Use of melanin in preparing medicine for treating chronic ulcerative colitis and its associated depressive-like behavior

Abstract

The present invention relates to the field of biomedicine technology, and discloses a use of melanin in the preparation of a drug for treating chronic ulcerative colitis and its associated depressive-like behavior. The present invention is the first to discover that melanin has the efficacy of treating chronic ulcerative colitis and its associated depressive-like behavior. MSPI can inhibit the LPS / TLR4 / NLRP3 / ASC / Caspase‑1 inflammatory signaling pathway, reduce oxidative stress, and restore the intestinal barrier, thereby improving UC symptoms. In addition, MSPI can also reduce glial cell activation, brain neuroinflammatory response and neuronal apoptosis, thereby reducing depressive-like behavior.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to use of melanin in preparing a medicine for treating chronic ulcerative colitis and its associated depressive-like behavior. Background Art

[0002] The clinical manifestations of inflammatory bowel disease (IBD) include paroxysmal abdominal pain, diarrhea, bloody stools, and weight loss. Pathological causes include neutrophils and macrophages passing through the intestinal barrier, abnormal cytokine secretion, and increased proteolytic enzymes and free radicals, leading to inflammation and ulcers. At the beginning of the 21st century, IBD had become a global disease, with an incidence rate higher than 0.3% in Western industrialized countries. In the past 20 years, the incidence and prevalence of IBD in developing countries such as South America, the Middle East, and Asia have risen sharply. According to epidemiological predictions, by 2025, the number of patients with inflammatory bowel disease in China will reach 1.5 million, which poses a severe challenge to the medical system. Inflammatory bowel disease, with an increasing prevalence and incidence year by year, has become a global disease with a long course, difficult treatment, and repeated attacks, which seriously affects the physical health and quality of life of patients.

[0003] In addition, studies have shown that in addition to the main manifestations such as intestinal damage, IBD patients are often accompanied by depressive-like behaviors, and this symptom is more obvious during the active stage of ulcerative colitis. However, most of the research on inflammatory bowel disease is currently focused on colon tissue lesions, and the research on depressive-like behaviors is not in-depth. The pathological characteristics of IBD are complex, and the existing conventional treatment drugs include antibiotics, immunosuppressants, etc., which cannot be completely cured and have a high recurrence rate, large toxic side effects, and can easily cause serious damage to the human body's physiology and psychology.

[0004] Natural active substances have the characteristics of high safety, few side effects, and diverse targets, and have been widely used in dietary supplements and natural medicines for chronic diseases. The catch of marine cephalopods has increased year by year, but byproducts such as ink sacs are discarded after processing, which is not conducive to resource utilization and ecological environment development. Ink sacs account for about 1.3% of the body weight of cephalopods, and its main chemical component, melanin, is the highest quality natural source of eumelanin. Therefore, the development of melanin is of great significance to improving the utilization rate of seafood processing waste and promoting the development of marine bioactive substances.

[0005] Melanin from cuttlefish ink (MSPI) is a natural bioactive macromolecule composed of 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid molecules. In earlier studies, MSPI was a spherical particle with a diameter of 90-140 nm and a molecular weight of 376. Melanin has a variety of pharmacological effects, including antioxidant, anti-inflammatory, anti-obesity, anti-aging, and reliable biocompatibility. However, the effects of MSPI on chronic ulcerative colitis (UC) and its accompanying depressive-like behavior have not been reported. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a use of melanin in preparing a medicine for treating chronic ulcerative colitis and its associated depressive-like behavior.

[0007] In order to achieve the above-mentioned purpose, the technical solution of the present invention is: use of melanin in preparing a medicine for treating chronic ulcerative colitis and its associated depressive-like behavior.

[0008] Furthermore, the drug contains a therapeutically effective dose of melanin and pharmaceutically acceptable excipients.

[0009] Furthermore, the dosage form of the drug is a tablet.

[0010] Furthermore, the dosage form of the drug is a capsule.

[0011] Furthermore, the dosage form of the drug is an injection.

[0012] Beneficial effects of the present invention: The present invention first discovered that melanin has the efficacy of treating chronic ulcerative colitis and its associated depressive-like behavior. MSPI can inhibit the LPS / TLR4 / NLRP3 / ASC / Caspase-1 inflammatory signaling pathway, reduce oxidative stress, and restore the intestinal barrier, thereby improving UC symptoms. In addition, MSPI can also reduce glial cell activation, brain neuroinflammatory response and neuronal apoptosis, thereby alleviating depressive-like behavior. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the experimental design diagram of the chronic UC model;

[0014] Figure 2 This is a comparison of the length of the mouse colon;

[0015] Figure 3 It is a comparison chart of the disease activity index (DAI) of mice;

[0016] Figure 4 is a comparison of H&E staining (×100) of distal mouse colon tissue;

[0017] Figure 5It is the AB staining (×100) image of mouse colon tissue and the colon tissue scoring image; the total score of AB staining is the sum of the staining intensity score and the positive cell ratio score; staining intensity score: 0 point: no staining, no expression of the target protein was detected; 1 point: weak staining, the target protein expression is weak, the color is light and difficult to identify; 2 points: moderate staining, the target protein expression is clearly visible, the color is moderate; 3 points: strong staining, the target protein expression is strong, the color is dark and obvious; positive cell ratio score: 0 point: 0%, no cells are positive; 1 point: less than 10%, a few cells are positive; 2 points: 10% to 50%, some cells are positive; 3 points: more than 50%, most cells are positive;

[0018] Figure 6 It is the expression diagram of LPS and inflammatory factors IL-Iβ, IL-6, IFN-γ and TNF-α in mouse colon tissue;

[0019] Figure 7 is a Western blot analysis of Caspase-1, ASC, TLR4, and NLRP3 levels in mouse colon tissue and their content bar graphs;

[0020] Figure 8 The immunohistochemistry and scoring diagrams of MCP-I and F4 / 80 in mouse colon tissue: A is the immunohistochemistry and scoring diagram of MCP-1 in mouse colon tissue, and B is the immunohistochemistry and scoring diagram of F4 / 80 in mouse colon tissue; Immunohistochemistry score = ∑ (pi × i) = (percentage of weakly positive area × 1) + (percentage of moderately positive area × 2) + (percentage of strongly positive area × 3); pi: pixel area of ​​positive signal; i: positive level;

[0021] Fig. 9 is the immunofluorescence staining of ROS in mouse colon tissue (×100) and the ROS level;

[0022] Fig.10 is a graph of MDA and 8-OHdG levels in mouse colon tissue;

[0023] Fig.11 The immunohistochemical and histological scoring diagram of proteins (Caspase-3, Bax, Bcl-2, ZO-1 and Occludin) in mouse colon: A is Caspase-3, B is Bax, C is Bcl-2, D is ZO-1, and E is Occludin; Immunohistochemical score = ∑ (pi × i) = (percentage of weakly positive area × 1) + (percentage of moderately positive area × 2) + (percentage of strongly positive area × 3); pi: pixel area of ​​positive signal; i: positive level;

[0024] Fig.12It is the alpha diversity analysis of mouse intestinal flora: A is the cluster analysis diagram of mouse intestinal flora based on PCA and PCoA scores, and B is the Venn diagram of mouse intestinal microbial species;

[0025] Fig.13 are Chao1, Coverage, Shannoneven, Shannon, and Simpson indices of mouse intestinal flora;

[0026] Fig.14 is the species composition of the mouse gut microbiota at the phylum level;

[0027] Fig.15 is the species composition of the mouse gut microbiota at the family level;

[0028] Fig.16 is the species composition of the mouse gut microbiota at the genus level;

[0029] Fig.17 It is the score graph of the depressive-like behavior (OFT, MBT, TST, FST and 8-arm maze) of mice;

[0030] Fig.18 These are the H&E staining and AB staining scoring images of mouse brain tissue: A is the brain tissue H&E staining (×100) image, and B is the brain tissue AB staining (×200) and scoring image; the AB staining score is the sum of the staining intensity score and the positive cell ratio score; staining intensity score: 0 point: no staining, no expression of the target protein was detected; 1 point: weak staining, the target protein expression is weak, the color is light and difficult to identify; 2 points: moderate staining, the target protein expression is clearly visible, the color is moderate; 3 points: strong staining, the target protein is strongly expressed, the color is dark and obvious; positive cell ratio score: 0 point: 0%, no cells are positive; 1 point: less than 10%, a few cells are positive; 2 points: 10% to 50%, some cells are positive; 3 points: more than 50%, most cells are positive;

[0031] Fig.19 The levels of brain inflammation and the expression of synapse-related proteins in mice: A is a bar graph of LPS content in brain tissue, and B is a Western blot and a bar graph of Caspase-1, ASC, TLR4, and NLRP3 content in brain tissue;

[0032] Fig. 20 It is the level of inflammation in the mouse brain and the expression of synapse-related proteins, and it is a graph of the levels of inflammatory factors IL-Iβ, IL-6, IFN-γ, and TNF-α in brain tissue;

[0033] Fig.21Figure 1 is the IHC staining and histological scoring of Syn, DCX, BDNF and PSD-95 in mouse brain tissue: A is Syn, B is DCX, C is BDNF, and D is PSD-95; Immunohistochemical score = ∑ (pi × i) = (percentage of weakly positive area × 1) + (percentage of moderately positive area × 2) + (percentage of strongly positive area × 3); pi: pixel area of ​​positive signal; i: positive level;

[0034] Fig. 22 is the expression of oxidative stress in mouse brain tissue: A is the immunofluorescence image of brain tissue ROS, B is the image of brain tissue ROS, MDA and 8-OHdG content levels, C is the immunohistochemical staining image and histological score image of Bax in brain tissue, and D is the immunohistochemical staining image and histological score image of Bcl-2 in brain tissue; Immunohistochemical score = ∑ (pi × i) = (percentage of weak positive area × 1) + (percentage of medium positive area × 2) + (percentage of strong positive area × 3); pi: pixel area of ​​positive signal; i: positive level;

[0035] Fig.23 is the expression of apoptosis and glial cells in mice: A is the immunohistochemical staining and histological scoring of Caspase-3 in brain tissue, B is the TNUEL staining and scoring of brain tissue, C is the immunohistochemical staining and histological scoring of IBA-1 in brain tissue, and H is the immunohistochemical staining and histological scoring of GFAP IHC in brain tissue; Immunohistochemical score = ∑ (pi × i) = (percentage of weakly positive area × 1) + (percentage of moderately positive area × 2) + (percentage of strongly positive area × 3); pi: pixel area of ​​positive signal; i: positive level;

[0036] In the figure: compared with the blank group, #P<0.05, ##P<0.01; compared with the model group, *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0038] The experimental materials used in the examples of the present invention but not specifically described are all conventional experimental materials in the art and can be purchased through commercial channels.

[0039] Embodiment 1:

[0040] Melanin for the treatment of chronic ulcerative colitis and its associated depressive-like behavior.

[0041] 1. Experimental Animals

[0042] Male C57BL / 6J mice (8 weeks old, 20-25 g) were purchased from Jinan Pengyue Experimental Animal Co., Ltd. Before the experiment, mice were raised under standard conditions (temperature 25±2°C, humidity 55±5%) for 7 days, four mice per cage, and fed corn feed at a dosage of 5g / mouse / day. After 7 days of adaptive feeding, the mice were randomly divided into three groups (8 mice / group), including a blank group, a model group, and an MSPI (150 mg / kg / day) group. The blank group and the model group were only given normal saline for seven consecutive days, and corn feed was fed at a dosage of 5g / mouse / day. The MSPI group (melanin group) was given a dose of 150 mg / kg / day of MSPI for seven consecutive days, and corn feed was fed at a dosage of 5g / mouse / day. Then, 2 mL of 2% DSS solution was added for 5 consecutive days in the model group and the MSPI group, and the DSS solution was stopped for the next 14 days, and only normal saline was given for gavage. Subsequently, 2 mL of 2% DSS solution was added for 5 days, and then the DSS solution was stopped for the next 14 consecutive days, and only saline was given for gavage. This cycle established a chronic UC model ( Figure 1 ). After the behavioral experiment, the mouse feces were collected under sterile conditions, and the mice were anesthetized with 10% ether and cervical dislocation was performed. The colon and brain tissues of the mice were quickly frozen in liquid nitrogen and stored at −80 °C, or collected in 4% paraformaldehyde for subsequent biochemical analysis. All animal experiments were approved by the Animal Ethics Committee of Qingdao University of Science and Technology (No. SYXK 2022-1009).

[0043] 2. Test methods

[0044] 1. Disease Activity Index (DAI) and Depression-like Behavior Test

[0045] The disease activity index score was used to assess the severity of chronic colitis. DAI is composed of three scores: weight change, stool bleeding, and stool consistency. There are five levels of weight loss (0, no weight loss or weight gain; 1, 1%-10%; 2, 10%-15%; 3, 15%-20%; 4, >20%), two levels of stool consistency (0, normal; 4, diarrhea), and two levels of stool bleeding (0, normal; 4, severe stool bleeding). After DSS intervention, DAI was calculated using the comprehensive scores of the three situations. That is, DAI = (body mass index + stool shape + bleeding) / 3. Subsequently, the mice were subjected to the open field test (OFT), marble burying test (MBT), eight-arm maze test (8-Arm-Maze), forced swimming test (FST), and tail suspension test (TST) to assess the depressive behavior of the mice.

[0046] (1) Open field test

[0047] In the open field test, mice were tested for open field anxiety and exploratory activity. The test equipment was a 50×50cm 2 A square test open field with a height of 40 cm. At the beginning of the experiment, pinch the mouse's tail at the proximal 1 / 2 to 1 / 3, and gently place it in the central grid of the open field test box. After 1 minute of adaptation, the mouse's behavior was recorded within the next 4 minutes. During the experimental interval, wipe the open field with 75% ethanol to eliminate odor interference. At the same time, the camera hanging on it records the video in real time. After the experiment, the total movement distance (total distance) and the distance moved in the center (central distance), as well as the peripheral movement distance (peripheral distance) of the mouse are recorded as indicators of exploratory activity and depression.

[0048] (2) Marble Burial Experiment

[0049] During the marble burying experiment, mice were placed in a sterile cage covered with 5 cm high corn cob bedding, on which 20 marbles were evenly arranged (arranged in 4 rows and 5 columns). Mice were allowed to explore freely for 30 minutes, and then photos were taken to record their marble burial (each marble was marked as buried if more than two-thirds of its volume was covered by bedding). After the mouse finished exploring, the marbles and the mouse cage were wiped with 75% ethanol and new bedding was replaced for the next mouse experiment.

[0050] (3) Eight-arm maze test

[0051] Spatial memory was assessed using an eight-arm radial maze, which consisted of eight equally spaced arms radiating outward from an octagonal central platform. The experiment lasted for five days. Before the experiment began, mice were restricted to a diet that was maintained at 85% of their normal body weight. The mice were first habituated to the radial arm maze for two days, 10 min per day, and then placed in the center of the maze, allowing each mouse to move freely. On the third day, bait was placed at the end of each arm and the mice were allowed to explore freely. On the fourth day, four arms were randomly selected to place food at the end, and the training trial ended after all eight arms were completed (if the entire body was on the arm, except for the tail, the mouse was considered to have entered an arm of the maze) or 10 min later. On the fifth day, the same actual test as on the fourth day was performed to observe whether the mice remembered the arm in which the food was placed during the experiment. The maze was wiped clean with 75% ethanol between each trial to prevent any odor clues. The activity behavior of the mice during the entire experiment was recorded using a camera. After the trial, the working memory errors and reference memory errors were analyzed to assess short-term and long-term memory errors. Only first entry into the baited arm was recorded as a correct choice, with reentry into a previously entered arm counted as a working memory error and entry into the non-baited arm counted as a reference memory error.

[0052] (4) Forced swimming test

[0053] During the experiment, mice were placed in a transparent cylinder with a diameter of 17.5 cm and a height of 20 cm. The water in the cylinder was 10 cm high and the water temperature was maintained at 25°C to prevent the mice from hypothermia. The mice were videotaped for 6 minutes. The total time the mice remained motionless was measured in the last 4 minutes of the test. Mobility was defined as a situation where the mouse was in a static posture, that is, there was no movement except for the movements necessary to keep the head above the water, and the stopwatch was used for manual scoring. At the end of the test, the mice were dried in a warm cage for 15 minutes and then returned to the cage. Clean water was replaced between each experiment.

[0054] (5) Tail suspension test

[0055] The mouse tail was fixed on a horizontal bar (about 1 cm from the tail tip) and suspended from the tail, with the distance between the nose tip of the mouse and the ground about 20 cm. The mouse remained suspended for 6 minutes and recorded. At the beginning, the mouse showed active activity to adapt to the environment, and after a period of time, there would be intermittent static behavior, which means that the mouse was in a desperate state at this time. After the experiment, the observer scored the time each individual spent immobile in the last 4 minutes of the 6 minutes.

[0056] 2. Hematoxylin-eosin (H&E) and Alcian blue (AB) staining

[0057] The colon and brain tissues fixed with formaldehyde were vertically embedded in paraffin, cut into 5 μm sections, dewaxed and hydrated, and frozen for later use. The sections were stained with H&E and AB, respectively. The tissues were observed under an optical microscope and scored by determining the degree of cell damage and inflammatory cell infiltration.

[0058] 3. Nissl staining

[0059] The frozen sections were fixed with 4% PFA fixative for 20 minutes, then rinsed with distilled water, stained with Nissl staining for 5 minutes, then rinsed twice with distilled water, and then rinsed with 70% ethanol. Finally, the hippocampal images were observed using a fluorescence microscope, and the neuronal cells with clear and complete hippocampal dentate gyrus and uniformly distributed Nissl vesicles around the nucleus were counted using Image Pro-Plus software.

[0060] 4. Cell apoptosis assay (TUNEL)

[0061] Cell apoptosis in the above frozen sections was detected using an apoptosis detection kit (Roche, Indianapolis, IN, USA). Microscope photography was performed (BX53, Olympus, Tokyo, Japan). The percentage of the total number of positive cells was calculated based on the average number of positive cells in five different fields of view. Apoptosis index = number of apoptotic cells / total number of counted cells × 100.

[0062] 5. Western blot (WB) detection

[0063] Colon and brain tissues were lysed separately with pre-chilled RIPA lysis buffer, and total protein was extracted according to the manufacturer's instructions. After 12% SDS-PAGE separation, the protein lysate was transferred to a PVDF membrane. The membrane was incubated overnight at 4°C with a specific primary antibody (1 / 1000), and then incubated with an appropriate secondary antibody. The image was displayed on a gel system using ECL.

[0064] 6. Enzyme-linked immunosorbent assay (ELISA)

[0065] Freshly excised colon and brain tissues were homogenized with potassium dihydrogen phosphate buffer (pH 6.8), centrifuged at 10,000 g for 15 min at 4 °C, and the supernatant was collected. The contents of TNF-α, IL-6, IFN-γ, IL-1β, MDA, and 8-OHdG in colon and brain tissues were detected by ELISA kits, and the protein content was detected by BCA protein detection kit. The absorbance was measured at a wavelength of 450 nm.

[0066] 7. Immunohistochemistry (IHC) assay

[0067] Freshly excised colon and brain tissues were fixed by immersion in 4% paraformaldehyde at 4°C and incubated with primary antibodies. After washing three times with PBS, colon and brain tissues were developed with appropriate secondary antibodies and photographed under an optical microscope. The number of cells was measured using ImageJ software. For immunofluorescence, the expression level of ROS in tissues was detected by double antigen immunofluorescence and analyzed by detecting fluorescein using a fluorescence microscope. The selected area within the threshold signal intensity was measured using ImageJ software. H-Score: H-SCORE = ∑ (pi × i) = (percentage of weak positive area × 1) + (percentage of medium positive area × 2) + (percentage of strong positive area × 3). pi: pixel area of ​​positive signal; i: positive level.

[0068] 8. 16S rRNA analysis of fecal microbial flora

[0069] Sequencing was performed on the Majorbio platform. Total DNA was obtained from each sample using the FastDNA spin kit, and the results of PCR amplification were collected for fluorescence quantification. Quantification was performed using a microplate reader (Flx800, BioTek, USA). The samples were proportionally distributed according to the results of fluorescence quantification and the amount of sequencing required. The v3-v4 highly variable region of the 16S rRNA gene was amplified and sequenced on an Illumina sequencer in PE150 mode.

[0070] 3. Experimental Results

[0071] 1. MSPI can improve the symptoms of chronic colitis caused by DSS

[0072] After DSS intervention, mice showed symptoms such as weight loss, diarrhea, and blood in feces. Compared with the blank group, mice in the model group showed obvious UC symptoms, such as shortened colon ( Figure 2 ) and DAI scores were significantly increased ( Figure 3 ). MSPI intervention significantly alleviated the UC symptoms of mice, reduced intestinal wall swelling, and restored intestinal length to normal. H&E staining ( Figure 4 ) showed that the colon mucosa of the blank group mice was not damaged, the villi were clearly visible, the crypts were intact, the glands were neatly arranged, and there was no inflammatory cell infiltration or ulcer. AB staining ( Figure 5 ) showed that the goblet cells in the colon tissue of the model group were significantly reduced, resulting in severe damage to the mucus layer and very little mucus left. Compared with the model group, MSPI intervention therapy helped to restore the integrity of the mucus layer (P < 0.01) and maintain the physical protective barrier.

[0073] 2. MSPI can reduce inflammatory response and intestinal macrophage infiltration

[0074] Lipopolysaccharide (LPS) can stimulate immune system cells and induce strong inflammatory responses. LPS in mouse tissues was extracted using an LPS kit to obtain an extract, and the absorbance was measured at 450nm using an enzyme-labeled detector. Figure 6 It can be seen that the LPS content in the colon tissue of the model group mice was significantly higher than that in the blank group, while that in the MSPI group was significantly lower than that in the model group (P<0.01). WB was used to detect the expression of TLR4, NLRP3, ASC and Caspase-1 inflammasome in mice ( Figure 7 ), ACTIN monoclonal antibody (ACTIN) was used as the standard reference. The results showed that the expression of the above proteins in the colon of the model group mice was significantly increased compared with the blank group (P<0.01). In addition, the immunohistochemical results showed that ( Figure 8 ), the high expression of monocyte chemoattractant protein-1 (MCP-1) and macrophage marker F4 / 80 in the colon tissue of mice in the model group was significantly higher than that in the blank group (P<0.01). On the contrary, after MSPI intervention, its high expression in the colon was significantly reversed.

[0075] 3. MSPI reduces oxidative stress and apoptosis and enhances intestinal barrier in UC mice

[0076] Oxidative stress is a typical feature of UC. Fig. 9As shown in the figure, compared with the blank group, the level of reactive oxygen species (ROS) in the model group mice was significantly increased, while the intervention of MSPI had a significant inhibitory effect on ROS (P < 0.01). ELISA results showed that compared with the blank group, the levels of MDA (malondialdehyde) and 8-OHdG in the model group were significantly increased, while the intervention of MSPI significantly reduced the expression of oxidative stress ( Fig.10 ). Moreover, the expressions of Caspase-3 and Bax in the model group were significantly higher than those in the blank group, while the expression of Bcl-2, a protein that inhibits cell apoptosis, was inhibited ( Fig.11 MSPI reversed the expression of the above proteins.

[0077] Apoptosis increases intestinal permeability and exacerbates intestinal mucosal barrier dysfunction. To further determine whether MSPI intervention can improve the integrity of the intestinal barrier, we used IHC staining to detect the expression levels of tight junction (TJ) proteins (Ocludin, ZO-1) in the mouse colon ( Fig.11 The expression levels of TJ proteins in the model group were significantly lower than those in the blank group (P<0.01), while after treatment with MSPI, the expression levels of the above TJ proteins were significantly restored (P<0.01).

[0078] 4. MSPI regulation in the gut microbiota

[0079] Disruption of the intestinal microbiota can lead to inflammation and abnormal intestinal environment. Fig.12 It can be seen that the composition of the intestinal microbiota in the model group changed significantly. Chao1, Coverage, Shannoneven, Shannon, and Simpson indices were used to evaluate the α-diversity of the microbiota ( Fig.13 ), compared with the model group, oral administration of MSPI significantly increased microbial diversity and relative abundance, compared with the blank group, the microbial diversity and relative abundance of the MSPI group were significantly reduced (P < 0.01), and the MSPI group was closer to the blank group. The high-throughput sequencing results of different groups were analyzed for the β diversity of intestinal microbiota using principal component analysis (PCA) and principal coordinate analysis (PCoA) Fig.12 Middle A), the results showed that the blank group and the MSPI group were clustered together and clearly separated from the model group.

[0080] Subsequently, by analyzing the relative abundance of various microorganisms at a specific taxonomic level, it was found that ( Fig.14 , Fig.15 , Fig.16), at the phylum level, the relative abundance of Bacteroidetes and Actinobacteria in model mice decreased by 22% and 4.6%, respectively, while the relative abundance of sessile bacteria and vermiform microorganisms in model mice increased by 20% and 5.6%, respectively. The intervention of MSPI reversed this trend. In addition, the probiotics in healthy mice were mainly Mussolini, Duboisella, Bifidobacterium and Lactobacillus. However, through the intervention of MSPI, their abundance was significantly restored, so that the composition of the intestinal microbiota tended to be normal.

[0081] 5. MSPI can significantly alleviate depressive-like behaviors

[0082] The results showed that in the OFT experiment, the average central activity distance of mice in the model group was significantly reduced, and the peripheral exploratory behavior increased ( Fig.17 ). MSPI intervention can effectively increase the central activity distance of mice. In the MBT, the mice in the model group could bury 76.7±3.6% of the marbles, while the mice in the MSPI group could bury 9.3±1.2% of the marbles, which significantly reduced the stereotyped repetitive behavior of the mice. When performing TST, the mice in the model group remained still for a significantly longer time (P<0.01), which represented a longer period of despair. The results of TST were comparable to those of FST. The reduction in stillness time and the increase in spontaneous activity time indicate that MSPI intervention is beneficial for depressive mice. Finally, the results of the 8-arm maze showed that the working memory errors and reference memory errors of the model mice increased significantly, while the MSPI intervention significantly reduced the error frequency.

[0083] 6. MSPI can reduce neuronal damage

[0084] H&E staining results ( Fig.18 Middle A) showed that the cells in the brain tissue of the model group mice were arranged irregularly, the number of normal cells decreased, the nucleus shrank, the infiltration of inflammatory cells increased, and the hippocampus showed mild edema, neutrophil fixation and other neuronal degeneration symptoms. MSPI intervention significantly reduced the number of infiltrating cells, restored cell morphology, and improved the neuronal degeneration symptoms in the hippocampus of mice. Similar results were also observed by Nissl staining ( Fig.18 Middle B). The neuronal cell structure of the blank group mice was intact, with regular morphology, obvious nuclei, abundant Nissl bodies in the cytoplasm, and obvious pyramidal neurons. The neuronal cell morphology of the MSPI group was improved, cell edema was reduced, the number of Nissl bodies increased, and both morphology and number were significantly improved.

[0085] 7. MSPI modulates inflammatory pathways in the mouse brain and reduces the expression of inflammatory cytokines

[0086] The mechanism of antidepressant treatment involves reducing the levels of neuroinflammatory factors in the brain. Fig.19), chronic UC significantly increased the levels of inflammatory proteins TLR4, NLRP3, ASC, and Caspase-1 in the mouse brain, while MSPI intervention significantly reduced their levels. In addition, the expression of proinflammatory cytokines in the brain of model mice was significantly increased, while MSPI intervention significantly inhibited the increase in proinflammatory cytokine levels ( Fig. 20 ).

[0087] 8. MSPI reverses synaptic protein damage caused by chronic UC

[0088] Neurons in the brain are connected by synapses. The results showed that postsynaptic density 95 (PSD-95) and synaptophysin (Syn) were significantly downregulated in the model group ( Fig.21 ), while it recovered in the MSPI group. In addition, compared with the blank group, doublecortin (DCX) and brain-derived neurotrophic factor (BDNF) in the model group were reduced (P<0.01). Therefore, we hypothesized that MSPI could improve cognitive dysfunction and depressive-like behavior by increasing the expression of synapse-related proteins.

[0089] 9. MSPI reduces oxidative stress in the mouse brain

[0090] Depression-like behaviors are often accompanied by increased oxidative stress in the brain. Data show that ( Fig. 22 The expression level of ROS in brain tissue of the model group was significantly higher than that of the blank group (P<0.01), and was significantly lower than that of the model group after MSPI intervention (P<0.01). ELISA test showed that the expression levels of 8-OHdG and MDA in brain tissue of the MSPI group were significantly lower than those of the model group (P<0.01).

[0091] 10. MSPI regulates apoptosis in brain tissue of chronic UC mice

[0092] By evaluating UC-induced brain cell apoptosis, it was found that the expression of Bcl-2 was reduced in the model group, while the expression of Caspase-3 and Bax was significantly increased. The intervention of MSPI significantly reversed this change ( Fig. 22 Medium CD, Fig.23 Then, the concentration of apoptotic cells in the mouse brain was measured by TUNEL staining ( Fig.23 Middle B), Depressive symptoms observed in model mice were accompanied by a significant increase in brain cell apoptosis. MSPI treatment reduced the increase in apoptosis to levels close to those of the control group.

[0093] 11. MSPI can reduce the activation of microglia and astrocytes in the mouse brain

[0094] Chronic activation of microglia and astrocytes can lead to the continuous production of proinflammatory cytokines, thus inducing neurodegenerative diseases. The immunohistochemical results of IBA-1 and GFAP showed that compared with the blank group, the microglia and astrocytes in the model group showed enlarged somites, retracted bifurcated processes, thickened processes, and increased number of processes ( Fig.23 ). In addition to morphological changes, quantitative analysis also found that the number of activated astrocytes and microglia in the model group was significantly higher than that in the blank group, while the number of activated microglia and astrocytes in the MSPI group was significantly lower than that in the model group (P<0.01). This suggests that the intervention of MSPI can regulate neuroinflammation by controlling the activation of glial cells.

[0095] 4. Results and Discussion

[0096] As the incidence and prevalence of IBD increase, its comorbidities depression and other central nervous system diseases are also gaining attention. The DSS-induced UC mouse model is similar to human UC and is a practical method to study the pathogenesis and drug treatment of UC. The mucus layer plays a key role in intestinal homeostasis, and the protective mucins and other proteins secreted by goblet cells are essential for the integrity of barrier function and the suppression of microbiota-driven intestinal inflammation. Their deficiency triggers inflammation. Pathological analysis of mouse colon confirmed that mucus-producing epithelial cells were severely lost after DSS-induced UC. MSPI can ameliorate inflammatory damage in the colon, help restore the integrity of the mucus layer and maintain the physical protective barrier.

[0097] LPS is a major component of the cell wall of most Gram-negative bacteria, which can stimulate immune system cells and induce a strong inflammatory response. Increased proinflammatory factors in the intestine trigger an inflammatory cascade, leading to colonic tissue damage and chronic inflammation. Macrophage infiltration is one of the histological features of UC and persists along with inflammation. The role of intestinal macrophages and their impact on the development of intestinal inflammation are regulated by multiple factors, such as the production of MCP-1, which causes macrophages to further aggregate and accelerate inflammation of colonic tissue, while the expression of cell surface glycoprotein F4 / 80 is limited to the macrophage lineage and is a marker of macrophages. Our study showed that MSPI intervention significantly reversed the DSS-induced increase in intestinal LPS and inflammatory factors, and played a role in alleviating intestinal inflammation.

[0098] Oxidative stress is usually defined as damage caused by an imbalance between the antioxidant system and toxic ROS. MDA is the end product of ROS-induced cell membrane lipid peroxidation, while 8-OHdG is a marker of DNA damage and oxidative stress. In addition, oxidative stress triggers epithelial cell apoptosis. Bcl-2 is the first regulatory protein found to prevent apoptosis. A reduced ratio of Bcl-2 to Bax can enhance mitochondrial apoptotic signaling by activating caspase-3, thereby inducing apoptosis, increasing intestinal permeability, and exacerbating intestinal mucosal barrier dysfunction, which is a major pathogenic factor in UC. The intestinal epithelium uses TJ proteins to maintain the integrity of the intestinal barrier, and the destruction of the intestinal barrier is mainly mediated by inflammatory factors. Intestinal epithelial cell apoptosis affects TJ proteins. These proteins are mainly composed of Ocludin and ZO-1, which can prevent bacterial adhesion and bacterial expansion into host tissue cells. Compared with the blank group, apoptosis-related proteins were significantly expressed in the model group, the expression of the apoptosis-inhibiting protein Bcl-2 was inhibited, and the expression level of TJ proteins was significantly lower than that in the blank group. Intervention with MSPI reversed the above changes, indicating its ability to reduce oxidative stress, decrease cell apoptosis, and repair the intestinal barrier.

[0099] When the intestinal mucosal barrier is damaged, the mucosal permeability increases, and small molecules such as LPS produced by the intestinal microbiota are more likely to enter the blood through the intestinal cavity, cross the blood-brain barrier, stimulate the brain inflammatory pathway, promote the release of inflammatory cytokines, lead to neuroinflammation and degenerative lesions, and thus aggravate cognitive impairment. One of the mechanisms of antidepressant treatment may be to reduce the level of LPS in the brain. Our results show that chronic UC significantly increases the level of LPS in the brain, activates the LPS / TLR4 / NLRP3 / ASC / Caspase-1 inflammatory pathway, and accelerates the expression of inflammatory factors in the model group, while MSPI can reduce inflammatory factors by inhibiting the inflammatory pathway.

[0100] Oxidative stress in the brain increases neuronal apoptosis and DNA damage, and is considered to be one of the pathogenic factors of depression. Organisms under oxidative stress will lead to increased secretion of proteases and produce a large number of oxidative compounds. At the same time, a large number of transcription factors related to inflammation are activated under oxidative stress, initiating the inflammatory process and increasing the secretion of proinflammatory cytokines. This is consistent with our results that chronic UC increases the level of oxidative stress in the body and causes oxidative damage to brain tissue, while MSPI can reduce tissue damage caused by oxidative stress and have a positive effect on the regulation of depressive-like behavior compared with the model group.

[0101] Excessive apoptosis is an important cause of depressive-like behavior. The experimental results confirmed that MSPI has neuroprotective ability against inflammation-induced apoptosis and depressive-like behavior. Synaptic-related proteins are involved in the regulation of synaptogenesis and neurotransmitter release, and play a key role in synaptic plasticity and memory formation. PSD-95 and SYN are two important scaffolding proteins with key functions in synaptic plasticity and synaptic enhancement. Compared with the blank group, these two proteins were significantly downregulated in the model group. DCX is a microtubule protein that is essential for the normal migration of neurons during development and has been widely used as a marker of postmitotic immature neurons and an indicator for judging brain damage. Our experimental results showed that the hippocampus of the model mice showed signs of neuronal lesions, and the levels of synaptic-related proteins were downregulated compared with the blank group. In addition, some key regulatory proteins related to synaptic plasticity, such as BDNF, were also significantly reduced in the model group. Therefore, our results show that MSPI can improve cognitive dysfunction and depressive-like behavior by restoring the expression of synaptic-related proteins.

[0102] Glial cells are resident immune cells in the brain and usually respond to pathological stimuli such as injury, stress, and infection. Learning and memory behaviors are also affected by glial cell activation. Astrocytes and microglia are brain cells that are mainly responsible for immune regulation and inflammatory responses. Activated microglia can destroy neurons through phagocytosis or produce neurotoxic cytokines and chemokines, leading to neurological dysfunction and apoptosis, thereby promoting neuronal death or inhibiting neuronal function. IBA-1 is a key player in microglial membrane actin cross-linking. Its expression increases with microglial activation and is a marker of microglial activation. Astrocytes are located between neurons and capillaries and are an important component of the blood-brain barrier of the normal brain nervous system. After brain injury, astrocytes are activated and show neurotoxic effects, inhibiting neuronal cell growth and axon regeneration. GFAP is a marker of astrocyte activation, and morphological changes of astrocytes can be observed after immunostaining. Our results show that compared with the model group, MSPI intervention can regulate neuroimmune responses by inhibiting the activation of glial cells.

[0103] In conclusion, this study provides strong evidence for the potential mechanism of MSPI in treating chronic UC and its accompanying depressive-like behaviors. The results showed that MSPI can inhibit the LPS / TLR4 / NLRP3 / ASC / Caspase-1 inflammatory signaling pathway, reduce oxidative stress, and restore the intestinal barrier, thereby improving UC symptoms. In addition, MSPI can reduce glial cell activation, brain neuroinflammatory response, and neuronal apoptosis, thereby alleviating depressive-like behaviors. This suggests that MSPI can be used as a drug ingredient to treat chronic UC and its accompanying depressive-like behaviors through the key microbiota-gut-brain axis pathway.

[0104] The above-described embodiments are only preferred solutions of the present invention and are not intended to limit the present invention in any form. Other variations and modifications are possible without exceeding the technical solutions described in the claims.

Claims

1. Use of melanin in cuttlefish ink in the preparation of a medicine for treating depressive-like behavior associated with chronic ulcerative colitis.

2. The use according to claim 1, characterized in that: The medicine comprises melanin in cuttlefish ink and pharmaceutically acceptable excipients.

3. The use according to claim 1, characterized in that: The dosage form of the drug is tablets.

4. The use according to claim 1, characterized in that: The dosage form of the medicine is capsule.

5. The use according to claim 1, characterized in that: The dosage form of the drug is injection.

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