An FGF20 modified protein and its application in improving ulcerative colitis

Abstract

The present invention discloses an FGF20 modified protein and its application in improving ulcerative colitis, belonging to the technical field of biomedicine. The amino acid sequence of the FGF20 modified protein is shown as SEQ ID NO: 2. The present invention constructs a non-mitogenic FGF20 modified protein with significantly weakened heparin affinity. It is experimentally confirmed that compared with ordinary FGF20, the FGF20 modified protein can better relieve DSS-induced ulcerative colitis, reduce the expression of pro-inflammatory factors, and significantly improve the intestinal barrier function damage induced by DSS, thus exerting a more excellent prevention and treatment effect on ulcerative colitis. It can be seen that the present invention provides new drugs and treatment plans for the clinical treatment of ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a FGF20 modified protein and its application in improving ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a chronic non-specific inflammatory bowel disease (IBD) that occurs in the colon and rectum, with unknown etiology and an increasing incidence year by year. It is estimated that the number of IBD patients in China will exceed 1.5 million by 2025. Most UC patients mainly rely on glucocorticoids for maintenance treatment. Treatment is carried out with 5-aminosalicylic acid, thiopurines, biological agents of tumor necrosis factor and integrin, as well as small molecule inhibitors (such as Janus kinase inhibitors) according to the severity of symptoms. However, these drugs often cause serious adverse reactions. When the disease is severe, up to 15% of patients need surgical treatment due to ineffective drug treatment or secondary long-term colitis leading to malnutrition.

[0003] Fibroblast growth factor 20 (FGF20) is a member of the FGF superfamily and has functions of promoting the development of dopaminergic neurons, hair growth and tissue repair. Some studies have shown that it can effectively improve the symptoms of UC induced by DSS. However, wild-type FGF20 has strong downstream signal activation activity, which will lead to strong cell proliferation promoting activity, thus affecting the treatment effect on UC. Summary of the Invention

[0004] The purpose of the present invention is to provide a FGF20 modified protein and its application in improving ulcerative colitis, so as to solve the problems existing in the above-mentioned prior art. Compared with ordinary FGF20, the FGF20 modified protein can better relieve DSS-induced ulcerative colitis, reduce the expression of pro-inflammatory factors, and significantly improve the intestinal barrier function damage induced by DSS, exerting a more excellent prevention and treatment effect on ulcerative colitis. The present invention provides new drugs and treatment plans for the clinical treatment of ulcerative colitis.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] The present invention provides a FGF20 modified protein, and the amino acid sequence of the FGF20 modified protein is as shown in SEQ ID NO: 2.

[0007] Further, the amino acid sequence as shown in SEQ ID NO: 2 has mutations at positions 170, 171, 180, 185 and 186, with alanine, cysteine, glutamate, histidine, glycine, tyrosine, proline, serine, methionine, valine, isoleucine, phenylalanine, leucine, tryptophan or threonine.

[0008] The present invention also provides an application of the FGF20 modified protein in the preparation of a drug for improving ulcerative colitis.

[0009] Further, the FGF20 modified protein improves ulcerative colitis by reducing the inflammation level and alleviating the damage of intestinal barrier function.

[0010] Further, the reduction of the inflammation level includes reducing the expression level of pro-inflammatory factors.

[0011] Further, the pro-inflammatory factors include IL-6, TNF-α and IL-1β.

[0012] Further, the alleviation of the damage of intestinal barrier function includes increasing the expression levels of tight junction proteins Occludin, ZO-1 and mucin MUC2.

[0013] The present invention also provides a drug for preventing and / or treating ulcerative colitis, which contains an effective dose of the FGF20 modified protein.

[0014] Further, the dosage form of the drug is tablet, capsule, granule, injection or spray.

[0015] Further, the administration method of the drug is oral administration or non-gastrointestinal administration.

[0016] The present invention discloses the following technical effects:

[0017] In the present invention, the structure of FGF20 is compared with that of the FGF20-FGFR1-Heparin ternary complex, and a structural simulation diagram of the FGF20-FGFR1-Heparin ternary complex is constructed. Through the analysis of this simulated structure, it is known that the important heparin binding sites of FGF20 are N170, K171, R180, Q185 and K186. After mutating these binding sites, a non-mitogenic FGF20 modified protein with significantly weakened heparin affinity is constructed. It is experimentally confirmed that compared with ordinary FGF20, the FGF20 modified protein can better relieve DSS-induced ulcerative colitis, reduce the expression of pro-inflammatory factors, and significantly improve the damage of intestinal barrier function induced by DSS, thus exerting a more excellent prevention and treatment effect on ulcerative colitis. It can be seen that the present invention provides a new drug and treatment plan for the clinical treatment of ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0019] Figure 1 It is a structural simulation diagram of the FGF20-FGFR1-Heparin ternary complex;

[0020] Figure 2 The FGF20 modified construct alleviates DSS-induced ulcerative colitis and reduces the inflammatory level; A: Comparison of colon lengths; B: Body weight changes; C: Diagram for comparing colon lengths; D: DAI score; E: H&E staining diagram; F: Statistical chart of IL-1β expression level; G: Statistical chart of TNF-α expression level; H: Statistical chart of IL-6 expression level;

[0021] Figure 3 The FGF20 modified construct alleviates DSS-induced intestinal barrier function damage; A: Statistical chart of intestinal barrier permeability; B: Expression results of tight junction proteins Occludin, ZO-1, and mucin MUC2; C: Statistical chart of MUC2 expression level; D: Statistical chart of Occludin expression level; E: Statistical chart of ZO-1 expression level;

[0022] Figure 4 The FGF20 modified construct alleviates DSS-induced intestinal barrier function damage; A: H&E staining diagram; B: Statistical chart of Occludin expression level; C: Statistical chart of ZO-1 expression level; D: Statistical chart of MUC2 expression level. Detailed implementation manners

[0023] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0026] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the description of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of the present invention are obvious to those skilled in the art. The description and examples of the present invention are merely exemplary.

[0027] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, that is, they are meant to include but not be limited to.

[0028] Example 1 Construction of FGF20 modified body

[0029] Previous studies of the present invention found that FGF20 can improve inflammatory bowel disease, that is, wild-type FGF20 activates a strong downstream signal and results in pro-cell proliferation activity. Based on the signal transduction characteristics of FGF20, it is known that FGF20 forms a dimer of the FGF20-FGFR1-Heparin ternary complex with FGFR1 under the assistance of heparin molecules. In this study, the structure of FGF20 was compared with that of the FGF20-FGFR1-Heparin ternary complex, and a structural simulation diagram of the FGF20-FGFR1-Heparin ternary complex was constructed (see Figure 1 ); from this simulated structure, it can be seen that the important heparin-binding sites of FGF20 are N170, K171, R180, Q185, and K186; finally, by mutating the above heparin-binding sites into alanine (A), cysteine (C), glutamic acid (E), histidine (H), glycine (G), tyrosine (Y), proline (P), serine (S), methionine (M), valine (V), isoleucine (I), phenylalanine (F), leucine (L), tryptophan (W), or threonine (T), or by freely combining the mutated amino acid types of the above heparin-binding sites, a non-mitogenic FGF20 modified body with significantly weakened heparin affinity was constructed.

[0030] The amino acid sequence of human FGF20 protein is as follows (SEQ ID NO: 1):

[0031] MAPLAEVGGFLGGLEGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARGGPGAAQLAHLHGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLGMNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGARS KRHQKFTHFLPRPVDPERVPELYKDLLMYT;

[0032] Note: The shaded positions are the important heparin-binding sites of FGF20.

[0033] The amino acid sequence of the FGF20 variant is as follows (SEQ ID NO: 2):

[0034] MAPLAEVGGFLGGLEGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARGGPGAAQLAHLHGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLGMNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALXXDGTPRDGAXSKRHXXFTHFLPRPVDPERVPELYKDLLMYT.

[0035] Note: The shaded positions are the mutation sites of the FGF20 variant, and X represents a mutation to A / C / E / H / G / Y / P / S / M / V / I / F / L / W / T.

[0036] Example 2 Therapeutic effect of FGF20 variant on ulcerative colitis

[0037] Taking the FGF20 variant with all mutation sites in Example 1 mutated to alanine (A) as an example, the following studies were carried out:

[0038] Experimental materials and methods

[0039] (1) Materials and reagents

[0040] The DSS was purchased from MP Biomedicals, USA; the GAPDH antibody was purchased from Chengdu Zhengneng Biotechnology, the ZO-1 antibody was purchased from Affinity Biosciences, the Occludin was purchased from proteintech, the MUC2 antibody was purchased from abcam; the rabbit secondary antibody was purchased from proteintech; the H&E and immunohistochemistry kits were both purchased from Thermo Fisher Scientific; the mouse ELISA kit was purchased from Jianglai Biotechnology, and the FITC-Glucose was purchased from sigma-Aldrich.

[0041] (2) Construction and experimental grouping of the ulcerative colitis mouse model induced by DSS

[0042] Twenty female SPF-grade C57BL / 6 mice aged 6 - 8 weeks were randomly divided into 4 groups:

[0043] ① Normal control group (NC group, n = 5);

[0044] ② UC model group (DSS group, n = 5);

[0045] ③ UC model + RE-FGF20 modified body treatment group (DSS + RE-FGF20 group, n = 5);

[0046] ④ UC model + unmodified FGF20 control group (DSS + FGF20 group, n = 5);

[0047] The C57BL / 6 background mice were purchased from the Experimental Animal Center of Wenzhou Medical University, aged 6 - 8 weeks; license number: SCXK(Zhe)2019 - 0001. They were raised, modeled and observed in the Experimental Animal Center of Wenzhou Medical University with free diet. After one week of adaptive feeding, the NC group was fed with pure water for 7 days; the DSS group was fed with a 3.5% DSS solution prepared with pure water for 7 days; the DSS + RE-FGF20 group was intraperitoneally injected with 3 mg / kg of the RE-FGF20 modified body protein every day on the basis of feeding with a 3.5% DSS solution; the DSS + FGF20 group was intraperitoneally injected with 3 mg / kg of the unmodified FGF20 protein every day on the basis of feeding with a 3.5% DSS solution.

[0048] (3) Evaluation of the ulcerative colitis model

[0049] Observe the fecal morphology and the presence of occult blood in the feces of the mice. The evaluation was carried out according to the colitis disease activity index (DAI), and the specific scoring rules are shown in Table 1.

[0050] Table 1

[0051]

[0052] Note: Normal stools are formed stools; loose stools are semi-formed, pasty stools, and stools that do not adhere to the anus;

[0053] Watery stools are watery stools that adhere to the anus. The sum of each score is the DAI score.

[0054] (4) Histopathology

[0055] Colon tissues were fixed with paraformaldehyde, embedded in paraffin, sectioned, and then stained with H&E and subjected to immunohistochemistry experiments.

[0056] (5) Detection of colon permeability

[0057] The mice to be tested were fasted overnight, and a FITC-Glucose solution was prepared with physiological saline and gavaged at a dose of 40 mg FITC-Glucose (sigma-Aldrich 3-5 kDa) / 100 g body weight; 4 hours later, the serum of the mice was collected, loaded onto a 96-well plate, a blank well was set with the serum of mice gavaged with an equal amount of physiological saline, and a standard curve was set at the same time. Subsequently, the fluorescence intensity was detected with a fluorescence microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0058] (6) Serum ELISA detection

[0059] Blood was collected from the mice in each group by orbital enucleation. After centrifugation at 3000 r / min at 4°C for 15 min, the supernatant was taken, and the contents of IL-6, TNF-α, and IL-1β in the serum were detected according to the ELISA detection kit (Shanghai Jianglai Biotechnology).

[0060] (7) Western blot

[0061] The total protein samples extracted from the mouse colon tissues were denatured and separated by SDS-PAGE, then the protein gel was transferred to a PVDF membrane, blocked for 1.5 hours, incubated with the corresponding primary antibody overnight at 4°C, washed, and incubated with the secondary antibody for 1 hour. The results were analyzed by gray scale analysis using ImageJ software.

[0062] (8) Statistical analysis

[0063] Experimental data were expressed as mean ± standard error of the mean (SEM). One-way ANOVA and t-tests were used to compare sample differences. Each experiment was repeated three times biologically. All data were statistically processed and graphed using GraphPad Prism 9.0 software. *p<0.05, **p<0.01, ***p<0.001 indicate statistically significant differences, highly significant statistical differences, and extremely highly significant statistical differences, respectively. Statistical analysis

[0064] 2 Result analysis

[0065] (1) The FGF20 modified construct can better alleviate DSS-induced ulcerative colitis and reduce the inflammation level compared with ordinary FGF20.

[0066] To detect the effect of the FGF20 modified construct in DSS-induced ulcerative colitis compared with ordinary FGF20, the morphological and histological changes of the mouse colon were evaluated. It was found that the therapeutic effect of the modified construct was better than that of ordinary FGF20, and it could better relieve the bloody stools, weight loss, decrease in colon length, and increase in DAI score caused by DSS in mice ( Figure 2 A-D in the figure). HE results showed that compared with the DSS+FGF20 group, the histopathological damage in the DSS+RE-FGF20 group was significantly reduced, the intestinal crypts were intact, the goblet cells were arranged neatly, the infiltration of inflammatory cells was less, and the intestinal mucosa was intact ( Figure 2 E in the figure). Subsequently, the inflammation levels of mice in each group were detected by ELISA. The results showed that compared with the DSS+FGF20 group, the treatment with the FGF20 modified construct reduced the contents of IL-6, TNF-α, and IL-1β in the serum of mice ( Figure 2 F-H in the figure). The above results indicate that the FGF20 modified construct can better alleviate DSS-induced ulcerative colitis and reduce the expression of pro-inflammatory factors compared with ordinary FGF20.

[0067] (2) The FGF20 modified construct can better relieve the intestinal barrier function damage induced by DSS compared with ordinary FGF20.

[0068] Colitis induced by DSS is often accompanied by damage to the intestinal barrier function. The intestinal permeability of each group was detected by FITC-Glucose. It was found that compared with the DSS+FGF20 group, the intestinal barrier permeability in the DSS+RE-FGF20 group decreased ( Figure 3 A in the figure). And Western Blot results showed that the expressions of tight junction proteins Occludin, ZO-1, and mucin MUC2 in the DSS+RE-FGF20 group increased significantly ( Figure 3 B-E in the figure), and immunohistochemistry further verified this result ( Figure 4 A-D in the figure).

[0069] The embodiments described above are only used to describe the preferred mode of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. An FGF20 modified protein, characterized in that, The amino acid sequence of the FGF20 modified protein is shown in SEQ ID NO: 2; The amino acid sequence shown in SEQ ID NO: 2 has alanine mutations at positions 170, 171, 180, 185 and 186.

2. Use of the FGF20 modified protein according to claim 1 in the preparation of a drug for improving ulcerative colitis.

3. The application according to claim 2, wherein The FGF20 modified protein improves ulcerative colitis by reducing the level of inflammation and alleviating the damage of intestinal barrier function.

4. The application according to claim 3, characterized in that The reduction of the level of inflammation includes reducing the expression level of pro-inflammatory factors.

5. The application according to claim 4, wherein The pro-inflammatory factors include IL-6, TNF-α and IL-1β.

6. The application according to claim 3, characterized in that The alleviation of the damage of intestinal barrier function includes increasing the expression levels of tight junction proteins Occludin, ZO-1 and mucin MUC2.

7. A drug for preventing and / or treating ulcerative colitis, characterized in that, The drug contains an effective dose of the FGF20 modified protein according to claim 1.

8. The drug according to claim 7, characterized in that, The dosage form of the drug is tablets, capsules, granules, injections or sprays.

9. The drug according to claim 8, characterized in that, The administration method of the drug is oral administration or non-gastrointestinal administration.

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