Application of combination of sophocarpidine and gallic acid in preparation of additive for relieving intestinal injury of piglets

Abstract

The invention discloses application of combination of sophocarpidine and gallic acid in preparation of an additive for relieving intestinal injury of piglets, and relates to the technical field of molecular biology. The invention provides an application of combination of sophocarpidine and gallic acid in preparation of an additive for relieving intestinal injury of piglets, the concentration of sophocarpidine in the additive for relieving intestinal injury of piglets is 1-3 [mu] g / mL, and the concentration of gallic acid in the additive for relieving intestinal injury of piglets is 0.3-0.5 [mu] g / mL. The injury is the injury caused by enterotoxigenic escherichia coli. The matrine and gallic acid are combined for use, so that the epithelial barrier function can be improved, the budding efficiency of group-like organs is improved, the gene abundance of the closed protein Occludin, the gene abundance of the goblet cell marker MUC2 and the gene abundance of the absorption cell marker Villin are recovered to the level of a control group, and the effect is better than that of single use; the problems that enterotoxigenic escherichia coli damages intestinal tracts of piglets and the effect of an existing single additive is poor are effectively solved.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, specifically to the application of matrine and gallic acid in the preparation of additives to alleviate intestinal damage in piglets. Background Technology

[0002] Enterotoxigenic Escherichia coli (ETEC) is a major pathogen causing diarrhea in piglets during swine production. It primarily colonizes the intestinal epithelium through adhesin-mediated processes and secretes virulence factors such as heat-sensitive enterotoxin (LT) and heat-stable enterotoxin (ST), leading to disease. ETEC infection can cause yellow and white scours in piglets (especially 1-3 days and 2-4 weeks old), manifesting as acute watery diarrhea, severe dehydration, and high mortality, resulting in economic losses for the swine industry. Regarding intestinal barrier disruption, ETEC not only induces apoptosis of intestinal epithelial cells and damages the integrity of the intestinal mucosa, but its enterotoxins also target and disrupt the tight junctions between intestinal epithelial cells, leading to increased intestinal permeability, loss of barrier function, and passive entry of electrolytes and body fluids into the intestinal lumen, exacerbating diarrhea. Furthermore, studies have confirmed that the heat-stable enterotoxin STp derived from ETEC interferes with the normal proliferation and differentiation of intestinal stem cells, causing them to abnormally differentiate into cystic organoids, hindering epithelial repair and renewal, thereby further damaging the structural and functional integrity of the intestinal mucosa. Therefore, developing a feed additive that effectively alleviates ETEC damage is crucial for improving pig farming efficiency.

[0003] Gallic acid (GA), a natural phenolic acid, has shown significant application potential in the fields of anti-inflammation and antibacterial properties, with its multi-target mechanism of action becoming increasingly clear. In terms of anti-inflammation, studies have shown that gallic acid can effectively alleviate various inflammatory models. In a zebrafish foodborne enteritis model, gallic acid can reduce intestinal and hepatic inflammation, improve gut microbiota structure, and increase beneficial bacteria by activating PPARγ-mediated immunomodulation and antioxidant processes. For inflammatory bowel disease, gallic acid can capture free ammonia in the gut, generating amination-modified products with anti-inflammatory activity, while simultaneously increasing beneficial metabolites such as short-chain fatty acids by regulating gut microbiota, achieving both symptomatic and radical treatment. Furthermore, in a paclitaxel-induced neuroinflammation model, gallic acid showed a relieving effect on neuropathic pain. In terms of antibacterial properties, gallic acid has inhibitory effects on a variety of pathogens. Optimized gallic acid extracts showed inhibitory activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis, with the most significant inhibitory effect against Staphylococcus aureus. Technological improvements, such as gallic acid-modified selenium nanoparticles, further enhanced its antibacterial activity, showing significantly better inhibitory effects against Staphylococcus aureus and Escherichia coli than gallic acid alone. Furthermore, research teams have discovered that multiple components of gallic acid (including gallic acid and its derivatives) have inhibitory effects on periodontal disease pathogens. These results indicate that gallic acid, through multiple pathways of inflammatory response regulation and multi-mechanism inhibition of pathogenic microorganisms, has the potential to improve animal gut health and reduce antibiotic use as a feed additive, providing a theoretical basis for its application in animal production.

[0004] Matrine (M), a natural alkaloid derived from the legume Sophora flavescens, exhibits significant efficacy in combating intestinal inflammation and promoting mucosal repair. Its anti-inflammatory effects manifest through multiple pathways of inflammatory inhibition: it not only reduces the expression levels of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β, and interleukin-6, but also enhances the activity of antioxidant enzymes, mitigating oxidative stress damage to intestinal tissues. Regarding mucosal repair, matrine directly promotes the proliferation and migration of intestinal epithelial cells, accelerating the re-epithelialization process at mucosal defects; simultaneously, it enhances goblet cell function, promotes mucin secretion, and improves the intestinal chemical barrier. Studies have shown that matrine effectively maintains the normal distribution of tight junction proteins in intestinal epithelium, reduces intestinal permeability, and prevents bacterial and toxin translocation. In a piglet diarrhea model, matrine intervention significantly improved intestinal morphology and structure, increased the ratio of villus height to crypt depth, and improved the efficiency of nutrient digestion and absorption. These properties make matrine a potential feed additive to replace antibiotics, providing an effective nutritional regulation strategy for alleviating intestinal inflammation and promoting mucosal integrity in livestock and poultry. Although matrine and gallic acid can effectively improve intestinal health, no researchers have yet evaluated the effects of their combined use. Summary of the Invention

[0005] To address the aforementioned shortcomings in the prior art, this invention provides the application of matrine and gallic acid in the preparation of additives to alleviate intestinal damage in piglets, effectively solving the problems of damage to the piglet intestines caused by enterotoxin-producing Escherichia coli and the poor efficacy of existing single additives.

[0006] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is: to provide the application of matrine and gallic acid in the preparation of an additive to alleviate intestinal damage in piglets.

[0007] Furthermore, the concentration of matrine in the additive for alleviating intestinal damage in piglets is 1-3 μg / mL.

[0008] Furthermore, the concentration of matrine in the additive for alleviating intestinal damage in piglets is 2 μg / mL.

[0009] Furthermore, the concentration of gallic acid in the additive for alleviating intestinal damage in piglets is 0.3-0.5 μg / mL.

[0010] Furthermore, the concentration of gallic acid in the additive for alleviating intestinal damage in piglets was 0.4 μg / mL.

[0011] Furthermore, the intestinal damage in piglets was caused by enterotoxin-producing Escherichia coli.

[0012] An additive comprising the aforementioned matrine, gallic acid, and medically acceptable excipients.

[0013] In summary, the present invention has the following beneficial effects: 1. The combined formulation of matrine and gallic acid provided by this invention, with the synergistic effect of 2 μg / mL matrine and 0.4 μg / mL gallic acid, can significantly improve the budding efficiency of porcine jejunal organoids after ETEC injury, increase the gene abundance of Occludin and Villin in the organoids, and restore the MUC2 gene to the control group level. The scheme provided by this invention can effectively protect piglet intestinal stem cells from ETEC damage and has great application potential in actual production. Attached Figure Description

[0014] Figure 1 Figures showing organoid growth under different concentrations of matrine treatment; Figure 2 Figure 1 shows the changes in budding efficiency and gene abundance of organoids under different concentrations of matrine treatment. Figure 3 Figures showing the growth of organoids under different concentrations of gallic acid treatment; Figure 4Figure 1 shows the budding efficiency and gene abundance changes of each group of organoids under different concentrations of gallic acid treatment. Figure 5 Figures showing organoid growth under treatment with different concentrations of matrine and gallic acid; Figure 6 Figure 1 shows the changes in budding efficiency and gene abundance of organoids under different concentrations of matrine and gallic acid combined treatment. Figure 7 Figure showing the growth of jejunal organoids in piglets after combined intervention with matrine and gallic acid in ETEC-induced damage; Figure 8 The figure shows the results of the combined intervention of matrine and gallic acid on the budding efficiency and gene abundance of ETEC-damaged jejunal organoids in piglets. Detailed Implementation

[0015] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0016] Example 1: Determining the optimal dosage of matrine and gallic acid in combination. 1. Isolation and culture of porcine jejunal crypts Collect 10 cm of fresh porcine jejunum tissue and place it in a culture dish containing ice-cold DPBS. Longitudinally dissect the intestine to expose the mucosa. Rinse the inside of the intestine repeatedly with pre-prepared, chilled DPBS, shaking vigorously or using a pipette to agitate, until most of the intestinal contents and mucus are removed. Use a sterile scalpel or forceps to scrape away excess mucus from the intestinal tissue. Then, cut the cleaned intestinal slices into small fragments approximately 0.5 cm × 0.5 cm, add ice-cold DPBS, and wash by hand for 5 minutes, changing the DPBS repeatedly until the supernatant is no longer significantly turbid. Transfer the tissue fragments to 50 mL centrifuge tubes and add pre-chilled crypt separation buffer (prepared with DPBS to a concentration of 2 mM EDTA buffer), at least 15-20 times the volume of the tissue. Place the centrifuge tubes in an icebox and gently shake on a shaker (120 rpm). After 30 minutes, remove the centrifuge tubes from the ice box, discard the supernatant (EDTA buffer), and add 10-20 mL of ice-cold DPBS to the centrifuge tube containing tissue fragments. Vigorously shake the centrifuge tubes by hand for 1-2 minutes, then let them stand for a few moments to allow large tissue fragments to settle. Transfer the crypt-rich supernatant to a new 50 mL ice-cold centrifuge tube. Incubate the collected crypt-rich supernatant on ice for 10 minutes to allow the crypts to accumulate at the bottom of the centrifuge tube. Finally, mix the crypts with Matrigel on ice, drop the mixture into a culture plate to form droplets, and incubate for 20 minutes to allow the Matrigel to solidify. Then, add preheated organoid complete culture medium.

[0017] 2. Passage of porcine jejunal organoids The optimal time for passage is when porcine organoids exhibit typical budding structures on Matrigel (usually 5 days after culture). First, carefully aspirate the old culture medium and add pre-chilled PBS to each well. Gently pipette the droplets to break them up, repeating this step to completely dissolve the Matrigel. Then, collect the organoid suspension into a 1.5 mL centrifuge tube and centrifuge at 4°C for 5 min (1000 rpm), discarding the supernatant to obtain the organoid pellet. Next, pipette the organoids using a 1 mL syringe until they are broken into suitable small cell clusters. After disruption, centrifuge again, discard the supernatant, and quickly mix with Matrigel thawed on ice. Next, spot 20 μL of the cell-Matrigel mixture into the center of each well and incubate at 37°C for 10–15 min to allow it to polymerize. Finally, carefully add pre-warmed organoid complete culture medium along the well walls and return the culture plate to a 37°C, 5% CO2 incubator for continued culture, observing organoid reformation and growth periodically.

[0018] 3. Dosage screening of matrine / gallic acid / matrine + gallic acid According to experimental requirements, the passaged organoids were divided into 4 groups, with 3 replicates per group (each replicate containing organoids from 2 cell culture wells). Stem cell culture medium containing 0, 1, 2, and 4 μg / mL matrine was used for treatment, and the growth of organoids in each group was recorded for 5 consecutive days. The growth on day 1 and day 5 is shown below. Figure 1 As shown in the figure. The budding efficiency and gene expression of each group are as follows: Figure 2 As shown. Figure 2 In the figure, AD represents the budding efficiency, Occludin mRNA abundance, Villinm RNA abundance, and MUC2 abundance results under different concentrations of matrine treatment.

[0019] Stem cell culture media containing 0, 0.4, 0.8, and 1.6 μg / mL gallic acid were used for treatment, and the growth of organoids in each group was recorded for 5 consecutive days. The growth on day 1 and day 5 is shown below. Figure 3 As shown in the figure. The budding efficiency and gene expression of each group are as follows: Figure 4 As shown. Figure 4 In the figure, AD represents the budding efficiency, Occludin mRNA abundance, Villin mRNA abundance, and MUC2 abundance under different concentrations of gallic acid treatment, respectively.

[0020] Stem cell culture media containing 0.2 μg / mL gallic acid + 2 μg / mL matrine, 0.4 μg / mL gallic acid + 1 μg / mL matrine, and 0.4 μg / mL gallic acid + 2 μg / mL matrine were used to treat the cells. The growth of organoids in each group was recorded for 5 consecutive days. The growth on day 1 and day 5 is shown below. Figure 5 As shown in the figure. The budding efficiency and gene expression of each group are as follows: Figure 6 As shown. Figure 6 In the figure, AD represents the germination efficiency, Occludin mRNA abundance, Villin mRNA abundance, and MUC2 abundance under different concentrations of matrine and gallic acid combined treatment, respectively.

[0021] RT-qPCR was used to detect the abundance of target genes in organoids. Organoid pellets were obtained by repeating the Matrigel washing step during organoid passage. RNA samples were obtained using an RNA extraction kit (Novizan, R711-01), followed by reverse transcription using a reverse transcription reagent (Novizan, R211-01) to convert the RNA into cDNA template. Primer sequences for the target genes (Occludin, Villin, MUC2) were designed on the NCBI website, as shown in Table 1. These primers were then synthesized at Sangon Biotech Co., Ltd. Finally, the cDNA template, SYBR dye, and primers were added to a quantitative plate, mixed, centrifuged, and then analyzed. After qPCR, raw data were obtained, and gene abundance was calculated using the 2–ΔΔCT method.

[0022] Depend on Figures 1-6 It was found that when the concentration of matrine exceeded 2 μg / mL or the concentration of gallic acid exceeded 0.4 μg / mL, the generation and budding efficiency of porcine jejunal organoids decreased, hindering organoid budding. Simultaneously, the gene abundance of Occludin, Villin, and MUC2 was downregulated. These results suggest that the barrier function of organoids was disrupted, and stem cell activity was inhibited. Therefore, 2 μg / mL and 0.4 μg / mL are the optimal concentrations for using matrine and gallic acid alone, respectively. Furthermore, treatments of 0.4 μg / mL gallic acid + 1 μg / mL matrine and 0.4 μg / mL gallic acid + 2 μg / mL matrine also significantly inhibited organoid expansion. Therefore, 0.2 μg / mL gallic acid + 2 μg / mL matrine was determined as the optimal concentration for combined use of the two treatments.

[0023] Table 1. Primer sequence list for detection genes

[0024] Example 2 evaluates the effect of the combined use of matrine and gallic acid in alleviating ETEC-induced damage to the jejunal organoids of piglets. Using passaged organoids as the research model, five groups were formed: a control group, an ETEC-damaged group, a matrine + gallic acid relief group, a matrine relief group, and a gallic acid relief group, with three replicates per group (each replicate containing organoids from two cell culture wells). After organoid seeding, the control group received normal stem cell culture medium, the damaged group received medium containing 1% ETEC culture supernatant, and the three relief groups received their respective optimal concentrations of stem cell culture medium. The growth of organoids in each group was recorded for five consecutive days. After completion, organoid samples were collected from each group. The gene abundance changes of Occludin, Villin, and MUC2 were detected according to the method described in Example 1. The growth on days 1 and 5 is shown below. Figure 7 As shown in the figure. The budding efficiency and gene expression of each group are as follows: Figure 8 As shown. Figure 8 In the figure, AD represents the results of budding efficiency, Occludin mRNA abundance, Villin mRNA abundance, and MUC2 abundance, respectively.

[0025] Depend on Figure 7 and Figure 8It was found that ETEC culture supernatant treatment reduced organoid budding efficiency, downregulated the gene abundance of Occludin and Villin in organoids, and upregulated the gene level of MUC2, suggesting that it disrupts the barrier function of porcine intestinal organoids, interferes with stem cell differentiation fate, and hinders their differentiation into absorptive cells. However, combined treatment with 0.2 μg / mL gallic acid + 2 μg / mL matrine improved organoid budding efficiency under ETEC exposure and reversed the changes in gene abundance of Occludin, Villin, and MUC2, indicating that the combined use of gallic acid and matrine can effectively alleviate the damage of ETEC to porcine jejunal stem cells. Furthermore, the alleviating effect of the combined use of gallic acid and matrine was significantly higher than that of matrine or gallic acid alone; therefore, the combined use of these two plant extracts is more valuable than adding either one alone.

[0026] The above description is only a preferred embodiment of the present invention and is 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 matrine and gallic acid in the preparation of additives to alleviate intestinal damage in piglets.

2. The application as described in claim 1, characterized in that, The concentration of matrine in the additive for alleviating intestinal damage in piglets is 1-3 μg / mL.

3. The application as described in claim 1, characterized in that, The concentration of gallic acid in the additive for alleviating intestinal damage in piglets is 0.3-0.5 μg / mL.

4. The application as described in claim 1, characterized in that, The intestinal damage in the piglets was caused by enterotoxin-producing Escherichia coli.

5. An additive, characterized in that, It includes matrine, gallic acid, and medically acceptable excipients as described in claim 1.

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