Application of pretreated mesenchymal stem cells in treatment of corneal alkali burn

Abstract

The invention relates to the technical field of biological medicines, and particularly provides application of pretreated mesenchymal stem cells (MSCs) in preparation of a medicine for promoting corneal alkali burn repair, and the MSCs are pretreated by adopting slow-release H2S to form H2S-MSCs. The H2S-MSCs inhibit corneal ferroptosis by activating an AMPK / Nrf2 / HO-1 pathway, and promote repair and regeneration of corneal tissues. In addition, the invention further provides a pharmaceutical composition containing H2S-MSCs, and the treatment effect of corneal alkali burn repair is further optimized.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of mesenchymal stem cells (MSCs) in preparing a medicine for promoting the repair of corneal alkali burns. Background Art

[0002] Corneal alkali burns, the leading cause of corneal blindness, often lead to serious complications such as persistent epithelial defects, corneal perforation, and limbal stem cell loss. Currently, there is a lack of effective curative treatment options. Traditional corneal transplantation, while the primary means of restoring vision, is limited by bottlenecks such as donor shortages and immune rejection. Therefore, the development of safe and effective new corneal repair treatment options is of great clinical value.

[0003] Recent studies have demonstrated that ferroptosis, a novel form of programmed cell death, plays a key role in ocular surface diseases. This pathological process is characterized by disturbed iron metabolism and iron-dependent lipid peroxidation. The cystine / glutamate antiporter (Solute Carrier Family 7 Member 11, SLC7A11) mediates cystine influx and participates in glutathione (GSH) biosynthesis, while glutathione peroxidase 4 (GPX4) consumes GSH to eliminate lipid peroxides, forming an antioxidant defense system. Notably, ferroptosis has been implicated in the pathological progression of various eye diseases, including glaucoma, dry eye, and age-related macular degeneration. Basic research has shown that the ferroptosis inhibitor ferrostatin-1 eye drops can significantly improve the prognosis of corneal alkali burns, providing a theoretical basis for interventional strategies targeting ferroptosis.

[0004] In the field of regenerative medicine, mesenchymal stem cells (MSCs) have become a hot topic in corneal repair research due to their multidirectional differentiation potential and ease of collection (derived from bone marrow, placenta, fat and other tissues). Existing evidence shows that MSCs improve the microenvironment of the cornea after alkali burns through multiple mechanisms such as promoting epithelial regeneration, inhibiting corneal cell apoptosis and regulating CD68+ macrophage infiltration. However, key issues such as low survival rate of MSCs after transplantation and functional decline during in vitro expansion seriously restrict their clinical translation effect. Based on this, the present invention focuses on the effect of the gas signal molecule hydrogen sulfide (H2S) on cell regulatory function - this endogenous molecule plays an important role in maintaining tissue homeostasis. By constructing an H2S pretreated MSCs delivery system, we explore its innovative strategy in enhancing the efficacy of corneal alkali burn repair.

[0005] In response to the core issues that restrict clinical efficacy, such as a sudden drop in survival rate due to microenvironmental stress after mesenchymal stem cells (MSCs) transplantation and functional decline during in vitro expansion (such as downregulation of paracrine factor expression), the present invention proposes an optimization scheme based on gas signaling molecule regulation. Specifically, this patent pre-treats MSCs with a sustained-release hydrogen sulfide donor, GYY4137 (morpholino-phosphonic acid disulfide, GYY4137), and uses its property of continuously releasing hydrogen sulfide to simulate a physiological concentration H2S environment to activate cell protective signaling pathways. This improves the survival rate and paracrine function of MSCs in harsh microenvironments, enhances the efficacy of MSCs in treating corneal alkali burns from at least three aspects: anti-inflammation, reduction of apoptosis, and inhibition of ferroptosis, and provides new ideas for the treatment of corneal alkali burns. Summary of the Invention

[0006] The present invention demonstrates that pre-treatment of MSCs with slow-release hydrogen sulfide can effectively improve the problems of low survival rate after MSC transplantation and functional impairment after repeated passages, thereby effectively improving the efficacy of MSCs on corneal alkali burns.

[0007] In order to achieve the above-mentioned purpose of the invention, the technical solution provided by the present invention is as follows:

[0008] The present invention provides an application of mesenchymal stem cells (MSCs) in the preparation of a drug for promoting the repair of corneal alkali burns, characterized in that the MSCs are pretreated with slow-release H2S to form H2S-MSCs.

[0009] In the present invention, the H2S-MSCs are transplanted into the corneal alkali burn site by subconjunctival injection.

[0010] In the present invention, the pretreatment uses a slow-release H2S culture medium with a concentration of 0.1-2.5 μmol / L to treat MSCs for 48 hours.

[0011] In the present invention, the pretreatment time is 36-60 hours.

[0012] In the present invention, the slow-release H2S is a water-soluble powder that can slowly release H2S.

[0013] In the present invention, the H2S-MSCs enhance the function of MSCs in inhibiting corneal alkali burn ferroptosis by activating the AMPK / Nrf2 / HO-1 pathway.

[0014] The present invention also provides a pharmaceutical composition for treating corneal alkali burns, characterized by comprising the H2S-MSCs prepared in the above manner and a pharmaceutically acceptable carrier.

[0015] In the present invention, the pharmaceutical composition further comprises a composite protective agent consisting of sodium hyaluronate (0.1-0.3% w / v), heparin (10-50 IU / mL) and vitamin C (50-100 μM).

[0016] The present invention has experimentally verified that the technical solution of pretreating mesenchymal stem cells (MSCs) with a slow-release hydrogen sulfide donor (GYY4137) can effectively improve the problems of low survival rate after MSC transplantation and functional decline after repeated passage, thereby effectively improving the efficacy of MSCs on corneal alkali burns. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In the following description of the drawings and examples, for convenience, the term “mesenchymal stem cells” is abbreviated as “MSCs”; the term “hydrogen sulfide” is abbreviated as “H2S”; the term “hydrogen sulfide preconditioned mesenchymal stem cells” is abbreviated as “H2S-MSCs”; the term “interleukin-1β” is abbreviated as “IL-1β”; the term “interleukin-10” is abbreviated as “IL-10”; the term “interleukin-6” is abbreviated as “IL-6”; the term “solute carrier family 7 member 11 (SLC7A11)” is abbreviated as “SLC7A11”; the term “glutathione peroxidase 4” is abbreviated as “glutathione peroxidase 4”. The term “ferritin heavy chain 1” is abbreviated as “FTH1”; the term “4-hydroxynonenal” is abbreviated as “4-HNE”; the term “AMP-activated protein kinase” is abbreviated as “AMPK”; the term “nuclear factor-E2-related factor 2” is abbreviated as “Nrf2”; and the term “heme oxygenase-1” is abbreviated as “HO-1”.

[0018] Figure 1 Schematic diagram of the effects of different concentrations of GYY4137 (slow-release H2S donor) on MSCs according to an embodiment of the present invention.

[0019] Figure 2 Schematic diagram of the effect of H2S pretreatment on MSCs under nutrient-deficient conditions according to an embodiment of the present invention.

[0020] Figure 3 Schematic diagram of the effect of H2S pretreatment on MSCs under oxidative stress damage according to an embodiment of the present invention.

[0021] Figure 4 Schematic diagram showing that H2S pretreated MSCs promote the repair of corneal alkali burns by inhibiting corneal inflammation and apoptosis according to an embodiment of the present invention.

[0022] Figure 5 Schematic diagram of H2S pretreatment enhancing the effect of MSC in inhibiting corneal alkali burn ferroptosis according to an embodiment of the present invention.

[0023] Figure 6 Schematic diagram of H2S-MSCs activating the AMPK / Nrf2 / HO-1 pathway in vivo according to an embodiment of the present invention. DETAILED DESCRIPTION

[0024] Hereinafter, the present invention will be described in detail based on exemplary embodiments, but the present invention is not limited to these embodiments. The present invention is embodied in various forms as follows, but should not be construed as being limited to the exemplary embodiments set forth herein. Therefore, the detailed description and embodiments of the present invention will convey the scope of the present invention to those of ordinary skill in the art and are to be construed as being within the scope of the present invention.

[0025] According to the present invention, the preparation of H2S-MSCs is achieved by pre-treating MSCs with slow-release H2S. The slow-release H2S is a water-soluble powder that can slowly release H2S. The cells are treated with a slow-release H2S culture medium at a concentration of 0.1-2.5 μmol / L, preferably 1 μmol / L, for 36-60 hours, preferably 48 hours. This pre-treatment process can significantly enhance the anti-ferroptosis function of MSCs and exert their role in corneal alkali burn repair by activating the AMPK / Nrf2 / HO-1 pathway.

[0026] In the present invention, H2S-MSCs are transplanted into the corneal alkali burn site via subconjunctival injection. This transplantation method ensures that the cells act directly on the damaged corneal tissue, improving the therapeutic effect.

[0027] The present invention also provides a pharmaceutical composition for treating corneal alkali burns, comprising the H2S-MSCs and a pharmaceutically acceptable carrier. Furthermore, the pharmaceutical composition also includes a composite protective agent consisting of sodium hyaluronate (0.1-0.3% w / v), heparin (10-50 IU / mL), and vitamin C (50-100 μM). This composite protective agent can provide a favorable microenvironment for the H2S-MSCs, enhancing cell survival and function.

[0028] Example 1 Establish a culture system for H2S pretreated MSCs.

[0029] Experimental method: H2S pretreatment of MSCs

[0030] Mesenchymal stem cells (MSCs) were cultured at a rate of 5×10 4 cells / cm 2 Cells were seeded at a density of 100 μg / L in a six-well plate and cultured in α-MEM complete medium containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator. When the cell confluence reached 80%-90%, the original culture medium was discarded and the cells were gently washed three times with 1× sterile phosphate buffered saline (PBS) pre-warmed to 37°C (1 mL each time for 30 seconds). Fresh complete culture medium containing gradient concentrations of GYY4137 (0 μmol / L, 0.5 μmol / L, 1.0 μmol / L, 1.5 μmol / L, and 2.0 μmol / L) was then added. Three replicates were set up for each group, and the following operations were performed at 24 and 48 hours:

[0031] Morphological observation: The cell morphology was photographed under an inverted optical microscope (200×), and the extension of pseudopodia and the degree of vacuolization were recorded.

[0032] Protein sample preparation: aspirate the culture medium, wash with pre-cooled PBS, add RIPA lysis buffer (containing 1% protease inhibitors), lyse on ice for 30 minutes, centrifuge at 4°C (12000 rpm, 15 minutes), collect the supernatant, and quantify the protein concentration by BCA method.

[0033] Functional detection: Western blot analysis of apoptosis markers Cleaved-caspase 3 and anti-inflammatory factor IL-10, β-actin as an internal control.

[0034] Experimental method: Construction of MSC oxidative stress injury model

[0035] Mesenchymal stem cells (MSCs) were collected at a rate of 5×10 4 cells / cm 2Cells were seeded at a high density in 6-well plates in α-MEM complete medium supplemented with 10% fetal bovine serum (FBS) and cultured at 37°C, 5% CO2 until the cells reached 80%-90% confluence. The culture medium was discarded and the cells were gently washed three times with prewarmed (37°C) sterile 1× PBS (2 mL each, 20 seconds) to remove residual serum. Cells were pretreated with complete medium supplemented with 1.0 μmol / L GYY4137 for 48 hours to simulate a slow-release H2S microenvironment.

[0036] After pretreatment, the cells were washed with PBS three times and replaced with complete culture medium containing 1 mmol / L H2O2. The cells were cultured for 24 or 48 hours to establish an oxidative stress injury model.

[0037] After terminating the culture, the cells were washed with pre-cooled PBS, gently scraped with a cell scraper, lysed on ice with RIPA lysis buffer (containing 1 mM PMSF) for 30 minutes, and centrifuged (12000 rpm, 4°C, 15 minutes) to collect the supernatant.

[0038] Western blot was used to detect apoptosis-related proteins Bax, Bcl-2, and Cleaved Caspase-9.

[0039] Experimental methods: Construction of MSC nutrient deprivation model

[0040] MSCs were seeded under the same conditions as above. When the cells reached 80%-90% confluence, they were washed three times with PBS and pretreated for 48 hours in complete culture medium containing 1.0 μmol / L GYY4137. The pretreatment medium was discarded, and after washing with PBS, the cells were replaced with reduced-serum medium containing only 1% FBS and cultured for an additional 24 or 48 hours to simulate the nutrient-depleted microenvironment in vivo.

[0041] Cell proteins were collected in the same way, and the expression levels of Bax, Bcl-2 and Cleaved Caspase-9 were detected by Western blot to analyze the regulatory effect of H2S pretreatment on cell apoptosis.

[0042] Experimental method: Western blot

[0043] Cells were lysed on ice using RIPA lysis buffer (containing protease / phosphatase inhibitors). Protein concentration was determined by BCA assay and adjusted to 2 μg / μL. A 30 μg protein sample was subjected to 10% SDS-PAGE gel electrophoresis (constant voltage 80 V, 30 minutes for stacking gel; 120 V, 60 minutes for separating gel) and wet-transferred to a PVDF membrane (300 mA, 90 minutes).

[0044] Block with 5% skim milk powder (prepared in TBST) at room temperature for 1 hour; incubate with primary antibody at 4°C overnight (dilution ratio as above), wash three times with TBST (10 minutes each); incubate with HRP-conjugated secondary antibody (1:5000) at room temperature for 1 hour, wash with TBST, and then develop with ECL chemiluminescence. ChemiDoc TM Images were acquired by the MP imaging system, and grayscale values were analyzed by Image Lab 6.0 software.

[0045] All experimental data are expressed as mean ± standard deviation (Mean ± SD), and significant differences are indicated by: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0046] Experimental results

[0047] Figure 1 The figure shows the regulatory effect of different concentrations of GYY4137 on MSCs apoptosis and anti-inflammatory factor expression. Figure 1 Figure (A) shows that to screen for an appropriate pretreatment concentration of GYY4137, MSCs were stimulated with a gradient of concentrations (0, 0.5, 1.0, 1.5, and 2.0 μmol / L) for 24 and 48 hours. Morphological observations revealed that in the high-concentration group (≥1.0 μmol / L), significant cell shedding occurred after 24 hours of stimulation, and the apoptotic area expanded after 48 hours. In the low-concentration group (0.5 μmol / L), the cells maintained intact morphology, with no obvious apoptotic features. In the 1.0 μmol / L group, localized cell detachment (a shedding rate of approximately 15%) occurred after 24 hours of stimulation. After 48 hours of culture, the remaining cells proliferated to fill the vacant areas, indicating that MSCs can tolerate sustained stimulation at this concentration (Scale bar: 50 μm).

[0048] Figure 1 B and 1C show the results of Western blot detection and quantitative analysis, which showed that GYY4137 induced MSC apoptosis in a concentration-dependent manner. At the same time, the anti-inflammatory factor IL-10 increased significantly after MSC stimulation with 1 μmol / L GYY4137 for 48 hours and decreased with the increase of GYY4137 concentration.

[0049] Considering the smaller apoptosis and the maximum IL-10 expression, 1 μmol / L GYY4137 pretreatment of MSCs for 48 hours was the optimal condition.

[0050] Figure 2 The results show that H2S pretreatment regulates MSCs apoptosis under nutrient-deficient conditions. Figure 2A simulates the ischemic and hypoxic microenvironment after transplantation. A nutrient deprivation model was constructed using low serum (1% FBS) culture medium. The expression of apoptosis-related proteins in MSCs pretreated with H2S (1.0 μmol / L GYY4137, 48 hours) and untreated MSCs was compared and analyzed. Figure 2 B: Quantitative analysis of caspase-9 in MSCs without pretreatment and H2S pretreatment after culture under nutrient-deprivation conditions for 24 and 48 h. Figure 2 C shows the quantitative analysis of the bax / bcl2 ratio in untreated and H2S-pretreated MSCs after culture under nutrient-deprivation conditions for 24 and 48 h.

[0051] The above experimental results show that H2S pretreatment inhibits the expression of MSC pro-apoptotic protein caspase 9 and the ratio of bax / bcl2, and significantly improves the survival of MSCs under nutrient deprivation for 24 hours and 48 hours.

[0052] To simulate the oxidative stress microenvironment caused by increased reactive oxygen species production after transplantation, an in vitro model of oxidative stress damage was constructed using 1 mmol / L H2O2. MSCs pretreated with 1.0 μmol / L GYY4137 for 48 hours and untreated MSCs were placed in a culture medium containing 1 mmol / L H2O2 and then cultured in a cell culture incubator for 24 and 48 hours. Figure 3 A shows the WB results of caspase 9, bax, and bcl2 in untreated and H2S-pretreated MSCs. Figure 3 B: Quantitative analysis of caspase 9 in untreated and H2S-pretreated MSCs after exposure to 1 mmol / L H2O2 for 24 and 48 hours. Figure 3 C shows quantitative analysis of the bax / bcl2 ratio in untreated and H2S-pretreated MSCs after 24 and 48 hours of exposure to 1 mmol / L H2O2. These results indicate that H2S pretreatment inhibits the expression of the pro-apoptotic protein caspase 9 in MSCs and the bax / bcl2 ratio, significantly improving the survival of MSCs after 24 and 48 hours of oxidative stress injury.

[0053] The above experimental results show that the optimal concentration of GYY4137 for MSC pretreatment is 1.0 μmol / L and the treatment time is 48 hours. Under this pretreatment condition, the expression level of the anti-inflammatory factor IL-10 in MSC is the highest, and the MSC is protected from damage caused by nutrient deprivation and oxidative stress.

[0054] Example 2 The dynamic experiment confirmed that H2S-MSC can promote the repair of corneal damage. Experimental method to construct mouse horn Membrane damage model

[0055] General anesthesia was induced with 2.5% (w / v) tribromoethanol (Avertin) at a dose of 240 mg / kg injected intraperitoneally, combined with 0.5% proparacaine eye drops for topical ocular anesthesia. A 2 mm diameter circular filter paper was soaked in 1.0 N NaOH solution for 30 seconds. Excess liquid was removed, and the paper was precisely applied to the center of the cornea for 20 seconds, with microtweezers gently pressing the edge of the filter paper to ensure full contact. After removal of the filter paper, the ocular surface was immediately rinsed with prewarmed (37°C) sterile PBS (30 mL) at a constant rate for 60 seconds to neutralize any residual alkaline substances.

[0056] After modeling, MSC and H2S-MSC treatment groups were injected subconjunctivally with 10ul of 1*10 5 MSCs or 1*10 5 H2S-MSCs were injected with PBS, and the control group was injected with an equal volume of PBS.

[0057] Experimental methods H&E staining

[0058] Sample collection: On days 7 and 14 after treatment, mice were killed by cervical dislocation, and the eyeballs were completely removed and fixed in 4% paraformaldehyde (prepared in PBS, pH 7.4) for 24 hours.

[0059] Dehydration and embedding: Dehydrate with graded ethanol (70%→80%→90%→100%, 1 hour each level), clear with xylene, and then embed in paraffin and prepare 5 μm sagittal sections.

[0060] Staining and observation: Hematoxylin staining for 5 minutes, 1% hydrochloric acid ethanol differentiation, eosin counterstaining for 2 minutes, neutral gum sealing, and photos under an optical microscope to observe the integrity of the corneal epithelium and matrix structure.

[0061] Experimental Methods Immunofluorescence staining

[0062] Sample processing: On the 7th day after treatment, the eyeballs were collected and fixed with 4% paraformaldehyde for 24 hours, dehydrated with gradient sucrose (10%→20%→30%, 24 hours each), and embedded in OCT before preparing 8 μm frozen sections.

[0063] Antigen retrieval and blocking: permeabilization with 0.1% Triton X-100 for 10 minutes, and blocking with 5% goat serum (in PBS) at room temperature for 1 hour.

[0064] Antibody incubation: primary antibody (such as anti-GPX4, 1:200) was incubated at 4°C overnight; Alexa Fluor 488 labeled secondary antibody (1:500) was incubated at room temperature in the dark for 2 hours; DAPI (1 μg / mL) was used for nuclear staining for 5 minutes, and anti-fluorescence quencher (ProLong TM Gold) coverslips.

[0065] Imaging analysis: Images were collected using a laser confocal microscope, and fluorescence intensity was quantified.

[0066] Experimental Method: Western Blot

[0067] On the 7th day after treatment, the mouse corneal tissue was precisely isolated and quickly ground into powder in liquid nitrogen. Pre-cooled RIPA lysis buffer (containing 1% protease inhibitor cocktail and 1 mM PMSF) was added and lysed on ice for 30 minutes. The supernatant was collected by high-speed centrifugation at 4°C (12,000×g, 15 minutes). The sample concentration was determined using a BCA protein quantification kit (standard curve R 2 >0.99). Equal amounts of protein (30 μg / well) were mixed with 4× Laemmli buffer and denatured in a metal bath at 95°C for 10 minutes. SDS-PAGE electrophoresis was then performed. The protein was transferred to a PVDF membrane using wet transfer (300 mA constant current, 90 minutes) and blocked with 5% skim milk powder (prepared with TBST) at room temperature for 1 hour. The primary antibody was incubated at 4°C overnight, washed with TBST, and the secondary antibody was incubated at room temperature using ChemiDoc. TM MP imaging system captures protein bands.

[0068] Experimental methods and statistical analysis

[0069] Statistical analysis All values are expressed as mean ± SD, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0070] Experimental results

[0071] The experiments in this example confirmed that H2S-MSC can better promote corneal damage repair by anti-inflammation, reducing apoptosis and inhibiting ferroptosis.

[0072] On the one hand, the experimental results showed that H2S pretreatment of MSCs improved corneal structure and inhibited corneal inflammation and apoptosis.

[0073] Figure 4 A shows photographs of the anterior segment of the mouse eyes taken 7 and 14 days after corneal alkali burn. The PBS group exhibited severe corneal opacity on days 7 and 14, with the iris unable to be seen. The results indicate that the H2S-MSC group had a stronger inhibitory effect on corneal opacity than the untreated MSC group.

[0074] Figure 4 B shows H&E staining, which reveals that the normal cornea has a regularly arranged structure, while alkali burns destroy the corneal structure and reveal a large number of inflammatory cells infiltrating the corneal stroma. The results show that compared with untreated MSCs, H2S-MSCs have a stronger inhibitory effect on inflammatory cell infiltration and a more regularly arranged corneal structure.

[0075] To evaluate corneal inflammation, corneal IL-1β immunofluorescence staining was performed, and the results were as follows Figure 4 C. A large number of IL-1β+ cells were detected in the PBS group, in stark contrast to the low number of IL-1β+ cells in the MSC-treated group. The results showed that H2S-MSCs exhibited a significant inhibitory effect on IL-1β expression compared with untreated MSCs.

[0076] Figure 4 C and 4D show the results of Western blot detection and quantitative analysis, which showed that the expression of inflammatory factors in corneal tissue changed significantly on the 7th day after treatment. The results showed that H2S-MSC could better inhibit the protein level of pro-inflammatory cytokine IL-6 and upregulate the expression of anti-inflammatory cytokine IL-10.

[0077] Figure 4 F shows the Western blot results of the expression of apoptotic proteins cleaved-caspase 3, bax, and bcl2 in the cornea 7 days after MSCs treatment. Figure 4 G. Quantitative analysis of cleaved-caspase 3 and bax / bcl2 in the cornea. The results showed that the level of the apoptotic protein cleaved-caspase 3 and the bax / bcl2 ratio in the cornea showed an increasing trend after corneal alkali burn. Compared with untreated MSCs, H2S-MSC treatment significantly reduced the expression of apoptotic proteins in the cornea.

[0078] On the other hand, the experimental results showed that H2S pretreatment of MSCs could inhibit corneal alkali burn ferroptosis.

[0079] Figure 5 A shows the results of Western blot detection of ferroptosis markers SLC7A11, GPX4, 4-HNE, and FTH1 in the cornea 7 days after corneal alkali burn. Figure 5 B shows the quantitative analysis of SLC7A11, GPX4, 4-HNE, and FTH1 in the cornea. The experimental results showed that corneal alkali burn inhibited the expression of GPX4 and SLC7A11 and promoted the expression of FTH1 and 4-HNE. H2S-MSC treatment inhibited the decrease in SLC7A11 and GPX4 proteins and the increase in FTH1 and 4-HNE proteins.

[0080] The experimental results further showed that H2S-MSCs can activate the AMPK / Nrf2 / HO-1 pathway in vivo. To further explore the specific mechanism by which H2S pretreatment of MSCs inhibits corneal alkali burn ferroptosis, Figure 6A shows that on day 7 after corneal alkali burn, the pAMPK / AMPK and Nrf2 protein levels in the PBS group were significantly lower than those in the other groups. In addition, the expression level of HO-1, a known downstream target of Nrf2, was also the lowest in the PBS group. Figure 6 B shows the quantitative analysis results of pAMPK / AMPK, Nrf2, and HO-1 in the cornea. The results indicate that H2S-MSC treatment upregulated the AMPK / Nrf2 / HO-1 signaling pathway.

[0081] The above experimental results show that compared with untreated MSCs, subconjunctival injection of H2S-MSCs has a more significant effect in inhibiting corneal alkali burn inflammation, apoptosis, and ferroptosis, thereby better promoting corneal damage repair. The potential mechanism may be through the activation of the AMPK / Nrf2 / HO-1 signaling pathway.

[0082] It should be understood that the exemplary embodiments described herein should be considered only in a descriptive sense and not as limiting the scope of the invention. The description of the technical solutions and embodiments of the present invention is intended to provide a comprehensive understanding of the present invention, but should not be construed as limiting the present invention to these specific embodiments. Those skilled in the art can, based on the teachings of the present invention, make appropriate modifications or adjustments to the embodiments without departing from the spirit and scope of the present invention.

[0083] The description of the features or aspects in each embodiment should generally be considered to be feasible for similar features or aspects in other embodiments, unless it is explicitly stated that they are not feasible. The specific technical means, parameter selection or experimental conditions mentioned in the present invention are only for illustrative purposes and should not be regarded as the sole limitation of the technical solution of the present invention. Without violating the basic principles of the present invention, those skilled in the art may replace, combine or optimize these features or aspects to achieve other specific embodiments of the present invention.

[0084] In addition, some technical features or operating steps mentioned in the present invention may not be described in detail in some embodiments, but this does not mean that these features or steps are not available or applicable in other embodiments. On the contrary, those skilled in the art can flexibly select and apply these technical features or operating steps according to specific needs and technical background to achieve the intended purpose of the present invention.

Claims

1. A use of mesenchymal stem cells (MSCs) in the preparation of a drug for promoting the repair of corneal alkali burns, characterized in that: The MSCs are pretreated with slow-release H2S to form H2S-MSCs.

2. The use according to claim 1, characterized in that The H2S-MSCs are transplanted into the corneal alkali burn site by subconjunctival injection.

3. The use according to claim 1, characterized in that The pretreatment uses a slow-release H2S culture solution with a concentration of 0.1-2.5 μmol / L.

4. The use according to claim 3, characterized in that Pretreatment times shown range from 36 to 60 hours.

5. The use according to claim 1, characterized in that The sustained-release H2S is a water-soluble powder that can slowly release H2S.

6. The use according to claim 1, characterized in that The H2S-MSCs enhance the function of MSCs in inhibiting corneal alkali burn ferroptosis by activating the AMPK / Nrf2 / HO-1 pathway.

7. A pharmaceutical composition for treating corneal alkali burns, characterized in that The method comprises the H2S-MSCs for use according to any one of claims 1 to 6, and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, wherein The invention further comprises a composite protective agent consisting of sodium hyaluronate (0.1-0.3% w / v), heparin (10-50 IU / mL) and vitamin C (50-100 μM).

Images