Application of food-derived bone soup nanoparticles in the establishment of an animal model of colitis

Abstract

The present application relates to the field of biological medicine, and more particularly to the application of foodborne bone soup nanoparticles in establishing a colonitis animal influence model, the foodborne pig bone soup nanoparticle component obtained by gel filtration chromatography separation and purification from pig bone soup can protect DSS-induced mouse ulcerative colitis and regulate intestinal flora disorder caused by colitis by reducing the production of inflammatory factors IL1beta, IL-6 and TNF-alpha and inhibiting inflammatory expression, and in the process of establishing the ulcerative colitis mouse influence model, it is found that the foodborne bone soup nanoparticles can increase the body weight and colon length of the colitis mice, improve the hematochezia and fecal form of the colitis mice, and improve the colon pathological structure of the colitis mice, and can be used as a potential drug for preventing and treating ulcerative colitis and can provide a new drug selection for clinic.

Description

[0001] The application is a divisional application of the invention patent with the title: Application of food-derived bone soup nanoparticles, application number: 202210046761.5, and the parent application date is January 17, 2022. TECHNICAL FIELD

[0002] The application relates to the field of biological medicine, and particularly relates to application of food-derived bone soup nanoparticles in establishing an animal model of colitis. BACKGROUND

[0003] Ulcerative colitis (UC) is a chronic nonspecific disease of the colorectal mucosa with unknown etiology. The disease has the characteristics of chronic progression, long course, and repeated attacks. The pathological features are abnormal production of cytokines, increased expression of adhesion molecules and cell infiltration, and eventually leading to epithelial cell apoptosis and mucosal damage. At present, there is no effective treatment method, and it is listed as one of the refractory diseases by WHO. In the past 20 years, the incidence and prevalence in China have continued to increase, and are closely related to the occurrence of colon cancer. The treatment of UC has become a thorny problem in clinical practice. Although some glucocorticoids and immunosuppressive agents can relieve symptoms, the effect is not very satisfactory, and long-term use will cause serious adverse reactions. Therefore, there is an urgent need for drugs with high safety and treatment of UC.

[0004] In recent years, naturally occurring nanoparticles widely present in some foods have attracted widespread attention. These food-derived naturally occurring nanoparticles have been consumed with food for a long time, have good safety, bioavailability and biocompatibility, and are more suitable for application in the food and pharmaceutical industries.

[0005] For example, the advantages of green tea extract assembling to form nanocarriers of anticancer chemotherapy drugs in safety and biocompatibility show good prospects as drugs.

[0006] Milk protein nanoparticles can improve the in vivo absorption efficiency of oral folic acid and omega-3 polyunsaturated fatty acids.

[0007] Nanoparticles self-assembled from gelatin can carry catechin EGCG and retain its biological activity, and are a future functional food additive.

[0008] Nanoparticles self-assembled from licorice protein can embed aconitine, and can effectively reduce the toxicity of aconitine to the body.

[0009] Even some plant-derived edible naturally occurring nanoparticles have been shown to have therapeutic effects on intestinal inflammation.

[0010] Pig bone contains bone marrow, rich in bone collagen, protein, lipid, polysaccharide, nucleic acid and mineral components, and the pig bone soup prepared by high-temperature boiling and slow simmering is not only delicious, but also rich in nutrition, and is more and more favored by people all over the world. It has been found through research that the pig bone soup contains a large amount of naturally occurring nanoparticles, which have been shown to regulate immune function and repair damaged intestinal barrier function. However, so far there has been no report on the application of food-derived bone soup nanoparticles in the prevention and treatment of intestinal inflammatory diseases. SUMMARY

[0011] The first object of the present application is to provide an application of food-derived bone soup nanoparticles in establishing a colonitis animal influence model.

[0012] To achieve the above-mentioned application purposes, the present application realizes the following technical solutions:

[0013] Application of food-derived bone soup nanoparticles in preparing anti-ulcerative colitis drugs.

[0014] Application of food-derived bone soup nanoparticles in regulating dysbiosis and repairing intestinal mucosal damage drugs.

[0015] Application of food-derived bone soup nanoparticles in reducing the production of inflammatory factors and inhibiting the expression of inflammatory factors.

[0016] As a preferred, the inflammatory factors include any one or a combination of IL1β, IL-6, TNF-α.

[0017] The preparation method of the above-mentioned food-derived bone soup nanoparticles comprises the following steps:

[0018] (1) preparing pig bone soup by heat processing pig bone;

[0019] (2) centrifuging the pig bone soup obtained in step (1) to obtain supernatant;

[0020] (3) separating the supernatant obtained in step (2) by gel exclusion chromatography combined with dynamic light scattering instrument to obtain pig bone soup functional nanoparticles.

[0021] As a preferred, the heat processing process of pig bone in step (1) is as follows: after soaking the pig bone in sodium citrate solution, the blood water is removed by washing with distilled water, and then the pig bone soup is obtained by filtering after boiling with distilled water.

[0022] As a preferred, the concentration of sodium citrate solution is 0.5-3%.

[0023] As a preferred, the solid-liquid ratio of pig bone to distilled water during boiling is 1: (1-3) kg / L.

[0024] As preferred, the boiling time is 0.5-3h.

[0025] As preferred, the centrifugal speed in step (2) is 5000-10000r•min -1 , and the centrifugal time is 10-20min.

[0026] As preferred, the separation step in step (3) is as follows: the supernatant is passed into a gel exclusion chromatography column, the functional nanoparticles are combined on the gel exclusion column, then the eluent is washed with a buffer solution, and the eluent is monitored by combining online dynamic light scattering at 280 nm ultraviolet band, the eluent of the light scattering peak is collected, and the eluent is dried to obtain the functional nanoparticles in the pig bone soup.

[0027] As preferred, the gel exclusion chromatography column is agarose or its derived cross-linking material.

[0028] The separation range thereof is 60KDa-20000 KDa, and the pore size is 45-165μm.

[0029] As preferred, the model of the gel exclusion chromatography column is Sephacryl S-1000 SF.

[0030] As preferred, the buffer eluent is a phosphate buffer solution with a concentration of 0.01~0.1M and a pH of 6.5~7.5.

[0031] As preferred, the particle size of the functional nanoparticles is 100-300nm.

[0032] Application of food-derived bone soup nanoparticles in establishing a colonitis animal influence model.

[0033] The method for establishing the colonitis animal influence model is as follows:

[0034] (S.1) The experimental animals are randomly divided into a normal group, a model group, a food-derived bone soup nanoparticle group, a bone soup group, and a positive control group, and the environment is adapted;

[0035] (S.2) DSS is prepared into a solution with distilled water, and the model group, the food-derived bone soup nanoparticle group, the bone soup group, and the positive control group are given free access to the solution to perform modeling;

[0036] During the modeling process:

[0037] The normal group and the model group are given distilled water by gavage every day;

[0038] The food-derived bone soup nanoparticle group and the bone soup group are given food-derived bone soup nanoparticle solution and bone soup by gavage, respectively;

[0039] The positive control group is given sulfasalazine solution by gavage every day;

[0040] (S.3) From the first day of administration, the body weight, stool shape, and stool with blood of the experimental animal are recorded daily at fixed time and place, and the stool shape and stool with blood are scored according to the standard of reference;

[0041] (S.4) After the administration, the experimental animal is decapitated, the colon is taken out, the stool in the colon is collected, and the effect of the food-derived bone soup nano-particle on the colonitis of the experimental animal is evaluated.

[0042] Preferably, the colonitis is ulcerative colitis.

[0043] Preferably, the experimental animal in step (S.1) is a five-week-old female BALB / c mouse (20±2g) of SPF level.

[0044] The environmental conditions are as follows: the temperature is 23±2℃, the humidity is 50±5%, and the adaptation time of the mouse is 7 days.

[0045] Preferably, the concentration of the food-derived bone soup particle solution and the bone soup is consistent in step (S.2).

[0046] Preferably, the concentration of the DSS solution is 5%.

[0047] Preferably, the food-derived bone soup particle group and the bone soup group are respectively administered intragastrically at a dose of 50 mL (food-derived bone soup particle solution or bone soup) / kg / day.

[0048] Preferably, the positive control group is administered intragastrically at a dose of 250 mg / kg / day of sulfasalazine solution.

[0049] Further, the food-derived bone soup nano-particle is used for increasing the body weight and colon length of the colitis mouse, improving the stool shape and stool with blood of the colitis mouse, and improving the pathological structure of the colitis mouse.

[0050] Therefore, the present application has the following beneficial effects:

[0051] (1) The food-derived pig bone soup nano-particle component obtained by gel filtration chromatography separation and purification from pig bone soup can reduce the production of inflammatory factors IL1b, IL-6, and TNF-α, inhibit the expression of inflammation, and restore the imbalance of inflammatory intestinal flora, so as to explore the potential application of the food-derived bone soup nano-particle in the treatment of ulcerative colitis.

[0052] (2) The application provides application of food-derived bone soup nanoparticles in prevention and treatment of ulcerative colitis, and the food-derived bone soup nanoparticles can increase the body weight and colon length of colitis mice, improve the hematochezia and stool shape of the colitis mice, and improve the pathological structure of the colitis mice, so that the food-derived bone soup nanoparticles can be used as a potential drug for prevention and treatment of ulcerative colitis and provide a new drug selection for clinic. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 TEM electron microscope observation diagram of the food-derived bone soup nanoparticles prepared in the application.

[0054] Figure 2 Result diagram of the influence of the food-derived bone soup nanoparticles on body weight, disease activity index (DAI), colon length, and colon weight to colon length ratio.

[0055] Figure 3 Result diagram of the influence of the food-derived bone soup nanoparticles on intestinal tissue pathological changes and intestinal tissue inflammatory factor secretion.

[0056] Figure 4 Result diagram of the influence of the food-derived bone soup nanoparticles on intestinal flora richness and diversity.

[0057] Figure 5 Result diagram of the comparative analysis of the microbial community composition of different samples.

[0058] Figure 6 Result diagram of the influence of the food-derived bone soup nanoparticles on intestinal microbial community structure. DETAILED DESCRIPTION

[0059] The application will be further described below in combination with the drawings and specific examples in the specification. Those skilled in the art can implement the application based on these descriptions. In addition, the examples of the application involved in the following description are generally only examples of a part of the application, rather than all examples. Therefore, based on the examples in the application, all other examples obtained by those skilled in the art without creative labor should belong to the protection scope of the application.

[0060] The preparation method of the food-derived bone soup nanoparticles in the application is as follows:

[0061] (1) The pig bone is soaked in 1% sodium citrate solution for 30 minutes, then washed with distilled water for 3 times to remove blood water. Distilled water is added according to the solid-liquid ratio of 1:2 kg / L, and boiled for 2 hours, then cooled to room temperature, and finally filtered with gauze to obtain pig bone soup;

[0062] (2) The crude filtrate is taken to a centrifuge, and the centrifugal speed is 8000 r•min -1Centrifugation at 40℃ for 10 min, and the supernatant was obtained;

[0063] (3) The supernatant was rapidly separated by using a gel exclusion chromatography column (10 mm*120 mm), the column filler was Sephacryl S-1000 SF, 0.05M phosphate buffer (pH 7.0) was used to fill the column and for equilibration, the flow rate was 0.3 mL / min, the sample amount was 1 mL, and the same phosphate buffer was used for elution, and the elution components were monitored by using ultraviolet 280 nm and DLS simultaneously. The peak with absorption under 280 nm and strong laser light scattering signal was collected, the peak time of the pig bone soup colloidal particles was 105-115 min, the functional nanoparticles were collected, and after permeation desalination in distilled water, freeze-drying was carried out for standby, the particle size distribution was 50-500 nm, the average particle size was 220 nm, the surface was negatively charged, the zeta potential was-15 mV, and the TEM electron microscope observation figure (Fig. 2) proved that the pig bone soup colloidal particles were uniform spherical. Figure 1

Specific embodiment

[0065] The experimental animals of the application are 30 SPF five-week-old female BALB / c mice (20±2 g) purchased from Zhejiang Province Experimental Animal Center. Under the condition that the temperature is 23±2℃ and the humidity is 50±5%, the mice will adapt to the new condition for 7 days.

[0066] The modeling drug of the application is DSS dextran sulfate sodium, which is purchased from MP Company, and the molecular weight is 36000-50000.

[0067] Experimental procedure:

[0068] The foodborne nanoparticles are dissolved in distilled water to be consistent with the light scattering value of the bone soup stock. 30 BALB / c mice are randomly divided into a normal group (marked as Control), a model group (marked as DSS), a foodborne bone soup particle group (marked as BSNPs), a bone soup group (marked as BS), and a positive control group (sulfasalazine, SASP, marked as SASP). After adapting to the environment for one week, the DSS is prepared into a 5% solution with distilled water, and the DSS group, the BSNPs group, the BS group, and the SASP group are allowed to drink freely for modeling, for 7 consecutive days. During the modeling period, the Control group and the DSS group are given distilled water for gavage every day; the BSNPs group and the BS group are given gavage at a dose of 50 mL (BSNPs or BS) / kg / day. The SASP group is given gavage at a dose of 250 mg / kg / day.

[0069] ​From the first day of administration, the body weight, stool shape, and stool with blood of mice were recorded daily at fixed time and place, and the stool shape and stool with blood were scored according to the standard of reference. Stool shape: normal, 0 points; soft and shaped, 1 point; very soft, 2 points; diarrhea, 3 points. Blood in stool: no blood in stool, 0 points; occult blood in stool, 1 point; obvious blood in stool, 2 points; diarrhea with blood and staining of the anus, 3 points. After 7 days of administration, the mice were sacrificed by cervical dislocation, the colon was removed, and the feces in the colon was collected for 16s rRNA determination. The colon length was measured with a ruler, the colon was washed with normal saline, and was divided into two parts. One part was fixed with paraformaldehyde, embedded in paraffin, cut into 4 μm sections, HE stained, and observed under an optical microscope for pathological changes of intestinal tissue. The other part was frozen in liquid nitrogen and stored at -80 degrees Celsius for mRNA expression analysis of inflammatory factors.

[0070] The degree of colitis in mice can be reflected by clinical indicators. Mice with severe colitis will have lighter body weight, increased disease activity index, and shorter colon length. In addition, the ratio of colon weight to colon length can also be used as an indicator to measure the degree of colitis, and the more severe the colitis, the larger the ratio will be.

[0071] Figure 2 Effects of food-derived bone soup nanoparticles on body weight, disease activity index (DAI), colon length, and the ratio of colon weight to colon length. From Figure 1 It can be seen that compared with the Control group, the body weight of the model DSS group decreased, the disease activity index increased, the colon length significantly shortened, and the ratio of colon weight to colon length significantly increased, indicating that the model was established. Compared with the model DSS group, the food-derived bone soup nanoparticles BSNPs group and the bone soup group BS significantly improved the body weight and colon length of mice, reduced the disease activity index and the ratio of colon weight to colon length, and the effect was comparable to or even slightly better than the positive control drug SASP, with statistical significance.

[0072] Figure 3The effect of food-derived bone soup nanoparticles on intestinal histopathological changes and intestinal tissue inflammatory factor secretion. The intestinal tissue condition was evaluated by HE staining, and the results showed that the normal group had complete intestinal mucosa epithelial cells, normal intestinal thread shape, and no inflammatory infiltration or damage to intestinal cells, and the intestinal tissue was in good condition. The model group showed obvious intestinal wall thickening, large areas of neutrophil infiltration in the lamina propria, irregular intestinal villi, partial villi and intestinal thread disappearance, and necrotic tissue and inflammatory cell infiltration, with obvious inflammatory status. In the food-derived bone soup nanoparticle group and the positive control group, the intestinal wall edema was converted to mild, the intestinal villi epithelium was complete, the neutrophil infiltration was significantly alleviated, and the inflammatory degree was reduced. Histological scores also showed that food-derived bone soup nanoparticles reduced histological scores, indicating that food-derived bone soup nanoparticles played a protective role in the intestine. TNF-α, IL-6, and IL-1β are important inflammatory factors related to intestinal inflammation. Analysis of the expression of inflammatory factors TNF-α, IL-6, and IL-1β in colon tissue showed that the model DSS group significantly increased the expression of TNF-α, IL-6, and IL-1β compared to the Control group, while the food-derived bone soup nanoparticle BSNPs group and the bone soup group BS significantly reduced the expression of these inflammatory factors, indicating that food-derived bone soup nanoparticles inhibited the production of inflammatory factors.

[0073] Figure 4 The effect of food-derived bone soup nanoparticles on intestinal flora richness and diversity. Chao1 and observed species indices reflect the richness of the flora, and the higher these two indices, the higher the richness; Shannon and Simpson indices reflect the diversity of the flora, and similarly, the higher these two indices, the higher the diversity. Intestinal inflammation reduces the richness and diversity of intestinal microbial communities. Compared with the Control normal group, the DSS model group significantly reduced the richness and diversity of intestinal flora. The food-derived bone soup nanoparticle BSNPs group and the bone soup group BS significantly improved the richness and diversity of intestinal flora, with biological statistical significance. This indicates that food-derived bone soup nanoparticles have the function of regulating intestinal microbial communities.

[0074] Figure 5 The microbial community composition of different samples was compared and analyzed. PCoA and NMDS analysis results showed that the food-derived bone soup nanoparticle BSNPs group, the bone soup group BS, and the positive control group SASP group were closer to the Control group, and the four groups were far from the model DSS group. Unweighted pair-group method with arithmetic means (UPGMA) analysis showed the same results. These results indicate that food-derived bone soup nanoparticles can restore the imbalance of intestinal inflammatory microbial communities.

[0075] Figure 6 The effects of food-derived bone broth nanoparticles (DSS) on gut microbiota structure were investigated. Results showed that DSS caused significant changes in gut microbiota structure. Compared with the normal control group, at the phylum level, DSS upregulated […]. Campilobacterota and Firmicutes The relative abundance was reduced, but the abundance was lowered. Relative abundance of Bacteroidota. In addition, DSS also significantly increased Firmicute / Bacteroidota (F / B) ratio. At the genus level, DSS significantly reduced Muribaculaceae (Figure 5D) , Alistipes (Figure 5H), and Alloprevotella The relative abundance of (Figure 5G) increased Helicobacter and Lachnospiraceae_NK4A136_group. In contrast, the BSNPs group (containing food-derived bone broth nanoparticles), the BS group (containing original bone broth), and the positive control group all showed a return to normal levels of these bacterial groups. These results indicate that food-derived bone broth nanoparticles have the function of regulating intestinal flora imbalance in enteritis.

[0076] Therefore, the above data indicate that the food-derived pork bone broth nanoparticles obtained by gel filtration chromatography from pork bone broth can protect against DSS-induced ulcerative colitis in mice and regulate intestinal flora imbalance caused by colitis by reducing the production of inflammatory factors IL1b, IL-6, and TNF-α and inhibiting inflammatory expression. This is intended to explore the potential application of food-derived bone broth nanoparticles in the treatment of ulcerative colitis.

[0077] Furthermore, during the establishment of a mouse model of ulcerative colitis, it was found that food-derived bone broth nanoparticles could increase the body weight and colon length of colitis mice, improve fecal bleeding and stool morphology, and improve the pathological structure of the colon in colitis mice. They can serve as a potential drug for the prevention and treatment of ulcerative colitis and provide a new drug option for clinical use.

Claims

1. Application of food-derived bone soup nanoparticles in establishing a colonitis animal influence model, characterized in that, the method for establishing the colonitis animal influence model comprises the following steps: (S.1) randomly divide experimental animals into a normal group, a model group, a food-derived bone soup nanoparticle group, a bone soup group, and a positive control group, and adapt to the environment; (S.2) prepare a DSS solution with distilled water, and allow the model group, the food-derived bone soup nanoparticle group, the bone soup group, and the positive control group to freely drink the solution to perform modeling; wherein, during the modeling process: the normal group and the model group are given distilled water for gavage every day; the food-derived bone soup nanoparticle group and the bone soup group are respectively given food-derived bone soup nanoparticle solution and bone soup for gavage; the positive control group is given a sulfasalazine solution for gavage every day; (S.3) starting from the first day of administration, record the body weight, fecal shape, and fecal blood of the experimental animals at fixed points and times every day, and score the fecal shape and fecal blood according to the standard in the reference; (S.4) after the administration is completed, the experimental animals are executed by decapitation, the colon is taken out, the feces in the colon are collected, and the influence of the food-derived bone soup nanoparticles on the colonitis of the experimental animals is evaluated.

2. The application according to claim 1, characterized in that, the colonitis is ulcerative colitis.

3. The application according to claim 2, characterized in that, in step (S.1), the experimental animals are SPF five-week-old female BALB / c mice; the environmental adaptation conditions are as follows: the temperature is 23±2℃, the humidity is 50±5%, and the mice are adapted for 7 days.

4. The application according to claim 2, characterized in that, in step (S.2), the concentration of the food-derived bone soup nanoparticle solution and the bone soup is consistent.

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