MHCII, IL-6, and TNF-α was significantly upregulated in the LPS group. P <0.01). In the Rg1 group, the mRNA expression levels of iNOS and TNF-α were decreased, and IL-1β and MHCII ( P <0.01), IL-6 ( P The mRNA expression of ginsenoside Rg1 (<0.05) was significantly increased. These results indicate that ginsenoside Rg1 can inhibit M1 polarization in piglet macrophages.

[0033] qPCR results of M2 macrophage polarization-related cytokines and genes are as follows: Figure 3As shown, compared with the control group, the mRNA expression levels of Arg-1 and IL-10 were decreased in the LPS group, and TGF-β ( P <0.01) and CD163 ( P <0.05) significantly reduced; compared with the LPS group, the Rg1 group significantly increased the expression of Arg-1 and TGF-β ( P The result was <0.01, indicating that ginsenoside Rg1 can inhibit M1 polarization and promote macrophage polarization towards M2 polarization.

[0034] qPCR results of TLR4-NF-κB pathway-related genes are as follows: Figure 4 As shown, in the TLR4-NF-κB signaling pathway, compared with the control group, the mRNA expression of TLR4, MyD88, and NF-κB was significantly upregulated in the LPS group. P <0.01), IKBα mRNA expression was significantly downregulated ( P <0.01); After Rg1 treatment, the expression of TLR4, MyD88, and NFκB was significantly reduced compared with the LPS group ( P <0.01), IKBα expression showed an upward trend, while p65 expression levels decreased, with no significant difference.

[0035] To investigate the direct molecular target of ginsenoside Rg1, molecular docking technology was used to simulate the binding of Rg1 to Toll-like receptor 4 (TLR4). Using AutoDock Vina software, the docking simulation showed that ginsenoside Rg1 stably binds to the protein pocket of TLR4, with a binding free energy (ΔG) of -4.93 kJ / mol, indicating a spontaneous and stable binding. This result suggests that TLR4 may be a key upstream target for ginsenoside Rg1. Rg1, by binding to TLR4, inhibits downstream NF-κB p65 nuclear translocation and p38 / JNK phosphorylation.

[0036] qPCR results of MAPK pathway-related genes are as follows: Figure 5 As shown, the mRNA expression of p38 was significantly increased in the LPS group ( P <0.01), SOCS1 mRNA expression was significantly downregulated ( P <0.01), MAPK1, JNK1 and STAT1 showed an upregulation trend; after Rg1 treatment, p38 expression was significantly reduced compared to the LPS group ( P<0.01), SOCS1 expression showed an upward trend, while MAPK1, JNK1, and STAT1 expression were downregulated. This result is consistent with the molecular docking results above, indicating that ginsenoside Rg1 effectively inhibits the overactivation of downstream MAPK signaling pathways (especially p38 and JNK) by targeting TLR4.

[0037] (4) Intestinal barrier function indicators: detect the mRNA expression level of tight junction proteins (ZO-1, Claudin, Occludin) and intestinal mucosal permeability indicators (DAO enzyme activity).

[0038] Expression levels of tight junction proteins in the jejunum of piglets, such as Figure 6 As shown, the mRNA levels of intestinal barrier damage markers (ZO-1, Claudin-1, Occludin, and DAO) in the LPS group were significantly lower than those in the control group. P <0.01); Conversely, the mRNA levels of ZO-1, Claudin-1, and Occludinm increased in the Rg1 group ( P >0.05). Notably, the expression level of DAO in the Rg1 group was significantly restored ( P The result was <0.001, indicating that LPS administration could cause intestinal barrier damage, and that ginsenosides could alleviate this damage.

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. The application of ginsenoside Rg1 in the preparation of products for alleviating or preventing intestinal barrier damage, characterized in that: The intestinal barrier damage is lipopolysaccharide-induced intestinal barrier damage, and the ginsenoside Rg1 functions by regulating the M1 / M2 polarization balance of macrophages.

2. The application of ginsenoside Rg1 according to claim 1 in the preparation of products for alleviating or preventing intestinal barrier damage, characterized in that: The intestinal barrier damage is intestinal barrier damage related to weaning stress in piglets, and the ginsenoside Rg1 exerts its effect by inhibiting the activity of the TLR4-NF-κB and MAPK pathways.

3. The application of ginsenoside Rg1 according to claim 1 or 2 in the preparation of products for alleviating or preventing intestinal barrier damage, characterized in that: The purity of the ginsenoside Rg1 is ≥98%.

4. The application of ginsenoside Rg1 according to claim 1 in the preparation of products for alleviating or preventing intestinal barrier damage, characterized in that: The dosage of ginsenoside Rg1 in the drug is 10 mg / kg body weight, administered via intraperitoneal injection, and administered continuously for 6 days before intestinal barrier injury modeling.

5. The use of ginsenoside Rg1 according to claim 1 or 2 in the preparation of products for alleviating or preventing intestinal barrier damage, characterized in that, The ginsenoside Rg1 regulates the polarization balance of macrophages M1 / M2 by downregulating the expression of M1 macrophage markers IL-1β, TNF-α, IL-6, iNOS, and MHC II, and upregulating the expression of M2 macrophage markers IL-10, TGF-β, Arg-1, and CD163.

6. The application of ginsenoside Rg1 according to claim 2 in the preparation of products for alleviating or preventing intestinal barrier damage, characterized in that, The ginsenoside Rg1 inhibits the activity of the TLR4-NF-κB pathway by reducing the expression of TLR4, MyD88, and NF-κB p65 proteins and inhibiting the nuclear translocation of NF-κB p65; it also inhibits the activity of the MAPK pathway by downregulating the phosphorylation levels of p38 and JNK.

7. The use of ginsenoside Rg1 according to claim 1 or 2 in the preparation of products for alleviating or preventing intestinal barrier damage, characterized in that, The protective effect of ginsenoside Rg1 on the intestinal barrier is also manifested in: upregulating the mRNA expression levels of intestinal tight junction proteins ZO-1, Claudin-1, and Occludin, and restoring the activity of DAO enzyme, an indicator of intestinal mucosal permeability.

8. The application of ginsenoside Rg1 according to claim 2 in the preparation of products for alleviating or preventing intestinal barrier damage, characterized in that: The feed additive is used to prevent weaning stress diarrhea or antibiotic-associated enteritis in piglets.

9. A veterinary preparation for alleviating or preventing intestinal barrier damage, characterized in that: The active ingredient is ginsenoside Rg1, and the purity of ginsenoside Rg1 is ≥98%. The veterinary preparation is used for piglets, and the dosage is 10 mg / kg body weight.

10. A veterinary preparation for alleviating or preventing intestinal barrier damage according to claim 9, characterized in that, The veterinary preparation alleviates or prevents LPS-induced intestinal barrier damage in piglets by regulating the M1 / M2 polarization balance of macrophages, the activity of the TLR4-NF-κB pathway and the MAPK pathway.

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