Application of phosvitin in preparation of medicine for treating inflammatory bowel disease

Abstract

The invention provides application of phosvitin in preparation of a medicine for treating inflammatory bowel diseases. The inflammatory bowel disease comprises ulcerative colitis, and the ulcerative colitis is colitis induced by DSS (Di-Sulfate Sodium Sulfate). The phosvitin is natural protein extracted from eggs and has no adverse reaction on human bodies, and discomfort caused by long-term anti-inflammatory drug treatment is avoided. The phosvitin provided by the invention has the property of high net negative charge, so that the phosvitin has an anti-proteolysis effect, can resist a stomach high-acid environment, reaches intestinal tracts and exerts a curative effect.

Description

Technical Field

[0001] The present application belongs to the pharmaceutical field, and specifically relates to the use of phosvitin in the preparation of medicaments for treating inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic and relapsing inflammatory disease of the digestive tract that includes Crohn's disease (CD) and ulcerative colitis (UC). The incidence of IBD has been increasing in recent years. UC is a chronic, nonspecific inflammatory condition of the colon that is highly prevalent. Common symptoms of UC include weight loss, diarrhea, blood in the stool, and pain.

[0003] At present, the pathogenesis of UC is not very clear, but many studies have reported that the possible mechanisms of UC include inflammation, intestinal flora imbalance and intestinal barrier dysfunction. Studies have reported that the intestinal barrier of UC patients is damaged, and there is a close connection between inflammation and the intestinal barrier. In addition, intestinal flora is also crucial for UC, which may be a new direction for the treatment of UC. Intestinal microbiota homeostasis helps maintain intestinal health. Once this balance is broken, inflammatory cells accumulate and infiltrate into the intestinal epithelium, leading to colon inflammation. Therefore, the imbalance of intestinal flora is related to the development of UC. The diversity of intestinal microbiota in UC patients is reduced, with an enrichment of potentially harmful bacteria and a decrease in beneficial bacteria. Fecal microbiota transplantation (FMT) has been used in the treatment of UC patients, which can protect the disease by transplanting flora containing beneficial bacteria into patients.

[0004] For the treatment of UC, traditional therapeutic drugs such as 5-aminosalicylic acid (5-ASA) are associated with severe side effects. Growing evidence suggests that dietary patterns and bioactive dietary components have potential impacts on the prevention and treatment of UC. Therefore, identifying new dietary intervention strategies to reduce the incidence of UC is crucial.

[0005] Phosvitin (PSV), a phosphoprotein found in egg yolk, has been shown to possess various biological activities, including anti-inflammatory and antioxidant properties. PSV constitutes approximately 4% of the dry weight of egg yolk and is composed of 216 amino acid residues, 56% of which are serine, 80% of which are phosphorylated. As the most highly phosphorylated natural protein, PSV exhibits excellent digestive stability and is not easily degraded in the intestine.

[0006] Therefore, this study aimed to investigate the effects of PSV supplementation on disease symptoms, intestinal permeability, inflammatory response, and intestinal flora in mice treated with dextran sodium sulfate (DSS), and to explore the role of intestinal flora through FMT experiments. Summary of the Invention

[0007] This application addresses the above-mentioned technical problems and provides the use of phosvitin in the preparation of drugs for the treatment of inflammatory bowel disease, aiming to solve the problems of severe side effects and poor therapeutic effects of existing drugs for the treatment of inflammatory bowel disease, as well as to improve intestinal permeability, inflammatory response and the effects on intestinal flora.

[0008] The present application provides the use of phosvitin in the preparation of a drug for treating inflammatory bowel disease.

[0009] The present application also provides a pharmaceutical composition comprising phosvitin and a pharmaceutically acceptable carrier.

[0010] Beneficial effects

[0011] The present application provides a use of phosvitin in the preparation of a drug for inflammatory bowel disease, thereby avoiding the problem of difficult to cure and easy relapse in the existing treatment technology.

[0012] While existing drugs for the treatment of inflammatory bowel disease (IBD) can control the condition to a certain extent, they still have the following problems: limited efficacy; individual variability: some patients do not respond well to the drugs, resulting in limited efficacy; low remission rates: some patients find it difficult to achieve or maintain clinical remission; side effects: immunosuppressants: may increase the risk of infection and malignancy; biologics: may trigger allergic reactions or increase the risk of infection; long-term use of hormones: may lead to side effects such as osteoporosis and diabetes.

[0013] Existing treatments for inflammatory bowel disease (IBD) are expensive, for example, biologics and small molecule drugs, which are expensive and impose a heavy financial burden on long-term use. The phosvitin in this application is a natural protein extracted from eggs that has no adverse reactions in the human body and avoids the discomfort associated with existing antimicrobial treatments. This reduces medication costs and alleviates the burden on patients.

[0014] The phosvitin provided in the present application has the property of high net negative charge, which makes it resistant to proteolysis and can withstand the high acid environment of the stomach, reach the intestine and exert therapeutic effects.

[0015] Existing drugs for treating inflammatory bowel disease (IBD) lack a radical cure: they can only manage symptoms but cannot completely cure IBD. This application provides the use of phosvitin in the preparation of a drug for treating inflammatory bowel disease, which simultaneously improves intestinal inflammation by regulating intestinal flora and promoting barrier recovery.

[0016] This application demonstrates that PSV supplementation effectively improves DSS-induced colitis by enhancing intestinal barrier integrity, inhibiting inflammatory responses, and regulating intestinal microbiota. This study provides a new reference for the treatment strategy of inflammatory bowel disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 PSV improves DSS-induced colitis in mice, including (A) experimental design; (B) mouse body weight changes; (C) DAI score; (D) representative images of mouse colon; (E) colon length; (F) spleen index (spleen weight / body weight).

[0018] Figure 2 Effects of PSV on histopathological damage and inflammatory response in mice with DSS-induced colitis, including (A) representative images of H&E, AB, and PAS staining of mouse colon; (B) histopathological score; (C) goblet cell number; (D) IL-6 mRNA expression level in mouse colon; (E) IL-1β mRNA expression level in mouse colon; (F) TNF-α mRNA expression level in mouse colon; (G) COX-2 mRNA expression level in mouse colon.

[0019] Figure 3 Effects of PSV on intestinal barrier integrity in mice with DSS-induced colitis, where (A) is a representative image of immunofluorescence staining of tight junction proteins in mouse colon tissue; (B) is the mRNA expression level of Muc-2 in mouse colon; (C) is the mRNA expression level of Claudin-1 in mouse colon; (D) is the mRNA expression level of Occludin in mouse colon; and (E) is the mRNA expression level of ZO-1 in mouse colon.

[0020] Figure 4 Effects of PSV on the intestinal microbial structure of mice with DSS-induced colitis, where (A) is the Shannon index; (B) is the Pielou e index; (C) is the Simpson index; (D) PCA analysis of intestinal flora; (E) LEfSe analysis; (F) LDA analysis of differential flora.

[0021] Figure 5 Effects of PSV on the intestinal microbial composition and specific microbial abundance in mice with DSS-induced colitis, including (A) phylum-level microbial composition analysis; (B) relative abundance difference of Firmicutes; (C) relative abundance difference of Proteobacteria; (D) relative abundance difference of Verrucomicrobiota; (E) family-level microbial composition analysis; (F) relative abundance difference of Lachnospiraceae; (G) relative abundance difference of Enterobacteriaceae; (H) relative abundance difference of Akkermansiaceae; (I) genus-level microbial composition analysis; (J) relative abundance difference of Escherichia-Shigella; (K) relative abundance difference of Akkermansia; and (L) relative abundance difference of Clostridium.

[0022] Figure 6 Effects of PSV on the production of short-chain fatty acids in the feces of mice with DSS-induced colitis, where (A) is the concentration of acetate, (B) is the concentration of propionate, (C) is the concentration of butyrate, and (D) is the concentration of isobutyrate.

[0023] Figure 7 Correlation analysis of experimental parameters in mice with DSS-induced colitis.

[0024] Figure 8 In one embodiment of the present application, an experimental design was conducted to investigate the improving effect of PSV on DSS-induced colitis in mice. DETAILED DESCRIPTION

[0025] The present invention will be further illustrated below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, modifications of various equivalent forms of the present invention made by those skilled in the art all fall within the scope defined by the claims attached to this application.

[0026] One embodiment of the present application provides the use of phosvitin in the preparation of a drug for treating inflammatory bowel disease.

[0027] In one embodiment, the inflammatory bowel disease comprises ulcerative colitis.

[0028] In one embodiment, the ulcerative colitis is induced by DSS.

[0029] In one embodiment, the intervention dose of phosvitin is 200 mg / day-330 mg / day.

[0030] In one embodiment, the intervention dose of phosvitin is 226.699 mg / day-302.401 mg / day.

[0031] One embodiment of the present application provides a pharmaceutical composition for treating inflammatory bowel disease, characterized by comprising phosvitin and a pharmaceutically acceptable carrier.

[0032] In one embodiment, the carrier includes one or more of a disintegrant, a diluent, a lubricant, a binder, a wetting agent, a flavoring agent, a filler, a suspending agent, a surfactant, and a preservative.

[0033] The experimental design of the improvement effect of PSV on DSS-induced colitis in mice in one embodiment of the present application is shown in Figure 8 .

[0034] Extraction of phosvitin

[0035] Carefully break the shell of a fresh egg, separate the egg white with an egg yolk separator, roll the egg yolk on filter paper to remove the egg white and ovary, cut the egg yolk membrane with a toothpick, collect a total of 200 mL of egg yolk liquid, and place the egg yolk liquid in an ice water bath to keep it cold; add 1 L of water to the egg yolk liquid, adjust the pH to 5, and stir at 4°C for 6 h.

[0036] After stirring, the egg yolk solution was centrifuged at 7000 rpm for 1 h, the precipitate was collected, 800 mL of 0.05 mol NaCl solution was added, and the mixture was stirred for 4 h. The stirred suspension was centrifuged at 7000 rpm for 30 min, and the precipitate was collected.

[0037] Place the collected precipitate in a beaker and add a mixture of 600 mL of n-hexane and 200 mL of ethanol. Stir in a fume hood at 4°C for 6 hours. After stirring, filter the mixture to form a cake. After the cake-like precipitate is collected and air-dried in a fume hood, add 400 mL of 1.74 M NaCl solution and stir at 4°C for 4 hours.

[0038] The supernatant was collected after centrifugation at 7000 rpm and 15° C. for 40 min, filtered, dialyzed for 48 h and freeze-dried to obtain phosvitin.

[0039] Example 1 PSV improves symptoms of DSS-induced colitis in mice

[0040] Animal experiments were conducted to investigate the protective effects and related mechanisms of PSV against DSS-induced inflammatory bowel disease in mice. The experimental animals were C57BL / 6 mice (8 weeks old) housed in a standard SPF environment (temperature 22±2°C, humidity 50±15%, 12-h light / dark cycle).

[0041] The experimental period is 5 weeks. The specific experimental steps and grouping are as follows: Figure 1 Shown in A are the control group (Control group), PSV alone treatment group (PSV group), DSS modeling group (DSS group), PSV intervention group (PSV+DSS group), and fecal microbiota transplantation intervention group (FMT+DSS group).

[0042] The specific processing methods for each group are as follows:

[0043] Control group: 0.2 mL of normal saline was gavaged daily for 1-5 weeks, and normal drinking water was provided throughout the intervention;

[0044] PSV group: 50 mg / kg PSV was administered daily by gavage during the 1-5 weeks intervention period, and normal drinking water was provided throughout the intervention period;

[0045] DSS group: 0.2 mL of normal saline was gavaged daily during the intervention period, normal drinking water was provided for 1-4 weeks, and 2.5% DSS (w / v) was replaced in the 5th week;

[0046] PSV+DSS group: 50 mg / kg PSV was administered orally daily during the 1-5 weeks of intervention, and normal drinking water was provided during the 1-4 weeks, and replaced with 2.5% DSS (w / v) in the 5th week;

[0047] FMT+DSS group: The intestinal flora of mice was eliminated by antibiotics for 1-2 weeks. Feces of mice in the PSV group were collected for preparation of fecal homogenate for 3-5 weeks, and 0.2 mL of fecal homogenate supernatant was gavage daily. Normal drinking water was provided for 1-4 weeks, and 2.5% DSS (w / v) was replaced in the 5th week.

[0048] Starting from the 28th day of the experiment (before the start of DSS intervention), the mice were weighed daily until the end of the experiment.

[0049] The experimental results showed that the body weight of mice in the DSS group was significantly lower than that in the Control group (p<0.001) ( Figure 2 However, PSV and FMT intervention significantly (p < 0.01) inhibited this trend. Disease activity index (DAI) is an important indicator for assessing the severity of inflammatory bowel disease. DAI is scored by weight, fecal status, and fecal occult blood level of mice ( Figure 2 In Figure C), the score of mice in the DSS group was significantly higher than that in the Control group (p<0.001), while PSV and FMT intervention significantly reduced the DAI score (p<0.001). Inflammatory bowel disease can lead to a shortening of colon length. The colon length of mice in the DSS group was significantly lower than that in the Control group (p<0.001), while PSV and FMT intervention significantly (p<0.001) inhibited this trend. The spleen index of mice in the DSS group was significantly higher than that in the Control group (p<0.05). PSV intervention restored it to normal levels, while there was no significant difference between the mice in the FMT intervention group and the DSS group.

[0050] The results showed that DSS intervention successfully induced inflammatory bowel disease in mice, while PSV and FMT intervention improved the symptoms of DSS-induced colitis in mice.

[0051] Example 2: Effect of PSV on the Improvement of Intestinal Inflammation in DSS-Induced Colitis Mice

[0052] The effect of PSV on improving intestinal inflammation in DSS-induced colitis mice was investigated by H&E staining, AB staining, PAS staining, and expression levels of inflammation-related genes. Animal grouping and intervention methods were the same as in Example 1.

[0053] Pathological analysis of the staining revealed that ( Figure 2 Middle A), mice in the DSS group showed severe colonic ulcers, and compared with the control group, the mucosal and submucosal crypt structures were destroyed and goblet cells were lost ( Figure 2In the middle C), a large number of inflammatory cells infiltrated the lamina propria, and PSV and FMT intervention improved these phenomena. Similarly, the pathological scoring analysis of H&E staining results found that ( Figure 2 In Figure B), the disease score of mice in the DSS group increased significantly (p<0.001), while PSV and FMT intervention reduced the pathology score by approximately 1.5 points. Furthermore, the levels of inflammatory factors IL-6, IL-1β, TNF-α, and COX-2 in mice in the DSS group were significantly higher than those in the control group (p<0.001). In contrast, the expression levels of inflammatory factors in the PSV+DSS and FMT+DSS groups were lower than those in the DSS group. These results suggest that PSV can alleviate DSS-induced intestinal inflammation.

[0054] Example 3: Effect of PSV on the intestinal barrier of mice with DSS-induced colitis

[0055] Tight junction (TJ) proteins play a crucial role in intestinal mucosal barrier integrity and intestinal permeability. Studies have shown that TJ proteins are disrupted in the intestines of mice with DSS-induced colitis. Therefore, this study analyzed the effects of PSV treatment on three major TJ proteins in mice: Claudin-1, Occludin, and ZO-1, using immunofluorescence staining and PCR. Animal grouping and intervention methods were the same as in Example 1.

[0056] like Figure 3 As shown in A, DSS reduced the expression of three tight junction proteins, while PSV and FMT intervention reversed this trend in terms of area and fluorescence intensity. Similarly, the mRNA expression levels of Claudin-1, Occludin, and ZO-1 ( Figure 3 Figures C, D, and E) also showed a similar trend.

[0057] According to the experimental results, it was also observed that the level of Muc-2 in mice in the DSS group was significantly decreased ( Figure 3 (B), whereas this change was reversed in PSV- and FMT-treated mice.

[0058] The experimental results showed that PSV could prevent intestinal barrier damage in DSS-induced colitis in mice.

[0059] Example 4 Effect of PSV on the intestinal flora structure of mice with DSS-induced colitis

[0060] To investigate the effects of PSV on the intestinal microbiota of mice with colitis, 16S rRNA gene sequencing was used to analyze changes in the intestinal microbiota of each group of mice. Alpha diversity measures the species richness and even distribution within a community. The Shannon, Pielou e, and Simpson indices reflect differences in alpha diversity. Animal grouping and intervention methods were the same as in Example 1.

[0061] from Figure 4 In AC, it can be seen that the α diversity index of the DSS group samples decreased, indicating that the richness and diversity of the intestinal flora in the DSS group decreased. PSV intervention and FMT treatment increased the α diversity index of mice and increased the diversity of the intestinal flora in colitis mice. According to the OTU level, the differences in the composition of the intestinal microbiota between different treatments were evaluated by principal component analysis (PCA) ( Figure 4 In Figure D), the bacterial community structure of the control group was similar to that of the PSV group, but significantly different from that of the DSS group, indicating significant differences in their bacterial community structures. The PSV and FMT groups were intermediate between the control and DSS groups, indicating that PSV and FMT interventions mitigated the DSS-induced differences in bacterial composition in mice.

[0062] The results were analyzed by linear discriminant analysis (LDA) and effect size analysis (LEfSe) ( Figure 4 E and F) The effect of CA on the composition of the intestinal flora of each group of mice was evaluated, and the intestinal flora of each group of mice was further analyzed to obtain the dominant microorganisms with significant differences in the intestinal tract of mice in different treatment groups. Figure 4 As shown in E and F, the abundance of Escherichia-Shigella and Akkermansia in DSS-treated mice was different from that in the other groups. The abundance of Eisenbergiella, Bacteroides, Streptococcus, and Clostridium in the PSV+DSS group was different from that in the other groups.

[0063] The experimental results showed that PSV intervention and FMT treatment can regulate the composition of the intestinal flora of colitis mice and increase the species diversity and richness of the intestinal flora. It has a significant effect on the treatment of inflammatory bowel disease and improving the intestinal flora environment.

[0064] Example 5 Effects of PSV on the Intestinal Microbial Composition and Abundance of Specific Microorganisms in Mice with DSS-Induced Colitis

[0065] The effects of PSV on the intestinal flora of colitis mice were further explored by analyzing the composition of the intestinal flora at the phylum, family, and genus levels, as well as the relative abundance of specific microorganisms. Animal grouping and intervention methods were the same as in Example 1. The species composition histogram showed that the microbial composition of different groups was quite different at the phylum, family, and genus levels, while the differences among samples within the groups were relatively small ( Figure 5 A, E, I). At the phylum level, the abundance of Firmicutes decreased, while the abundance of Proteobacteria and Verrucomicrobiota increased in the DSS group, while PSV intervention and FMT treatment reversed this trend ( Figure 5BD). At the family level, the abundance of Lachnospiraceae decreased, while the abundance of Enterobacteriaceae and Akkermansiaceae increased in the DSS group, while PSV intervention and FMT treatment reversed this trend ( Figure 5 At the genus level, the abundance of Clostridium decreased, while the abundance of Escherichia-Shigella and Akkermansia increased in the DSS group, while PSV intervention and FMT treatment reversed this trend ( Figure 5 JL).

[0066] The experimental results showed that Escherichia-Shigella and Akkermansia may be closely related to the occurrence of colitis, and PSV intervention and FMT treatment can improve the abnormal abundance of specific intestinal microorganisms in colitis mice.

[0067] Example 6: Regulatory Effect of PSV on Short-Chain Fatty Acids in Feces of Mice with DSS-Induced Colitis

[0068] Short-chain fatty acids (SCFAs) are formed by intestinal microorganisms from dietary fiber and indigestible carbohydrates and play an important role in intestinal homeostasis. SCFAs can reduce intestinal inflammation and improve intestinal barrier function. To investigate the differences in SCFA content between groups, the animal grouping and intervention methods were the same as in Example 1.

[0069] Depend on Figure 6 It can be seen that the concentrations of acetic acid, propionic acid, butyric acid and isobutyric acid in the feces of mice in the DSS group were significantly reduced, while the content of SCFA increased to varying degrees after PSV and FMT intervention.

[0070] In order to explore the correlation between intestinal flora, proinflammatory factors and colitis disease indicators, we performed Spearman correlation analysis based on the experimental indicators ( Figure 7 ). It can be seen that the relative abundance of specific intestinal microbes is significantly correlated with other indicators. Experimental results show that PSV improves the intestinal barrier and immune imbalance in DSS colitis by remodeling the intestinal microbiota and restoring SCFA levels. FMT experiments confirm that the microbiota mediates the primary therapeutic effect of PSV.

[0071] These results demonstrate that PSV supplementation effectively ameliorates DSS-induced colitis by enhancing intestinal barrier integrity, inhibiting inflammatory responses, and regulating the gut microbiota. FMT experiments demonstrate that the gut microbiota plays a key role in this process. This study provides new insights into therapeutic strategies for inflammatory bowel disease.

[0072] Example 7 PSV human equivalent dose conversion

[0073] The experimental animals were C57BL / 6 mice, and a good protective effect was achieved at an intervention dose of 50 mg / kg / day. Using the human-animal body surface area method for conversion, according to the formula provided in the US FDA document "Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers," the human equivalent dose = mouse no observed adverse dose (NOAEL) / factor = 50 / 12.3 (mg / kg) ≈ 4.07 (mg / kg). According to data published in the "Fifth National Physical Fitness Monitoring Bulletin" (2022) by the National Physical Fitness Monitoring Center, the average weight of Chinese citizens aged 20-59 is 70.4-74.3 kg for men and 55.7-60.8 kg for women. The calculated human equivalent dose is 226.699 mg-302.401 mg / day.

[0074] The above are only preferred embodiments 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 principles 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 phosvitin in the preparation of drugs for the treatment of inflammatory bowel disease.

2. The use according to claim 1, characterized in that The inflammatory bowel disease includes ulcerative colitis.

3. The use according to claim 2, characterized in that The ulcerative colitis was induced by DSS.

4. The use according to claim 1, characterized in that The intervention dose of the phosvitin is 200 mg / day-330 mg / day.

5. A pharmaceutical composition for treating inflammatory bowel disease, characterized in that: The invention comprises phosvitin and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, characterized in that The carrier includes one or more of a disintegrant, a diluent, a lubricant, a binder, a wetting agent, a flavoring agent, a filler, a suspending agent, a surfactant and a preservative.

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