Application of recombinant walnut derived peptide rWDP

Abstract

The invention discloses application of a recombinant walnut derived peptide rWDP in preparation of a medicine for promoting ackermania muciniphila proliferation, a medicine for improving colon injury, a medicine for relieving neuroinflammation, a medicine for improving neurotransmitter level and a medicine for relieving systemic inflammation. Experimental results show that the rWDP can significantly promote Ackermania muciniphila proliferation, regulate intestinal flora balance, improve colon injury, enhance intestinal barrier function, relieve neuroinflammation, improve neurotransmitter level and relieve systemic inflammation. The rWDP has wide application prospects in the fields of functional food, precise nutrition, biological medicine and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the application of recombinant walnut-derived peptide rWDP. Background Art

[0002] In recent years, studies have shown that there is a close connection between the composition of the gut microbiota and various health functions, although the specific mechanisms are still being explored. The gut microbiota not only regulates the metabolic functions of the host but also affects the overall health status of the host through pathways such as immune signaling, metabolic regulation, and inflammation levels. In aspects such as metabolic diseases, immune regulation, and inflammation control, the role of the gut microbiota has received increasing attention. Intervention measures in this field, such as probiotics, prebiotics, and dietary adjustments, have shown the potential to improve metabolic functions and regulate immune responses in multiple experimental studies. However, the effects of these intervention measures vary due to individual microbiota differences, and their consistency in the population has not been verified through large-scale clinical trials.

[0003] Among the many gut microbiota members, Akkermansia muciniphila has received extensive attention due to its important role in maintaining gut homeostasis, immune regulation, and metabolic control. Studies have shown that Akkermansia muciniphila mainly inhabits the intestinal mucus layer, can degrade mucin, promote mucus layer renewal, enhance intestinal barrier function, and play a potential role in reducing systemic inflammation, regulating immune responses, and affecting metabolic levels. Some animal experiments have found that the abundance of Akkermansia muciniphila may be associated with the improvement of metabolic health and the alleviation of inflammation, but its direct role in overall health still lacks large-scale clinical verification. In addition, some studies have shown that the level of Akkermansia muciniphila is lower in some patients with metabolic diseases, but it is not clear whether this association is causal and further research is still needed.

[0004] To enhance the abundance of Akkermansia muciniphila in the host gut, current intervention strategies mainly include prebiotic supplementation and direct oral administration of probiotic - Akkermansia muciniphila. Traditional prebiotics, such as fructooligosaccharides, inulin, and chicory fiber, have been proven to promote the proliferation of Akkermansia muciniphila, but these substances usually act on the entire gut microecology and may simultaneously promote the growth of other symbiotic bacteria (such as Bifidobacterium, Lactobacillus), thus lacking specificity. In addition, the effect of prebiotics depends on the host's microbiota ecological environment, its proliferation efficiency is greatly affected by individual differences, and usually a relatively high dose needs to be ingested to achieve a significant effect, limiting its application in precisely regulating Akkermansia muciniphila.

[0005] On the other hand, direct oral administration of probiotic Akkermansia muciniphila is considered another potential strategy. However, as a strict anaerobe, Akkermansia muciniphila has a low survival rate in an oxygen-exposed environment, and the production, storage, and application of its live bacterial preparations face significant technical challenges. Even strains prepared under special anaerobic packaging conditions may still have their activity decreased due to factors such as gastric acid and bile salts after entering the digestive tract, limiting their bioavailability. Therefore, there is currently no widely used probiotic - Akkermansia muciniphila supplementation program on the market, and there is an urgent need for new methods to efficiently, stably, and specifically promote the proliferation of Akkermansia muciniphila.

[0006] To address this research gap, the present invention provides a novel strategy, namely promoting the selective proliferation of Akkermansia muciniphila through recombinant walnut-derived peptide rWDP. Different from traditional prebiotics that rely on providing fermentation substrates, recombinant walnut-derived peptide may directly act as a signaling molecule to regulate the gut microbiota, specifically promoting the proliferation and metabolic activity of Akkermansia muciniphila, thereby enhancing its beneficial effects on the host. In addition, as a peptide molecule, recombinant walnut-derived peptide has higher stability and bioavailability compared to traditional probiotic supplementation programs, and can avoid problems such as low survival rate and susceptibility to the environment during probiotic supplementation, thus becoming a more efficient gut microecological intervention strategy. The proposal of the present invention provides a new technical means for precise microecological regulation and health improvement, and has broad application prospects in the fields of functional foods, precision nutrition, and biomedicine. Summary of the Invention

[0007] The object of the present invention is to provide the application of recombinant walnut-derived peptide rWDP to solve the deficiencies of the prior art.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] In the first aspect of the present invention, there is provided the application of recombinant walnut-derived peptide rWDP in the preparation of a drug for promoting the proliferation of Akkermansia muciniphila.

[0010] In the second aspect of the present invention, there is provided the application of recombinant walnut-derived peptide rWDP in the preparation of a drug for improving colon injury.

[0011] In the third aspect of the present invention, there is provided the application of recombinant walnut-derived peptide rWDP in the preparation of a drug for reducing neuroinflammation.

[0012] In the fourth aspect of the present invention, there is provided the application of recombinant walnut-derived peptide rWDP in the preparation of a drug for improving neurotransmitter levels.

[0013] In the fifth aspect of the present invention, there is provided the application of recombinant walnut-derived peptide rWDP in the preparation of a drug for reducing systemic inflammation.

[0014] Advantages of the present invention:

[0015] The experimental results of the present invention show that the recombinant walnut-derived peptide rWDP has significant effects in the following aspects:

[0016] 1. Promote the proliferation of Akkermansia muciniphila and regulate the balance of intestinal flora: rWDP significantly increased the abundance of Akkermansia muciniphila.

[0017] 2. Improve colon injury and enhance intestinal barrier function: rWDP significantly improved the colon tissue structure injury induced by D-galactose and restored the crypt morphology and epithelial integrity.

[0018] 3. Alleviate neuroinflammation: rWDP significantly inhibited the activation of microglia and alleviated the neuroinflammation induced by D-galactose.

[0019] 4. Improve neurotransmitter levels: rWDP significantly restored the levels of neurotransmitters such as glutathione, kynurenine, 4-aminobutyric acid, histidine, adrenaline, and 5-hydroxyindoleacetic acid, and improved the disorder of neurotransmitter metabolism.

[0020] 5. Alleviate systemic inflammation: rWDP significantly reduced the levels of VCAM-1 and IFN-γ, increased the levels of G-CSF and CXCL1, and alleviated systemic inflammation.

[0021] The present invention has broad application prospects for rWDP in the fields of functional foods, precision nutrition, and biomedicine. Brief Description of the Drawings

[0022] Figure 1is the gut microbiota composition. A is the heatmap of the relative abundances of 11 genera in the cecal feces of mice in different groups, showing significant differences. The heatmap uses a color gradient from orange to light blue to represent the changes in relative abundances at the genus level, with orange indicating higher abundances and light blue indicating lower abundances. Gray represents genera with no significant differences between the two groups, red represents genera with significant differences between the two groups (P < 0.05), and dark red represents genera with extremely significant differences (P < 0.01). Upward and downward arrows indicate more and fewer relative abundances of genera in the control group and rWDP group, respectively, compared to the model group. The taxonomic information (family and phylum) of these genera is shown on the right. The abundance differences of genera were evaluated using Metastats. B is a cladogram highlighting key microbiota alterations. The circles radiating from the inside out represent taxonomic levels from phylum to species. Each small circle at different taxonomic levels represents a taxon at that level, and the diameter of the small circle is proportional to the relative abundance. Coloring principle: Species with no significant differences are uniformly colored yellow, and differential species Biomarkers are colored according to the group. Green nodes represent microbial taxa that play important roles in the control group, orange nodes represent microbial taxa that play important roles in the model group, and purple nodes represent microbial taxa that play important roles in the rWDP group. The species names represented by numbers in the figure correspond to C. C is a bar chart of the LDA value distribution. The numbers in the figure correspond to the species identified in B, that is, species with an LDA Score greater than the set value (default setting is 4), showing species with significant abundance differences in different groups. The length of the bar chart represents the effect size of the differential species (i.e., the LDA Score).

[0023] Figure 2 is the result of colon histopathology (HE staining).

[0024] Figure 3 is the immunofluorescence analysis of microglia in the hippocampus and cerebral cortex. The immunofluorescence images show sections of the hippocampus (A) and cerebral cortex (B), stained with an anti-IBA1 antibody (red, microglia marker) and counterstained with DAPI (blue, nucleus). The merged image (Merge) shows the co-localization of microglia and the nucleus.

[0025] Figure 4 is the heatmap of neurotransmitters. It shows the expression levels of differential neurotransmitters among the control group (Control), model group (Model), and rWDP group (rWDP). Each row represents a neurotransmitter, and each column represents a sample. The color gradient ranges from orange to light blue, where orange indicates a higher expression level and light blue indicates a lower expression level.

[0026] Figure 5Effect of rWDP on cytokine levels in the serum of D-galactose-induced senescent mice. A is the level of VCAM-1 (vascular cell adhesion molecule-1), B is the level of G-CSF (granulocyte colony-stimulating factor), C is the level of CXCL1 (CXC motif chemokine ligand 1), D is the level of IFN-γ (interferon-γ), E is the level of IL-1β (interleukin-1β), F is the level of IL-6 (interleukin-6), and G is the level of TNF-α (tumor necrosis factor-α).

[0027] Figure 6 For the results of liver and kidney histopathology (HE staining). Detailed implementation manners

[0028] The present invention will be further explained below in conjunction with embodiments and the accompanying drawings. The following embodiments are only used to illustrate the present invention, but do not limit the scope of implementation of the present invention.

[0029] Embodiment 1

[0030] 1. Preparation of rWDP

[0031] The recombinant walnut-derived peptide rWDP involved in the present invention is the GS-(YZY)18 recombinant tandem protein prepared by the pET28a-(EY10) / Rosetta(DE3) recombinant engineering bacteria in Example 9 of CN118930657A, hereinafter simply referred to as rWDP. The rWDP prepared by the method described in Example 9 of CN118930657A was detected for its endotoxin content by the Limulus reagent (LAL) method to ensure that its endotoxin content ≤ 0.50 EU / mg, meeting the endotoxin limit requirements for injectable drugs. 18 2. Animal experiment design

[0032] The following mouse studies were approved by the Experimental Animal Ethics Committee of Zhejiang A&F University (No. ZAFUAC202429) and followed the "Guide for the Care and Use of Laboratory Animals" issued by the European Commission.

[0033] Male C57BL / 6J mice at 6 weeks of age (body weight 20.0 ± 2.0 g) were selected for the experiment and purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (license number SYXK (Beijing) 2022-0006). The mice were housed in a standardized environment in the Experimental Animal Center of Zhejiang A&F University, with a light cycle of 12 hours (lights on at 7 am), a constant temperature of 22 ± 2 °C, a relative humidity of 55 ± 5%, and provided with autoclaved feed and water for free intake.

[0034] The experiment was divided into three groups, with 4 mice in each group:

[0035]

[0036] ​Control group: Each mouse was intragastrically administered 0.2 mL of sterile normal saline per day and subcutaneously injected with 0.2 mL of sterile normal saline at the posterior neck for 60 days.

[0037] Model group: Each mouse was intragastrically administered 0.2 mL of sterile normal saline per day and subcutaneously injected with 0.2 mL of D-galactose (400 mg D-galactose / kg mouse / day, prepared with sterile normal saline) at the posterior neck for 60 days to establish an aging model.

[0038] rWDP group (rWDP): Each mouse was intragastrically administered 0.2 mL of rWDP (60 mg rWDP / kg mouse / day, prepared with sterile normal saline) per day and subcutaneously injected with 0.2 mL of D-galactose (400 mg D-galactose / kg mouse / day, prepared with sterile normal saline) at the posterior neck for 60 days.

[0039] 3. Sample collection and processing

[0040] After the experiment, the mice were fasted for 4 hours and deeply anesthetized by intraperitoneal injection of tribromoethanol. Blood was collected from the heart, allowed to stand at room temperature for 30 minutes, and then centrifuged at 3000 rpm for 20 minutes at 4°C to obtain serum, which was stored at -80°C for cytokine determination. At the same time, brain, colon, liver, and kidney tissues were collected. Some tissues were fixed in 4 m / v% paraformaldehyde for 24 hours for histological analysis and immunofluorescence analysis, and the remaining tissues were immediately frozen in liquid nitrogen and stored at -80°C for neurotransmitter analysis. In addition, feces were collected from the cecum, placed in a sterile centrifuge tube, immediately frozen in liquid nitrogen, and stored at -80°C for intestinal flora analysis.

[0041] 4. Data analysis

[0042] The measured data were expressed as mean ± standard deviation (SD), and statistical analysis was performed using GraphPad Prism 9.0. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test was used for inter-group comparison, and the Kruskal-Wallis test was used for non-normally distributed data.

[0043] 5. Regulatory effect of rWDP on intestinal flora

[0044] The specific method is as follows: DNA was extracted from mouse cecal feces using the CTAB method. After evaluating the DNA quality by agarose gel electrophoresis, the V3-V4 region of the 16S rRNA gene was amplified using primers 341F (shown in SEQ ID NO:1: CCTACGGGNGGCWGCAG) and 806R (shown in SEQ ID NO:2: GGACTACHVGGGTATCTAAT). The library was constructed and sequenced on the Illumina NovaSeq 6000 platform. The raw data was processed using fastp, FLASH, and vsearch to generate amplicon sequence variants (ASVs), and taxonomic annotation was performed through the SILVA database.

[0045] The results are as Figure 1 shown. The rWDP group significantly increased the abundance of Akkermansia and decreased the abundance of harmful bacteria such as Rikenella, Oscillibacter, and Lachnoclostridium ( Figure 1 A). Further analysis by linear discriminant analysis effect size (LEfSe) found that Akkermansia muciniphila was enriched in the rWDP group ( Figure 1 B and 1C). In summary, rWDP significantly regulated the intestinal microbiota composition.

[0046] 6. Improvement effect of rWDP on colon injury

[0047] The specific method is as follows: The colon tissues were fixed in 4 m / v% paraformaldehyde for 24 hours, embedded in paraffin, cut into 4-μm thick sections, stained with hematoxylin and eosin (HE), scanned using a Pannoramic 250 FLASH scanner, and the images were analyzed by ImageJ software.

[0048] The results are as Figure 2 shown. Through histopathological analysis, it was found that the colon tissue structure of the mice in the model group was severely damaged, the crypt structure was destroyed, and the epithelial integrity was reduced. The rWDP group significantly improved the colon tissue structure and restored the crypt morphology and epithelial integrity.

[0049] 7. Alleviating effect of rWDP on neuroinflammation

[0050] The specific method is as follows: The brain tissues were fixed in 4 m / v% paraformaldehyde for 24 hours, embedded in paraffin, cut into 4-μm thick sections, stained with IBA1 immunofluorescence to label microglia, scanned using a Pannoramic 250 FLASH scanner, and analyzed by ImageJ software.

[0051] The results are as Figure 3As shown, through immunofluorescence analysis, it was found that the activation of microglia in the hippocampus and cerebral cortex of mice in the model group increased significantly, while the rWDP group significantly reduced the activation of microglia, indicating that rWDP can alleviate D-galactose-induced neuroinflammation.

[0052] 8. Improvement effect of rWDP on neurotransmitter levels

[0053] The specific method was as follows: The brain tissue was quickly frozen in liquid nitrogen and ground into powder, and neurotransmitters were extracted using 70 v / v% aqueous methanol solution, and the neurotransmitter levels were analyzed using UPLC-MS / MS.

[0054] The results were as Figure 4 shown. Through neurotransmitter analysis, it was found that the levels of glutathione, kynurenine, 4-aminobutyric acid, histidine, adrenaline, and 5-hydroxyindoleacetic acid in the model group of mice were significantly decreased, while the rWDP group significantly restored the levels of these neurotransmitters, indicating that rWDP can improve the disorder of neurotransmitter metabolism.

[0055] 9. Alleviating effect of rWDP on systemic inflammation

[0056] The specific method was as follows: The levels of CXCL1, G-CSF, IL-1β, IL-6, TNF-α, VCAM-1, and IFN-γ in serum were measured using the ABplex mouse cytokine 7-plex detection kit.

[0057] The results were as Figure 5 shown. Through serum cytokine determination, it was found that the levels of VCAM-1 and IFN-γ in the model group of mice were significantly increased, and the levels of G-CSF and CXCL1 were significantly decreased, while the rWDP group significantly decreased the levels of VCAM-1 and IFN-γ, and increased the levels of G-CSF and CXCL1 at the same time, indicating that rWDP has a systemic anti-inflammatory effect.

[0058] 10. Toxicity of rWDP to the liver and kidney

[0059] The specific method was as follows: The liver tissue and kidney tissue were fixed in 4 m / v% paraformaldehyde for 24 hours, embedded in paraffin and cut into 4-μm thick sections, stained with hematoxylin and eosin (HE), scanned using a Pannoramic250FLASH scanner, and the images were analyzed using ImageJ software.

[0060] The results were as Figure 6 shown, indicating that rWDP has no toxicity to the liver and kidney.

Claims

1. Use of recombinant walnut-derived peptide rWDP in the preparation of a drug for promoting the proliferation of Akkermansia muciniphila.

2. Use of recombinant walnut-derived peptide rWDP in the preparation of a drug for improving colon injury.

3. Use of recombinant walnut-derived peptide rWDP in the preparation of a drug for reducing neuroinflammation.

4. Use of recombinant walnut-derived peptide rWDP in the preparation of a drug for improving neurotransmitter levels.

5. Use of recombinant walnut-derived peptide rWDP in the preparation of a drug for reducing systemic inflammation.

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