阅读说明：本技术 Ank三肽及其应用 (ANK tripeptide and application thereof ) 是由 涂勇刚 李媛 赵燕 徐明生 姚瑶 吴娜 于 2021-06-19 设计创作，主要内容包括：本发明公开了一种ANK三肽,其氨基酸序列为Ala-Asn-Lys。通过试验验证发现本发明的ANK三肽能够有效降低促炎因子mRNA表达水平,具有一定的医学价值,可用于开发预防和/或辅助治疗炎症性肠病的食品或药物。(The invention discloses an ANK tripeptide, the amino acid sequence of which is Ala-Asn-Lys. Experiments prove that the ANK tripeptide can effectively reduce the expression level of proinflammatory factor mRNA, has a certain medical value, and can be used for developing foods or medicines for preventing and/or assisting in treating inflammatory bowel diseases.)

An ANK tripeptide characterized in that its amino acid sequence is Ala-Asn-Lys.

2. The ANK tripeptide according to claim 1, characterized in that it has a molecular weight of 331 Da.

3. The ANK tripeptide according to claim 1, characterized in that it is obtained by solid phase synthesis.

4. Use of the ANK tripeptide of any one of claims 1 to 3 in the manufacture of a food or a medicament for preventing and/or alleviating inflammatory bowel disease.

5. A pharmaceutical composition characterized by comprising the ANK tripeptide according to any one of claims 1 to 3 as an active ingredient for use in the prevention and/or alleviation of inflammatory bowel disease.

6. The pharmaceutical composition of claim 5, further comprising a solvent or a pharmaceutically acceptable adjuvant.

Technical Field

The invention belongs to the technical field of biology, particularly relates to the field of bioactive peptides, and particularly relates to an ANK tripeptide and application thereof.

Background

Crohn's Disease (CD) and Ulcerative Colitis (UC) are the two leading types of inflammatory bowel disease, IBD, one of the severe diseases worldwide except cardiovascular disease, with an increasing incidence and prevalence profile over time. To date, the pathogenesis of IBD is not fully understood, and there are numerous factors that influence IBD, such as: environmental factors, immune response factors, genetic factors, microbial factors, etc. (Arora et al, 2015). While there have been many advances in the treatment of IBD, it remains an incurable disease. Clinically, the main treatment method of UC is aminosalicylate, patients with severe UC can use short-term steroid therapy, and if the above treatment methods cannot relieve intestinal inflammation of UC, the patients are administered with immunomodulators and anti-tumor necrosis factor alpha (anti-TNF alpha) inhibitors to relieve intestinal inflammation. Aminosalicylate is almost ineffective in CD, and can be used to induce remission in patients with distal ileum, ileocecum or right colon disease using enteric budesonide, and prednisolone can be used to reduce intestinal inflammation in severe CD patients. If the above treatment regimen still fails to alleviate intestinal inflammation in CD patients, an anti-TNF α inhibitor is administered thereto, but about 30% of patients are ineffectual in using anti-TNF α. Of the patients who are effective against inhibitors of tumor necrosis factor alpha, up to 10% of the patients respond to the drug each year. In addition, current IBD drugs are associated with serious side effects, including loss of immune tolerance and drug resistance, infection complications, endocrine damage, kidney and liver damage (Nielsen, Bjerrum, Herfarth, & Rogler, 2013; Poitras, Gougeon, Binn, & Bouin, 2008). The current treatment scheme has low curative effect, and the rise of adverse reaction is a main concern of clinical treatment. Therefore, the search for new food-derived natural products with anti-inflammatory activity as alternative drugs or nutritional supplements for the treatment of IBD is an important focus of researchers.

Research shows that RPIV can inhibit TNF-alpha to induce IL-8 secretion of Caco-2 cells and inhibit expression of phosphorylated protein in NF-kappa B, MAPK signal path. CR, FL, HC, LL and MK obviously inhibit the expression of TNF-alpha induced de Caco-2 cell proinflammatory cytokine mRNA and up-regulate the expression of inflammatory factor IL-10. PAY effectively inhibits LPS-induced NO and COX-2 secretion in RAW264.7 cells. VPY significantly inhibit the secretion of IL-8 and TNF-alpha.

Disclosure of Invention

The invention aims to solve the defects of the prior art and provides an ANK tripeptide, the amino acid sequence of which is Ala-Asn-Lys, and the molecular weight of which is 331 Da. The ANK tripeptide is obtained by a solid phase synthesis method, can effectively prevent and relieve inflammatory bowel diseases, and can be applied to preparation of food or medicines for preventing and/or relieving inflammatory bowel diseases.

It is another object of the present invention to provide a pharmaceutical composition, the active ingredient of which comprises the ANK tripeptide, for preventing and/or alleviating inflammatory bowel disease. The pharmaceutical composition may also include a solvent or a pharmaceutically acceptable adjuvant.

The anti-inflammatory activity of the tripeptide is studied at concentrations of 2, 4, 8 and 16mM by using an in vitro model of inflammation induced by TNF-alpha on human intestinal epithelial cells (HT-29). The content of the typical cytokine IL-8 is determined, and the cell viability is determined by using a WST-1 kit. The invention adopts RT-PCR to combine Western-blot to verify the anti-inflammatory action mechanism. The invention adopts a DSS-induced Balb/c mouse acute colitis model to research the anti-inflammatory activity of the tripeptide in vivo.

The invention has the beneficial effects that: the invention provides an ANK tripeptide which is proved by experiments to be capable of effectively reducing the expression level of proinflammatory factor mRNA, has a certain medical value and can be used for developing foods or medicines for preventing and/or assisting in treating inflammatory bowel diseases.

Drawings

FIG. 1 is a graph showing the effect of ANK on TNF- α -induced IL-8 secretion from HT-29 cells and on cell viability;

FIG. 2 is a graph showing the effect of ANK on TNF- α induced mRNA expression levels of HT-29 cytokines;

FIG. 3 is a graph showing the effect of ANK on TNF- α induced levels of expression of key proteins in the NF- κ B pathway in HT-29 cells;

FIG. 4 is a graph showing the combined effect of ANK on mice, wherein (A) is a graph showing the effect of ANK on weight loss in mice induced by DSS, (B) is a graph showing the change in weight of mice in each group on day 14, (C) is a graph showing the effect of ANK on clinical symptoms in mice induced by DSS, and (D) and (E) are graphs showing the effect of ANK on colon length induced by DSS;

FIG. 5 is a graph showing the effect of ANK on the degree of lesion in mouse colon and a graph showing the colon tissue scores of groups of mice, wherein (A) is a graph showing the effect of ANK on the degree of lesion in mouse colon induced by DSS, and (B) is a graph showing the colon tissue scores of groups of mice;

FIG. 6 is a graph showing the effect of ANK on the expression level of MPO in colon tissue of DSS-induced mice;

FIG. 7 is a graph showing the effect of ANK on the levels of the proinflammatory cytokines TNF- α and IL-6 in colon tissue of mice induced by DSS;

FIG. 8 is a graph showing the effect of ANK on the expression levels of proinflammatory cytokine mRNA in colon tissue of mice induced by DSS.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments and the accompanying drawings to fully understand the objects, aspects and effects of the present invention.

Verification of anti-inflammatory activity of tripeptide ANK in-vitro inflammation model and discussion of action mechanism of tripeptide ANK

1. Inflammation model establishment

At 3X 10⁵cells are planted on a cell culture plate at the density of cells/mL, the culture solution is changed every 24h, the cells are cultured for 5-7 days until the cells are fused to about 85%, the culture solution is discarded, and the cells are washed for 2 times by HBSS buffer solution. Tripeptide samples at different concentrations were preincubated for 2h with fresh medium DMEM-F12 containing 5% FBS, followed by an additional incubation of the plates with TNF- α (to a final concentration of 5ng/mL) for 4 h. After incubation, cell supernatants were collected for ELISA assay, and the underlying cells were processed according to different experimental requirements.

2. Cell viability assay

The treated cells were subjected to viability assay with reference to the method described by Mengya Zhang (Zhang et al, 2018), et al. After the experimental cells are washed by HBSS for three times, adding an equivalent amount of WST-1 diluent into each hole to uniformly cover the cell culture plate, putting the cell culture plate into a cell culture box, incubating the cell culture plate for about 15 minutes in a dark place, measuring an OD (optical density) value at 450nm when the color change of the solution is obvious, and calculating the cell activity according to the following formula:

cell viability (%) - (As450/Ac450) x 100%, where As450 represents the sample-treated group and Ac 450-negative untreated group.

Determination of IL-8 content

The IL-8 content was determined with reference to the method of Schaujen et al (Schaujen, 2015). The samples were diluted five times and used for detection.

RNA-PCR assay

After the experimental cells were washed three times with ice-cold HBSS, the surface liquid was removed, and appropriate amount of transZol lysate was added, after sufficient lysis, total RNA in HT-29 was extracted according to the TransZOL Up Plus RNA Kit. RNA concentration and purity were assessed by determining the ratio at A260/A280 nm. cDNA was synthesized using Easy Script One-Step gDNA Removal and cDNA Synthesis SuperMix kit. RT-PCR was amplified using Perfect StartTM Green qPCR SuperMix kit with denaturation at 94 ℃ for 5s, annealing at 60 ℃ for 15s and extension at 72 ℃ for 10 s. The whole reaction is kept for 40 amplification cycles, expression Change of key genes is determined, GAPDH is used as an internal reference gene, POS positive model group is used as a control, and Fold Change value expression percentages of other groups are calculated, and primer sequences are shown in the table 1.

TABLE 1

5. Western blot

The cultured cells are pretreated to obtain denatured total protein. The cellular proteins were separated by 10% SDS-PAG gel and transferred to PVDF membrane activated with methanol, blocked with 5% skimmed milk powder at room temperature for 1.5 h. After washing, the membranes were immersed well in primary antibody dilution and incubated overnight at 4 ℃. After washing, the membrane was then incubated for 1h by thoroughly immersing in a secondary antibody dilution. After washing, the PVDF membrane was taken out to a ChemiDocTMTouch apparatus, and an ECL developing solution was dropped by a pipette to develop color, and photographed and recorded. And (4) performing gray level analysis by using Image J software, and calculating the relative expression quantity of each histone.

Secondly, in vivo anti-inflammatory activity verification is carried out on tripeptide ANK by adopting an in vivo model, and the action mechanism is discussed

1. Mouse acute colitis model

A test was carried out after housing and raising for one week 80 healthy female Balb/c mice, 6-8 weeks old and 18-20g average body weight, supplied by the Hunan Seklineda company. The captive breeding temperature is 25-27 ℃, the humidity is 74%, the illumination and the darkness alternate in 12h period, and food and water are freely obtained. The mice were randomly divided into 10 groups, as described in table 2 below, with 10 mice per group. TABLE 2

2. Evaluation of clinical symptoms of colitis in mice

Mice were observed and evaluated daily for weight change, stool consistency and stool bleeding, according to disease activity index scoring criteria set forth in Cooper (Cooper, Murthy, Shah, & Sedergran,1993) in table 3.

TABLE 3

DAI ═ (body mass index score + stool trait score + bleeding)/3.

3. Colon tissue HE score

The distal colon was washed with saline and fixed with 4% paraformaldehyde, paraffin embedded and sectioned, stained with hematoxylin and eosin staining solution, photographed under an optical microscope and evaluated for histological scoring.

4. TNF-alpha, IL-6 assay in colonic tissue

The colon tissue is washed by normal saline, total protein of the colon tissue is extracted according to the instruction of an animal tissue whole protein extraction kit, the concentration of the supernatant protein is determined by a BCA protein quantitative kit, the concentration of TNF-alpha and IL-6 proinflammatory factors is determined according to a mouse ELISA kit, and the relative percentage is calculated by taking POS as a control.

5. Antioxidant enzyme MPO assay

The colon tissue was washed with normal saline and homogenized, the enzyme activity was measured according to MPO kit instructions, and the relative percentage was calculated using POS as a control.

6. Gene expression assay in colonic tissue

The colon tissue is washed by normal saline, ground by liquid nitrogen, added with 1mL Trizol lysate for lysis for 5min, centrifuged at 12000rpm and 4 ℃ for 10min, and the total RNA of the tissue is extracted according to the kit. cDNA was synthesized using Easy Script One-Step gDNA Removal and cDNA Synthesis SuperMix kit. RT-PCR was amplified using Perfect StartTM Green qPCR SuperMix kit with denaturation at 94 ℃ for 5s, annealing at 60 ℃ for 15s and extension at 72 ℃ for 10 s. The entire reaction was maintained for 40 amplification cycles. The expression Change of the key gene was determined, GAPDH was used as an internal reference gene, and the POS positive model group was used as a control to calculate the percent expression of the other groups' Fold Change values, with the primer sequences shown in table 4.

TABLE 4

Third, in vitro test results

1. Verification of the anti-inflammatory Activity of tripeptide ANK

IL-8 is a neutrophil attractant, which induces migration of neutrophils from peripheral blood to inflamed tissues, and significantly increases IL-8 in tissues of IBD patients compared to healthy people. Therefore, IL-8 was selected as an inflammatory marker and tripeptides with potential anti-inflammatory activity were initially screened (Mahida, Ceska, Effenberger, Kurlark, Lindley, & Hawkey, 1992). As shown in FIG. 1, the anti-inflammatory activity of tripeptide ANK at concentrations of 2, 4, 8, 16mM was investigated by measuring the inflammatory marker IL-8 using a model of TNF- α induced inflammation of HT-29 cells. The cell viability is measured by combining the WST-1 method, and no significant difference is found in the cell viability between the model group and the blank control group. Tripeptide-pretreated HT-29 inflammatory cells effectively inhibit IL-8 secretion (p <0.05) and are non-cytotoxic. When the ANK concentration was as high as 16mM, the IL-8 secretion was decreased by 99%, respectively, as compared with the model group.

2. Effect of tripeptide ANK on TNF-alpha induced expression levels of HT-29 cytokine mRNA

Based on previous studies, ANK was found to have anti-inflammatory effects. To further investigate the anti-inflammatory mechanism, RT-PCR was used to analyze mRNA expression in HT-29 cells treated with the tripeptide ANK (16 mM). As shown in FIG. 2, ANK was effective in inhibiting the expression of the proinflammatory factors TNF- α, IL-6, IL-8, IL-1 β, IL-12 and cyclooxygenase (COX-2) mRNA.

3. Effect of tripeptide ANK on NF- κ B Signal pathway

NF-KB is a key transcription factor that regulates the expression of genes encoding pro-inflammatory mediators. Western-blot was used to detect the levels of p-p65 and p-I κ B α in the cells of each test group. The results show (as shown in FIG. 3) that ANK exerts its anti-inflammatory effects by blocking TNF- α induction of phosphorylation of I κ B α and phosphorylation of p65 in HT-29 cells.

Results of in vivo experiments

1. Effect of tripeptide ANK on DSS-induced clinical symptoms and weight loss in mice acute colitis

As shown in fig. 4(a), there was no significant difference in initial body weight for each treatment group, and there was no significant decrease in body weight for each group of mice before the DSS-induced inflammation model, indicating that the tripeptide ANK neither affected body weight nor caused diarrhea. The mice in each group except the blank group underwent a significant weight loss on day 10 and continued to decline until day 14. As shown in fig. 4(B), the body weight of the mice in the model group ingested with only 4.5% DSS decreased by 15.56%, and the administration of ANK at concentrations of 50mg/mL and 150mg/mL effectively slowed the body weight loss of the mice with DSS-induced acute colitis compared to the model group.

Numerous scholars have shown that the DSS-induced acute colitis model in mice is associated with symptoms such as weight loss, sticky stool, rectal bleeding, and listlessness (ZHao, Yao, Xu, Wang, Wang, & Tu, 2017). We evaluated the status of mice according to the DAI scoring system established by predecessors. As shown in fig. 4(C), the DAI values of each group, except the blank group, increased with the progression of the disease. Compared with the model group, after ANK gavage, the DAI value of each group of mice is smaller than that of the model group, and the clinical symptoms are relatively light.

In the DSS-induced mouse model of colitis, shortening of the mouse colon is closely related to the severity of colitis. As shown in FIG. 4(D), in the present study, the colon length was (4.56. + -. 0.22) cm in the model group, while that in the blank group was (7.58. + -. 1.04) cm. ANK (50mg/mL and 150mg/mL) intervention was effective in improving the degree of colon shortening (P < 0.05).

2. Effect of tripeptide ANK on Colon tissue lesions in mice with acute colitis

In order to continuously explore the influence of the tripeptide ANK on the DSS-induced acute colitis tissue damage of mice, the colon tissue is subjected to HE section staining analysis. Similar to other scholars, the pathological histological evaluation of mouse colon tissue induced by DSS only shows that the colon is severely abnormal in colon tissue structure and the crypt structure of the mucosa is almost completely disappeared; massive and fibrous tissue proliferation, as indicated by the black arrows in the POS of FIG. 5 (A); partial mucosal epithelial cells were absent and the lamina propria was uncovered, as indicated by the red arrow; submucosa edema and inflammatory cell infiltration were seen as indicated by yellow arrows. When colon tissues of each group were scored according to the histopathological scoring method described in the previous person, the colon histological score of the mice subjected to ANK stem prognosis was significantly decreased compared to that of the model group as shown in FIG. 5 (B). Proves that ANK plays a beneficial role in preventing and intervening acute colitis of mice.

3. Effect of tripeptide ANK on DSS-induced MPO Activity in mouse Colon tissue

MPO content is reported to be closely related to the development of inflammation, and neutrophil aggregation can lead to the release of large amounts of MPO (Joh & Kim, 2011). The influence of the tripeptide ANK on the accumulation of neutrophils in colon tissues of DSS-induced mouse acute colitis was investigated by measuring the MPO enzyme activity. As shown in FIG. 6, the MPO content of the model group was significantly increased after the induction of DSS, compared to the blank control group. Compared with the model group, ANK-50 is reduced by 58%, ANK-150 is reduced by 78%, so that ANK can effectively inhibit the secretion of MPO in colon tissues of mice with acute colitis induced by DSS.

4. Effect of tripeptide ANK on content of proinflammatory cytokines TNF-alpha and IL-6 in colon tissue of mouse

The anti-inflammatory activity of the tripeptide ANK in mice was investigated by measuring the TNF-alpha and IL-6 content in the colon of mice. Results other groups of proinflammatory factor secretion were observed using the model group as a control. As shown in FIG. 7, TNF-. alpha.and IL-6 expression was significantly elevated in colon tissue of mice induced with DSS alone (p <0.05) compared to the blank group. Compared with the model group, the tripeptide ANK can effectively inhibit the secretion of TNF-alpha (p <0.05) and IL-6(p <0.05), wherein the ANK-50 is reduced by 56.88% and 48.23% respectively, and the ANK-150 is reduced by 72.63% and 66.26% respectively.

5. Effect of tripeptide ANK on proinflammatory cytokine mRNA expression

To further investigate the anti-inflammatory activity of ANK in vivo, inflammatory cytokine mRNA in colon tissue was assayed. The results were compared with the model group to observe the expression of proinflammatory factor mRNA in other groups. As shown in FIG. 8, compared to the model group, the tripeptide ANK is effective in dose-dependently inhibiting the expression levels of the proinflammatory factors TNF-alpha (p <0.05), IL-1 beta (p <0.05), IL-6(p <0.05), IL-17(p <0.05), MCP-1(p <0.05), IFN (p <0.05) mRNA.

In conclusion, the tripeptide ANK has good anti-inflammatory activity both in vivo and in vitro. Therefore, the tripeptide ANK can be used for preventing or slowing down the development of health care products or medicines for inflammatory bowel diseases.

The above description is only a preferred embodiment of the present invention, and the present invention is not limited to the above embodiment, and the present invention shall fall within the protection scope of the present invention as long as the technical effects of the present invention are achieved by the same means. The invention is capable of other modifications and variations in its technical solution and/or its implementation, within the scope of protection of the invention.