Application of bone marrow mesenchymal stem cell-derived apoptotic body in preparation of medicine for preventing / treating keratitis

Abstract

The invention belongs to the field of biological medicines, and particularly relates to application of apoptotic bodies derived from mesenchymal stem cells in preparation of medicines for preventing / treating keratitis. According to the invention, the bone marrow mesenchymal stem cells are induced by a chemical induction method, so that the bone marrow mesenchymal stem cells are subjected to apoptosis and apoptotic bodies are generated. Experiments prove that the apoptotic body derived from the bone marrow mesenchymal stem cells provided by the invention can realize inflammation regulation in bacterial keratitis by regulating polarization of macrophages, and a novel prevention and treatment method which is safer, easy to store and has a practical application effect is provided for treatment of the disease. The invention further provides a medicine for preventing / treating keratitis, the medicine takes the bone marrow mesenchymal stem cell-derived apoptotic body as an effective component, the medicine can be applied to high-risk patients who are prone to bacterial infection after corneal trauma in a pretreatment mode, and a prevention measure is provided for the high-risk exposure patients.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the use of apoptotic bodies derived from bone marrow mesenchymal stem cells in the preparation of drugs for preventing / treating keratitis. Background Art

[0002] Bacterial keratitis is an acute inflammatory disease caused by bacterial infection. As an ophthalmic emergency, severe bacterial keratitis can lead to corneal ulcers, perforations, and even permanent vision loss. The disease occurs worldwide. Common pathogens include Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Gram-negative bacilli. Pseudomonas aeruginosa is the primary pathogen in severe cases due to its highly invasive nature and drug resistance.

[0003] Currently, common treatments for bacterial keratitis include medication and surgery. Medication generally involves antibiotic eye drops or eye ointments. However, long-term or inappropriate use of antibiotic eye drops can lead to the development of drug-resistant bacteria, making subsequent treatment more difficult. The use of eye ointments can also cause blurred vision, impacting patients' daily lives. Surgical treatment is generally reserved for patients with deeper corneal ulcers who are ineffective with medication. However, the procedure can damage corneal tissue, causing corneal thinning and increasing the risk of corneal perforation.

[0004] In view of this, searching for keratitis treatment drugs that are safe, effective, and unlikely to induce drug resistance, and that have both preventive and therapeutic functions, has become an important research direction and a technical problem that needs to be solved urgently in the current clinical intervention of bacterial keratitis. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide the use of apoptotic bodies derived from bone marrow mesenchymal stem cells in the preparation of drugs for preventing / treating keratitis, which has solved the problems in the prior art.

[0006] To achieve the above-mentioned purpose, the present invention adopts the following technical solutions.

[0007] The present invention provides the use of apoptotic bodies derived from bone marrow mesenchymal stem cells in the preparation of a medicine for preventing / treating keratitis.

[0008] Bone marrow mesenchymal stem cells (BMSCs) are a type of adult stem cell found in virtually all organs and tissues after birth, possessing potent immunomodulatory, self-renewal, and multidirectional differentiation potential. BMSCs are easy to isolate, culture, and expand, offering promising applications in regenerative cell therapy. The extracellular vesicles (EVs) derived from BMSCs have garnered significant attention in recent years due to their similar biological properties to their parental cells. These include exosomes (Exos), microvesicles (MVs), and apoptotic bodies (ApopBs). Apoptotic bodies are vesicles secreted by apoptotic cells that target macrophages for clearance of debris left behind by cell death. Due to limitations in isolation techniques, exosomes are currently the most studied type of EV. In contrast, the therapeutic applications of apoptotic bodies in various diseases have remained largely unexplored.

[0009] Studies have shown that bone marrow mesenchymal stem cells undergo extensive apoptosis shortly after transplantation. Apoptotic BMSCs exhibit even greater immunoregulatory capacity than surviving BMSCs, potentially indicating that apoptosis enhances BMSC immunoregulation. The ultimate consequence of apoptosis is the production of apoptotic bodies (ApopBs). Clearance of ApopBs primarily relies on two cell types: professional phagocytes, such as macrophages and immature dendritic cells; and non-professional neighboring cells. Clearance of apoptotic bodies by different cell types often triggers distinct physiological effects. Ingestion by macrophages can promote macrophage polarization toward the M2 phenotype, thereby achieving anti-inflammatory effects. Conversely, ingestion by immature dendritic cells can result in proinflammatory effects. Ingestion by neighboring cells often promotes tissue repair and cell proliferation. Existing studies have shown that under natural conditions, ApopBs in the body are more likely to be phagocytosed by macrophages and exert anti-inflammatory effects. The mechanism may be that the phosphatidylserine exposed on the surface of the apoptotic bodies makes them more targeted to macrophages. In normal cells, phosphatidylserine is mainly distributed on the cytoplasmic side of the cell membrane. When cells undergo apoptosis, it flips to the outside of the cell membrane and expresses an "eat me" signal, targeting macrophages for recognition and clearance.

[0010] In some embodiments of the present invention, the method for preparing the apoptotic bodies comprises: inducing bone marrow mesenchymal stem cells to undergo apoptosis and produce apoptotic bodies using a chemical induction method.

[0011] In some embodiments of the present invention, the method for preparing apoptotic bodies comprises the following steps:

[0012] Step 1: using an apoptosis-inducing reagent to induce apoptosis of bone marrow mesenchymal stem cells to produce apoptotic bodies, and collecting the cell supernatant;

[0013] Step 2: taking the cell supernatant of step 1 and performing a first centrifugation to remove large cell debris, and then retaining the cell supernatant after the first centrifugation;

[0014] Step 3, taking the cell supernatant after the first centrifugation, performing a second centrifugation to remove medium-sized organelles and vesicles, and then retaining the cell supernatant after the second centrifugation;

[0015] Step 4: taking the cell supernatant after the second centrifugation, performing a third centrifugation, retaining the precipitate, and obtaining apoptotic bodies derived from bone marrow mesenchymal stem cells.

[0016] In some embodiments of the present invention, the conditions for the first centrifugation are: 750g~850g, centrifugation for 10min~15min.

[0017] In some embodiments of the present invention, the second centrifugation is performed at 2000 g to 3500 g for 25 min to 35 min.

[0018] In some embodiments of the present invention, the third centrifugation is performed at 15,000 g to 18,000 g for 25 min to 35 min.

[0019] In some embodiments of the present invention, in step 1, bone marrow mesenchymal stem cells are added to a cell culture medium containing an apoptosis-inducing agent and cultured for 14 to 18 hours to induce the bone marrow mesenchymal stem cells to produce the apoptotic bodies.

[0020] In some embodiments of the present invention, the conditions for the first centrifugation are: 800g, centrifugation for 10 min; the conditions for the second centrifugation are: 3000g, centrifugation for 30 min; the conditions for the third centrifugation are: 16000g, centrifugation for 30 min.

[0021] In some embodiments of the present invention, the diameter of the apoptotic body is 180 nm to 210 nm.

[0022] In some specific embodiments of the present invention, the average diameter of the apoptotic bodies is 200 nm.

[0023] In some embodiments of the present invention, the drug is used to improve the inflammatory response of keratitis.

[0024] In some embodiments of the present invention, the drug is used to promote corneal epithelial healing.

[0025] In some embodiments of the present invention, the dosage forms of the drug include tablets, sprays, granules, capsules, oral solutions, injections, and suspensions.

[0026] The invention also discloses a medicine for treating keratitis, wherein the medicine contains the apoptotic body as the only effective ingredient.

[0027] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable carrier.

[0028] In some embodiments of the present invention, the content of the pharmaceutical apoptotic bodies is 1 wt% to 99 wt%.

[0029] In some embodiments of the present invention, the pharmaceutically acceptable carrier includes at least one of a diluent, a binder, a wetting agent, a lubricant, a disintegrant, a solvent, an emulsifier, a solubility aid, a preservative, a pH regulator, an osmotic pressure regulator, a surfactant, a coating material, an antioxidant or a buffer.

[0030] Based on the technical solution of the present invention, compared with the prior art, the present invention has the following beneficial effects:

[0031] The bone marrow mesenchymal stem cell-derived apoptotic bodies provided by the present invention can alleviate the inflammatory response in bacterial keratitis by influencing the polarization of macrophages. Experimental validation demonstrates that apoptotic bodies can modulate inflammation in bacterial keratitis by regulating macrophage polarization, providing a novel, safer, easily stored, and practically effective preventive and therapeutic approach for the disease.

[0032] The apoptotic bodies provided by this invention can actively target sites of inflammation, inducing the induction of regulatory immune cells (such as macrophages) to restore immune balance. Furthermore, the apoptotic bodies are phagocytosed through a "silent clearance" mechanism without inducing an inflammatory response. Furthermore, the apoptotic bodies provided by this invention can be used as a pretreatment for patients at high risk of bacterial infection after corneal trauma, providing a preventive measure for patients at high risk of exposure. Therefore, they offer advantages in both disease prevention and treatment.

[0033] Compared with drug treatment, the present invention uses apoptotic bodies derived from bone marrow mesenchymal stem cells for the treatment of keratitis. This method does not rely on antibiotics and can avoid the generation of drug-resistant bacteria. Compared with surgical treatment, this method does not have the risk of corneal perforation. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Figure 2 shows the morphological observation results of bone marrow mesenchymal stem cells.

[0035] Figure 2 Identification of bone marrow mesenchymal stem cells after induction of apoptosis.

[0036] Figure 3 The figure shows the results of cell apoptosis detection by flow cytometry.

[0037] Figure 4 This is the result of Western Blot detection of the expression of apoptosis-related proteins caspase-3 and cleaved-caspase-3; Figure 4 A in the figure represents the electrophoresis results of apoptosis-related proteins caspase-3 and cleaved-caspase-3; Figure 4 B in the figure shows the statistical results of the expression of apoptosis-related proteins caspase-3 and cleaved-caspase-3.

[0038] Figure 5 Flow chart for the extraction of apoptotic bodies from bone marrow mesenchymal stem cells.

[0039] Figure 6 The results of projection electron microscopy observation of apoptotic bodies are shown in Figure 2.

[0040] Figure 7 This is a statistical graph showing the number of particles of various sizes in the sample detected by nanoparticle tracking technology of apoptotic bodies.

[0041] Figure 8 The anterior segment of the eye was modeled in mice for different treatments and on different days.

[0042] Figure 9 The following are the statistical results of corneal opacity score, corneal inflammatory response score and corneal epithelial fluorescent staining area / corneal area under different treatments and different treatment days; Figure 9 A in the figure represents the bar graph of the corneal opacity scores under different treatments and different treatment days; Figure 9 B in the figure represents the bar graph of the corneal inflammation response scores under different treatments and different treatment days; Figure 9 C in the figure represents the bar graph of the statistical results of corneal epithelial fluorescent staining area / corneal area under different treatments and different treatment days.

[0043] Figure 10 The results of HE staining of the cornea of ​​mice in different treatment groups are shown.

[0044] Figure 11 The statistical graphs show the detection results of pro-inflammatory genes, immune regulation-related genes, macrophage M1 (pro-inflammatory phenotype) polarization markers, and macrophage M2 (anti-inflammatory phenotype) polarization markers in each group of mouse samples; Figure 11 A in the figure represents the statistical results of the expression of the pro-inflammatory gene iNOS in the corneal tissues of mice in different treatment groups; Figure 11B in the figure represents the statistical results of the expression of pro-inflammatory gene Arg-1 in corneal tissues of mice in different treatment groups; Figure 11 C in the figure represents the statistical results of the expression of the pro-inflammatory gene TGF-β1 in the corneal tissues of mice in different treatment groups; Figure 11 D in the figure represents the statistical results of the expression of pro-inflammatory gene IL-6 in the corneal tissues of mice in different treatment groups; Figure 11 E in the figure represents the statistical results of the expression of the pro-inflammatory gene IL-10 in the corneal tissues of mice in different treatment groups.

[0045] Figure 12 The electrophoresis results show the expression of macrophage polarization iNOS protein and Arg-1 protein in protein samples of mice in different treatment groups.

[0046] Figure 13 The bar graph shows the statistical results of the detection of macrophage polarization iNOS protein and Arg-1 protein expression in the protein samples of mice in different treatment groups; Figure 13 A in the figure represents the bar graph of statistical results of Arg-1 protein expression detection; Figure 13 B in the figure represents the bar graph of statistical results of iNOS protein expression detection.

[0047] Figure 14 The results of the detection of macrophage polarization CD86 protein in the protein samples of mice in different treatment groups are shown in FIG. Figure 14 A in the figure represents the electrophoresis result of CD86 protein; Figure 14 B in the figure represents the bar graph of statistical results of CD86 protein expression level. DETAILED DESCRIPTION

[0048] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0049] The following describes the details in conjunction with specific embodiments.

[0050] 1. Experimental Materials

[0051] The bone marrow mesenchymal stem cells used in the present invention have been disclosed in Mesenchymal Stem Cell-Derived Exosomes as Drug Carriers for Delivering miRNA-29b to Ameliorate Inflammation in Corneal Injury Via Activating Autophagy.

[0052] Example 1: Preparation of apoptotic bodies derived from bone marrow mesenchymal stem cells

[0053] The apoptotic bodies provided in this example are obtained by inducing apoptosis in bone marrow mesenchymal stem cells and extracting the apoptotic bodies after successful apoptosis. The bone marrow mesenchymal stem cells in this example are mouse bone marrow mesenchymal stem cells. The specific preparation method is as follows:

[0054] 1. Isolation, culture and passage of bone marrow mesenchymal stem cells

[0055] Four-week-old clean-grade female C57BL / 6J mice (purchased from Beijing Sibeifu Biotechnology Co., Ltd.) were selected and killed by cervical dislocation and immersed in a beaker filled with 75% ethanol. The beaker was quickly transferred to an ultra-clean workbench, and the skin and subcutaneous tissue of the bilateral hind limbs were carefully peeled off. The bilateral hind limbs of the mice were completely separated while ensuring the integrity of the femoral and tibial bone marrow cavities. The culture dish with the mouse hind limbs was placed on a table pre-irradiated with ultraviolet light, and transferred to a sterile PBS solution for washing twice. The muscle tissue was carefully separated with ophthalmic scissors to expose the femur and tibia. The peeled femur and tibia were separated at the knee joint, the epiphyses at both ends were cut open to expose the bone marrow cavity, and a 1 mL sterile syringe was used to draw the pre-prepared BMSCs special culture medium (Oricell MUXMX-90012 mouse bone marrow mesenchymal stem cell culture medium + 10% fetal bovine serum + 1% penicillin / streptomycin) was used to gently flush the bone marrow from the bone marrow cavity into a 10 cm culture dish. The bone marrow cavity was repeatedly flushed until the bone became white and translucent.

[0056] Add an appropriate amount of complete culture medium (Manufacturer: Oricell Biotechnology Co., Ltd.; Catalog No.: MUXMX-90012) to the dish again, mix the cells evenly using the cross-mixing method, and incubate in a constant-temperature incubator at 37°C, 5% CO2, and relative humidity. After one week of culture, adherent cells will form dozens of dense colonies, at which point they can be passaged. After several passages, BMSCs will be gradually purified. When passaged to P3-P5, they can be used for apoptosis induction and subsequent experiments.

[0057] 2. Phenotypic identification and morphological observation of bone marrow mesenchymal stem cells

[0058] After long-term research by the inventors, the above-mentioned BMSCs isolation and purification method has been used for a long time and has shown good stability and repeatability. It can efficiently enrich BMSCs. At the same time, its phenotypic characteristics have been identified and analyzed by flow cytometry in previously published articles (i.e., positive expression of CD90, CD44, CD105, negative expression of CD34, CD45), and its phenotype has been strictly confirmed. At the same time, the isolated BMSCs were morphologically observed and photographed using a cell imaging microplate detection system. The results are as follows. Figure 1 As shown: Under the light microscope, it can be observed that BMSCs passaged to P3-P5 are spindle-shaped or polygonal, growing in a single layer adherent to the wall, and can grow in a vortex shape in denser areas.

[0059] 3. Induction of apoptosis and identification of bone marrow mesenchymal stem cells

[0060] Use dimethyl sulfoxide (DMSO) to prepare staurosporine (STS) working solution with a DMSO concentration of 500 μM, and add it to the prepared bone marrow mesenchymal stem cell complete culture medium (manufacturer: Oricell Biotechnology Co., Ltd.; product number: MUXMX-90012) to make the final STS concentration 500 nM.

[0061] P3-P5 cells were pre-seeded in a six-well plate. When the cells grew to a density of 80%, the old culture medium was discarded, the cells were rinsed 2-3 times with sterile PBS, and cell culture medium containing 500nM STS was added. The cells were placed in a cell constant temperature incubator at 37°C, 5% CO2, and relative saturated humidity for 16 hours to complete the induction of cell apoptosis.

[0062] 4. Identification of bone marrow mesenchymal stem cells after apoptosis induction

[0063] (1) Fully automatic live cell imaging system to dynamically observe the morphological changes of apoptotic cells

[0064] Dynamic observation of cell morphology after apoptosis induction was performed using a fully automated live-cell imaging system, set for 16 hours of continuous recording, with images automatically acquired every hour. The culture environment was maintained using a built-in CO2 and temperature control module (37°C, 5% CO2).

[0065] The results are as follows Figure 2 As shown: In the unmedicated state, mouse BMSCs were spindle-shaped or polygonal, and some cell membranes shrank 2 hours after administration; vesicle-like structures were visible 4 hours after administration, and after 6 hours, there were almost no BMSCs with normal morphology. The cell morphology further shrank and became round, and a large number of vesicles aggregated around the cells; after 12 hours, the number of vesicle aggregations continued to increase compared with before, and the cell morphology had no obvious changes, indicating that STS can successfully induce typical morphological changes of apoptosis in BMSCs and produce apoptotic bodies.

[0066] (2) Determination of cell apoptosis by flow cytometry

[0067] After the apoptosis-induced treatment, the cells were digested and prepared into a cell suspension with 1×Annexin V Binding Solution. The suspension was thoroughly mixed with FITC and PI dyes. After incubation at room temperature in the dark, 1×Annexin V Binding Solution was added to make up 500 μL of the system. The apoptosis rate was detected by flow cytometry.

[0068] The results are as follows Figure 3 As shown in A to D: After BMSCs were induced to apoptosis by staurosporine, the apoptosis rate was approximately 65.4%, and there was a significant statistical difference compared with the untreated control group cells, indicating that under STS induction, the apoptosis rate of BMSCs was approximately 65.4% (Q2+Q3), and there was a significant statistical difference compared with the untreated control group cells (P < 0.001).

[0069] (3) Western Blot detection of the expression of apoptosis-related proteins caspase-3 and cleaved-caspase-3

[0070] Western blotting was performed to examine the expression of proteins involved in the apoptosis pathway in cells induced by apoptosis. To prepare total protein samples, discard the culture medium and add RIPA buffer containing protease inhibitors to the culture dish containing cells induced by apoptosis. The cells were lysed on ice. Cells were thoroughly scraped and lysed by sonication on ice. The protein concentration of the samples was determined according to the instructions of the BCA protein quantification kit. 5× protein loading buffer was added to the samples, mixed thoroughly, and then boiled at 100°C for 10 minutes to prepare the protein loading sample. Western blotting was used to examine the expression of apoptosis-related proteins, caspase-3 and cleaved-caspase-3, in the protein samples from the cells induced by apoptosis.

[0071] The results are as follows Figure 4 As shown in Figures A and B, there was no statistically significant difference in Caspase-3 protein expression between the normal and STS-treated groups. However, cells in the STS-treated group significantly overexpressed Cleaved-Caspase-3 protein, demonstrating that STS treatment activates the apoptotic pathway and cleaves Caspase-3, indicating that these samples have entered the apoptosis-related signaling cascade mediated by the Caspase family. Combined with the aforementioned validation results, this further demonstrates the successful induction of apoptosis by this protocol.

[0072] 5. Extraction method of bone marrow mesenchymal stem cell-derived apoptotic bodies (BMSCs-ApopBs)

[0073] After inducing apoptosis in BMSCs, collect the cell culture supernatant in a centrifuge tube. Store the supernatant at 4°C for short-term storage and at -80°C for longer-term storage. Minimize the storage time of the supernatant and complete extraction as quickly as possible. Studies have shown that the number of extracellular vesicles in the supernatant decreases with storage time.

[0074] like Figure 5 Extract apoptotic bodies using the gradient centrifugation method shown: Centrifuge the collected cell supernatant at 800g for 10 minutes at 4°C to remove large cell debris. Transfer the supernatant to a new sterile centrifuge tube and centrifuge at 3000g for 30 minutes at 4°C to remove medium-sized extracellular vesicles. Collect the supernatant again and centrifuge at 16000g for 30 minutes at 4°C to obtain apoptotic body pellet. Discard the supernatant, resuspend the pellet in sterile PBS, and repeat the centrifugation at 16000g for 30 minutes at 4°C to wash the pellet. Finally, resuspend the pellet in 100 μL of sterile PBS to obtain a BMSCs-ApopBs-PBS suspension, which can be stored at -80°C.

[0075] 6. Identification of apoptotic bodies

[0076] like Figure 6 A and B in Figure 7 As shown in Table 1, transmission electron microscopy showed that the prepared BMSCs-ApopBs had a double-layer membrane structure; nanoparticle tracking technology detected that the vesicle diameter was about 200 nanometers; compared with untreated cells, the bone marrow mesenchymal stem cells after STS-induced apoptosis highly expressed the apoptosis-related protein Cleaved-Caspase-3, indicating that the BMSCs induced by STS underwent typical cell apoptosis and produced apoptotic bodies. The structure and particle size distribution of the extracted apoptotic bodies were consistent with the description in the literature.

[0077] Table 1 Comparison of particle size and particle concentration statistics (baseline value vs. mean ± standard error)

[0078] index Basic statistics Mean ± standard error average value 200.2nm 200.8 ± 5.1 nm majority 164.7nm 149.8 ± 13.5 nm D10 86.3nm 81.7 ± 3.1 nm D50 173.8nm 174.4 ± 2.7 nm D90 315.9nm 322.4 ± 15.7 nm Particle concentration ± standard error - 1.98e+08 ± 1.20e+07 particles / ml Single field particle count ± standard error - 33.1 ± 1.8 particles / frame

[0079] Note: “-” in the table means that the item does not exist.

[0080] Example 2: Anti-inflammatory effect of BMSCs-ApopBs in lipopolysaccharide-induced bacterial keratitis model in mice

[0081] 1. Experimental Animals

[0082] 6-8 week old female C57BL / 6J mice (purchased from Beijing Sibeifu Biotechnology Co., Ltd.).

[0083] 2. Grouping and Dosing

[0084] Before the experiment, each mouse underwent routine ophthalmological examination under slit lamp. After excluding individuals with ocular infection and corneal damage, the mice were randomly divided into three groups, with 6 mice in each group: normal group (N group), PBS group, and ApopBs group. The mice in the normal group were not treated. The mice in the PBS group and the BMSCs-ApopBs-PBS suspension (ApopBs group) were treated with 1×10 9 ApopBs at a concentration of particles / mL were used for subconjunctival injection pretreatment. Sterile PBS was used as the treatment control group. 30 minutes before modeling, 5 μL of sterile PBS solution (PBS modeling control group) and BMSCs-ApopBs-PBS suspension (ApopBs group) were injected into the subconjunctiva of the right eyes of mice in the PBS group and ApopBs group, respectively, using a sterile microsyringe.

[0085] 3. Establishment of a mouse bacterial keratitis model

[0086] Only the right eye of each mouse was used for modeling. All procedures were performed under sterile conditions, and the periocular skin of the right eye was routinely disinfected with iodine. Six- to eight-week-old C57BL / 6J mice were anesthetized with an intraperitoneal injection of 70 mg / kg sodium pentobarbital solution combined with oxybuprocaine hydrochloride eye drops. The mice were fixed under a stereomicroscope, and a central corneal incision was made using a 2.5 mm diameter corneal trephine. The corneal epithelium in the centrally marked area was scraped off using an AlgerBrush II to expose the corneal stroma. A bacterial keratitis model was induced in mice by applying 3 μL of a 10 μg / μL LPS solution to the scraped corneal surface. Slit-lamp anterior segment photography was then used to assess the degree of corneal inflammation, opacity, and corneal epithelial healing. The successful establishment of the bacterial keratitis model was determined by clinical findings.

[0087] Immediately after modeling (D0), on the third day after modeling (D3), the fifth day after modeling (D5), and the seventh day after modeling (D7), the mice in each group were anesthetized. The modeled eyes of the mice in each group were observed and photographed under a slit lamp to evaluate the following three aspects: the degree of corneal opacity, the degree of inflammatory response, and the healing of the corneal epithelium. The specific methods are as follows:

[0088] (1) Scoring of corneal opacity: The model mouse eyes were observed and photographed using the diffuse light illumination method of a slit lamp microscope. The corneal opacity of the mice was graded according to the scoring criteria developed in previous studies. The specific criteria were: completely transparent cornea with no opacity (grade 0); mild corneal opacity with clear iris details (grade 1); corneal opacity with slightly blurred iris texture (grade 2); diffuse moderate corneal opacity, affecting the observation of some anterior chamber structures and iris texture, with blurred pupil margin (grade 3); severe corneal opacity with no visible iris texture and anterior chamber structure (grade 4).

[0089] (2) Scoring of corneal inflammation: The scoring method is generally used under bright field observation. The three observation indicators are divided into none (0 points) - mild (1 point) - moderate (2 points) - severe (3 points) according to the severity. The specific scoring indicators are: degree of ciliary body congestion, degree of central corneal edema, and degree of peripheral corneal edema. The final inflammation score = total score (0-9 points) / 9.

[0090] (3) Corneal epithelial healing: Before staining, use a cotton swab dipped in sterile saline to gently wipe away secretions and foreign matter on the surface of the mouse cornea and around the eye. Turn off the ambient light, use a pipette to draw 5 μL of sodium fluorescein reagent and drop it onto the surface of the mouse cornea. After evenly covering the cornea, adjust the slit lamp light source to cobalt blue light, and observe, record, and photograph the fluorescent staining of the mouse corneal epithelium. The obtained images were analyzed using Image J software to calculate the fluorescent staining area of ​​the corneal epithelium of each group of mice / total corneal area to reflect the corneal epithelial healing status.

[0091] 4. Paraffin section preparation and HE staining

[0092] Seven days after modeling, mice in each group were killed by cervical dislocation. The intact eyeballs were removed using sterile curved forceps, retaining the optic nerve as a landmark. The eyeballs were then rinsed two to three times with sterile saline to remove blood and hair. Under a stereomicroscope, surrounding muscle and connective tissue were removed. A small incision was made at the posterior pole of the eyeball using a 1 mL disposable sterile syringe needle to preserve the eye's morphology after fixation. The eyeballs were then immersed in a tissue fixative (10% paraformaldehyde) at 4°C for 24 hours. Subsequently, the eyeballs were dehydrated and waxed using a gradient of xylene and varying concentrations of ethanol. After waxing, the eyeballs were paraffin-embedded, sectioned, and dried. The dried sections were stained with hematoxylin-eosin and mounted for observation.

[0093] 5. Quantitative reverse transcription polymerase chain reaction (RT-qPCR)

[0094] (1) Total RNA was extracted from the mouse cornea using the Redzol one-step total RNA extraction kit. On the second day after modeling, the mice in each group were killed by cervical dislocation. The intact eyeballs were removed with sterile curved forceps. The eyeballs were rinsed 2-3 times with sterile saline to clean the blood and hair on the surface. The eyeballs were placed in a 10 cm culture dish filled with sterile saline. The culture dish was placed on ice to prevent RNA degradation. Under a stereomicroscope, the corneas were separated with sterile ophthalmic microscissors and placed in a 1.5 mL enzyme-free ep tube. 1 mL of Redzol reagent was added to each cornea. After standing on ice for 15 minutes, the cornea was crushed with an ultrasonic tissue disruptor. Subsequently, the RNA extracted from each cornea was dissolved in 30 μL sterile enzyme-free water according to the kit instructions.

[0095] (2) A 40 μL reverse transcription system was used. The specific steps were as follows: Pre-denaturation: Calculate the sample volume required for 1 μg RNA in each group based on the measured RNA concentration, and prepare a system of 1 μg RNA + 2 μL Oligo dT primer, and make up 25 μL with ddH2O. After low-speed centrifugation, place the system in a PCR amplification device. The program was set to: 70°C pre-denaturation for 5 minutes, and immediately place the system on ice to cool to 0°C. After the amplification, add 8 μL 5×M-MLV Buffer, 4 μL dNTP, and 1 μL M-MLV reverse transcriptase to each tube. After thorough mixing, place the PCR tube in the amplification instrument. The program was set to: 42°C reaction for 1 hour; 94°C reaction for 10 minutes, and then cool to 4°C to terminate the reaction. At this time, cDNA samples in each group were obtained.

[0096] (3) RT-qPCR experiments were performed using the SYBR Green I method (20 μL) reaction system. Each sample well was added with: 10 μl SYBR Premix (2×), 0.5 μl of upstream and downstream primers, 1 μl of cDNA template, and 20 μl of ddH2O. The liquid in each well was thoroughly shaken and mixed, and bubbles were removed before loading onto the machine. The amplification program was set as follows: pre-denaturation: 95°C for 3 min; cycle stage: 95°C for 15 s—60°C for 30 s—75°C for 30 s (55 cycles in total)—4°C for termination. After the reaction was completed, the data were exported and analyzed. The data obtained for each group were used with GAPDH as the internal reference, 2 -ΔΔCt The expression level of the target gene in each group relative to the N group was calculated.

[0097] The primers used in this experiment were designed using Primer 5.0 and synthesized by Shanghai Bioengineering. The species of origin was mouse. The primer sequences are shown in Table 1:

[0098] Table 1 Primer sequences

[0099]

[0100] 6. Western Blot

[0101] To further observe the role of mouse bone marrow mesenchymal stem cell-derived apoptotic bodies in bacterial keratitis in mice, the mice in each group were killed by cervical dislocation and the intact corneal tissue was immediately removed to complete corneal tissue protein extraction. The subsequent steps were the same as the Western Blot detection method in Example 1 to detect the expression of macrophage polarization-related proteins in the corneal protein samples of each group of mice.

[0102] 7. Results

[0103] (1) Modeling results

[0104] The results of corneal opacity score, corneal inflammation score and corneal epithelial healing were as follows: Figure 8 and Figure 9 As shown in Figures A to C, the corneal opacity scores of the PBS control group were significantly higher than those of the BMSCs-ApopBs treatment group on the 3rd, 5th, and 7th day after the model was established, and the differences were statistically significant. The corneal inflammation scores of the PBS control group were significantly higher than those of the BMSCs-ApopBs treatment group on the 3rd, 5th, and 7th day after the model was established, and the differences were statistically significant. The corneal epithelial healing of mice in the BMSCs-ApopBs treatment group was significantly better than that in the PBS control group on the 3rd, 5th, and 7th day after the model was established, and the differences were statistically significant. In summary, this demonstrates that the bacterial keratitis animal model of mice was successfully established in this embodiment. At the same time, compared with PBS pretreatment, ApopBs pretreatment can improve the inflammatory response of bacterial keratitis in mice and promote corneal epithelial healing.

[0105] (2) Paraffin section preparation and HE staining results

[0106] The results are as follows Figure 10As shown, HE staining of corneal sections from mice in the normal group (N group) revealed uniform corneal thickness, tightly packed and neatly arranged epithelial cells, flat surface cells, cuboidal basal cells, and evenly arranged nuclei at the base. Collagen fibers in the corneal stroma were regularly arranged in lamellar patterns, with scattered fibroblasts. Stromal edema and neovascularization were absent. The endothelium consisted of a single layer of flat cells, uniformly distributed and neatly arranged. HE staining of the corneas from mice in the control group (PBS group) revealed localized corneal thickening, disorganized epithelial cell arrangement, and an increased nuclear-to-cytoplasmic ratio, indicating active proliferation. The corneal stroma showed significant edema and thickening, widened intercellular spaces, and numerous inflammatory cell infiltration within the epithelium and stroma. Endothelial cells were partially detached, irregular in morphology, and disorganized. HE staining of the corneas from mice in the drug-treated group (ApopBs group) revealed slight corneal thickening in the central region, with disorganized epithelial cells. The corneal stroma was slightly thickened and edematous, with widened intercellular spaces and scattered inflammatory cell infiltration within the stroma. Endothelial cells were irregularly arranged. On the one hand, the above results further illustrate that the bacterial keratitis animal model in mice was successfully established. At the same time, ApopBs pretreatment can significantly improve corneal edema, loose and disordered stromal layer, and inflammatory cell infiltration caused by modeling.

[0107] (3) Detection results of pro-inflammatory genes, immune regulation-related genes, macrophage M1 (pro-inflammatory phenotype) polarization markers, and macrophage M2 (anti-inflammatory phenotype) polarization markers in each group of mouse samples

[0108] The results are as follows Figure 11 As shown in Figures A to E, two days after modeling, the expression of the proinflammatory gene IL-6 in the corneal tissue of mice in the PBS group was significantly higher than that in the N group. Compared with the PBS group, the expression of immune-regulatory genes IL-10 and TGF-β1 in the corneal tissue of mice in the ApopBs-treated group was significantly increased, while the expression of the proinflammatory gene IL-6 was significantly decreased. Furthermore, the gene expression level of iNOS, a marker of macrophage M1 (proinflammatory phenotype) polarization, in the corneal tissue of mice in the ApopBs-treated group was significantly decreased compared with the PBS group, while the gene expression level of Arg-1, a marker of macrophage M2 (anti-inflammatory phenotype) polarization, was significantly increased. These results suggest that ApopBs exerts a significant anti-inflammatory effect in the treatment of bacterial keratitis in mice. ApopBs can promote the polarization of macrophages in tissues toward the M2 anti-inflammatory phenotype and inhibit the polarization of macrophages toward the M1 pro-inflammatory phenotype, suggesting that BMSCs-ApopBs achieve their anti-inflammatory effect by affecting macrophage polarization.

[0109] (4) Detection results of macrophage polarization-related protein expression in mouse corneal protein samples

[0110] The results are as follows Figure 12 、 Figure 13 A and B in Figure 14As shown in Figures A and B, after the second day of treatment, the expression of iNOS and Arg-1 in samples from the PBS and ApopBs groups was significantly increased compared with that in the N group. Furthermore, the expression of iNOS and CD86 in the PBS group was significantly increased compared with that in the ApopBs group, while the expression of Arg-1 was significantly decreased. After the third and seventh days of treatment, the expression of iNOS in the PBS group was not significantly different from that in the ApopBs group. However, the expression of Arg-1 in the PBS group was still significantly decreased compared with that in the PBS group on the third and seventh days of treatment. These differences were statistically significant. These results indicate that LPS-induced corneal inflammation in mice promotes the infiltration of macrophages in the corneal tissue. However, the PBS group showed an increase in M1 macrophage-related proteins, while the ApopBs group showed an increase in M2 macrophage-related proteins. In addition, the above results also show that the expression of M1 macrophage-related proteins such as iNOS in each group of samples was most significant on the second day after treatment, and then decreased rapidly, and ApopBs treatment can inhibit the transformation of macrophages recruited in corneal tissue to the M1 pro-inflammatory phenotype; the expression of M2 macrophage-related proteins such as Arg-1 in each group of samples gradually increased after treatment, and ApopBs treatment can promote the transformation of macrophages recruited in corneal tissue to the M2 anti-inflammatory phenotype.

[0111] The technical features of the above-described embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0112] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Application of apoptotic bodies derived from bone marrow mesenchymal stem cells in the preparation of drugs for the prevention / treatment of keratitis.

2. The use according to claim 1, characterized in that The method for preparing the apoptotic body comprises: using a chemical induction method to induce bone marrow mesenchymal stem cells to cause apoptosis and generate apoptotic bodies.

3. The use according to claim 1, characterized in that The diameter of the apoptotic body is 180 nm to 210 nm.

4. The use according to claim 1, wherein The drug is used to improve the inflammatory response of keratitis.

5. The use according to claim 1, characterized in that The drug is used for promoting corneal epithelial healing.

6. The use according to claim 1, wherein The dosage forms of the medicine include tablets, sprays, granules, capsules, oral solutions, injections, and suspensions.

7. A drug for preventing / treating keratitis, characterized in that: The drug contains the apoptotic body described in claim 1 as the only effective ingredient.

8. The drug according to claim 7, wherein The drug further includes a pharmaceutically acceptable carrier.

9. The drug according to claim 7, wherein The content of the apoptotic bodies for medicine is 1 wt% to 99 wt%.

10. The drug according to claim 7, wherein The pharmaceutically acceptable carrier includes at least one of a diluent, a binder, a wetting agent, a lubricant, a disintegrant, a solvent, an emulsifier, a solubility aid, a preservative, a pH regulator, an osmotic pressure regulator, a surfactant, a coating material, an antioxidant or a buffer.

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