Application of exogenous mitochondria in preparation of medicine for treating colitis

Abstract

The invention belongs to the technical field of biological medicines, and relates to application of exogenous mitochondria in preparation of a medicine for treating colitis, and the exogenous mitochondria is derived from mammary epithelial cells. The exogenous mitochondria derived from mammary epithelial cells is used for treating young individual colitis for the first time, and the symptoms including inhibition of weight loss, improvement of diarrhea and hemafecia, repair of colonic mucosal lesion (such as crypt structure damage and inflammatory cell infiltration) and / or reduction of disease activity index (DAI score) can be remarkably relieved; the problems that in the prior art, mitochondria sources are limited, oral stability is poor, and the curative effect verification of a young model is insufficient are solved, and a new strategy is provided for colitis treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of exogenous mitochondria in the preparation of drugs for treating colitis. Background Technology

[0002] Colitis is a disease characterized by inflammation and ulceration of the intestinal mucosa. It is common in infants, young mammals, and immunocompromised individuals. The causes are closely related to pathogen infection, immune abnormalities, and impaired intestinal barrier function. Clinical data shows that the incidence of acute colitis in infants is approximately 15%-20% (highest incidence in infants under 6 months of age). In large-scale farms, the incidence of enteritis in young calves, piglets, and lambs reaches 30%-50%, 25%-40%, and 20%-35%, respectively. Clinical manifestations include diarrhea, bloody stools, and weight loss. In severe cases, it can lead to intestinal perforation and sepsis, resulting in high mortality and posing a significant challenge to medical care and animal husbandry. This situation underscores the urgent need to develop treatment programs tailored to the specific physiological characteristics of young individuals. Current colitis treatment techniques have significant shortcomings, as detailed below: 1. Insufficient targeted treatment: In human clinical practice, aminosalicylic acid drugs (such as mesalazine) and glucocorticoids are commonly used. Although they can suppress the inflammatory response, they cannot repair the damaged intestinal mucosa, and long-term use can easily lead to immunosuppression and liver and kidney damage. Treatment of young animals relies on antibiotics, which can easily cause dysbiosis and drug resistance, and are not suitable for non-bacterial enteritis. 2. Poor stability of biological agents: Existing intestinal repair biological agents (such as epidermal growth factor and stem cell exosomes) are sensitive to the digestive environment and are easily degraded by gastric acid and pancreatic enzymes after oral administration. They require complex processes such as microencapsulation and coating, resulting in high costs and low bioavailability (usually <20%), making it difficult to widely apply them.

[0003] 3. Disconnect between animal models and clinical translation: Existing studies mostly use adult animal models, which differ significantly from young individuals in terms of intestinal barrier maturity, digestive enzyme activity, and immune system development. For example, the intestinal mucosal barrier of young individuals is not fully developed, and their response to pathogens and inflammatory factors differs from that of adults, making it difficult to directly translate research findings into applications for young populations. Exogenous mitochondria, as a novel "energy supplement," can repair mitochondrial dysfunction in damaged cells by directly providing ATP. However, currently, mitochondria are mostly extracted from liver or muscle tissue, which presents problems such as difficulty in obtaining raw materials, large differences in activity, and instability. Furthermore, there is a lack of efficacy verification for juvenile colitis models.

[0004] Therefore, there is an urgent need in this field for a cell-derived exogenous mitochondria that does not rely on tissue extraction and can be applied to the treatment of colitis in young individuals. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing treatments for colitis in young individuals, such as slow mucosal repair, significant side effects, and poor conversion rates, and to provide an application of mitochondria derived from mammary epithelial cells in the preparation of drugs for treating colitis.

[0006] In order to achieve the above-mentioned objectives, in a first aspect, the present invention provides the use of exogenous mitochondria in the preparation of a medicament for treating colitis in young individuals.

[0007] Optionally, the exogenous mitochondria are derived from mammary epithelial cells.

[0008] Optionally, the method for preparing the exogenous mitochondria includes the following steps: culturing mammary epithelial cells to a confluence of 80%-90%, and then extracting the exogenous mitochondria using a cell-mitochondrial separation kit.

[0009] Optionally, the mammary epithelial cells are the mouse immortalized mammary epithelial cell line HC11.

[0010] Optionally, the activity of the exogenous mitochondria can be verified by Jenners Green B staining and / or Mito-Tracker Green fluorescence staining.

[0011] Optionally, active mitochondria appear blue-green under the Jenners Green B staining method, and / or the fluorescent particles are clear under the Mito-Tracker Green fluorescence staining method, indicating that the mitochondria are of qualified activity.

[0012] Optionally, the drug is an oral formulation.

[0013] Optionally, the drug comprises exogenous mitochondria as an active ingredient and optional pharmaceutical excipients.

[0014] Optionally, the pharmaceutical excipient includes skim goat milk.

[0015] Optionally, the young individuals are selected from young mice, calves, piglets, lambs, and infants.

[0016] Optionally, the treatment can improve at least one of the following symptoms: weight loss, diarrhea, rectal bleeding, colon and rectal shortening, and mucosal damage.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention uses exogenous mitochondria derived from mammary epithelial cells, whose activity remains stable after short-term storage at 4°C for 6 hours. Compared with exogenous mitochondria from other sources (such as tissues or stem cells), it is more stable, proving that it is more suitable for oral formulation storage and transportation. 2. Using a DSS-induced colitis model in young mice, oral administration of exogenous mitochondria derived from mammary epithelial cells can significantly alleviate colitis symptoms. The specific effects are as follows: weight maintenance and nutritional improvement, control of diarrhea and hematochezia, improvement of comprehensive inflammatory indicators, protection of colorectal tissue, and repair of mucosal pathology. 3. The whey protein in the skimmed goat milk in the mitochondrial preparation of the present invention can maintain the stability of the mitochondrial membrane, so that the obtained mitochondrial preparation can play a role in treating enteritis through multiple mechanisms such as energy supplementation and membrane structure protection, providing a biomaterial for clinical treatment of enteritis that combines energy supply, mucosal repair and function maintenance. Attached Figure Description

[0018] Figure 1 The image shows the results of activity detection of HC11 cell-derived mitochondria extracted in Example 1 using the Jenners Green B staining method. Figure 2 The image shows the results of activity detection of HC11 cell-derived mitochondria extracted in Example 1 using the Mito-Tracker Green fluorescence staining method. Figure 3 This is a comparison chart of body weight and body weight change rate trends in mice of the blank control group, model control group, and mitochondrial intervention group during the experimental period in Case 3. Figure 4 The image shows a comparison of the fecal characteristics of mice in the blank control group, model control group, and mitochondrial intervention group on Day 8 in Case 3. Figure 5 To illustrate the trend of disease activity index (DAI) scores during the experimental period in mice of the blank control group, model control group, and mitochondrial intervention group in Case 3. Figure 6 The graph shows the comparison and quantitative statistical results of the colorectal length of mice in the blank control group, model control group and mitochondrial intervention group after sacrifice on Day 8 in Case 3. Figure 7 HE staining images of colorectal tissue sections from mice in the blank control group, model control group, and mitochondrial intervention group in Case 3. Detailed Implementation

[0019] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: Unless otherwise specified in the examples, the conditions shall be performed in accordance with conventional conditions or the conditions recommended by the manufacturer; if the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased commercially.

[0020] Example 1: Preparation of mitochondria from HC11 cells This embodiment uses the immortalized mouse mammary epithelial cell line HC11 (ATCC number: CRL-3063) as raw material, and extracts highly active mitochondria using the Thermo Fisher Scientific Mitochondrial Extraction Kit (catalog number: 89874). The specific steps are as follows: 1.1 Preparation of Experimental Materials ① Cell raw materials: HC11 cells in the logarithmic growth phase, cultured in 10 cm cell dishes, with cell confluence reaching 80%-90% (under the microscope, the cells cover the bottom of the flask without obvious gaps, and the morphology is spindle-shaped epithelial-like). ② Kit components: Thermo Fisher Scientific Mitochondrial Extraction Kit (catalog number: 89874), containing reagents A, B, and C; ③Auxiliary instruments and consumables: ultra-clean workbench, 37℃ constant temperature water bath, 4℃ benchtop high-speed centrifuge (maximum speed 15000g), optical microscope (equipped with 100× objective lens), sterile 2 mL centrifuge tubes, sterile pipette tips (10μL, 100μL, 1000μL), glass cell scraper.

[0021] 1.2 HC11 cell collection ① Discard the old culture medium in the cell culture dish, add 1 mL of pre-cooled sterile PBS buffer (pH 7.4), gently shake the culture flask for 10 seconds, wash away the residual culture medium on the cell surface, repeat the washing twice to ensure that there is no culture medium residue; ② Add 1 mL of PBS to the culture flask and gently scrape the bottom of the culture flask with a sterile glass cell scraper to ensure that all cells are detached and a cell suspension is formed; ③ Transfer the cell suspension to a 2 mL sterile centrifuge tube, place it in a 4℃ benchtop high-speed centrifuge, centrifuge at 850 g for 5 min, discard the supernatant, and leave the HC11 cell pellet at the bottom of the tube.

[0022] 1.3 Kit-based mitochondrial extraction and purification ① Cell lysis: Add 800 μL of pre-chilled reagent A from the kit to the HC11 cell pellet, vortex for 5 seconds, and place on ice for no more than 2 minutes; ②Preliminary separation: Add 10 μL of reagent B from the kit to the cell lysis buffer, vortex for 5 seconds, and incubate on ice for 5 minutes, vortexing for 5 seconds every minute during this period; add 800 μL of reagent C from the kit to the liquid, gently invert the centrifuge tube 5-8 times to mix thoroughly; place the mixture in a 4℃ benchtop high-speed centrifuge and centrifuge at 700 g for 10 minutes; ③Mitochondrial purification: After centrifugation, transfer the supernatant to a new 2 mL centrifuge tube, and then place it in a benchtop high-speed centrifuge at 4℃ and centrifuge at 12000 g for 15 min; after centrifugation, a white precipitate can be seen at the bottom of the centrifuge tube. Discard the supernatant, which is the crude mitochondrial product. ④Mitochondrial washing: Add 500 μL of pre-cooled reagent C from the kit to the crude mitochondrial product, gently pipette 5-8 times with a 100 μL pipette to resuspend the precipitate; centrifuge again at 12000g speed and 4℃ for 5 min, discard the supernatant, and retain the purified mitochondria at the bottom of the tube; ⑤ Preparation of oral mitochondrial solution: Add 200 μL of diluent containing 2% skim goat milk to the purified mitochondria, and gently pipette with 100 μL pipette until completely resuspended to form a mitochondrial suspension. Store at 4℃ for a short period of time (≤24h).

[0023] Example 2: Activity verification of HC11 cell-derived mitochondrial extract. 2.1 Preparation of experimental reagents James Green B staining solution (0.5%, prepared with physiological saline, freshly prepared and used immediately), Mito-Tracker Green mitochondrial green fluorescent probe (Beyotime Biotechnology Co., Ltd., catalog number C1048), physiological saline, and anti-fluorescence quenching mounting solution.

[0024] 2.2 Verification of mitochondrial activity using Jenners green B staining method Jenners Green B is a mitochondrial-specific active dye. It can only enter active mitochondria and bind to cytochrome oxidase (the core functional enzyme of mitochondria), turning blue-green. Inactivated mitochondria cannot bind the dye and remain colorless or pale pink. The specific operation is as follows: ① Staining treatment: Take 10 μL of mitochondrial sample extracted in Case 1, add 10 μL of 0.5% Jenners Green B stock solution, gently invert the centrifuge tube 3 times to mix, and incubate at room temperature (25±2℃) in the dark for 15 min. During this period, gently tap the bottom of the tube once every 5 min to ensure that the dye is fully bound to the mitochondria. ②Observation under an optical microscope: Take 10 μL of the stained sample, drop it onto a glass slide, cover it with a coverslip (to avoid air bubbles), and observe it under an optical microscope. Randomly select 5 fields of view for each group to observe the number of active mitochondria (blue-green). 2.3 Mito-Tracker Green fluorescence staining method to verify mitochondrial activity Mito-Tracker Green is a mitochondrial membrane-specific fluorescent probe that can penetrate the active mitochondrial membrane and bind to inner membrane lipids, emitting green fluorescence. The fluorescence intensity is positively correlated with mitochondrial membrane integrity (a core indicator of activity). The specific operation is as follows: ① Staining solution preparation: Prepare a mitochondrial stock solution of Mito-Tracker Green to a final concentration of 1 mM using anhydrous DMSO according to the instructions. After preparation, it can be stored at -20°C or lower, protected from light. Take a small amount of 1 mM Mito-Tracker Green stock solution and add it to PBS at a ratio of 1:5000 to obtain a final concentration of 20-200 nM, thus preparing the Mito-Tracker Green working solution. Prepare and use immediately (store protected from light).

[0025] ② Sample incubation: Take 10 μL of the mitochondrial sample extracted in Example 1, add 10 μL of Mito-TrackerGreen working solution, mix gently, and incubate at room temperature in the dark for 20 minutes (to ensure that the dye fully enters the mitochondria). ③ Fluorescence intensity detection: Microscopic observation: Take 10 μL of the incubated sample and drop it onto a special glass slide for a fluorescence microscope. Seal the slide with anti-fluorescence quenching mounting solution and observe it under a fluorescence microscope (488nm excitation, 520nm emission). Randomly select 5 fields of view for each group and take fluorescence images.

[0026] 2.4 Activity Validation Results and Analysis Figure 1 shows the results of activity detection of mitochondria derived from HC11 cells extracted in Example 1 using the Jenners Green B staining method. Figure 1A shows the staining results of the freshly extracted sample at 0h, and Figure 1B shows the staining results of the sample stored at 4℃ for 6h. As can be seen from the figures, the mitochondria in Figure 1A are uniformly blue-green, and the proportion of active mitochondria is high. In Figure 1B, the mitochondria still maintain the blue-green staining characteristics, with only a few individuals showing a slight decrease in staining intensity. There is no obvious swelling or colorless inactivation phenomenon, and the activity status meets the qualified standard.

[0027] Figure 2 shows the results of activity detection of mitochondria derived from HC11 cells extracted in Example 1 using the Mito-Tracker Green fluorescence staining method. Figure 2A shows the fluorescence image of the sample after fresh extraction at 0h, and Figure 2B shows the fluorescence image of the sample after short-term storage at 4℃ for 6h. As can be seen from the figures, the fluorescent particles in Figure 2A are clear and bright, evenly distributed, and have no diffuse fluorescence signal, indicating that the mitochondrial membrane is intact. In Figure 2B, the fluorescent particles still maintain a clear shape. Although the fluorescence intensity is slightly lower than that in Figure 2A, there is no obvious signal attenuation or aggregation.

[0028] Example 3: Experimental study on the improvement of enteritis in mice by oral administration of mitochondria This study used 3-week-old SPF-grade C57 mice as the research subjects. An acute colitis model was constructed using 2% (by weight / volume) sodium dextran sulfate (DSS). Simultaneously, the mice were gavaged with HC11 cell-derived mitochondria prepared in Example 1 (whose activity was verified in Example 2). The physiological status, weight changes, and diarrhea of ​​the mice were observed for 8 consecutive days to verify the intervention effect of mitochondria on colitis in young mice. The specific experimental protocol is as follows: 3.1 Preparation of Experimental Materials ① Experimental animals: 3-week-old SPF grade C57 mice (weight 12-15g), the housing environment was controlled at temperature 22±2℃, humidity 50±5%, and light cycle 12h / 12h (light 8:00-20:00). After 3 days of acclimatization (free access to standard mouse feed and sterile distilled water), the experiment began. ② Modeling reagent: Sodium dextran sulfate (DSS, MP Biomedical, catalog number: 160110), prepared as a 2% (w / v) DSS aqueous solution with sterile distilled water, sterilized by 0.22 μm filter membrane, and stored at 4℃ protected from light (use within 24 hours); ③ Intervention reagent: Mitochondria derived from HC11 cells prepared in Example 1 were used to prepare an oral solution of mitochondria using a sterile aqueous solution containing 2% skim goat milk. The solution was stored at 4°C and equilibrated to room temperature (25±2°C) before use. ④ Auxiliary instruments and consumables: Electronic balance (accuracy 0.01g), 1mL sterile gavage syringe (needle diameter 0.5mm to avoid damaging the mouse esophagus), sterile water bottle, animal feces collection box, mouse weight recording table, and Disease Activity Index (DAI) scoring table. 3.2 Experimental Group Design Three-week-old mice after acclimatization were randomly divided into three groups of 5-6 mice each, ensuring that the weight and sex ratio of mice in each group were consistent. The specific grouping is as follows: ① Blank control group (H2O): Free access to sterile distilled water throughout the process, and 0.2 mL of sterile aqueous solution containing 2% skim goat milk (excluding mitochondria) was administered by gavage daily for 7 consecutive days; ② Model control group (DSS): Received free access to 2% DSS aqueous solution throughout the entire process, and were given 0.2 mL of sterile aqueous solution containing 2% skim goat milk (excluding mitochondria) by gavage daily for 7 consecutive days; ③ Mitochondrial intervention group (DSS+Mito): Free drinking of 2% DSS aqueous solution throughout the course of treatment, and oral administration of 0.2 mL of the mitochondrial solution prepared in Example 1 by gavage daily for 7 consecutive days; (Note: All mice in all groups were given free access to standard feed. The gavage time was fixed at 13:00 every day to avoid affecting the experimental results due to time differences. The DSS aqueous solution and distilled water were changed every two days to ensure freshness.) 3.3 Colitis Model Construction and Mitochondrial Intervention Procedure The experiment lasted for 8 days, and the specific operating steps are as follows: ①Day 0 (Experiment Start Day): Each of the three groups of mice was numbered, and their initial weight was measured using an electronic balance (denoted as W0) and recorded in a weight table; The blank control group was replaced with a sterile distilled water bottle, and the model control group and mitochondrial intervention group were replaced with a 2% DSS aqueous solution water bottle. The mice were given their first gavage according to their groups: the blank control group and the model control group were given skim goat milk solution without mitochondria, and the mitochondrial intervention group was given oral mitochondrial solution. During gavage, the mice were gently fixed and the gavage needle was slowly inserted into the esophagus (to a depth of about 1 cm) to avoid choking or esophageal damage. ②Day 1-Day 7 (Mid-Experiment): The drinking water was changed every two days at 8:00 AM (distilled water for the blank control group, and fresh 2% DSS solution for the other two groups). The gavage procedure was repeated daily at 13:00 in groups to ensure that the single gavage dose for each mouse was precisely 0.2 mL. Observe and record the mouse's condition at 5:00 PM every day. See "Step 4" for specific observation details. ③Day 8 (Experiment End Date): At 8:00 AM, the final body weight of each group of mice was measured (referred to as W7), and the rate of change in body weight was calculated. The final gavage and patient status observation were completed at 9:00 AM; After euthanizing the mice, colon tissue was collected, colon length was measured, and HE staining was performed to verify the pathological features of colitis. 4. Observation Indicators and Judgment Criteria During the experiment (Day 0-Day 7), mice in the three groups were observed uniformly at 5:00 PM every day. The core observation indicators included the general condition of the mice, weight changes, and diarrhea. The specific judgment criteria are as follows: ① General state observation: Activity level: Record the frequency of spontaneous activity in mice (active: frequent running and grooming; lethargic: curled up in a corner and moving little; drowsy: slow response to external stimuli). Hair condition: Observe the hair's luster (shiny, messy, dry) and fluffiness (fluffy, close-fitting); Mental state: Assessed by the mouse's response to touch (sensitive: quickly avoids; sluggish: reacts slowly; unresponsive: does not move when touched). ② Observation of weight changes: Weigh the mice daily (accurate to 0.01g) and calculate the daily weight change rate: Weight change rate = (daily weight - initial weight W0) / initial weight W0 × 100%; If the mouse's weight decreases by ≥10% from its initial weight, the observation frequency should be increased (to once in the morning and once in the evening) to avoid severe dehydration; ③ Observation of colitis condition: Stool characteristics: scored according to the following criteria (0-4 points): 0 points = normal formed stool (granular); 1 point = slightly loose stool; 2 points = semi-loose stool; 3 points = loose stool; 4 points = watery stool; Fecal occult blood: Detected using fecal occult blood test strips, scored according to the following criteria (0-2 points): 0 points = negative (test strip does not change color); 1 point = weakly positive (test strip is light pink); 2 points = strongly positive (test strip is dark red). ④ Disease Activity Index (DAI) score (to comprehensively determine the severity of colitis): The total score is calculated based on "weight change (0-4 points) + stool characteristics (0-4 points) + fecal occult blood (0-2 points)" (out of 10 points). The higher the score, the more severe the colitis: 0 points = no inflammation; 1-3 points = mild inflammation; 4-6 points = moderate inflammation; 7-10 points = severe inflammation.

[0029] 5. Recording and Preliminary Analysis of Experimental Results The experimental results of each group are presented visually through the attached figures. Combined with quantitative data and pathological characteristics, the intervention effect of HC11 cell-derived mitochondria on DSS-induced colitis in young mice is systematically verified. The specific results are as follows: ① Results of weight change Figure 3 Figure 3A shows a comparison of the body weight (indicated by the weight change rate) and the trend of body weight change rate in mice from the blank control group, model control group, and mitochondrial intervention group during the experimental period (indicated by Figure 3B). The results show that: The mice in the blank control group showed continuous weight gain without any weight loss; the mice in the model control group showed significant weight loss starting from Day 5; the mice in the mitochondrial intervention group only showed slight weight fluctuations starting from Day 8, with a significantly lower weight loss than the model control group, and a weight gain trend close to that of the blank control group.

[0030] These results indicate that oral administration of HC11 cell-derived mitochondria can effectively alleviate DSS-induced weight loss in young mice and improve nutrient absorption disorders caused by enteritis.

[0031] ② Results of diarrhea Figure 4 shows a comparison of the fecal characteristics of the three groups of mice on Day 8. The results show that the blank control group had no diarrhea throughout the course, the feces were regular granular, and the fecal occult blood test was negative. The feces of the model control group were mostly soft, unformed, and had obvious blood. The feces of the mitochondrial intervention group were mostly regular granular and had no obvious blood.

[0032] These results confirm that mitochondria can significantly improve DSS-induced intestinal dysfunction in young mice and reduce diarrhea and intestinal mucosal bleeding.

[0033] ③ DAI scoring results Figure 5 shows the trend of Disease Activity Index (DAI) scores in the three groups of mice during the experimental period. The DAI score comprehensively reflects changes in body weight, fecal characteristics, and occult blood status. The results show: The blank control group had a DAI score of 0 throughout the entire process and showed no signs of inflammation. The model control group showed a significant increase in DAI score starting from Day 44, while the mitochondrial intervention group only showed a slight increase in DAI score starting from Day 7. The DAI score was significantly lower than that of the model control group and was close to the non-inflammatory state of the blank control group.

[0034] These results further validate the anti-inflammatory effect of mitochondria on colitis in young mice from the perspective of comprehensive inflammatory indicators.

[0035] ④ Results of colorectal length measurement Figure 6 shows a comparison of colon and rectum lengths after sacrifice in the three groups of mice on Day 8 (Figure 6A) and a quantitative statistical result diagram (Figure 6B). Shortened colon and rectum length is a typical pathological feature of DSS-induced colitis. The results show: The length of the colon and rectum in the blank control group mice was 7.1±0.2cm, with intact morphology and no shortening; In the model control group, the colorectal region of mice was significantly shortened due to inflammatory stimulation, with a length of only 6.1±0.3 cm, which was 14.1% shorter than that of the blank control group; The length of the colon and rectum in the mitochondrial intervention group mice was 7.3±0.2cm, which was not significantly different from that in the blank control group.

[0036] These results indicate that mitochondria can alleviate the inflammatory damage of DSS to the colorectal tissue of young mice and inhibit colorectal shortening.

[0037] ⑤ HE staining results Figure 7 shows HE staining images of histopathological sections of colorectal tissue from three groups of mice. The results show: The blank control group showed intact colorectal mucosal epithelium, regular gland arrangement, no inflammatory cell infiltration, and clear mucosal and muscular layer structures. In the model control group, the colorectal mucosa was severely damaged, with epithelial cell shedding, glandular structure destruction, and a large number of neutrophils and lymphocytes infiltrating the mucosal layer, accompanied by the formation of mucosal ulcers. The colorectal mucosal epithelium of the mitochondrial intervention group was basically intact, the glands were arranged in a relatively regular manner, there was only a small amount of inflammatory cell infiltration, no obvious mucosal ulcers, and the degree of inflammatory damage was significantly milder than that of the model control group.

[0038] From a pathological morphological perspective, these results confirm that mitochondria can repair DSS-induced colorectal mucosal damage in young mice and reduce inflammatory responses.

[0039] ⑥ Research Conclusions The results above indicate that feeding 3-week-old mice with 2% DSS aqueous solution for 7 consecutive days can successfully establish an acute colitis model with physiological characteristics similar to those of young individuals (human infants, young mammals). The model control group showed significant weight loss, diarrhea, bloody stools, shortened colon and rectum, and mucosal damage. In contrast, the intervention group, which was simultaneously gavaged with HC11 cell-derived mitochondria, showed excellent performance in terms of weight maintenance, improved fecal characteristics, reduced DAI score, preservation of colon and rectum length, and repair of mucosal damage. This fully demonstrates that mitochondria have a significant intervention effect on DSS-induced colitis in young mice.

[0040] This embodiment, by precisely controlling the core parameters of "3-week-old young mice + 2% DSS concentration + 7-day intervention period", not only verified the effectiveness of the colitis model, but also deeply integrated the "HC11 cell mitochondrial extraction - activity verification" technology mentioned above with the model application, and improved the technical closed loop of "raw material preparation - quality control - efficacy verification". It provides reliable model support and data basis for subsequent research on the mechanism of mitochondrial therapy for colitis (such as energy supplementation, inflammatory factor regulation) and cross-species application (calves, piglets, infants).

[0041] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. Use of exogenous mitochondria in the preparation of a medicament for treating colitis in a young individual.

2. Use according to claim 1, characterized in that, The exogenous mitochondria are derived from mammary epithelial cells.

3. Use according to claim 1, characterized in that, The preparation method of the exogenous mitochondria comprises the following steps: culturing mammary epithelial cells to a confluence of 80-90%, and then extracting the exogenous mitochondria using a cell mitochondria separation kit.

4. Use according to claim 2 or 3, characterized in that, The mammary epithelial cells are a mouse immortalized mammary epithelial cell line HC11.

5. Use according to any one of claims 1 to 4, characterized in that, The activity of the exogenous mitochondria is verified by Janus green B staining and / or Mito-Tracker Green fluorescent staining.

6. The use according to claim 5, characterized in that, Under the Janus green B staining, the active mitochondria are blue-green, and / or under the Mito-Tracker Green fluorescent staining, the fluorescent particles are clear, indicating that the mitochondria activity is qualified.

7. Use according to claim 1, characterized in that, The medicament is an oral preparation comprising exogenous mitochondria as an active ingredient and optionally a pharmaceutical excipient.

8. Use according to claim 7, characterized in that, The pharmaceutical excipient includes skimmed goat milk.

9. The use according to claim 1, characterized in that, The young individual is selected from a young mouse, a calf, a piglet, a lamb and an infant.

10. The use according to claim 1, characterized in that, The treatment can improve at least one symptom of weight loss, diarrhea, hematochezia, shortening of the colorectum and mucosal injury.

1. Use of exogenous mitochondria in the preparation of a medicament for treating colitis in a young individual. The exogenous mitochondria are derived from mammary epithelial cells. The preparation method of the exogenous mitochondria comprises the following steps: culturing mammary epithelial cells to a confluence of 80-90%, and then extracting the exogenous mitochondria using a cell mitochondria separation kit. The mammary epithelial cells are a mouse immortalized mammary epithelial cell line HC11. The activity of the exogenous mitochondria is verified by Janus green B staining and / or Mito-Tracker Green fluorescent staining. Under the Janus green B staining, the active mitochondria are blue-green, and / or under the Mito-Tracker Green fluorescent staining, the fluorescent particles are clear, indicating that the mitochondria activity is qualified. The medicament is an oral preparation comprising exogenous mitochondria as an active ingredient and optionally a pharmaceutical excipient. The pharmaceutical excipient includes skimmed goat milk. The young individual is selected from a young mouse, a calf, a piglet, a lamb and an infant. The treatment can improve at least one symptom of weight loss, diarrhea, hematochezia, shortening of the colorectum and mucosal injury.

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