The QGK tripeptide, characterized in that its amino acid sequence is Gln-Gly-Lys.

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

3. The QGK tripeptide according to claim 1, wherein the QGK tripeptide is obtained by a solid phase synthesis method.

4. Use of the QGK tripeptide according to any one of claims 1 to 3 for the preparation of a food or a medicament for the prevention and/or alleviation of inflammatory bowel disease.

5. A pharmaceutical composition comprising the QGK tripeptide according to any one of claims 1 to 3 as an active ingredient for preventing and/or alleviating inflammatory bowel disease.

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

Background

Ulcerative Colitis (UC) is a chronic, idiopathic inflammatory disease of the colonic mucosa, starting in the rectum and usually spreading in a continuous manner towards the proximal rectum and even the colon, with clinical symptoms typified by bloody diarrhea. UC is more prevalent than crohn's disease. According to statistics, the prevalence rate of the colitis patients in North America and northern Europe is the highest, 9-20 patients with ulcerative colitis exist in 10 ten thousand people, and the prevalence rate of the colitis patients in the southern hemisphere and eastern region is relatively low. In recent years, with westernization of eating habits and increase of living pressure of people, the UC prevalence rate is remarkably increased and gradually increased year by year, which brings heavy burden to public health service. The incidence of ulcerative colitis is bimodal, with the major peak between the ages of 15-30 and the second, smaller peak between the ages of 50-70.

The intestinal epithelium is the first physical barrier against foreign pathogens. Normally, the epithelial layer is covered by a mucosal layer, which is a physical barrier between the host immune cells and the microbes in the lumen, and in combination with antimicrobial peptides (defensins) helps the host to defend against invasion by pathogenic microbes. In UC patients, the synthesis of certain colonic mucin subtype (mucin-2) sulfides is reduced, the epithelial barrier is damaged, the permeability is increased, and the mucosal reaction is abnormal. Continued epithelial damage and a decrease in defensins during inflammation leads to exposure of the intestinal flora and amplification of inflammation (Frank, Amand, Feldman, Boedeker, Harpaz, & Pace, 2007; Simms, Doecke, Walsh, Huang, Fowler, & Radford-Smith, 2008). The lamina propria of the intestine is composed of dendritic cells and macrophages, which present antigens to B and T cells, which in turn activate and acquire a new immune response. In patients with UC, the number of activated and mature dendritic cells increases with increasing external stimuli, and their number is closely related to the development of the disease.

In the invention, the applicant discloses an amino acid sequence of an anti-inflammatory tripeptide, and detects the anti-inflammatory effect of the anti-inflammatory tripeptide in an in vitro inflammation model and an in vivo inflammation model, and the result shows that the tripeptide can inhibit the expression of proinflammatory factors in vivo and in vitro and has good anti-inflammatory effect.

Disclosure of Invention

The invention aims to solve the defects of the prior art and provides a QGK tripeptide, the amino acid sequence of which is Gln-Gly-Lys and the molecular weight of which is 331 Da. The QGK 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 QGK 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 QGK tripeptide is proved by tests 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 QGK on TNF- α -induced IL-8 secretion from HT-29 cells and on cell viability;

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

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

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

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

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

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

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

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 QGK in-vitro inflammation model and discussion of action mechanism of tripeptide QGK

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, 2017; M.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 Ac450 represents the 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 gDNAremoval 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 QGK 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-StepgDNA 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 anti-inflammatory activity of tripeptide QGK

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 QGK 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 QGK concentration was as high as 16mM, the IL-8 secretion amount was decreased by 99%, respectively, as compared with the model group.

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

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

3. Effect of tripeptide QGK on NF-kB 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 QGK exerts its anti-inflammatory effects by blocking TNF- α -induced phosphorylation of I κ B α and phosphorylation of p65 in HT-29 cells.

Results of in vivo experiments

1. Effect of tripeptide QGK on DSS-induced clinical symptoms and weight loss of 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 QGK 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 weight loss was most significant in the model group mice compared to the blank group, and the administration of QGK at concentrations of 50mg/mL and 150mg/mL was effective in reducing weight loss in DSS-induced acute colitis mice.

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 QGK intragastric administration, the DAI value of each group of mice is smaller than that of the model group, and the clinical symptoms are relatively mild.

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. QGK (50mg/mL and 150mg/mL) intervention was effective in improving the degree of colon shortening (P < 0.05).

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

In order to continuously explore the influence of the tripeptide QGK 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 QGK stem prognosis was significantly decreased compared to that of the model group, as shown in FIG. 5 (B). The beneficial effect of QGK on the prevention and intervention of the acute colitis of the mice is proved.

3. Effect of tripeptide QGK 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 tripeptide QGK on the accumulation of neutrophils in colon tissues of DSS-induced mouse acute colitis is researched through the determination of 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, the QGK-50 is reduced by 64 percent, and the QGK-150 is reduced by 44 percent, so that the QGK can effectively inhibit the secretion of MPO in colon tissues of mice with acute colitis induced by DSS.

4. Influence of tripeptide QGK on content of proinflammatory cytokines TNF-alpha and IL-6 of colon tissue of mouse

The contents of TNF-alpha and IL-6 in the colon of the mice are measured, and the anti-inflammatory activity of tripeptide QGK in the mice is researched. 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 QGK can effectively inhibit the secretion of TNF-alpha (p <0.05) and IL-6(p <0.05), wherein the QGK-50 is reduced by 57.15 percent and 58.64 percent respectively, and the QGK-150 is reduced by 45.61 percent and 35.1 percent respectively.

5. Effect of tripeptide QGK on proinflammatory cytokine mRNA expression

To further investigate the anti-inflammatory activity of QGK 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, the tripeptide QGK 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 compared with the model group.

In conclusion, the tripeptide QGK has good anti-inflammatory activity both in vivo and in vitro. Therefore, the tripeptide QGK 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.