CN113461776B - Small peptide with enteritis preventing and treating function and application thereof - Google Patents
Small peptide with enteritis preventing and treating function and application thereof Download PDFInfo
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- CN113461776B CN113461776B CN202110774011.5A CN202110774011A CN113461776B CN 113461776 B CN113461776 B CN 113461776B CN 202110774011 A CN202110774011 A CN 202110774011A CN 113461776 B CN113461776 B CN 113461776B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
The invention provides a small peptide with the function of preventing and treating enteritis and application thereof, belonging to the technical field of biology. The small peptide has the function of preventing and treating enteritis, and the amino acid sequence of the small peptide is shown as SEQ ID NO:1 and the amino acid sequence is MDTSAPSPF. The small peptide can effectively prevent and treat enteritis related diseases. The small peptide is derived from wheat germs, and has the characteristics of safety, high efficiency and wide sources. The small peptide is simple and convenient to extract and purify, has wide prospects in the fields of foods, medicines and the like, and can be applied to preparing medicines or functional foods for preventing and treating enteritis.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a small peptide with anti-inflammatory activity and application thereof in preparing medicines for preventing and treating enteritis.
Background
The enteritis is frequently seen and common, seriously affects the physical health of people, and is difficult to cure, long in course of disease and easy to repeatedly attack. Among them, colitis is more in enteritis, and is classified into two types, acute and chronic. If the acute stage patients are properly controlled, the symptoms can be relieved and finally cured, otherwise, the inflammation repeatedly attacks, finally changes into chronic, increases the risk of changing into colon cancer, is one of the most serious diseases of the gastrointestinal tract, and is listed as one of the modern refractory diseases by the world health organization. However, there is a lack of efficient small peptides in the prior art for the treatment and prevention of enteritis.
Disclosure of Invention
The invention aims to provide a small peptide with better enteritis prevention and treatment functions.
The invention also aims to provide the application of the small peptide in preparing medicines for preventing and treating enteritis.
The invention adopts the following technical scheme:
a small peptide with the function of preventing and treating enteritis has an amino acid sequence shown in SEQ ID NO: 1.
The application of the small peptide in preparing a medicament for preventing and treating enteritis.
The enteritis includes colitis and other inflammatory diseases.
The small peptide has stronger anti-inflammatory activity through in vivo colon inflammation injury test, is derived from wheat germ protein, is an ideal natural active substance, and has the characteristics of safety, high efficiency, wide sources and low price. The small peptide can be applied to the preparation of medicines for preventing and treating enteritis diseases.
The invention has the beneficial effects that: the functional characteristics of the small peptides of the invention are determined by DSS-induced colitis experiments in mice. Firstly, in vitro experiments show that the small peptide has no influence on the proliferation activity of normal colon epithelial cells NCM460, and has good safety. In-vivo experimental results show that the small peptide can obviously improve colon pathology and inflammation level of a colonitis model mouse and improve the health level of the mouse. Therefore, the small peptide can effectively prevent and treat enteritis related diseases. The small peptide is derived from wheat germs, and has the characteristics of safety, high efficiency and wide sources.
Drawings
FIG. 1 is a secondary mass spectrum of a small peptide of the present invention, wherein the abscissa represents mass-to-charge ratio values (m/z) of ions and the ordinate represents the intensity of ion flow.
FIG. 2 is a graph showing the effect of the small peptides of the invention on NCM460 proliferative activity, wherein the small peptides are the small peptides of the invention, and the control group is a control well.
FIG. 3 is the colon length of each group of mice, wherein the H-small peptide group, L-small peptide group are model+small peptide high dose group and model+small peptide low dose group, respectively.
FIG. 4 shows the results of HE staining of colon tissue from each group of mice, wherein the H-small peptide group and L-small peptide group are model + small peptide high dose group and model + small peptide low dose group, respectively.
FIG. 5 shows the IL-1. Beta. And TNF-. Alpha.levels in serum of mice from each group, wherein the H-small peptide group and L-small peptide group were model + small peptide high dose group and model + small peptide low dose group, respectively.
Description of the embodiments
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Materials: the wheat germ protein small peptide used in the experiment is synthesized by the gold Style biotechnology Co-Ltd according to the identification result of LC-MS/MS by using the FlexPeptide polypeptide synthesis technology, and the sequence is as follows: MDTSAPSPF. Caco-2 cell lines and NCM460 cell lines were purchased from Shanghai department of science cell banks; fetal bovine serum (Fetal Bovine Serum, FBS), DMEM medium, EDTA-pancreatin (0.05%), penicillin-Streptomycin (100×), PBS phosphate buffer from Gibco company of usa; miRNA chemicals, RNase-free H 2 O was purchased from bioengineering (Shanghai) stock Co.Ltd; lipofectamine 3000, opti-MEM medium available from Invitrogen, inc., USA; dimethyl sulfoxide (DMSO) was purchased from Sigma, usa; thiazole blue (MTT), 1% paraformaldehyde solution was purchased from beijing solibao technologies limited; c57BL/6 male mice were purchased from Bodhisattva biomedical technologies Co., ltd; formalin was purchased from Sigma, usa; dextran sodium sulfate (Dextran sodium sulfate, DSS, MW:36000-50000 Da) is available from MPbio corporation of America; hematoxylin, eosin, CD11b antibody, secondary antibody (HRP-labeled goat anti-rabbit) were purchased from wuhansai wil biotechnology limited; other reagents are domestic analytical pure.
Main instruments and equipment: biosafety cabinet (Thermo Fisher, china); MZE multifunctional microplate reader (Molecular Devices (usa); SL16R bench centrifuge (Thermo Fisher, china); carbon dioxide incubator (Thermo Fisher, china); fluorescence microscopy (Cal Cai Siyou, germany).
Wheat germ protein is digested by simulating gastrointestinal tract and ultrafiltered to separate several small peptides, and one of the small peptides has the amino acid sequence MDTSAPSPF (hereinafter referred to as the small peptide of the present invention, fig. 1). The sequence of the small peptide is shown as SEQ ID NO. 1. The small peptides of the invention were synthesized by Nanjing Jinsri biotechnology limited. Through a large number of screening work, the small peptide disclosed by the invention has good anti-inflammatory activity, and can effectively relieve colon inflammation injury of mice.
The effect of the small peptides of the invention on the proliferative activity of NCM460, a normal colon epithelial cell, was examined by MTT assay.
NCM460 cells were cultured in 96-well plates, and sample wells and control wells were provided with 3X 10 wells per well 3 Individual cells. 100. Mu.L of DMEM medium containing 0, 20, 80 and 320. Mu.M of small peptides of the invention and 10% fetal bovine serum are respectively added into each sample hole, and 6 parallel holes are arranged at each small peptide concentration; 100. Mu.L of DMEM medium containing 10% (volume percent) fetal bovine serum was added to each control well, and 6 parallel wells were set without any drug. A blank well was additionally provided, without cells, and 6 parallel wells were set by adding 100. Mu.L of PBS buffer (0.01M, pH 7.2-7.4). After incubating the 96-well plates in a CO2 incubator at 37 ℃ for 24 h, the medium was removed and 100 μl of dimethyl sulfoxide (DMSO) was added to each well, and after incubation for 15 min, the absorbance of each well was measured at 570 nm, and the average value was calculated. The cell proliferation rates of each group were calculated as follows: cell proliferation rate= (average sample well OD value-average blank well OD value)/(average control well OD value-average blank well OD value).
The results are shown in FIG. 2, compared with the control hole, the small peptide has no significant effect (p > 0.05) on the proliferation activity of NCM460, and has no toxic effect on normal colon epithelial cells of human and good safety.
1) Moulding and administration
Male C57BL/6 mice of 6 weeks old were purchased from Bodhamia biomedical technology Co., ltd (China without tin). 50C 57BL/6 mice were randomly divided into 5 groups of 10 mice each, which were respectively a normal group, a model group, a model+small peptide high dose group, a model+small peptide low dose group, and a positive control group. In addition to the normal group, other groups used the following methods to construct a mouse model of colitis: the experimental mice were free to drink 2.5% DSS (dextran sodium sulfate) in water for 7 days to construct a colitis mouse model.
In the process of constructing the colonitis mouse model, the model group mice have no other medicines for lavage. Model + small peptide high dose group and model + small peptide low dose group in addition to administration of colitis model drug DSS, the model + small peptide high dose group was lavaged daily with small peptide of the invention, dose 200 mg/kg·d; model + small peptide Low dose groups were perfused daily with small peptides of the invention at a dose of 50 mg/kg.d. Positive control mice were perfused daily with mesalamine at a dose of 60 mg/kg.d, except for the colitis model drug. In addition, normal mice were kept normally without any drug administration. The diet drinking method of each mouse in the model group, the model+small peptide high dose group, the model+small peptide low dose group and the positive control group is the same as that of the mice in the normal group.
After the modeling was completed, mice were sacrificed, colon length of the mice was measured, then colon tissues were collected for HE staining and immunohistochemical analysis, and plasma was collected to detect inflammatory factors.
(2) Results of colon length detection in mice
The experimental results in fig. 3 show that the normal colon length is 7.0±0.4. 0.4 cm and the model colon length is 4.4±0.7 cm. The colon length of the model group was significantly reduced compared to the normal group (p < 0.01). The colon length of the positive control group was 6.0±1.0 cm, and the colon lengths of the model+small peptide high dose group and the model+small peptide low dose group were 5.3±0.5 cm and 5.6±1.0 cm, respectively. Thus, the model + small peptide high dose group and the model + small peptide low dose group significantly restored colon length (p < 0.05) in enteritis mice. The experimental result shows that the small peptide can obviously improve the colon length of a colonitis mouse.
(3) HE staining results
Colon tissues of each group of mice were HE stained as follows: colonic tissue was fixed with formalin, then washed 3 times with PBS, and gradient dehydrated in ethanol for 1 h. Then, the colon tissue was taken out, placed in xylene solution for transparency treatment 2 times, allowed to stand for 1. 1 h each time, and then the tissue was transferred into 54 ℃ paraffin for wax impregnation. Paraffin embedding at 56-58 deg.c, cooling to room temperature, slicing, spreading in warm water, fishing with glass slide and stoving. Then the slices are placed in xylene solution again for dewaxing treatment, and the slices are placed in 100%, 95%, 85% and 75% ethanol solution for rehydration for 1 min respectively. Then, the sections were stained with hematoxylin for 5 min, differentiated with 1% hydrochloric acid solution for 5-10s, washed twice with water, and stained with eosin for 2 min. And (3) carrying out ethanol dehydration again, and then respectively treating for 3 min and 5 min by using a xylene solution. Finally, the gel was blocked with neutral resin and histological analysis was performed under an optical microscope according to the pathological criteria.
As can be seen from fig. 4, compared with the normal group, the intestinal mucosa in the model group is severely damaged, the crypt disappears, and the model+small peptide high dose group and the model+small peptide low dose group significantly improve the damage of the intestinal mucosa, and the crypt recovers, which proves that the small peptide of the invention can prevent the damage of the intestinal tissue in the colonitis mice.
5) Effect of the small peptides of the invention on IL-1 beta and TNF-alpha content in mice with a model of this invention
The amounts of IL-1 beta and TNF-alpha in the serum of each group of mice were determined using mouse IL-1 beta and TNF-alpha ELISA kits (Hangzhou Union Biotechnology Co., ltd.) according to the manufacturer's instructions.
As can be seen from fig. 5, the levels of IL-1 beta and TNF-alpha in the serum of the model group were significantly increased (p < 0.01) compared to the normal group, while the model + small peptide high dose group, the model + small peptide low dose group significantly improved the inflammatory level in the mice with colitis (p < 0.01), and the model + small peptide high dose group mice serum was close to the positive group, indicating the important role of the small peptides of the invention in preventing and improving colitis.
SEQUENCE LISTING
<110> university of financial and financial institutions in Nanjing
<120> a small peptide having the function of preventing and treating enteritis and the use thereof
<130> 202107071
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 9
<212> PRT
<213> wheat germ
<400> 1
Met Asp Thr Ser Ala Pro Ser Pro Phe
1 5
Claims (2)
1. A small peptide with the function of preventing and treating colonitis, which is characterized in that: the amino acid sequence is shown in SEQ ID NO: 1.
2. Use of the small peptide of claim 1 in the manufacture of a medicament for the prevention and treatment of colitis.
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| CN202110774011.5A CN113461776B (en) | 2021-07-08 | 2021-07-08 | Small peptide with enteritis preventing and treating function and application thereof |
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| CN113461776B true CN113461776B (en) | 2023-05-16 |
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| US20130261066A1 (en) * | 2010-08-31 | 2013-10-03 | The Research Foundation Of State University Of New York | Treatment of inflammatory bowel diseases using a tripeptide |
| KR20150014483A (en) * | 2012-05-11 | 2015-02-06 | 주식회사 카엘젬백스 | Anti-inflammatory Peptides and Composition comprising the same |
| CN111249299B (en) * | 2020-03-31 | 2022-03-22 | 南京财经大学 | Application of soybean RNA extract in preparing medicine for preventing and treating enteritis |
| CN113073126A (en) * | 2021-04-01 | 2021-07-06 | 昆明理工大学 | Application of linseed active polypeptide in preparation of products for preventing, intervening/treating colitis |
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