Application of o-nitroso-benzaldehyde group-modified bio-gelatin in the preparation of drugs for preventing or treating inflammatory bowel disease

Abstract

The present invention discloses the application of bio-gelatin modified with o-nitroso benzaldehyde group in the preparation of drugs for preventing or treating inflammatory bowel disease; the bio-gelatin modified with o-nitroso benzaldehyde group is prepared by the following method: S1. Add 4-(4-hydroxymethyl-3-nitro-2-methoxyphenoxy) butyric acid into the aqueous gelatin solution, and react the carboxyl group in 4-(4-hydroxymethyl-3-nitro-2-methoxyphenoxy) butyric acid with the amino group in gelatin to obtain bio-gelatin modified with o-nitrobenzyl alcohol group; S2. After irradiating the bio-gelatin modified with o-nitrobenzyl alcohol group with a near-ultraviolet light source, it is converted into bio-gelatin modified with o-nitroso benzaldehyde group. The present invention uses bio-gelatin modified with o-nitroso benzaldehyde group for preventing or treating inflammatory bowel disease, which can form a uniform, dense and stable coating on the intestinal surface and has good preventive and therapeutic effects on inflammatory diseases.

Description

(1) Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of bio - gelatin modified with o - nitroso - benzaldehyde group in the preparation of drugs for preventing or treating inflammatory bowel disease. (2) Background Art

[0002] Disclosing the information of this background art section is only intended to increase the understanding of the overall background of the present invention, and it is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Inflammatory bowel disease (IBD) is an immune - mediated chronic inflammatory disease, including Crohn's disease and ulcerative colitis. Clinically, inflammatory bowel disease is often accompanied by symptoms such as abdominal pain, fever, intestinal obstruction, or diarrhea, mucopurulent bloody stools, etc., and often due to repeated attacks of the disease. The current treatment plan for inflammatory bowel disease is mainly based on the traditional anti - inflammatory drug 5 - aminosalicylic acid. Although it can play an anti - inflammatory role at the target site, its effect of inducing remission of inflammation is not satisfactory. Only 23% of patients enter the remission stage after 6 weeks of clinical application. Therefore, finding an effective means to treat inflammatory bowel disease is urgently needed. (3) Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide the application of bio - gelatin modified with o - nitroso - benzaldehyde group in the preparation of drugs for preventing or treating inflammatory bowel disease.

[0005] To solve the above - mentioned technical problems, the present invention adopts the following technical solutions:

[0006] The present invention provides the application of bio - gelatin modified with o - nitroso - benzaldehyde group in the preparation of drugs for preventing or treating inflammatory bowel disease;

[0007] The bio - gelatin modified with o - nitroso - benzaldehyde group is prepared by the following method:

[0008] S1. Add 4 - (4 - hydroxymethyl - 3 - nitro - 2 - methoxyphenoxy) butyric acid to the aqueous gelatin solution, and react the carboxyl group in 4 - (4 - hydroxymethyl - 3 - nitro - 2 - methoxyphenoxy) butyric acid with the amino group in gelatin to obtain bio - gelatin modified with o - nitrobenzyl alcohol group;

[0009]

[0010] S2. Irradiate the bio - gelatin modified with o - nitrobenzyl alcohol group with a near - ultraviolet light source, and convert it into bio - gelatin modified with o - nitroso - benzaldehyde group.

[0011]

[0012] Preferably, the step S1 is specifically implemented as follows:

[0013] 4-(4-Hydroxymethyl-3-nitro-2-methoxyphenoxy)butyric acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are added to dimethyl sulfoxide (DMSO) to obtain a mixed solution; then the mixed solution is slowly added to the gelatin solution, and the reaction is carried out at 40-50 °C (preferably 45 °C) for 3-5 hours (preferably 4 h). The resulting reaction solution is dialyzed with distilled water for 4-6 days (preferably 5 days), and the collected product is freeze-dried to obtain gelatin modified with o-nitrobenzyl alcohol groups; the molar ratio of 4-(4-hydroxymethyl-3-nitro-2-methoxyphenoxy)butyric acid to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:1, and the feeding ratio of 4-(4-hydroxymethyl-3-nitro-2-methoxyphenoxy)butyric acid to gelatin is 2:1.

[0014] Preferably, the step S2 is specifically implemented as follows: The gelatin modified with o-nitrobenzyl alcohol groups is diluted with PBS buffer to obtain a solution, and irradiated with a near-ultraviolet light source, with a power density of 30-35 mW / cm 2 (more preferably 30 mW / cm 2 ), and the irradiation time is 15 s - 2 min (more preferably 1 min) to obtain gelatin modified with o-nitroso benzaldehyde groups. Further preferably, the wavelength of the near-ultraviolet light source is 365 nm.

[0015] The inflammatory bowel disease described in the present invention includes Crohn's disease (CD) and ulcerative colitis (UC).

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention uses gelatin modified with o-nitroso benzaldehyde groups for the prevention or treatment of inflammatory bowel disease, which can form a uniform, dense and stable coating on the intestinal surface, and has good preventive and therapeutic effects on inflammatory diseases, providing a new solution strategy for the treatment of inflammatory bowel disease. (IV) BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0018] Figure 1:Fourier transform attenuated total reflection infrared spectra of gelatin, bio-gelatin modified with o-nitrobenzyl alcohol groups, and bio-gelatin modified with o-nitroso benzaldehyde groups under the irradiation of near-ultraviolet laser in the embodiments of the present invention; wherein ①: gelatin, ②: bio-gelatin modified with o-nitrobenzyl alcohol groups, ③: bio-gelatin modified with o-nitroso benzaldehyde groups.

[0019] Figure 2 : SEM images of the colon surface of the untreated group and the groups treated with gelatin and GelNB molecular coatings respectively (scale bar: 30 μm (upper figure); 3 μm (lower figure, magnified)).

[0020] Figure 3 : Fluorescence images of the mouse colon surface detected by fluorescence microscopy after 2 hours of treatment with untreated (control), fluorescently labeled gelatin, and GelNB molecules (scale bar: 100 μm).

[0021] Figure 4 : Detection of the distribution and retention time of untreated, fluorescently labeled gelatin, and GelNB molecular coatings in the colon of mice by IVIS Spectrum. Among them, ***p < 0.001.

[0022] Figure 5 : (C-D) Measurement of amino acid (C) and ion (D) absorption using dialysis bags pretreated with gelatin and GelNB molecular coatings. Among them, *p < 0.05 and **p < 0.01.

[0023] Figure 6 : Absorption of arginine by dialysis bags treated with gelatin and GelNB molecular coatings. Data are expressed as mean ± SEM. **P < 0.01 compared with gelatin.

[0024] Figure 7 : Zeta potential of gelatin and GelNB molecular coating solutions. Data are expressed as mean ± SEM. **P < 0.01 compared with gelatin.

[0025] Figure 8A : Live / dead staining results of NCM460 cells loaded with gelatin and GelNB molecular coatings on day 0 and day 3.

[0026] Figure 8B : Detection of CCK-8 at different time points in the control, gelatin, and GelNB groups to quantify cell viability in NCM460 cell cultures. (Compared with the Control group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with the PBS group, #p < 0.05, ##p < 0.01, p < 0.001.)

[0027] Figure 8C: Expression differentiation of genes related to the physical intestinal barrier function in NCM460 cells cultured for 2 days by qPCR analysis. The left side is the gelatin group, and the right side is the GelNB group. (*p < 0.05, **p < 0.01, ***p < 0.001 compared with the gelatin group, ns indicates no significant difference statistically)

[0028] Figure 8D : Morphological changes of macrophages cultured in vitro after loading gelatin and GelNB molecular coatings.

[0029] Figure 8E : GelNB induces macrophages to differentiate into M2 rather than M1 at the mRNA expression level after 48 h. Among them, the left side is the gelatin group, and the right side is the GelNB group; *p < 0.05, **p < 0.01, ***p < 0.001, ns indicates no significant difference statistically.

[0030] Figure 9 : Wound healing ability of NCM460 cells in plates pretreated with negative control (control), gelatin, and GelNB molecular coatings (scale bar: 200 μm).

[0031] Figure 10 : The model of DSS-induced mice and the treatment processes of three groups.

[0032] Figure 11 : Quantitative ratios of daily regular water intake (A) and daily regular food intake (B) to the body weight of mice when treated with PBS (control) and GelNB groups for 7 days. Data are expressed as mean ± SEM. Compared with the PBS group, nsP > 0.05.

[0033] Figure 12 : Percentages of body weight loss in mice after treatment in the control group, PBS, GelNB, and mesalazine groups. Data are expressed as mean ± SEM. n = 15. Compared with the control group, *p < 0.05, **p < 0.001, ***p < 0.001; compared with the PBS group, #p < 0.05, ##p < 0.01, p < 0.001; compared with the mesalazine group, &p < 0.05.

[0034] Figure 13 : Measurement results of DAI scores after treatment. Calculate the body weight loss index, fecal blood index, and stool consistency index. Data are expressed as mean ± SEM. n = 15. Compared with the control group, *p < 0.05, ***p < 0.001; compared with the PBS group, #p < 0.05, ##p < 0.01, p < 0.001; compared with the mesalazine group, &p < 0.05.

[0035] Figure 14: Colonic injury scores after treatment of DSS-induced mice. Data are presented as mean ± SEM. Compared with the control group, **P<0.01,***P<0.001; compared with the PBS group, P<0.001; compared with the mesalazine group, &P<0.05.

[0036] Figure 15 : (A) Macroscopic images of colonic tissues on the 12th day of treatment. (B) Quantitative length of the colon after 12 days of treatment. (C) Quantitative ratio of colon weight to mouse body weight after 12 days of treatment. Among them, compared with the control group, *p<0.05, **P<0.01,***P<0.001; compared with the PBS group, #P<0.05, ##P<0.01, P<0.001; compared with the mesalazine group, &P<0.05.

[0037] Figure 16 : Survival curves of each group of mice treated with negative control, PBS, GelNB, and mesalazine. Among them, compared with the control group, *p<0.05, **P<0.01,***P<0.001; compared with the PBS group, #P<0.05, ##P<0.01, P<0.001; compared with the mesalazine group, &P<0.05.

[0038] Figure 17 : Representative histological sections of the colon stained with H&E (scale bar: 50 μm).

[0039] Figure 18 : Results of MPO activity detected by the MPO detection kit. Data are presented as mean ± SEM. Compared with the control, *P<0.05, **P<0.01; compared with mesalazine, &P<0.05.

[0040] Figure 19 : Results of qPCR analysis of distal colon of genes related to intestinal inflammation. Data are presented as mean ± SEM. Compared with the control group, *p<0.05, ***p<0.001; compared with the PBS group, #p<0.05, ##p<0.01; compared with the mesalazine group, &P<0.05.

[0041] Figure 20 : Results of qPCR analysis of genes (A) ZO-1 and (B) Occludin-1 related to the physical intestinal barrier function of the distal colon in DSS-induced mice. n = 3, data are presented as mean ± SEM. Compared with the control group, *P<0.05, **P<0.01; compared with the PBS group, #P<0.05 compared with PBS; compared with the mesalazine group, &&P<0.01.

[0042] Figure 21: (A) ROS activity levels in mice of the Control, PBS, GelNB, gelatin, and mesalazine groups; (B) Quantitative analysis of ROS signals, from left to right are the Control, PBS, GelNB, gelatin, and mesalazine groups. Data are expressed as mean ± SEM, n = 3. **p < 0.01, ***p < 0.001, ns p ≥ 0.05.

[0043] Figure 22 : Representative expression of Ki67, ZO-1, and Occludin-1 in colon sections by immunohistochemical staining (scale bar: 50 μm).

[0044] Figure 23 : In vivo GelNB molecular coating promotes the recovery of TNBS-induced mice. (A): Schematic diagram of four different treatments for TNBS-induced mice. (B): Percentage of body weight loss in mice after treatment with negative control, PBS, mesalazine, and GelNB. (C): DAI scores of mice after treatment, including body weight loss index, fecal blood index, and stool consistency index. (D): Macroscopic images of colon tissues on the 7th day after initial treatment. (E): Measurement and comparison of colon lengths between groups. (F): Quantitative ratio of colon weight to body weight in the TNBS model. (G): Representative tissue sections of mouse colon stained with H&E (scale bar: 50 μm). (H) and (I): Images of ROS activity and quantitative analysis of ROS signals in mouse colitis. (J): Representative expression of Ki67, ZO-1, and Occludin-1 in colon sections by immunohistochemical staining (scale bar: 50 μm). Data are expressed as mean ± SEM. n = 5. Compared with the control group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with PBS, #p < 0.05, ##p < 0.01, p < 0.001; ns no significant difference statistically.

[0045] Figure 24 : Colon injury scores in TNBS-induced mice after treatment. Data are expressed as mean ± SEM. Compared with the control group, **P < 0.01; ***P < 0.001; compared with the PBS group, #P < 0.05, P < 0.001; compared with the mesalazine group, &&P < 0.01.

[0046] Figure 25: qPCR analysis of intestinal inflammation-related genes in the distal colon of mice induced by TNBS, where (A): IL 1α, (B): IL 1β, (C): IL 6, (D): TNFα, (E): iNOS, (F): IFN, (G): IL 4, (H): IL 10. Data are expressed as mean ± SEM. Compared with the control group, *P < 0.05, **P < 0.01; ***P < 0.001; compared with the PBS group, #P < 0.05, ##P < 0.01, P < 0.001; compared with the mesalazine group, &P < 0.05.

[0047] Figure 26 : qPCR analysis of physical intestinal barrier function genes in the distal colon of mice induced by TNBS, where (A): ZO-1, (B): Occludin-1. Data are expressed as mean ± SEM. Compared with the control group, *P < 0.05, **P < 0.01; compared with the PBS group, #P < 0.05, ##P < 0.01. (V) Specific implementation manners

[0048] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

[0049] The present invention will be further described in conjunction with specific examples. The following examples are only for explaining the present invention and do not limit its content.

[0050] Example 1: Preparation of bio-gelatin modified with o-nitroso-benzaldehyde group

[0051] 4-Hydroxy-3-(methoxy-D3) benzaldehyde (8.90 g, 58.5 mmol, 1.06 eq.), potassium carbonate (10.2 g, 73.8 mmol, 1.34 eq.) and methyl 4-bromobutyrate (9.89 g, 55.0 mmol, 1.0 eq.) were dissolved in dimethylformamide (DMF) for reaction. The mixture was deposited with cold water (200 ml) to obtain the product. Then, the product was dissolved in DMF and dried to obtain methyl 4-(4-formyl-2-methoxyphenoxymethoxyphenyl) butyrate.

[0052] Methyl 4-(4-formyl-2-methoxyphenoxymethoxyphenyl)butyrate (9.4 g, 37.3 mmol, 1 eq.) was added to cold (2 °C) nitric acid solution (70%) for 3 h. To deposit the solid product, the mixture was filtered with cold water and then purified in DMF. After hydrolysis with 100 ml of 10% aqueous trifluoroacetic acid at 90 °C, it was dried under reduced pressure and the solvent was removed to obtain a pale yellow powder. Then the product (7.4 g, 23.8 mmol, 1.0 eq.) was dissolved in THF / EtOH (1:1 v / v). After 3 h, NaBH4 (1.43 g, 35.7 mmol, 1.5 eq.) was slowly added. Then the product in the aqueous layer was extracted with dichloromethane, and the organic layer was dried over magnesium sulfate. The crude product was purified by silica gel column chromatography using DCM / MeOH = 10:1 (1% TEA), and finally 5.31 g (18.6 mmol, 78.3%) of 4-(4-hydroxymethyl-3-nitro-2-methoxyphenoxy)butyric acid as a pale yellow powder was obtained.

[0053] Preparation of GelNB: A 10% w / v homogeneous aqueous gelatin solution (Sigma-Aldrich) was maintained at 37 °C. While 106 mg of 4-(4-hydroxymethyl-3-nitro-2-methoxyphenoxy)butyric acid was dissolved in 5 mL of dimethyl sulfoxide (DMSO), 56 mg of 1-(3-dimethylaminopropyl)–3-ethylcarbodiimide hydrochloride (EDC) (Aladdin) and 33 mg of N-hydroxysuccinimide (NHS) (Aladdin) were added to the DMSO solution of 4-(4-hydroxymethyl-3-nitro-2-methoxyphenoxy)butyric acid as catalysts respectively to obtain a mixed solution. Then the mixed solution was slowly dropped into 200 mL of 10% aqueous gelatin solution and reacted at 45 °C for 4 h. To purify the product, the resulting solution was dialyzed against distilled water for 3 days. It was collected and lyophilized to obtain bio-gelatin modified with o-nitrobenzyl alcohol groups, which was stored at 4 °C for further use.

[0054] Preparation of enema solution: The lyophilized bio-gelatin modified with o-nitrobenzyl alcohol groups was diluted with 1×PBS buffer solution at 37 °C to obtain a 100 mg / mL solution. After activation with ultraviolet light (365 nm, 30 mW / cm 2 ) for 1 min, the solution became a bio-gelatin solution modified with o-nitroso-benzaldehyde groups (denoted as GelNB) solution, which was used as the enema solution.

[0055] Fourier transform infrared (FTIR)-attenuated total reflection (ATR) spectroscopy (Nicolet iS50; Thermo, USA) was used to test the gelatin solution, the unactivated bio-gelatin solution modified with o-nitrobenzyl alcohol groups, and the UV-activated bio-gelatin solution modified with o-nitroso-benzaldehyde groups. The results are as Figure 3As shown. Compared with gelatin, the enhanced absorption bands of the primary hydroxyl groups of the bio-gelatin modified with o-nitrobenzyl alcohol groups at around 1280 and 1170 cm -1 indicate the successful grafting of 4-(4-hydroxymethyl-3-nitro-2-methoxyphenoxy)butyric acid onto gelatin, belonging to -OH deformation vibration and C-O stretching vibration respectively. However, in the bio-gelatin modified with o-nitroso benzaldehyde groups, the intensities of these two peaks decrease, while the stretching vibrations of C-O (1650 cm -1 ) and C-C (1550 cm -1 ) show red-shift. The results indicate that the primary hydroxyl groups of 4-(4-hydroxymethyl-3-nitro-2-methoxyphenoxy)butyric acid gradually transform into phenolic aldehyde conjugated groups after UV irradiation.

[0056] Example 2: In vitro intestinal wall modification and integration

[0057] Referring to Example 1, the freeze-dried bio-gelatin modified with o-nitrobenzyl alcohol groups was diluted with 1×PBS buffer solution at 37 °C to obtain a 100 mg / mL solution. After activation with ultraviolet light (365 nm, 30 mW / cm 2 ) for 1 minute, the solution became a bio-gelatin solution modified with o-nitroso benzaldehyde groups, which was the enema solution. Gelatin was diluted with 1×PBS at 37 °C to obtain a 100 mg / mL gelatin solution as a control.

[0058] The enema solution was gently injected into the anus of the mice and pinched for 2 minutes to prevent outflow. The colon was excised from the euthanized mice, washed 3 times with 1×PBS buffer solution and fixed overnight with 2.5% glutaraldehyde. Liquid nitrogen was used to break the colon into small pieces. After extraction with different concentrations of ethanol (50%, 70%, 80%, 90% and 100% twice) for 10 minutes in turn, all samples were observed under SEM (Nova Nano 450; Thermo) to compare the surface morphology. The results are as Figure 2 shown.

[0059] As Figure 2 shown: The normal colon consists of a layer of columnar epithelium covered with minute protrusions. Although the changes in the gelatin treatment group were not obvious, the coating in the GelNB treatment group was dense, indicating that GelNB adhered strongly to the colon surface and formed a physical protection barrier on the mucosal surface.

[0060] Example 3: Fluorescence evaluation of GelNB.

[0061] Reference [H. Tan, D. Huang, L. Lao, C. Gao, RGD modified PLGA / gelatin microspheres as microcarriers for chondrocyte delivery, J Biomed Mater Res B Appl Biomater 91(1)(2009)228 - 238.], Rhodamine B (RB) was added to GelNB as a fluorescent indicator to quantify the adhesion ability of the coating on the intestinal wall. Rhodamine - labeled gelatin (Gel - NB) and GelNB (GelNB - RB) were produced by mixing rhodamine B isothiocyanate with gelatin and GelNB respectively at 48 °C for two days. The lyophilized Gel - NB and GelNB - RB foams were then dissolved in 1×PBS at 37 °C to produce enema solutions with a concentration of 100 mg / mL. As described in Example 2, the enema solution was gently injected into the anus of mice and pinched for 2 minutes to prevent outflow. Detection was performed using a fluorescence microscope and IVIS Spectrum (PerkinElmer, USA) at 0, 2, 24, and 48 hours after enema respectively. Mice untreated with the enema solution were used as the control group. The detection results are as Figure 3 and Figure 4 shown. Figure 3 showed that the fluorescence - labeled GelNB was able to adhere to the intestinal wall and form a thin and dense coating on the scale of hundreds of nanometers. The fluorescence intensity of the gelatin group was very weak, similar to that of the control group, indicating that gelatin could not firmly attach to the intestinal wall. Figure 4 showed that at 48 h, GelNB maintained stronger adhesion in the mouse colon. In contrast, mice treated with gelatin showed only weak fluorescence in the colon, and most of it had been excreted. This indicates that GelNB has better retention performance.

[0062] Figure 4 showed that GelNB maintained more viscosity in the mouse colon at 48 hours. In contrast, mice treated with gelatin showed only weak fluorescence in the colon, and most of it had been excreted. This indicates that GelNB has better retention performance, which is the basis for our subsequent application in mice.

[0063] These results indicate that GelNB can form a uniform and stable coating on the intestinal surface.

[0064] Example 4: Equilibrium dialysis experiment.

[0065] Gelatin and freeze - dried o - nitrophenyl methanol - group - modified bio - gelatin were respectively diluted with 1×PBS buffer solution at 37 °C to obtain solutions with a concentration of 100 mg / mL. Respectively with ultraviolet light (365 nm, 30 mW / cm2 ) After activation for 1 minute, a dialysis bag with a molecular weight cut-off of 3500 Da was immersed in the obtained gelatin solution and GelNB solution. After soaking for 30 min, the dialysis bag was taken out. Deionized water was filled into the dialysis bags with a molecular weight cut-off of 3500 Da coated with gelatin and GelNB respectively, then sealed with clips and immersed in 500 mL of D-glucose (Meilun Biology, China), L-arginine (Sangon Biotech, China), L-glutamic acid (Macklin, China), L-tryptophan (Macklin), glycine (Macklin), L-glutamine (Macklin), L-cysteine (Macklin) or Ringer's solution. As a control, deionized water was placed in a separate clean dialysis bag. The liquid in the dialysis bag was periodically collected in a vacuum tube as a dialysis test sample. The amino acid concentration was measured using a total amino acid assay kit (Nanjing Jiancheng Bioengineering Institute, China). The glucose concentration was evaluated using a glucose colorimetric assay kit (Elabscience, China). The ion concentration was measured using an automatic biochemical analyzer (Beckman Coulter AU5800, USA).

[0066] Compared with the gelatin-treated dialysis bag, the infiltration of glucose in the GelNB-treated dialysis bag was significantly inhibited within half an hour, and the inhibition lasted for 2 hours (p < 0.01). It has been reported that high glucose can induce macrophages and other immune cells to develop in a pro-inflammatory direction. Therefore, the low permeability of GelNB to glucose may affect the intestinal immune response. Free fatty acids (NEFAs), such as butyrate, are small molecule compounds produced by the decomposition of dietary fiber by gut microbiota. GelNB significantly promoted the permeability of NEFAs (p < 0.05). It has been reported that butyrate can regulate the production of Tregs and enhance the activity of macrophages to prevent colitis. The uptake of amino acids is also closely related to the progression of IBD. In this context, four amino acids were selected for the permeability experiment, and among them, glutamine, glutamic acid, and glycine could easily penetrate into the dialysis bag through GelNB in the intestinal environment (p < 0.05)( Figure 5 C). Studies have shown that glutamine can increase the expression of tight junction proteins and promote intestinal repair, while glycine can reduce the MPO activity in colon tissue and can also reduce the expression of vascular endothelial growth factor (VEGF-A) and its receptor, inhibit the increase in vascular density caused by inflammation, and reduce the aggregation of intestinal granulocytes. This indicates that the selective absorption of amino acids by GelNB may contribute to the recovery of IBD. In addition, Figure 5 Figure D shows that GelNB can prevent the exchange of electrolytes in the intestine because the cations (such as Ca 2+ , Na + and K + ) in the dialysis bag seem to be higher in gelatin than in GelNB (P < 0.05).

[0067] Ca 2+ 、Na + 、K + and arginine are all positively charged and blocked by GelNB, while glutamine, glutamate and glycine are all negatively charged and tend to be absorbed by the intestine covered by GelNB( Figure 6 ). Therefore, we speculate that the GelNB molecular coating may selectively absorb nutrients, thereby reducing the stimulation of nutrients to the intestine.

[0068] To further explore the mechanism of GelNB selectively permeating metabolites in the intestinal lumen, the present invention measured the zeta potential on the surface of gelatin and GelNB solutions (100 mg / mL) dissolved in 1XPBS. We found that the zeta potential on the surface of GelNB was 6.91 mV( Figure 7 ), which indicates that GelNB may act as a physical barrier, representing a charge barrier, thus affecting material exchange.

[0069] Example 5: In vitro cell experiments to determine the mechanism by which GelNB affects cell viability and proliferation.

[0070] 1. NCM460 cells

[0071] NCM460 is a common colon mucosal cell line purchased from the Shanghai Cell Bank. The mouse macrophage cell line RAW264.7 is from ATCC. All cell lines were cultured in DMEM containing 10% fetal bovine serum and 100 mg / mL penicillin-streptomycin (double antibody) at 37 °C in a 5% CO2 incubator.

[0072] Immerse the sterilized clean glass slides in an aqueous solution of 1% (3-aminopropyl)triethoxysilane (Macklin) for half an hour and then dry. Add 500 μL of the o-nitrobenzyl alcohol group-modified gelatin solution (100 mg / mL) dissolved in 1XPBS buffer to the dried glass slides to form a molecular coating. After activating with ultraviolet light (30 mW / cm 2 ) for 1 minute, a GelNB coating is obtained on the glass slides. Using the same operation as above, a gelatin coating is formed on the glass slides with a gelatin solution (100 mg / mL) dissolved in 1X PBS buffer. As a control, the dried glass slides are not coated. Then place the glass slides with GelNB coating, gelatin coating and uncoated into 6-well plates. Inoculate cells at a density of 1×10 5 cells per well.

[0073] Cell viability was measured using the live / dead staining assay and the CCK-8 assay. The live / dead staining assay was performed using a live cell / dead cell double staining kit (Solarbio) on day 0 and day 3. The CCK-8 assay was performed daily from day 1 to day 3. The CCK-8 reagent (Yeasen) was diluted to 1:10 (v / v) in DMEM (Gibco), 100 μL of the CCK-8 dilution was added to each well, and incubated at 37 °C for 1 hour. Absorbance represents live cells, and the optical density was measured at 450 nm using a microplate reader (Thermo MK3, USA).

[0074] As Figure 8A The live / dead staining of NCM460 cells as shown in

[0075] As Figure 8B The results of the CCK-8 assay as shown in

[0076] Both of the above two experiments indicated that the GelNB molecular coating could significantly promote the proliferation of intestinal epithelial cells and was beneficial to intestinal repair.

[0077] IBD is often accompanied by the destruction of the intestinal mucosal structure and the increase of intestinal mucosal permeability. As the structural basis of mucosal healing, cytoskeletal remodeling can restore and maintain the intact barrier function of intestinal epithelium. Among them, tight junction-related proteins ZO1, Occludin-1, and Claudins are key regulators of the cytoskeleton. After culturing NCM460 cells for 2 days, quantitative RT-PCR was used to analyze the expression and differentiation of these genes related to the physical intestinal barrier function in the cells. The results showed that GelNB could significantly increase the expression of tight junction-related proteins ZO1 and Claudins in NCM460 cells at the mRNA level (P<0.001)( Figure 8C ), indicating that GelNB has the effect of enhancing intestinal barrier integrity and promoting mucosal healing.

[0078] 2. Macrophages

[0079] Referring to the culture method of NCM460 cells, macrophages were cultured on culture dishes loaded with GelNB and gelatin coatings. The morphological changes of the cells were examined by optical microscopy on day 0 and day 2, and the levels of markers such as TNF, TLR4, CLEC4D, TLR1, MRC1, and ARG1 in macrophages were detected by quantitative RT-PCR after culturing for 2 days. It was found that the GelNB coating significantly promoted the expression of MRC1 (P<0.05) and ARG1 (P<0.001), which are characteristics of macrophage M2 differentiation( Figure 8D and8E ) while the expression of TLR1 (P<0.001), TLR4 (P<0.05) and other genes with characteristics of macrophage M1 differentiation were significantly inhibited. Meanwhile, the expression level of CLEC4D also showed a downward trend in the GelNB group. These results indicate that compared with gelatin, GelNB can further promote the phenotypic transformation of macrophages into M2, shift the balance towards the M2 type, and reduce the inflammatory response.

[0080] Example 7: Wound Scratch Assay

[0081] Cell migration was measured using the wound scratch assay. A sterilized clean glass slide was immersed in an aqueous solution of 1% (3-aminopropyl)triethoxysilane (Macklin) for half an hour and then dried to obtain an aminated glass slide. The freeze-dried o-nitrobenzyl alcohol group-modified bio-gelatin was diluted with 1×PBS buffer at 37 °C to obtain a 100 mg / mL solution, which was activated with ultraviolet light (365 nm, 30 mW / cm 2 ) for 1 minute to obtain the GelNB solution. Gelatin was diluted with 1×PBS buffer to obtain a 100 mg / mL gelatin solution. 500 μL of the GelNB solution and the gelatin solvent were respectively added to the aminated glass slide to form a coating. The uncoated aminated glass slide was used as a control. The aminated glass slides loaded with the GelNB coating, gelatin coating and no coating were respectively placed into 6-well plates. Approximately 1×10 5 NCM460 cells were collected for each group and seeded into 6-well plates. Two days after the cells reached confluence, a sterile pipette tip was used to scratch the wound from the top to the bottom of the well through the middle. Then the cells were washed twice with 1×PBS buffer to remove potential debris. The migration area was photographed twice every 12 hours and then measured using ImageJ software (National Institutes of Health, USA).

[0082] The results Figure 9 showed that after applying GelNB to the surface of the culture dish, at 12 h, the migration speed of the cells was significantly faster than that of the control group and the gelatin-treated group, and almost covered the scratched area within 24 h ( Figure 9 ). This indicates that the GelNB molecular coating enhances cell migration and aggregation, recruiting surrounding cells to repair damaged epithelial cells.

[0083] Example 8: In Vivo Animal Study Induction of IBD Mouse Model and Treatment of IBD.

[0084] 1. Eight-week-old male C57 BL / 6 (C57) mice were used for the experiments. All experiments were carried out strictly in accordance with the institutional guidelines of the Sir Run Run Shaw Hospital Animal Care and Use Committee of Zhejiang University School of Medicine, and complied with the NIH Guide for the Care and Use of Laboratory Animals (Ethics Code SRRSH202107106). Colitis was induced by DSS (Yeason, China) according to the methods described in the literature [C. Trilleaud, V. Gauttier, K. Biteau, I. Girault, L. Belarif, C. Mary, S. Pengam, G. Teppaz, V. Thepenier, R. Danger, G. Robert-Siegwald, M. Néel, S. Bruneau, A. Glémain, A. Néel, A. Poupon, J. F. Mosnier, G. Chêne, M. Dubourdeau, G. Blancho, B. Vanhove, N. Poirier, Agonist anti-ChemR23 mAb reduces tissue neutrophil accumulation and triggers chronic inflammation resolution, Sci Adv 7(14)(2021).] and the literature [N. He, X. Chen, D. Wang, K. Xu, L. Wu, Y. Liu, H. Tao, Q. Zhao, X. Cao, Y. Li, N. Liu, X. Qi, Z. Han, D. Kong, J. Yang, Z. Li, VE-Cadherin regulates the self-renewal of mouse embryonic stem cells via LIF / Stat3 signaling pathway, Biomaterials 158(2018)34 - 43.].

[0085] The mice were divided into four groups: control group, PBS group, GelNB group and mesalazine group, with 15 mice in each group. DSS was dissolved in sterile distilled water at 50 mg / mL to obtain an aqueous DSS solution. As Figure 10As shown, all mice were first adaptively raised in a SPF-class animal house for one week and had free access to water and food. Then, mice in the PBS group, GelNB group, and mesalazine group were fed an aqueous DSS solution for 7 days, while the control group mice drank sterile distilled water, which was changed every two days. Starting from the third day after inducing colitis with DSS, mice in the PBS group, GelNB group, and mesalazine group were given 500 μL of enema solution every 48 h (the enema solutions were: 1×PBS buffer, 100 mg / mL GelNB solution dissolved in 1×PBS buffer, 0.5% mesalazine solution dissolved in 1×PBS buffer), and the administration continued until the 11th day. On the 12th day, the mice were sacrificed.

[0086] After the mice were euthanized, the colon from the cecum to the anus was removed, the length of each colon was recorded, and then each colon was washed with 1×PBS buffer. The part near the anus (0.5 cm) was removed, that is, the distal colon tissue was used for MPO activity measurement and RT-qPCR analysis. The remaining colon tissue was fixed in 4% formaldehyde for further histological and immunohistochemical analysis.

[0087] 2. To exclude the potential influence of GelNB or PBS treatment on DSS modeling, the present invention explored the effects of GelNB or PBS treatment on the water intake and diet of mice. That is, after mice were adaptively raised in a SPF-class animal house for one week, they were divided into two groups: the PBS group and the GelNB group, with 5 mice in each group. Then, mice in the PBS group and the GelNB group were given 500 μL of enema solution once a day (the enema solutions were: 1×PBS buffer, 100 mg / mL GelNB solution dissolved in 1×PBS buffer) for 7 consecutive days, and the regular daily water intake and food intake of the mice were recorded. The results are as Figure 11 shown, confirming that there was no significant difference between the two groups.

[0088] 3. Clinical evaluation of IBD in DSS-induced mice:

[0089] Refer to the literature [L.R. Fitzpatrick, J. Wang, T. Le, In vitro and in vivo effects of gliotoxin, a fungal metabolite: efficacy against dextran sodium sulfate-induced colitis in rats, Dig Dis Sci 45(12)(2000)2327-2336.] and the literature [G. Can, S. Ayvaz, H. Can, S. Demirtas, H. Aksit, B. Yilmaz, U. Korkmaz, M. Kurt, T. Karaca, The Syk Inhibitor Fostamatinib Decreases the Severity of Colonic Mucosal Damage in a Rodent Model of Colitis, J Crohns Colitis 9(10)(2015)907-917.], and calculate the DAI score and colonic injury score for each animal. Each group of mice was observed once a day during the treatment period, and the changes in body weight, diarrhea, and bleeding were recorded in a timely manner. The blood in the feces was detected using a hemolysis assay kit (Nanjing Jiancheng Bioengineering Institute), and the detection operation was carried out according to the instructions of the kit. The DAI consists of scores for weight loss, diarrhea, and bloody stools. The colonic injury score was determined based on the infiltration and loss of goblet cells and crypts shown in the sections.

[0090] According to previous studies, DSS generally takes 7-9 days to induce acute UC in mice. The first to three days are the initial stage of inflammatory injury, during which the clinical symptoms and pathological changes of the mouse colon are usually not obvious. To evaluate whether GelNB has a preventive effect on the progression of IBD, in the present invention, PBS, GelNB, and mesalazine were administered by enema to mice on the 3rd day after receiving DSS drinking water. Considering that GelNB maintained a relatively high adhesiveness at 48 h, in order to reduce the damage caused by enema to the mice, the present invention administered the drug once every 48 h. The drug was continuously administered until the 11th day ( Figure 10 ).

[0091] (1) As Figure 12As shown, the body weight of the mice in the PBS treatment group continued to decline, decreasing by approximately 20% on the 8th day, and the body weight decline of the mice in the other two groups was less than that of the PBS group (P<0.001). This indicates that both GelNB and mesalazine have a protective effect on the weight loss of DSS-induced IBD mice. Surprisingly, the GelNB group had a lower weight loss amplitude than the mesalazine group before the 8th day, and the body weight significantly recovered after the 8th day, indicating that GelNB can prevent weight loss as the pathogenic factors continue to be severe.

[0092] (2) The present invention continuously measured the disease activity index (DAI, the specific indicators and scoring criteria are shown in Table 1, and the DAI value is obtained by dividing the total score of the three results by 3) to evaluate the severity of colitis.

[0093] Table 1 Disease Activity Index Scoring Criteria

[0094] Score Weight loss Stool character Stool occult blood test (benzidine method) 0 points 0-1％ Normal Negative 1 point 1-5％ Soft stool Weak positive (light blue) 2 points 5-10％ Mucus-like stool Positive (blue) 3 points 10-20％ Watery stool Strong positive (dark blue) 4 points ＞20％ Grossly bloody stool

[0095] Results Figure 13 As shown. The score in the GelNB treatment group began to decline after the 8th day. Notably, the median DAI score of the mice on the 12th day recovered to 4 points and had an obvious downward trend. According to the body weight of the mice in the GelNB treatment group on the 12th day, the "weight loss" score corresponding to DAI was 2 points. Interestingly, based on the weight recovery trend from the 8th to the 12th day, we hypothesized that the body weight of the mice would still maintain a significant upward trend. On the 12th day, the body weight of the mice in the GelNB treatment group would further recover until it approached the value of the control group. In addition, the DAI of the mice in the GelNB treatment group would also continuously decline to approach or equal the value of the control group. This result also indicates that using GelNB treatment in the early stage of IBD can rapidly slow down the trend of the disease developing into a severe disease and quickly recover to the normal level.

[0096] (3) The colon injury scoring criteria for the DSS-induced mice after treatment refer to Table 2.

[0097] Table 2 Colon Injury Scoring Criteria

[0098] Score Scoring criteria 0 points Normal histological morphology 1 point Histological damage limited to endothelial cells, mild inflammatory cell infiltration 2 points Focal ulcer changes, tissue structure destruction limited to the mucosal layer, pancreatic-enteric intestinal wall gland structure, mild inflammatory cell infiltration 3 points Focal, transmural ulcers and inflammation, mild to moderate inflammatory cell infiltration 4 points Larger transmural ulcers and inflammation, severe inflammatory cell infiltration 5 points Large ulcers and inflammation, lesions extending from the mucosa to the serosa, severe inflammatory cell infiltration

[0099] Results are as Figure 14 shown, the degrees of inflammation, inflammation depth, and glandular injury in the GelNB group were lower than those in the other groups.

[0100] 4. The ratio of colon weight to length is an indicator of inflammatory edema. After the mice were euthanized on the 12th day, the present invention conducted a general intestinal examination and found that GelNB could reverse the shortening of the colon length caused by colitis. Compared with other treatments, the colon morphology of the mice in the GelNB group was also closest to that of the normal control group mice ( Figure 15 A, 15B and 15C).

[0101] 5. Severe UC has a certain mortality rate. As Figure 16 shown, when the experiment lasted until the 12th day, the survival rate of the GelNB treatment group was as high as 90%, while the survival rates of the PBS treatment group and the mesalazine treatment group decreased to 50% and 60% respectively.

[0102] 6. Histological examination based on H&E staining

[0103] Colon tissues of the negative control, PBS, GelNB, and mesalazine groups were taken respectively, and histological examination was performed on them using hematoxylin (Solarbio) and eosin (Solarbio). They were dewaxed successively in xylene and alcohol solutions, stained with hematoxylin for 3 minutes, stained with eosin for 1 minute, dehydrated successively in alcohol and xylene solutions, and finally mounted. The results are shown in Figure 17 .

[0104] As Figure 17 shown, histological examination based on H&E staining showed typical histopathological features of colitis in the PBS treatment group, including severe epithelial ulcers, goblet cell loss, and excessive infiltration of intraepithelial lymphocytes. In contrast, the GelNB and mesalazine treatment groups had less mucosal inflammation, less colon congestion and edema. Moreover, compared with the mesalazine treatment group, the histological manifestations of the GelNB treatment group were more similar to those of the normal control group.

[0105] (7) Measurement of MPO activity:

[0106] MPO is a heme protein rich in neutrophils, which is synthesized by granulocytes in the bone marrow before entering the circulation and stored in phagocytic granules. External inflammatory stimuli can lead to the accumulation of neutrophils, thereby releasing MPO.

[0107] To measure the degree of colon inflammation, distal colon tissues near the anus of the negative control, PBS, GelNB, and mesalazine groups were taken respectively, and the expression level of MPO was detected using an MPO detection kit (Nanjing Jiancheng Bioengineering Institute) to evaluate the number of neutrophils. The results showed that after DSS induction, compared with the mice treated with GelNB, the MPO activities of the mice treated with PBS and mesalazine remained at a relatively high level ( Figure 18 ).

[0108] (8) RT-qPCR analysis

[0109] ① The working mechanism of mesalazine in the clinical treatment of IBD includes inhibiting the NF-κB pathway, regulating the PPARγ receptor, and inhibiting the expression of inflammatory factors such as TNFα, IL1, and IL-6.

[0110] To investigate the working mechanism of GelNB in the clinical treatment of IBD, distal colon tissues near the anus from the negative control, PBS, GelNB, and mesalazine groups were collected for RT-qPCR analysis, and the results are as Figure 19 shown.

[0111] As Figure 19 shown, after treatment with GelNB, the expressions of pro-inflammatory factors such as IF-1α (P<0.05), IF-1β (P<0.001), IL-6 (P<0.05), TNF-α, iNOS, and IFN-γ in the tissues were significantly lower than those in the mesalazine treatment group, while the expressions of anti-inflammatory factors such as IL-4 and IL-10 increased. This indicates that GelNB has a better effect than mesalazine, probably due to its stronger inhibitory effect on local inflammatory responses.

[0112] ② Intestinal epithelial tight junctions (TJs) are the key structural basis of intestinal barrier function, and damage to TJs leads to increased intestinal permeability. The early onset of IBD is often accompanied by an increase in intestinal mucosal barrier permeability, resulting in changes in the proteins (ZO-1 and Occludin-1) representing TJ expression.

[0113] To investigate the effect of GelNB on the expressions of ZO-1 and Occludin-1 in colon tissues, distal colon tissues near the anus from the negative control, PBS, GelNB, and mesalazine groups were collected for RT-qPCR analysis, and the results are as Figure 20 shown.

[0114] Figure 20 The results showed that IBD could damage the intestinal mucosal barrier, leading to decreased expressions of ZO-1 and Occludin-1. However, after enema with GelNB, the damage to TJs was reversed, promoting the expressions of Occludin-1 and ZO-1 to return to normal levels, while the effect of mesalazine was not obvious.

[0115] (9) Measurement of ROS release:

[0116] On the 3rd and 12th days after DSS induction, the mice in the negative control, PBS, GelNB, and mesalazine groups were intraperitoneally injected with luminol (injection dose: 10 mg / kg, 5-amino-2,3-dihydro-1,4-phthalazinedione, Yeasen). Bioluminescence imaging (BLI) was used to evaluate the ROS levels in the mice to examine the severity of IBD. The injected mice were subjected to bioluminescence imaging using an IVIS Lumina imaging system (PerkinElmer), and regions of interest (ROIs) in the fixed abdominal area were generated to quantify the average radiance of the peak BLI signal.

[0117] The results are as Figure 21As shown in Figures 21A and 21B, on the 3rd day after DSS induction in the present invention, it was found that the ROS level remained basically unchanged, while on the 12th day, compared with other groups, the detected ROS level in the PBS group was 5 times higher; the ROS in the GelNB treatment group was less than that in the mesalazine treatment group. This indicates that the GelNB molecular coating can significantly reduce the ROS level (P<0.001).

[0118] (10) Histology and immunohistochemical markers:

[0119] In the present invention, the expressions of intestinal epithelial proliferating cell marker protein (Ki67) and intestinal epithelial tight junction (TJ) proteins (ZO1 and Occludin1) in the mouse intestine were detected by immunohistochemical staining, and the degree of cell proliferation was analyzed at the tissue level.

[0120] Fixed colon tissues of the negative control, PBS, GelNB, and mesalazine groups were taken respectively. The fixed colon tissues were dehydrated and embedded in paraffin. The paraffin-embedded colon tissues were sectioned to obtain continuous transverse tissue sections, and the sections were subjected to antigen retrieval in citrate buffer (pH = 6) for 10 minutes. Goat serum was used to block the samples for 30 minutes, and then incubated overnight at 4°C with primary antibodies against Ki67 (Abcam, USA), Occludin-1 (Abcam), or ZO-1 (Cell Signaling Technology). The secondary antibody (Shanghai Gene Technology Co., Ltd., China) was incubated for 30 minutes. Images were taken using NANO ZOOMER S60 (Hamamatsu, Japan).

[0121] The expression of Ki67 in the mouse intestine was detected by immunohistochemical staining, and the staining results are as Figure 22 shown. Compared with the GelNB group and the mesalazine group, the PBS group had fewer Ki67-positive cells, indicating that compared with the PBS group, the GelNB group and the mesalazine group had significantly enhanced ability to improve the proliferation of intestinal columnar epithelial cells.

[0122] The expressions of ZO1 and Occludin1 were detected by immunohistochemical method, and the staining results are as Figure 22 shown. IBD can damage the intestinal mucosal barrier, resulting in decreased expressions of ZO1 and Occludin1, indicating that the intercellular TJs are damaged and the intestinal epithelial permeability is increased. However, after GelNB enema, the damage of TJs was reversed, promoting the expressions of Occludin1 and ZO1 to return to normal levels, and the effect of mesalazine was not obvious.

[0123] Example 9: TNBS-induced colitis

[0124] 1. Eight-week-old male C57 BL / 6 (C57) mice were used for the experiments. All experiments were performed strictly in accordance with the institutional guidelines of the Sir Run Run Shaw Hospital Animal Care and Use Committee of Zhejiang University School of Medicine, and were in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (ethical code SRRSH202107106). Colitis was induced by TNBS (Meilunbio) according to the methods described in the literature [C. Trilleaud, V. Gauttier, K. Biteau, I. Girault, L. Belarif, C. Mary, S. Pengam, G. Teppaz, V. Thepenier, R. Danger, G. Robert-Siegwald, M. Néel, S. Bruneau, A. Glémain, A. Néel, A. Poupon, J. F. Mosnier, G. Chêne, M. Dubourdeau, G. Blancho, B. Vanhove, N. Poirier, Agonist anti-ChemR23 mAb reduces tissue neutrophil accumulation and triggers chronic inflammation resolution, Sci Adv 7(14) (2021).] and the literature [N. He, X. Chen, D. Wang, K. Xu, L. Wu, Y. Liu, H. Tao, Q. Zhao, X. Cao, Y. Li, N. Liu, X. Qi, Z. Han, D. Kong, J. Yang, Z. Li, VE-Cadherin regulates the self-renewal of mouse embryonic stem cells via LIF / Stat3 signaling pathway, Biomaterials 158 (2018) 34 - 43.].

[0125] The mice were divided into four groups: control group, PBS group, GelNB group and mesalazine group, with 5 mice in each group. As Figure 23As shown in Figure A, TNBS (100 mg / kg) was dissolved in 50% ethanol to obtain a TNBS solution with a concentration of 2.5%. All mice were first acclimated in a SPF-class animal house for one week and had free access to water and food. Then, the TNBS solution was slowly injected into the rectums of mice in the PBS group, GelNB group, and mesalazine group through a catheter equipped with a 1 mL syringe. The mice were placed in a vertical position for about 1 minute to ensure that the therapeutic reagent could be evenly distributed throughout the colon and cecum. The control group mice were given a 50% ethanol solution. Starting from the second day after TNBS-induced colitis, the mice in the PBS group, GelNB group, and mesalazine group were given 500 μL enema solution every other day (respectively: 1×PBS buffer, 100 mg / mL GelNB solution dissolved in 1×PBS, 0.5% mesalazine solution dissolved in 1×PBS). The mice were sacrificed on the 7th day.

[0126] The colon from the cecum to the anus of the sacrificed mice was taken out, the length of each colon was recorded, and then each colon was washed with 1×PBS buffer. The part near the anus (0.5 cm), that is, the distal colon tissue, was removed for MPO activity measurement and RT-qPCR analysis. The remaining colon tissue was fixed in 4% formaldehyde for further histological and immunohistochemical analysis.

[0127] 2. Clinical evaluation of IBD:

[0128] The evaluation method refers to Example 8.

[0129] In the present invention, the continuous observation one week after TNBS induction was selected as the evaluation time window because there is research showing that the injury can completely disappear on the 7th day after TNBS induction, making it difficult to compare the curative effects among groups. Therefore, the 7-day observation time was considered the best. As Figure 23 shown in Figure B, in the first 5 days before the experiment, the body weights of the three groups of mice all decreased; however, the body weight of the mice in the GelNB group was always the highest among the three groups. In addition, three days after TNBS induction, the weights of the mice in the PBS group and mesalazine group continued to decline, while the weight of the mice in the GelNB group tended to be stable and showed an upward trend, suggesting that the GelNB molecular coating can delay the progression of CD to severe diseases. Correspondingly, as Figure 23 shown, although the DAI scores of both the GelNB and mesalazine groups decreased, the preventive effect of GelNB was higher than that of mesalazine. After euthanasia on the 7th day, the general intestinal examination results of TNBS-induced mice were similar to those of DSS-induced mice. The GelNB molecular coating can also reverse the pathological shortening of the colon length caused by colitis, restoring the colon / body weight to a level close to normal ( Figure 23D, E, F). The colon injury score also demonstrated that GelNB treatment could significantly cure TNBS-induced IBD (P<0.001), and the treatment effect was significantly better than that of mesalazine (P<0.001)( Figure 24 ). Histological examination based on H&E staining showed that the intestinal epithelium in the PBS group was more severely damaged with ulceration and more inflammatory cell infiltration, while the intestinal epithelium in the GelNB group had less severe edema and the histological appearance was closest to normal( Figure 23 G).

[0130] Similarly, the present invention investigated whether the GelNB molecular coating could also act as a physical barrier to regulate the inflammatory response in TNBS-induced IBD. The fluorescence imaging results showed that on the 7th day, the fluorescence intensity of ROS in the peritoneal cavity of mice in the GelNB group was significantly lower than that in the PBS group and the mesalazine group, indicating that the inflammatory level in mice basically decreased to the normal level after treatment with GelNB( Figure 23 H and 23I).

[0131] The present invention also detected the expression levels of a series of pro-inflammatory factors using RT-qPCR, and the results are as Figure 25 shown. The results showed that GelNB treatment significantly reduced the expression of IL1α, IL1β, IL-6, TNF-α, iNOS, and IFN-γ. While mesalazine treatment only alleviated the high-level expression of pro-inflammatory factors.

[0132] Immunohistochemical staining and RT-qPCR analysis of the proliferation index (Ki67) and intestinal barrier index (ZO1 and Occludin1) also showed similar effects to the DSS group( Figure 23 J, Figure 26 A and 26B).

[0133] Note:

[0134] The quantitative RT-PCR involved in the examples of the present invention was carried out by the following method:

[0135] Total RNA was extracted from cells or the colon by adding TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions, and the total RNA was synthesized into cDNA using oligo dT primers with reverse transcriptase (Yeason). Subsequently, real-time PCR reactions were carried out on a 480II (Roche, USA) with a reaction volume of 10 μL. 2 -ΔΔCt -ΔΔCt method was used to measure the relative mRNA fold change. The primers used in the real-time PCR reactions are listed in Table 1.

[0136] Table 1 Primer sequences for real-time reverse transcription polymerase chain reaction (qRT-PCR).

[0137]

[0138]

[0139] In the examples of the present invention, IBM SPSS statistical software version 22 (IBM Corporation, USA) was used for statistical analysis. Student's t-test and one-way analysis of variance were used to analyze the between-group variables. The results were expressed as the median ± standard error of the mean (SEM). Spearman or Pearson correlation analysis was used for correlation analysis. Statistical significance was set at P < 0.05.

Claims

1. Use of bio-gelatin modified with o-nitroso benzaldehyde group in the preparation of drugs for preventing or treating inflammatory bowel disease; The bio-gelatin modified with o-nitroso benzaldehyde group is prepared by the following method: S1. Add 4-(4-hydroxymethyl-3-nitro-2-methoxyphenoxy)butyric acid into the aqueous gelatin solution, and react the carboxyl group in 4-(4-hydroxymethyl-3-nitro-2-methoxyphenoxy)butyric acid with the amino group in gelatin to obtain bio-gelatin modified with o-nitrobenzyl alcohol group; S2. After irradiating the bio-gelatin modified with o-nitrobenzyl alcohol group with a near-ultraviolet light source, convert it into bio-gelatin modified with o-nitroso benzaldehyde group.

2. The application according to claim 1, characterized in that The specific implementation of step S1 is as follows: Add 4-(4-hydroxymethyl-3-nitro-2-methoxyphenoxy)butyric acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide into dimethyl sulfoxide to obtain a mixed solution; then slowly add the mixed solution into the gelatin solution, react at 40-50 °C for 3-5 hours, dialyze the obtained reaction solution with distilled water for 4-6 days, collect and lyophilize to obtain bio-gelatin modified with o-nitrobenzyl alcohol group; the molar ratio of 4-(4-hydroxymethyl-3-nitro-2-methoxyphenoxy)butyric acid to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:1, and the feeding ratio of 4-(4-hydroxymethyl-3-nitro-2-methoxyphenoxy)butyric acid to gelatin is 2:

1.

3. The application according to claim 1, characterized in that The specific implementation of the step S2 is as follows: Dilute the bio-gelatin modified with o-nitrobenzyl alcohol groups with PBS buffer solution to obtain a solution, and irradiate it with a near-ultraviolet light source with a power density of 30-35 mW / cm 2 , and the irradiation time is 15 s - 2 min to obtain the bio-gelatin modified with o-nitroso-benzaldehyde groups.

4. The application according to claim 1, wherein: The wavelength of the near-ultraviolet light source is 365 nm.

5. The application according to any one of claims 1 to 3, characterized in that The inflammatory bowel disease is Crohn's disease or ulcerative colitis.

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