Application of IGFBP-6 in preparation of medicine for treating ulcerative colitis

Abstract

The invention discloses application of IGFBP-6 in preparation of a medicine for treating ulcerative colitis, and relates to the technical field of biological medicine. The invention specifically relates to a medicine for treating ulcerative colitis by taking IGFBP-6 as an effective component, and the IGFBP-6 comprises IGFBP-6 recombinant protein, a purified form of the IGFBP-6 recombinant protein and a derivative obtained by chemically modifying the IGFBP-6 recombinant protein. Based on the fact that the serum expression level of IGFBP-6 in the ulcerative colitis period is increased and generation and development of the ulcerative colitis are hindered, the treatment effect of the IGFBP-6 recombinant protein on the ulcerative colitis is further researched and discussed, and it is found that the IGFBP-6 recombinant protein can remarkably inhibit expression and release of inflammatory factors mediated by the ulcerative colitis; the pyroptosis of colon macrophages is effectively relieved, and the intestinal injury of ulcerative colitis is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of IGFBP-6 in the preparation of a drug for treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a type of inflammatory bowel disease (IBD), characterized by a chronic inflammatory disease affecting the colon and rectum. Epidemiological data show that in the early years, ulcerative colitis was mainly prevalent in Western countries. In recent years, the incidence in Asian countries has gradually increased, and China has become one of the countries with the highest incidence in Asia (3.44 / 10,000). Due to the lack of effective cures, some UC patients are prone to recurrent attacks, resulting in a serious decline in their quality of life. Nearly 30%-60% of patients eventually require surgical removal of diseased intestinal tissue. In addition, UC patients are also at a significantly higher risk of colorectal cancer than normal people.

[0003] The etiology of UC has not been fully elucidated, and its occurrence and development involve multiple factors and processes. Studies have shown that immune response disorders are a key step in its occurrence and development. Currently, the clinical recommendation is for patients with acute complications (perforation, massive bleeding, etc.) and those who are ineffective with drug treatment to undergo surgical procedures, while the rest are mainly treated with conservative medical treatment. Although medical immunotherapy based on aminosalicylic acids and glucocorticoids is relatively mature, it still has many defects and shortcomings. For example, aminosalicylic acids have limited efficacy in severe patients, and glucocorticoids cannot be used for maintenance treatment due to hormone resistance or dependence. For patients who are insensitive to traditional treatment, new biological agents (anti-TNF-α monoclonal antibodies, IL-12 / 23 inhibitors, etc.) can quickly relieve inflammation and alleviate symptoms, but long-term use may affect the body's normal immune function, increase the risk of infection, and induce autoimmune diseases.

[0004] Insulin-like growth factor binding protein-6 (IGFBP-6) is a 23kD small bioactive polypeptide that is widely expressed in the body. IGFBP-6 is a member of the insulin-like growth factor binding protein family (IGFBPs), a class of secreted proteins that exert diverse functions in different tissues. They act on target cells through autocrine or paracrine pathways, regulating cell proliferation, apoptosis, and migration. Studies have shown that IGFBP-6 can induce chemotaxis of various immune cells and play a key role in communication between immune cells.

[0005] Recent studies have found that the expression level of IGFBP-6 is increased in the serum of clinical patients with ulcerative colitis. Further animal experiments and cell experiments have revealed that in an experimental UC mouse model, IGFBP-6 reduces intestinal tissue damage by inhibiting pyroptosis of colonic macrophages, suggesting that it is a new target for UC immunotherapy. Summary of the Invention

[0006] The purpose of the present invention is to solve the shortcomings of the prior art and to propose the use of IGFBP-6 in the preparation of a drug for treating ulcerative colitis.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The use of IGFBP-6 in the preparation of a drug for treating ulcerative colitis includes:

[0009] A drug for treating ulcerative colitis with IGFBP-6 as an active ingredient, wherein the types of IGFBP-6 include IGFBP-6 recombinant protein, purified form of IGFBP-6 recombinant protein, and derivatives obtained by chemical modification of IGFBP-6 recombinant protein;

[0010] The ulcerative colitis therapeutic drug has at least one of the following three effects: inhibiting the expression and release of inflammatory factors mediated by ulcerative colitis, reducing pyroptosis of intestinal macrophages induced by ulcerative colitis, and alleviating intestinal damage.

[0011] Preferably, the inflammatory factors that inhibit ulcerative colitis include interleukin-6 and tumor necrosis factor-α.

[0012] Furthermore: the ulcerative colitis therapeutic drug reduces intestinal epithelial cell pyroptosis by downregulating the expression or activation level of pyroptosis-promoting factors, and the pyroptosis-promoting factors include GSDMD, Cleaved-caspase 1, and IL-1β.

[0013] As a preferred embodiment of the present invention, when the drug for treating ulcerative colitis is used, the effective dosage of the active ingredient is greater than or equal to 50 μg / kg.

[0014] On the basis of the above scheme: the drug for treating ulcerative colitis is a single-component substance or a compound preparation.

[0015] Preferably, the drug for treating ulcerative colitis further comprises a pharmaceutically acceptable carrier or excipient.

[0016] Furthermore: the material form of the ulcerative colitis therapeutic drug includes solid, liquid, gel, semi-fluid, and aerosol.

[0017] The beneficial effects of the present invention are:

[0018] Based on the fact that the serum expression level of IGFBP-6 increases during UC and hinders the occurrence and development of UC, the present invention further studies and explores the therapeutic effect of IGFBP-6 recombinant protein on UC. It is found that IGFBP-6 recombinant protein can significantly inhibit the expression and release of inflammatory factors mediated by UC, effectively reduce the pyroptosis of colonic macrophages, and significantly improve UC intestinal damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is an ELISA bar graph of the IGFBP-6 expression levels in serum and intestinal tissue of UC model mice and the control group in Example 1 of the present invention;

[0020] Figure 2 For the UC model mice in Example 2 of the present invention, IGFBP-6 (- / -) Body weight curves and disease activity index scores of gene-deficient mice and UC model mice injected intraperitoneally with recombinant IGFBP-6 protein (rIGFBP-6);

[0021] Figure 3 For the UC model mice in Example 2 of the present invention, IGFBP-6 (- / -) A bar graph comparing the appearance and length of the colon in gene-deficient mice, UC model mice injected intraperitoneally with rIGFBP-6 protein, and the control group;

[0022] Figure 4 For the UC model mice in Example 2 of the present invention, IGFBP-6 (- / -) HE staining of the colon of gene-deficient mice, UC model mice injected intraperitoneally with rIGFBP-6 protein, and the control group;

[0023] Figure 5 For the UC model mice in Example 2 of the present invention, IGFBP-6 (- / -) PAS staining images of gene-deficient mice, UC model mice injected intraperitoneally with rIGFBP-6 protein, and control group;

[0024] Figure 6 This is a bar graph of the expression levels of IL-6 and TNF-α in intestinal tissues of UC model mice, UC model mice intraperitoneally injected with rIGFBP-6, and a control group in Example 2 of the present invention;

[0025] Figure 7 The UC model mice in Example 3 of the present invention, the UC model mice intraperitoneally injected with rIGFBP-6, and IGFBP-6 (- / -) Immunofluorescence images of UC model mice;

[0026] Figure 8The control group, WT UC model mice, IGFBP-6 (- / -) Mice and IGFBP-6 (- / -) Immunoblotting analysis of the expression levels of the pyroptosis pathway in colon tissue of UC model mice, as well as the control group, UC model mice and UC model mice injected intraperitoneally with rIGFBP-6. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be further described in detail below in conjunction with specific implementation methods.

[0028] The following describes embodiments of the present invention in detail. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended only to explain the present invention and are not to be construed as limiting the present invention.

[0029] The use of IGFBP-6 in the preparation of drugs for treating ulcerative colitis includes: drugs for treating ulcerative colitis (UC) with IGFBP-6 as an active ingredient, wherein the types of IGFBP-6 include IGFBP-6 recombinant protein, purified form of IGFBP-6 recombinant protein, and derivatives obtained by chemical modification of IGFBP-6 recombinant protein.

[0030] The chemical modification will change the structure of the protein, thereby affecting the stability, biological activity and function of the protein, but will not affect the main function of the protein; the purpose of the chemical modification is to further effectively control and improve the quality and safety of the drug, such as oxidation modification, glycosylation, phosphorylation, acetylation, etc.

[0031] The ulcerative colitis (UC) therapeutic drug has at least one of the following three effects: inhibiting the expression and release of inflammatory factors mediated by ulcerative colitis, reducing pyroptosis of intestinal macrophages induced by ulcerative colitis, and alleviating intestinal damage.

[0032] The inflammatory factors mediated by ulcerative colitis (UC) that are inhibited include but are not limited to interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), etc.; the ulcerative colitis treatment drug reduces intestinal epithelial cell pyroptosis by downregulating the expression or activation level of pyroptosis-promoting factors, and the pyroptosis-promoting factors include but are not limited to GSDMD, Cleaved-caspase 1, and IL-1β.

[0033] When the ulcerative colitis therapeutic drug is used, the effective dosage of the active ingredient is greater than or equal to 50 μg / kg; the ulcerative colitis therapeutic drug is a single-ingredient substance or a compound preparation; the ulcerative colitis therapeutic drug also includes a pharmaceutically acceptable carrier or excipient; the material form of the ulcerative colitis therapeutic drug includes solid, liquid, gel, semi-fluid, and aerosol.

[0034] The drug for treating ulcerative colitis is mainly targeted at mammals, preferably rodents, artiodactyls, perissodactyls, lagomorphs, primates, etc., and preferably monkeys, apes or humans.

[0035] The administration forms of the ulcerative colitis treatment drug are relatively diverse, and can be either gastrointestinal or parenteral. It is generally recommended to administer the drug via known routes for each chemical drug, such as intravenous injection, intraperitoneal injection, intramuscular injection, and subcutaneous injection. In animal experiments, intraperitoneal injection or tail vein injection is usually used, as intraperitoneal injection is simple to operate. For the treatment of human UC, intravenous injection or other methods are usually used.

[0036] Example 1:

[0037] Expression of IGFBP-6 in UC model mice

[0038] S1: Materials and Methods

[0039] S1.1: Construction of mouse UC model

[0040] Six- to eight-week-old C57BL / 6 male mice were randomly divided into two groups. The mice in the UC group were fed with 2.5% DSS for six days and then with normal drinking water. The mice in the control group were fed with normal drinking water until the end of the experiment on the eighth day, when the mice were euthanized.

[0041] S1.2: Detection of IGFBP-6 in mouse serum and intestinal tissue

[0042] The mouse heart blood was collected and centrifuged to obtain serum. The mouse small colon tissue was obtained, longitudinally dissected, the contents washed with PBS, homogenized, centrifuged and the supernatant collected. The IGFBP-6 level was detected by ELISA according to the kit instructions and detected using a microplate reader.

[0043] S1.3: Statistical Methods

[0044] GraphPad Prism 8 (GraphPad Software, La Jolla, CA) was used for all statistical analyses and data analysis. Continuous data were expressed as mean ± standard deviation, and ordinal data were expressed as median and interquartile range (IQR). Comparisons between two or more groups were performed with unpaired t-test or analysis of variance. p < 0.05 was considered statistically significant.

[0045] S2: Results

[0046] S2.1: If Figure 1 As shown in the figure, the expression level of IGFBP-6 in the serum of UC model mice was significantly higher than that in the intestinal tissue of the control group.

[0047] S2.2: If Figure 2 As shown in the figure, the expression level of IGFBP-6 in the intestinal tissue of UC model mice was significantly lower than that in the intestinal tissue sections of the control group.

[0048] S3: Conclusion

[0049] The expression levels of IGFBP-6 in mouse serum and intestinal tissue were detected by ELISA. The results showed that the expression of IGFBP-6 in model mice was significantly changed, suggesting that IGFBP-6 may be related to the occurrence and development of UC.

[0050] Example 2

[0051] This example found that IGFBP-6 is a key protective internal factor in the development of UC. IGFBP-6-deficient mice with UC model have more severe intestinal damage, while supplementation with rIGFBP-6 can alleviate intestinal damage.

[0052] The specific implementation process is as follows:

[0053] S1: Materials and Methods

[0054] S1.1: Modeling and Intervention

[0055] The modeling method was the same as S1.1 in Example 1; UC+rIGFBP-6 group: During the UC modeling period, rIGFBP-6 1000 μg / kg was intraperitoneally injected every other day for 3 consecutive times; UC+IGFBP-6 (- / -) Group: Using self-bred IGFBP-6 (- / -) Knockout mice; the control group was fed with normal drinking water and had free access to water; the experiment ended on the 8th day after UC induction.

[0056] S1.2: UC Disease Activity Index Score

[0057] During the modeling period, body weight, diarrhea, and rectal bleeding were measured daily, and the disease activity index (DAI) score was calculated using a well-established system: (a) weight loss: 0 = none; 1 = 1-5%; 2 = 5-10%; 3 = 10-15%; 4 = more than 15%; (b) stool consistency: 0 = normal; 2 = loose stools; 4 = diarrhea; (c) gross bleeding: 0 = normal; 2 = occult blood detection; 4 = severe bleeding.

[0058] S1.3: Detection of inflammatory factors in mouse intestinal tissue

[0059] The small intestinal tissue of mice was obtained, homogenized, centrifuged and the supernatant was collected. The inflammatory factors IL-6 and TNF-α were detected by ELISA according to the instructions of the kit and detected using an enzyme marker.

[0060] S1.4: Statistical Methods

[0061] GraphPad Prism 8 (GraphPad Software, La Jolla, CA) was used for all statistical analyses and data; continuous data are expressed as mean ± SD, and ordinal data are expressed as median and IQR. Comparisons between two or more groups were performed with unpaired t-test or analysis of variance; P < 0.05 was considered statistically significant.

[0062] S2: Results

[0063] The present experiment found that the body weight and disease activity index of UC mice injected with IGFBP-6 deficiency were significantly reduced (eg Figure 2 HE staining and PAS staining showed that intestinal damage was aggravated. However, these symptoms could be significantly alleviated in UC mice injected with rIGFBP-6 (as shown in Figure 2). Figures 3 to 5 shown).

[0064] Persistent inflammation is another important feature of UC and one of the causes of intestinal damage during UC. Therefore, this study evaluated intestinal inflammation by detecting the expression of intestinal inflammatory factors. After UC induction, proinflammatory cytokines such as IL-6 and TNF-α in intestinal tissues increased significantly, while rIGFBP-6 significantly inhibited the expression and release of inflammatory factors such as IL-6 and TNF-α mediated by UC (e.g., Figure 6 shown).

[0065] S3: Conclusion

[0066] Through the detection and analysis of disease activity index, weight change curve, HE staining, PAS staining and inflammatory factors, the results showed that IGFBP-6 plays an inhibitory role in the pathogenesis of UC. IGFBP-6 is a key protective internal factor in the occurrence and development of UC. rIGFBP-6 can reduce intestinal damage to UC, while IGFBP-6 (- / -) Can aggravate intestinal damage.

[0067] Example 3

[0068] This example found that rIGFBP-6 alleviates pyroptosis of intestinal macrophages, and IGFBP-6 (- / -) It can significantly aggravate the pyroptosis of intestinal macrophages.

[0069] The specific implementation process is as follows:

[0070] S1: Materials and Methods

[0071] S1.1: Modeling and Intervention

[0072] The modeling method was the same as that in S1.1 of the Example 1. UC+rIGFBP-6 group: During the UC modeling period, rIGFBP-6 1000 μg / kg was intraperitoneally injected every other day for 3 consecutive times; UC+IGFBP-6 (- / -) Group: Using self-bred IGFBP-6 (- / -) Knockout mice; the control group was fed with normal drinking water and had free access to water; the experiment ended on the 8th day after UC induction.

[0073] S1.2: Immunofluorescence staining

[0074] The mouse colon was fixed with 4% paraformaldehyde, embedded in paraffin, frozen in liquid nitrogen, and then sectioned; 8-mm-thick frozen sections of the colon were first rinsed three times with PBS and then incubated with blocking buffer containing 1% bovine serum albumin (BSA), 10% goat serum, and 0.3% TritonX-100 at room temperature for 1 hour; then, the blocking solution was removed, and the colon sections were incubated with F4 / 80 and Caspase-1 or IL-1β antibodies at 4°C overnight; the next day, the specimens were rinsed three times with PBS and incubated with the corresponding fluorescent secondary antibodies at room temperature for 2 hours; finally, the sections were counterstained with DAPI for 5 minutes at room temperature; and images were acquired using a fluorescence microscope.

[0075] S1.3: Western blot analysis

[0076] Mouse ileum tissue was added with RIPA buffer, ground with a homogenizer, and centrifuged at 13000 rpm for 15 min. The supernatant was used as tissue lysis buffer. Proteins were separated by SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane. fluoride, PVDF membrane); 5% skim milk was added at room temperature for 2 hours, and the membrane was washed three times with TBST. NLRP3 (1:1000), NLRC4 (1:1000), GSDMD (1:1000), IL-1β (1:1000), Caspase-1 (1:1000), Cleaved-caspase1 (1:500), and β-actin (1:5000) were added. The membrane containing the primary antibody was then incubated on a shaker at 4°C overnight, and then horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG antibody (1:10000) was added and incubated at room temperature for 1 hour. After washing the membrane three times, the PVDF membrane was developed using an ultrasensitive ECL chemiluminescence kit and quantitatively analyzed using Image J (National Institutes of Health, Bethesda, MD).

[0077] S1.4: Statistical Methods

[0078] GraphPad Prism 8 (GraphPad Software, La Jolla, CA) was used for all statistical analyses and data; continuous data are expressed as mean ± SD, and ordinal data are expressed as median and IQR. Comparisons between two or more groups were performed with unpaired t-test or analysis of variance; P < 0.05 was considered statistically significant.

[0079] S2: Results

[0080] S2.1: If Figure 7 As shown in the results, the proportion of pyroptotic cells in the UC group was significantly greater than that in the control group. IGFBP-6 deficiency aggravated UC-induced pyroptosis of macrophages, while rIGFBP-6 treatment almost completely abolished UC-induced pyroptosis of macrophages.

[0081] S2.2: If Figure 8 As shown in the results, changes in pyroptosis-related factors reflect the severity of pyroptosis in UC intestinal cells; therefore, the expression of NLRP3, NLRC4, GSDMD, Caspase-1, Cleaved-caspase 1 and IL-1β in the intestine was also evaluated in colon tissue samples collected after the UC model was established; compared with the control group, the expression of pro-pyroptosis factors in the UC group was upregulated; the upregulation of the above pyroptosis factors in the colon samples of IGFBP-6-deficient mice was more significant; and rIGFBP-6 significantly reversed this phenomenon, even returning to a level similar to that of the control group.

[0082] S3: Conclusion

[0083] Immunofluorescence and immunoblotting results showed that IGFBP-6 plays a key role in the course of UC by affecting pyroptosis of intestinal macrophages. IGFBP-6 deficiency can aggravate pyroptosis of intestinal macrophages; while rIGFBP-6 plays a protective effect on UC and significantly reduces pyroptosis of intestinal macrophages.

[0084] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. The use of IGFBP-6 in the preparation of a drug for treating ulcerative colitis, characterized in that: include: A drug for treating ulcerative colitis with IGFBP-6 as an active ingredient, wherein the types of IGFBP-6 include IGFBP-6 recombinant protein, purified form of IGFBP-6 recombinant protein, and derivatives obtained by chemical modification of IGFBP-6 recombinant protein; The ulcerative colitis therapeutic drug has at least one of the following three effects: inhibiting the expression and release of inflammatory factors mediated by ulcerative colitis, reducing pyroptosis of intestinal macrophages induced by ulcerative colitis, and alleviating intestinal damage.

2. Use of IGFBP-6 according to claim 1 in the preparation of a medicament for treating ulcerative colitis, characterized in that: The inflammatory factors inhibiting ulcerative colitis include interleukin-6 and tumor necrosis factor-α.

3. Use of IGFBP-6 according to claim 1 in preparing a medicament for treating ulcerative colitis, characterized in that: The ulcerative colitis therapeutic drug reduces intestinal epithelial cell pyroptosis by downregulating the expression or activation level of pyroptosis-promoting factors, wherein the pyroptosis-promoting factors include GSDMD, Cleaved-caspase 1, and IL-1β.

4. The use of IGFBP-6 according to claim 1 in the preparation of a medicament for treating ulcerative colitis, characterized in that: When the drug for treating ulcerative colitis is used, the effective dosage of the active ingredient is greater than or equal to 50 μg / kg.

5. Use of IGFBP-6 according to claim 1 in preparing a medicament for treating ulcerative colitis, characterized in that: The ulcerative colitis therapeutic drug is a single-component substance or a compound preparation.

6. Use of IGFBP-6 according to claim 1 in preparing a medicament for treating ulcerative colitis, characterized in that: The ulcerative colitis treatment drug further includes a pharmaceutically acceptable carrier or excipient.

7. Use of IGFBP-6 according to claim 1 in preparing a medicament for treating ulcerative colitis, characterized in that: The material forms of the ulcerative colitis therapeutic drug include solid, liquid, gel, semi-fluid, and aerosol.

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