CN112107536A

CN112107536A - Preparation method of polyphenol-amyloid fiber hydrogel for relieving acute intestinal inflammation of mice

Info

Publication number: CN112107536A
Application number: CN201910546359.1A
Authority: CN
Inventors: 胡冰; 于诗婕
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2019-06-20
Filing date: 2019-06-20
Publication date: 2020-12-22

Abstract

The invention relates to a preparation method of polyphenol-amyloid fiber hydrogel for relieving acute intestinal inflammation of mice. The egg white lysozyme amyloid fiber is prepared by heating at high temperature under an acidic condition, and then polyphenol is simply added into the amyloid fiber to form the reversible hydrogel through self-assembly. By constructing an IBD animal model and using the hydrogel for gastric lavage to treat mice, the hydrogel is found to be capable of remarkably relieving DSS-induced acute colonic inflammation. All the materials used in the invention are food grade, and are safer compared with the medicines for preventing or treating intestinal inflammation, thereby providing a new idea for developing tea polyphenol products and medicines with anti-inflammatory activity and improved bioavailability.

Description

Preparation method of polyphenol-amyloid fiber hydrogel for relieving acute intestinal inflammation of mice

Technical Field

The invention relates to a preparation method of polyphenol-amyloid fiber hydrogel and the technical field of relieving acute intestinal inflammation of mice induced by DSS.

Background

Inflammatory Bowel Disease (IBD) is a chronic non-specific inflammatory disease including Ulcerative Colitis (UC) and Crohn's Disease (CD). The symptoms resulting from IBD, including weight loss, hemorrhagic diarrhea and severe abdominal pain, are currently incurable, and the most commonly used drugs such as aminosalicylates and immunomodulators all show severe side effects, such as affecting kidney function. Dietary polyphenols in foods and plants have attracted a wide interest in the prevention and treatment of colitis in recent years, and many have been used in the prevention and treatment of chronic and acute colitis in rodent models. In previous studies polyphenolic compounds have been shown to induce the progression of amyloid peptides to disordered and amorphous aggregates, but in the present invention the simple addition of EGCG to amyloid fibrils present in the nematic phase successfully drives the formation of hydrogels through self-assembly, completing the theory of interaction of polyphenols with protein amyloid fibrils. According to the invention, under the condition of high-concentration amyloid fibers, polyphenol is added to drive self-assembly of the amyloid fibers, so that the loading capacity of EGCG is greatly improved, and meanwhile, the hydrogel has an anti-inflammatory effect on mouse intestinal inflammation induced by Dextran Sodium Sulfate (DSS). The materials used in the invention are all food grade, and have good biocompatibility and safety.

Disclosure of Invention

The invention aims to provide a preparation method of polyphenol-amyloid fiber hydrogel for relieving acute intestinal inflammation of mice.

The technical scheme of the method is as follows:

(1) the preparation of lysozyme amyloid fiber is characterized in that: the egg white lysozyme is dialyzed and purified by ultrapure water at 4 ℃, and then the lysozyme solution after dialysis is subjected to freeze-drying treatment. Weighing a certain mass of egg white lysozyme freeze-dried powder, dissolving the egg white lysozyme freeze-dried powder in ultrapure water to prepare a 2 wt% egg white lysozyme solution, adjusting the pH to 2 by using HCl, and magnetically stirring and incubating for 8 hours at 90 ℃ to prepare an egg white lysozyme amyloid fiber solution (2 wt%, pH 2).

(2) The preparation method of the polyphenol-amyloid fiber hydrogel is characterized by comprising the following steps: weighing a certain mass of EGCG and dissolving the EGCG in Bis-Tris buffer solution (10mM, pH 7.2), mixing the egg white lysozyme amyloid fibers obtained in the step (1) with the EGCG solution according to a certain volume ratio, magnetically stirring for 30s, and standing to form hydrogel.

(3) The construction and treatment of the mouse IBD model are characterized in that: 30C 57BL/6J male mice were randomly assigned to the normal diet blank group (NC), DSS-induced group, DSS-induced + hydrogel group 3 groups of 10 mice each. All mice were acclimatized for one week and were initially tested in 14-day-old IBD animals. On days 1-7 of the experiment, 1.5% DSS solution was given as drinking water to the remaining mice, except for the blank mice which normally drunk water. On days 8-14 of the experiment, administration of the samples of the sterile water for intragastric administration (200. mu.L/day) was discontinued for mice of the NC group and the DSS group, and the intragastric hydrogel samples of the DSS + hydrogel group. During the test period, the weight of the mice, the feed and drinking water quality and the solid feces quality were recorded daily. After the test is finished, blood is taken from eyeballs, and serum obtained after the blood is processed is stored at the temperature of minus 80 ℃. The mice were then sacrificed by cervical dislocation, dissected and the tissues and organs were collected. During the dissection, the colon tissue (from the cecum to the anus) was removed intact and its length measured and photographed. The colon tissue was then flushed with normal saline and split into two halves by longitudinal shearing, with the left half of the colon tissue rolled into Swiss rolls (Swiss-Roll) and fixed in 4% paraformaldehyde for pathological analysis. Animal test results show that the hydrogel remarkably relieves DSS-induced acute colitis.

The invention has the following advantages:

the reversible hydrogel formed by self-assembly of the egg white lysozyme amyloid fiber induced by polyphenol is prepared for the first time, and the loading capacity of EGCG is greatly improved.

The reversible hydrogel can remarkably relieve intestinal inflammation of mice induced by DSS, and the used materials are all food-grade, so that the reversible hydrogel has good biocompatibility and safety.

Drawings

FIG. 1 Effect of different treatment groups on mouse body weight and solid stool quality

FIG. 2 influence of different treatment groups on colon length and contents of inflammatory factors IFN-gamma and IL-1 beta in serum of mice

FIG. 3 Colon hematoxylin and eosin (H & E) stained section views of mice from different treatment groups

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are further described below with reference to the embodiments of the present invention. The described embodiments are some, but not all embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The first embodiment is as follows:

(1) the egg white lysozyme is dialyzed and purified by ultrapure water at 4 ℃, and then the lysozyme solution after dialysis is subjected to freeze-drying treatment. Weighing a certain mass of egg white lysozyme freeze-dried powder, dissolving the egg white lysozyme freeze-dried powder in ultrapure water to prepare a 2 wt% egg white lysozyme solution, adjusting the pH to 2 by using HCl, and magnetically stirring and incubating for 8 hours at 90 ℃ to prepare an egg white lysozyme amyloid fiber mother solution.

(2) Weighing a certain mass of EGCG in a Bis-Tris buffer solution (10mM, pH 7.2), mixing the egg white lysozyme amyloid fibers obtained in the step (1) with the EGCG solution according to a certain volume ratio, magnetically stirring for 30s, and standing to form polyphenol-amyloid fiber hydrogel (the concentration of EGCG is 25 mg/mL).

(3) 30C 57BL/6J male mice were randomly assigned to the normal diet blank (NC), DSS-induced and DSS-induced + hydrogel groups, 10 per group. All mice were acclimatized for one week and were initially tested in 14-day-old IBD animals. On days 1-7 of the experiment, 1.5% DSS solution was given as drinking water to the remaining mice, except for the blank mice which normally drunk water. On days 8-14 of the experiment, administration of the samples of the sterile water for intragastric administration (200. mu.L/day) was discontinued for mice of the NC group and the DSS group, and the intragastric hydrogel samples of the DSS + hydrogel group. During the test period, the weight of the mice, the feed and drinking water quality and the solid feces quality were recorded daily. After the test is finished, blood is taken from eyeballs, and serum obtained after the blood is processed is stored at the temperature of minus 80 ℃. The mice were then sacrificed by cervical dislocation, dissected and the tissues and organs were collected. During the dissection, the colon tissue (from the cecum to the anus) was removed intact and its length measured and photographed. The colon tissue was then flushed with normal saline and split into two halves by longitudinal shearing, with the left half of the colon tissue rolled into Swiss rolls (Swiss-Roll) and fixed in 4% paraformaldehyde for pathological analysis. Animal test results show that the hydrogel remarkably relieves DSS-induced acute colitis.

Example two:

(1) detection of inflammatory factors in serum

According to the instruction of a commercial Xinbo Sheng ELISA kit, the contents of interferon-gamma (IFN-gamma) and interleukin (IL-1 beta) which are factors related to inflammation in serum are detected.

(2) Histopathological study

Fresh colon tissue was fixed in 4% paraformaldehyde for more than 24 hours, Swiss-Roll embedded in paraffin and cut into 4 μm full-thickness sections. Sections were then stained with hematoxylin and eosin (H & E) to characterize histological changes and observed by Nikon ECLIPSE TI-SR (Tokyo, Japan) fluorescence microscopy.

As can be seen from fig. 1 to fig. 3, the polyphenol-amyloid fiber hydrogel can significantly relieve DSS-induced acute intestinal inflammation in mice.

Claims

1. A preparation method of polyphenol-amyloid fiber hydrogel for relieving acute intestinal inflammation of mice is characterized by comprising the following operation steps: preparing egg white lysozyme amyloid fibers; preparing polyphenol-amyloid fiber hydrogel; and (3) constructing and treating a mouse IBD model.

2. The preparation method of the egg white lysozyme amyloid fiber according to claim 1, which is characterized in that: the egg white lysozyme is dialyzed and purified by ultrapure water at the temperature of 4 ℃, and then the lysozyme solution after dialysis is subjected to freeze-drying treatment. Weighing a certain mass of egg white lysozyme freeze-dried powder, dissolving the egg white lysozyme freeze-dried powder in ultrapure water to prepare a 2 wt% egg white lysozyme solution, adjusting the pH to 2 by using HCl, and magnetically stirring and incubating for 8 hours at 90 ℃ to prepare an egg white lysozyme amyloid fiber solution (2 wt%, pH 2).

3. The preparation of polyphenol-amyloid fibril hydrogel according to claim 1, characterized in that: weighing a certain mass of EGCG and dissolving the EGCG in Bis-Tris buffer solution (10mM, pH 7.2), mixing the egg white lysozyme amyloid fibers obtained in the step (1) with the EGCG solution according to a certain volume ratio, magnetically stirring for 30s, and standing to form hydrogel.

4. Construction and treatment of a mouse model of IBD according to claim 1, characterized by: 30C 57BL/6J male mice were randomly assigned to the normal diet blank group (NC), DSS-induced group, DSS-induced + hydrogel group 3 groups of 10 mice each. All mice were acclimatized for one week and were initially tested in 14-day-old IBD animals. On days 1-7 of the experiment, 1.5% DSS solution was given as drinking water to the remaining mice, except for the normal drinking water of the NC group mice. On days 8-14 of the experiment, administration of the samples of the sterile water for intragastric administration (200. mu.L/day) was discontinued for mice of the NC group and the DSS group, and the intragastric hydrogel samples of the DSS + hydrogel group. During the test period, the weight of the mice, the feed and drinking water quality and the solid feces quality were recorded daily. After the test is finished, blood is taken from eyeballs, and serum obtained after the blood is processed is stored at the temperature of minus 80 ℃. The mice were then sacrificed by cervical dislocation, dissected and the tissues and organs were collected. During the dissection, the colon tissue (from the cecum to the anus) was removed intact and its length measured and photographed. The colon tissue was then flushed with normal saline and split into two halves by longitudinal shearing, with the left half of the colon tissue rolled into Swiss rolls (Swiss-Roll) and fixed in 4% paraformaldehyde for pathological analysis. Animal test results show that the hydrogel remarkably relieves DSS-induced acute colitis.